Doctor of Philosophy in Biochemistry

Molecular Studies on Plant Based Cyclotides for Protein- Protein Interaction

By Zahid Mushtaq M.Phil. (UAF)

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY IN BIOCHEMISTRY

DEPARTMENT OF BIOCHEMISTRY

FACULTY OF SCIENCES UNIVERSITY OF AGRICULTURE, FAISALABAD PAKISTAN 2016 DECLARATION

I hereby declare that the contents of the thesis, “Molecular studies on plant based cyclotides for protein-protein interaction” are product of my own research and no part has been copied from any published source (except the references, standard mathematical or genetic models/equations/formulae/protocols etc). I further declare that this work has not been submitted for award of any diploma/degree. The University may take action if the information provided is found inaccurate at any stage.

Zahid Mushtaq 2005-ag-238

The Controller of Examinations, University of Agriculture, Faisalabad.

“We the Supervisory Committee, certify that the contents and form of thesis submitted by Zahid Mushtaq, Regd. No. 2005-ag-238, have been found satisfactory and recommend that it be processed for evaluation, by the External Examiner (s) for the award of degree”.

SUPERVISORY COMMITTEE

1. CHAIRMAN (Prof. Amer Jamil)

2. MEMBER (Prof. Tahira Iqbal )

3. MEMBER (Dr. Nisar Ahmed) ACKNOWLEDGMENT

Words are bound and knowledge is limited to praise Allah, the omnipotent, the beneficent and merciful. Peace and blessings be upon Holy Prophet Muhammad (SAW), the everlasting source of guidance and knowledge for humanity. With genuine humanity, I acknowledge your aid, God. Please bless this work with your acceptance.

I have a pleasure to ensure my sincere gratitude and deepest thanks to Prof. Amer Jamil, whose stimulating supervision, guidance and support made this work possible. I heartily thank him very much for his valuable help and for his kindness.

I express my gratitude to Dr. Julio A Camarero, who guided me to the fascinating world of cyclotides during my research work in University of Southern California, Los Angeles, CA, USA. His support, encouragement, inspiring attitude and enthusiasm were impressive during IRSIP, HEC scholarship. The support, help and company of the whole group are appreciated, especially Dr. Jagadish Khrishnappa and Dr. Teshome for their help in research work.

I wish to thank Prof. Tahira Iqbal, Department of Biochemistry and Dr. Nisar Ahmad, Centre of Agricultural Biochemistry and Biotechnology (CABB) for their guidance, encouragement and help throughout my PhD studies.

I am also thankful to my genius friends and fellows Ghulam Mustafa, Muhammad Naeem, Dr. Kashif Jilani and Dr. Muhammad Shahid for their love providing amenities and friendship.

Many thanks are due to my dearest family for giving me so much joy and happiness outside lab. Special thanks to my brothers M. Awais, Shahid Mushtaq and all my beloved near ones for their prayers, guidance and best wishes at each and every fraction of my life.

I owe immense feelings of love and thanks for my affectionate mother and father, as their prayers are always behind my each success and my loving sisters, my fiancy, in laws and bhabhies for their continuous encouragement, untiring efforts and their patience while I did this work.

Zahid Mushtaq TABLE OF CONTENTS

Sr. No. TITLE Page No. Chapter 1 Introduction 1-9 1.1. Bioactive peptides and proteins 1 1.2. Cyclic peptides and cyclotide 2 1.3. Sources and distribution of bioactive cyclotides 3 1.4. Structural features of Cyclotide 3 1.5. Genetics of cyclotides 4 1.6. Candidates of enzymatic synthesis of cyclotides in plants 5 1.7. Production of recombinant Cyclotide 6 1.8. Labeling cyclotide using unnatural amino acid Uaa 7 1.9. Research objectives 8 Chapter 2 Review of literature 10-51 2.1. History of peptide 10 2.2. Cyclic peptides as stable bioactive compounds 10 2.3. Types of Cyclic peptides/proteins 12 2.4. Sources of cyclic peptides 14 2.4.1. Terrestrial plants 15 2.4.2. Marine plant 16 2.5. Microbial cyclic antimicrobial peptides 16 2.6. Food derived peptides 17 2.6.1 Other examples and functions of cyclic peptides 18 2.7. Cyclotides 21 2.7.1. Discovering Cyclotides 21 2.7.2. Structure of cyclotides 23 2.7.3. The Möbius, bracelet and trypsin inhibitor cyclotides 25 2.7.4. Cyclotide isolations 26 2.7.5. Cyclotides bioactive action and Mechanisms 31 A bioinformatics based approach for Cyclotides variants 2.8. 35 and features 2.8.1. CyBase 36 2.9. Candidate Genes invovlved in cyclotide synthesis 37 2.9.1. Asparginyl Endopeptidases (AEP) 37 2.9.2. Protein Disulphide Isomerases (PDI) 38 2.10. Production strategies of Cyclotide 40 2.10.1. Cyclotide expression in yeast cells 41 Engineered scaffold of cyclotides with grafted epitopes for 2.10.2. 42 enhanced bioactive role 2.11. Labeling of Cyclotide 43 Incorporation of non-natural amino acids by orthogonal t-RNA 2.11.1. 43 in peptides 2.11.2. Click chemistry for proteins labeling 45 2.11.3. Labeling Cyclotides 47

2.12. The Future of peptide-based drugs 50 Chapter 3 Materials and methods 52-78 SECTION-I 52 3.1. Screening of plant extracts and cyclotide for bioactive potential 3.1.1. Selection of plant material 52 3.1.2. Preparation of plants extracts 53 3.1.3. Proteinase K treatment 53 3.1.4. Protein estimations 53 3.2. Antioxidant studies 53 3.2.1. Total Phenolic contents (TPC) 54 3.2.2. Total Flavonoid contents (TFC) 54 3.2.3 Reducing power assay 55 3.2.4. DPPH scavenging assay 55 3.3. Determination of DNA damaging protection activity 56 Evaluation of antimicrobial activity crude extract and its 3.4. 57 polar fractions 3.5. Formation and hydrolysis of biofilm potential 58 Cytotoxicity of plant extracts/MCOTI-I by Hemolytic 3.6. 59 Activity Evaluation of thrombolytic activity of protein/peptide 3.7. 60 extract 3.8. Ames test or mutagenicity test 60 SECTION-II 3.9. 63 Cyclotide Genes isolation studies 3.9.1. DNA isolation 63 3.9.2. PCR amplification 63 3.9.3. PCR product extraction and purification 65 3.9.4. Plasmid DNA isolations 65 3.9.5. Sequencing and bioinformatics analysis of the sequences 65 SECTION-III

(completed in USC, Los Angeles, USA) 3.10. 66 Fluorescent labeling of cyclotide MCOTI-I proteins for studying protein-protein interactions 3.10.1. Instrumentation and materials used 66 3.11. Synthesis of DBCO-AMCA 67 3.12. Cloning of E. coli expression plasmids 67 3.13. Bacterial expression and purification 69 3.14. Expression and purification of intein precursor 1b 70

Invitro labeling of Texas red Succinamyl ester with Lys- 3.15. 72 MCOTI-I 3.16. In vitro labeling of MCoTI-AziF with DBCO-TxRD 73 3.17. Expression of trypsin-S195A-EGFP 73 Measurement of affinity constant between trypsin and 3.18. 74 TxRD-labeled MCoTI-AziF Western blot based optimization induction conditions of 3.19. 74 inteins expression for MCOTI-I production 3.20. Expression of cyclotide MCOTI-I in yeast cells (Published). 76 Results and discussions

Section –I Chapter 4 79-174 Bioactive potential of indigenous cyclotide bearing plants along with MCOTI-I. Extraction of plant extracts with proteins and other 4.1. 80 photochemical using two different buffer systems. 4.1.1 Protein estimation by Bradford method 82 4.2. In vitro antioxidant potential of selected medicinal plants 83 4.2.1. Total phenolic contents (TPC) 83 4.2.2. Total flavonoids contents (TFC) 85 4.2.3. Reducing power assay 86 4.2.4. DPPH radical scavenging assay 88 4.2.5. DNA damage protection assay 90 4.3. Antimicrobial activity of plant extract 93 4.4. Biofilm formation inhibition/hydrolysis 98 4.5. In-vitro cytotoxicity assay 100 4.6. Mutagenicity assay by Ames test 102 4.8. Thrombolytic assay 105 Section –II 108 4.9. Isolation of Cyclotide genes from selected local plants Section – III

(completed in USC, Los Angeles, USA) 4. 10. 117 Labeling of MCOTI-I cyclotide with florescent dye Texas Red for studying protein-protein interaction studies HPLC analysis of Cu++ free click chemistry Reaction of Texas 4.10.1. 120 red and DBCO amine and Texas Red DBCO amine

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4.11. Cloning and expression of wild type MCOTI-I 123 Labeling of MCOTI-I-AziF with Texas Red for protein- 4.12. 132 protein interaction studies Labeling of wild type (WT) MCOTI-I-Lys-NH with Texas 4.13. 2 138 Red succinimyl ester Preparation of trypsin-sepharose beads for capturing MCOTI-I 4.13.1. 141 or MCOTI-I-AziF Optimization and HPLC Monitoring of Cu ++ free click reaction 4.14.1 148 in buffered Guandium-HCl Mass spectrophotometric analysis of each reactant and product 4.14.2. of azido and alkyne reaction for optimization of buffered 151 GdmHCl in future labeling MCOTI-I-AziF Expression and quantification of MCOTI-I-AziF at large scale 4.14.3. 153 for labeling reaction after optimization steps 4.14.4. Labeling reaction of MCOTI-I-AziF : Texas Red-DBCO 159 HPLC Purification and M/S confirmation of labelled peptide 4.14.5. 161 MCOTI-I-Azide-TxRD 4.14.6. Protein-protein interaction studies based on FRET analysis. 162 Expression optimization of Wild Type MCOTI-I 4.15. 168 production (Wester blot analysis) Expression of Cyclotide MCOTI-I in yeast cells (Published, 4.16. 169 Jagadish et al., 2015) Summary 175-177 Literature cited 178-200 Different antioxidant Activities of leaf and seed extracts of Appendix-I selected medicinal plants prepared in PBS and protein 201-202 extraction buffer with and without proteinase K treatment. Antibacterial and biofilm formation inhibition activity of different plants extracts prepared in Protein extraction buffer PEB (with and without pretreatment of Proteinase K enzyme), Appendix- Phosphate buffer saline PBS and purified Cyclotide MCOTI-I 204-205 II protein (with and without pretreatment of Proteinase K enzyme) against Gram positive (Staphylococcus aureus and Bacillus subtilis strains) and gram negative (Escherichia coli and Pasturella multocida strains) Hemolytic activity (Percentage) of selected plants prepared in Appendix- PBS and protein extraction buffer with and without treatment of 207 III proteinase K enzyme Percentage mutagenicity by Ames test of selected plant extracts Appendix- prepared in PBS and protein extraction buffer (PEB) with and 209-210 IV without treatment of proteinase K enzyme Percentage thrombolytic activity of selected plants and Appendix- MCOTI-I prepared in protein extraction buffer with and 212 V without treatment of proteinase K enzyme Appendix- The characters predicted through different bioinformatics tools 214

VI of Cliotide sequence Composition of amino acids with number and molecular Appendix- percentage (135 amino acids) for complete CDS vs cyclotide 215 VII domain only (29 residues)

LIST OF FIGURES

Pages Figure No. Title No. Cyclic Cysteine Knot (CCK) framework of cyclotide MCoT-I 1.1 4 with 6 different loops and disulphide linkages. 1.2 Overview of Cyclotide labeling with Texas red dye. 9 A general structural representation of a cyclic structure of 2.1 10 cyclotide Structural scaffold and features of Cyclotide with loops 2.2 24 presented in different ways by Pedesrson et al., (2012) 2.3 The in vivo biosynthesis of cyclotides 41 2.4 A Cu free click reaction between an azide and Alkyne 46 Schematic illustration of the miniprotein scaffold approach to 2.5 51 peptide-based drug design. Standard curve for total phenolic contents using gallic acid as 3.1 54 standard where concentration is taken in mg/g of gallic acid. Standard curve for total flavonoid contents using catechin as 3.2 55 standard Protein estimation by Bradford assay of plant extracts prepared 4.1 83 in two different buffers (A graphical representation). Total phenolic contents of selected plants using two different 4.2 84 buffer system Total flavonoid contents of selected plants using two different 4.3 86 buffer system Percentage Reducing Power activity of selected plant extracts 4.4 87 using two different buffer systems. Percentage DPPH scavenging activity of selected plants using 4.5 89 two different buffer systems. DNA damage protection assay for selected plants using pBR322 4.6 91 plasmid DNA. DNA damage protection assay for selected plants using 4.7 92 ctDNA(calf thymus DNA) Antibacterial activity of different plants extracts prepared in Protein extraction buffer and purified Cyclotide MCOTI-I protein with and without proteinase K treatment against Gram 4.8 95 positive (Staphylococcus aureus and Bacillus subtilis strains.) and Gram negative (Escherichia coli and Pasturella multocida strains). Antibacterial activity of different plants extracts prepared in and Purified Cyclotide MCOTI-I protein with and without proteinase 4.9 K treatment against Gram positive (Staphylococcus aureus and 96 Bacillus subtilis strains.) and Gram negative (Escherichia coli and Pasturella multocida strains). 4.10 Antibacterial activity of different plants extracts prepared in 97

Protein extraction buffer (with and without pretreatment of Proteinase K enzyme), Phosphate buffer saline PBS and purified Cyclotide MCoTI-I protein (with and without pretreatment of Proteinase K enzyme) against Gram positive (Staphylococcus aureus and Bacillus subtilis strains) and gram negative (Escherichia coli and Pasturella multocida strains) Biofilm formation inhibition of protein extracts prepared in 4.11 protein extraction buffer and purified MCOTI-I cyclotide by 99 petri plate method with slight modification Percentage Biofilm formation inhibition of protein extracts 4.12 prepared in protein extraction buffer and purified MCOTI-I 100 cyclotide by microtiter plate assay method. Percent hemolytic activity of selected plants prepared in PBS 4.13 and protein extraction buffer with and without treatment of 101 proteinase K enzyme Percent mutagenicity by Ames test of selected plant extracts 4.14 prepared in PBS and protein extraction buffer (PEB) with and 103 without treatment of proteinase K enzyme Representation of Ames test of mutagenicity at selected plant 4.15 extracts prepared in PBS and protein extraction buffer (PEB) 104 with and without treatment of proteinase K enzyme Percentage thrombolytic activity of selected plants and MCOTI- 4.16 I prepared in protein extraction buffer with and without 106 treatment of proteinase K enzyme General representation of thrombolytic activity of selected 4.17 plants and MCOTI-I prepared in protein extraction buffer with 106 and without treatment of proteinase K enzyme 4.18 PCR amplification of cyclotide genes from Viola plants 108 Genomic DNA isolation and Isolation of Panitide gene from 4.19 110 panicum plant Isolation of Cliotide gene i.e. cyclotide genes from Clitoria 4.20 111 plants. Cliotide gene of 683bp submitted in Genbank (accession 4.21 112 number KP889219) CDS of Cliotide gene with protein Translation and its 4.22 113 comparison Prediction of signal peptide with the CDS by signal-4.1 4.23 114 Prediction (euk networks) sequence 4.24 Results of conserved domains search from NCBI. 115 Probabilities of Trans membrane domains ( 4.25 116 http://www.cbs.dtu.dk/services/TMHMM) 4.26 Possible Motifs present in the cliotide sequence. 117 4.27 HPLC analysis of pure texas red succinimyl ester 121 4.28 HPLC analysis of DBCO-Amine pure 121 HPLC analysis results of the click chemistry reaction between 4.29 122 Texas Red and DBCO amine

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4.30 M/S of both DBCO amines and TxRd-DBCO amines 123 4.31 Constructs pASK and pTXB1 124 PCR amplification, plasmid DNA isolation, restriction and 4.32 125 ligation of MCOTI-I in pTXB1 and pASK vectors SDS-PAGE confirmation of inteins cleaved during MCOTI-I 4.33 126 expression and cyclization 4.34 HPLC of pASK product MCOTI-I after trypsin capture 127 4.35 SDS-PAGE results showing EPL based expression of MCOTI-I 128 Analytical HPLC showing progress for GSH based (in vitro) 4.36 129 cyclization/folding of intein-fusion linear precursors A to C. HPLC of captured MCOTI-I for checking trypsin elutions after 4.37 130 GdmHCl to detach till no protein remains behind. 4.38 Mass of purified MCOTI-I from EPL method 131 pVLOmeRS and pET28a constructs used in expression of 4.39 133 MCOTI-I-AziF LC/MS analysis of expressed mutant MCOT-I-AziF captured on 4.40 134 trypsin beads LC/MS analysis results showing reaction of Texas Red-DBCO 4.41 135 amine dye with MCOTI-I-AziF 4.42 SDS-PAGE results of inteins expressed for MCOTI-I-AziF 137 4.43 HPLC based attempt to pool out all the MCOTI-I-AziF-TxRD 137 Schematic representation of ester dye with amino group of a 4.44 139 peptide. HPLC of MCOTI-I-Lys-TxRD labeled in phosphate (PO4) 4.45 139 buffer and in the presence of DMF 4.46 Mass spec results of MCOTI-I-Lys-TxRD 140 4.47 HPLC of trypsin capture tested using MCOTI-I 141 HPLC results of different volumes of trypsin beads capturing 4.48 142 same amount of MCOTI-I HPLC Analysis showing different solvents and beads types 4.49 144 optimized for maximum elutions of captured peptides 4.50 HPLC results of MCOTI-I-Lys-NH-TxRd interactions 146 4.51 HPLC of Buffered GdmHCl and DBCO amine 150 Reaction of 5-azido pentanoic acid with DBCO amine in 4.52 151 buffered GdmHCl Results of mass spec analysis of reactants and products of click 4.53 152-153 reaction optimized Summarized view of Intein mediated protein-trans splicing, in 4.54 cell folding of mutant cyclotide and its labeling with TxRd for 154 interactions with Trypsin-EGFP. 4.55 Results of SDS PAGE of the MCOTI-I-Azide expressed 155 LC/MS analysis of MCOTI-I-AziF expressed showing NH and 4.56 156 N3 forms LCMS based quantification standard WT-MCoTI-I represented 4.57 157 by 3rd charged state of peaks area selected (871.6 amu).

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4.58 LC/MS based quantification of MCOTI-I-AziF 158 LC/MS of labeling reaction completing between MCOTI-I-AziF 4.59 160 and TxRd-DBCO 4.60 HPLC purification of MCOTI-I-AziF-TxRd-DBCO 161 4.61 M/S of fluorescently labeled MCOTI-I-AziF-TxRd-DBCO 162 4.62 Expression of Trypsin EGFP 164 Absorption spectra for Trypsin-EGFP of different 4.63 165 concentrations FRET analysis shows MCOTI-I-AziF-TxRD-DBCO and 4.64 166 Trypsin-EGFP interactions 4.65 Calculation of Binding efficiency KD 167 Western blot analysis of inteins production at 1 mM IPTG 4.66 169 inductions In-cell expression of MCoTI-I based cyclotides in S. cerevisiae 4.67 171 cells using Npu DnaE intein-mediated PTS. Mass spectrum of in-cell generated (yeast cell) cyclotide 4.68 172 MCoTI-I.

LIST OF TABLES

Table No. Title Pages Structures, characteristics and references of 3 families of cyclotides 2.1 26 modifications Advantages and disadvantages of peptides as drugs (Vlieghe et al., 2.2 50 2010) 3.1 Common and scientific names of different family plants used 52 Scheme followed for Ames test to evaluate mutagenecity, showing 3.2 trearments given for each category of test sample and test strains 61-62 with mixture preparations (in mL) General PCR reaction mixture for amplification of cyclotide genes 3.3 64 (optimized separately for each gene) Grading of results for antimicrobial activity of different plant 4.1 95 extracts by disc/well diffusion method. HPLC yields of MCOTI-I-Lys-TxRd using different sepharose 4.2 147 beads and solvents for elutions.

Molecular Studies on Plant Based Cyclotides for Protein- Protein Interaction ABSTRACT Bioactive agents like secondary metabolites and peptides are gaining much interest while addressing the issues of agricultural and health threats including pests and pathogens and in drug development. Among the plant bioactive peptides, cyclotides being disulphide rich, stable, resistant and having ability to graft epitopes on it or allow sequence variations in its different loops that are very important regarding biological application and research interests for drug development and delivery. The present study was therefore focused on evaluating the bioactive potential of cyclotide bearing indigenous plants including Viola odorata, Viola tricolor, Viola hybrid, Petunia, Clitoria ternatea, pansy F1, Panicum vigatum, Panicum laxam, Panicum maximum and Hamelia patens. The extracts were prepared in phosphate buffer saline (PBS) and protein extraction buffer (PEB). Ptunia and MCOTI-I both separately showed highest DPPH activity (antioxidant) due toas the activity was reduced significantly after treating with Proteinase K. Hamelia possessed highest reducing power, hemolytic, thromobolytic and antimicrobial activites. Mutagenic responses of the (Ames test) medicinal plants were not significant. DNA demage protection assays were also done on all protein extracts including MCOTI-I. Cyclotide genes were also isolated from the selected plant’s DNA using specific primers. It was found that the chimeric arrangement of cliotide gene (from Clitoria plant) as most attractive with conserved sequences in cyclotide and Albumin-1 domains and little intronic variations. Moreover, for cyclotide-protein interaction studies, Texas Red-DBCO amine dye was synthesized to label MCOTI-I-AziF with p- azidophenylalanine Uaa in origami DE3. Optimizations were first done by using the MCOTI- I and its label form MCOTI-I-Lys-TxRd. The click reaction to bind TxRd-DBCO with MCOTI-I-AziF was finally done in buffered guianidinum HCl at a molar ratio of 1:100 and analysed through LC/MS and MCOTI-I-AziF-TxRd-DBCO was confirmed by mass spectrometry. For binding assay FRET analysis was done with Trypsin-EGFP, saturation was achieved with KD value 29.7 ± 1.08 nM. Optimization of same amount of MCOTI-I inteins (using westernblot) was taken in 3-4 h. The IPTG induction avoided the overnight incubations and unwanted backgrounds. The MCOTI-I a cyclotide was expressed for the first time in yeast cells. Current research opens new understandings towards the bioactive

xi potential of cyclotide bearing plants with/without peptide content, genetics of cyclotides genes from indegenous plants, optimizations of expression and in vitro labeling studies of MCOTI-I for optical studies regarding drug development and targeting.

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Chapter 1 Introduction

1.1. Bioactive peptides and proteins The occurrence of peptides in plants has been known for a long time (Samuelssons, 1959). Plants are persistently exposed to a large group of organisms that are damaging, pathogenic in nature and their existence conditions need quick defense replies from the plants by synthesizing agents like ROS, phenolics, phytoalexins, jasmonic, salicyclics, and sometimes antimicrobial peptides and proteins. These defensive responses comprise novel candidates that could be engineered to develop disease related resistance in plants (Castro and Fontes, 2005). Peptidal agents like defensins are known to be involved in host defense of plants and animals (Borregaard et al., 2000). Antimicrobial peptides are need of the day to combat against rapidly growing resistant strains affecting adversely the major areas of food, health and agriculture. Today’s research is mainly focused on the development of different strategies for selection, isolation and characterization of compounds with bioactive potential like peptides. Emphasis is laid on discovering distinctive novel chemical structures with drug potential, and to expose unknown targets, by investigating the structure based on evolutionary studies and optimizing their naturally existing activity (Keefe, 2001; Bohlin et al., 2010).

Major yield losses, losses in quality and safety of fresh and processed foods in agricultural production are due to fungal/microbial diseases of plants. Until recently the control of fungal or microbial diseases relied mainly on chemical fungicides, however, public concerns about their side effects on human health and the environment, has motivated research to develop new antimicrobial agents that meet current safety and health standards (Lee and Kim, 2015). Analysis in early 2013 has also revealed that almost 70% of the developmental pharmaceutical agents in clinical trials were of smaller size out of which >21% were antibodies i.e. may be proteins in nature. Contrarily besides development in the field of antibodies and small molecules, there is still somewhat limited interest in the peptide based drug development; approximately 7% of development products in the year 2013 were peptides although from historic perspective peptides have successful stories comparably in development products (Bhat et al., 2015). Peptides are potentially rich pharmacological

1 candidates as they can serve up as antagonists, agonists, or allosteric modulators across a wide range of target classes. They can also serve us as outstanding targeting agents to deliver grafted items and as transporters to create therapeutics that are bifunctional. Limitations like poor membrane permeability & oral bioavailability, susceptibility to proteolytic degradation and little spans of circulating half-life are often hurdles considered for developing peptide therapeutics.

1.2. Cyclic peptides and cyclotide

A number of cyclic peptides with potential pharmacological applications have been isolated from seeds, latex and roots of various plants. Such peptides possess different bioactivities such as immunosuppressive, cytotoxicity, vasorelaxant, antimalarial, acetylcholine esterase, cyclooxygenase, and tyrosinase inhibitor (Devappa et al., 2010).

A unique family of plant cyclic peptides/ macrocyclic peptides, cyclopeptides or cyclotides (Craik, 2001) are approximately 30 amino acids disulphide rich miniproteins with N- and C-terminal fused for cyclization. Due to its unique cyclic cystine knot structural motif (six absolutely conserved cysteine residues) becomes extremely stable to heat, enzymatic digestions and chemical treatments thus having important role in plant protection against pathogens and pests (Craik et al., 2010). In this motif, two disulfide bonds and their connecting backbone segments forms an embedded ring that penetrated by the third disulfide bond. Cyclotides in contrast to other circular poylpeptides, have a well-defined three- dimensional structure, and despite its small size, can be called miniproteins. Their unique circular backbone topology and knotted arrangement of three disulfide bonds make them exceptionally stable to thermal and enzymatic degradation. However having a number of attractive features there are still underappreciated from the drug development perspective. Interestingly the peptide macrocyclics can even change the duration of action by either formulation technologies or by conjugating with higher molecular weight scaffolds (Bhat et al., 2014). They have the potential to block different protein-protein interactions and also modulate targets that are not easily addressed by other small molecules and targets. Cyclization in macrocyclics can also bring better selectivity, potency and permeabilities especially when unique target profiles are considered. Moreover leading optimizations over a

2 broad range of cyclization chemistries are needed. Synthetically accessible, amenable to analoging and property based strategies need to be focused (Bhat et al., 2014).

1.3. Sources and distribution of bioactive cyclotides

Cyclotides have been reported from almost all kingdoms including bacteria and cyanobacteria. Cyclotides have been found from different plant families like Cucurbitaceae, Violaceae, Fabaceae, asteraceae and apocynaceae. The plant cyclotides are found in different plant parts like nodules, seeds, pods, shoots, flowers, leaves, roots and stems, with individual plants expressing a variety of cyclotides (Craik et al., 2010; Nguyen et al., 2011b, Slazak et al., 2015). Cyclotides are insecticidal, molluscicidal or anthelmintic activities, uterotonic, anti-HIV, cytotoxic, neurotensin inhibitory activity, anti-microbial, anti-tumour, antifouling, hemolytic and trypsin inhibition (Goransson et al., 2003; Craik et al., 2010; Poth et al., 2012), and still much more to be known in future regarding drug designing. In recent studies cyclotide analogues and variants were also tested for inhibition against dengue being specific and competitive (Kumar et al., 2012). Concept also exists that distributed hydrophobic and hydrophilic patches of amino acids in cyclotides are one of the reasons of its antimicrobial or insecticidal properties causing membrane disruptions (Craik et al., 2010).

1.4. Structural features of Cyclotide

Cyclotides structural features of cyclotides are strikingly interesting that are also correlated with their functions and diversity in types around the CCK structural motif. The CCK motif (Figure 1.1) is decorated in a systemic way by possessing six interconnecting segments called loops, from loop 1 to 6 successively numbered, starting at CysI. In most cyclotides, loops 1 and 4 are most conserved in both composition and length. In contrast other loops show greater variations in their sequences and size. Loop 5 that induces a local 180° backbone twist contains a basic residue cis-proline making it like a Möbius strip. Both major subfamilies mobius and bracelet (without cis-proline twist) contain some conserved amino acid residues such as the natural site for cyclization, Asn/Asp in loop 6 and Glu in loop 1 (Gould et al., 2011) with only a conservative change i.e. either glycine or alanine and makes hydrogen bonding with amino acid residues from loops 3 and 5 (https://en.wikipedia.org/wiki/Cyclotide). Loop 6 is flexible in composition and is not required for biological activity or folding. This loop is also not present in a related acyclic

3 trypsin inhibitor/linear trypsin inhibitors from the squash family (Camarero et al., 2007). The ubiquitous presence of just a single amino acid in loop 4 makes it unique too. Conserved (- OH)-containing residues and a glycine is present in loop 3 (Dutton et al., 2004). Several trypsin inhibitors have been isolated from M. cochinchinensis, including MCoTI-I, MCoTI- II, and MCoTI-III. Unusually, MCoTI-I and MCoTI-II are head-to-tail cyclized peptides, whereas MCoTI-III is an acyclic peptide. The structure of MCoTI-II has been determined and contains a cystine knot motif (Felizmenio-Quimio et al., 2001).

Figure 1.1 Cyclic Cysteine Knot (CCK) framework of cyclotide MCOTI-I with 6 different loops and disulphide linkages.

Cyclotides that share properties of both bracelet and Mobius (psyle C, kalata B8 etc) are also starting to become known as hybrid cyclotides (Gould et al., 2011).

1.5. Genetics of cyclotides

More than 200 cyclotide genes have so far been discovered and expected by some scientists to have more than 50,000 members as genes in only plants. Perhaps believed to be one of the lasgest gene ecoding plant peptides. The cyclotide genes encode linear 11–14 kDa precursor proteins, that contains 1-3 domains of mature cyclotides, having a pro-domain, endoplasmic reticulum (ER) signal sequence and a region of hydrophobic amino acid residues at C-terminal (Craik et al., 2010). Some genes encode only a few linear variants that occur naturally are known as uncyclotides. Cyclotide variants with shortest transcripts from petunia of solanaceae (Poth et al., 2012) and linear variants panitides are reported from panicum of family poaceae (Nguyen et al., 2012 b). Cyclotide genes are known to be differentially expressed either in inducible or constitutive forms in different parts (Poth et al., 2012). A dozen gene sequences exists as hybrid sequences between two subfamilies mobius

4 and bracelet (Nguyen et al., 2012a). Our studies (unpublished) and studies by Nguyen et al., (2011a) some genes of the cyclotides like in family fabaceae of Clitoria exists or encodes for precursors are chimeras, cyclotide domain and A1b domain (albumin-1). These genes might e emerged from either horizontal gene transfer or convergent evolution in plant genomes.

1.6. Candidates of enzymatic synthesis of cyclotides in plants

Cyclotides cyclization occurs as a post-translational modification although poorly understood. Extensive studies have recognized two major enzymes named AEP & PDI (asparaginyl endopeptidases & protein disulfide isomerases) that are believed to be involved in the processing of cyclotide, from cyclization events towards folding perspectives (Saska et al. 2007; Gillon et al. 2008). Vacuolar processing enzyme VPEs or AEP are inhabitants of the large plant valcuole present in vegetative parts whose major role is to degrade or immobilize protein by using different proteases and to recycle nutrients. This enzyme is also involved in the conversions of proproteins to their respective mature functional/structural forms (Chen et al,. 2008). AEP, a ubiquitous enzyme in plants, involves in normal role in catabolism of protein substrates after Asn residues hijacked by cyclotide precursors utilize them for the formation of peptide bond also. These VPE / AEPs can also serve us as a tool to regulate or alter different plant growth stages alongwith maturation of different plant peptides and proteins reported a genetically encoded enzyme from potato, sweet potato asparaginyl endopeptidases (SPAE), transformed in model plant Arabidopsis through Agrobacterium mediated transformation. It was found that the transgenic plant with AEP showed earlier floral transitions and leaves senescence than controls without transgenes alongwith few incompletely-developed siliques/plants. AEP is responsible for catalyzing in a single processing event both peptide bond cleavage and ligation of cyclotides. Evidence of such catalysis by AEP was seen in Niotiana benthamiana (not producer of cyclic peptides) when transformed by KB1 cDNA produced mature cyclotide and no linear forms were present. (Saska et al., 2012). Such methods can be useful in future by making transgenics to manipulate plants to produce peptides like cyclotides where these are not naturally present (Chen et al., 2008).

Protein disulphide isomerase (PDI) is an oxidoreductase enzyme produced in higher amounts in the endoplasmic reticulum. Proteins like prolamins that are >90% of the wheat

5 grain protein content are dependent on maturation for this type of enzyme (Dhanapal, 2012). Interestingly PDI enzymes enhanced the proper folding yield of cyclotide-like molecules, including a linear cyclic molecule and a reduced cyclotide (Gruber et al., 2007) and mopping up some misfolded cyclotide proteins and refold them appropriately (Craik et al., 2010). It is speculated that the NTRs are involved in the proper folding of precursor protein before cleavage and cyclization. Experiments explained the mechanism of enzyme dependent folding of plant cyclotides by comparing the absence and presence of folding of cyclotide kalata B1 derivatives in O. affinis PDI (OaPDI) which improved the correct oxidative folding of cyclotide at physiological pH. S-S isomerization is one of the key role of plant PDI for the production of insecticidal cyclotides. The in vivo relevance of this mechanism still remains to be established. Presumably the PDI interacts usually with the precursor protein, rather than the processed cyclotide domains (Craik et al., 2010).

1.7. Production of recombinant Cyclotide

Even though the synthesis of several cyclotides chemically by using solid-phase peptide synthesis has been already reported yet biosynthesis of cyclotides can be done by recombinant DNA technology. An attempt to produce a cyclotide KB1 by recombinant methods was unsuccessful due to issues related to isolation of the same folded cyclotide from the soluble cellular fractions that were too complex. Despite KB1 was the first cyclotide to be produced by recombinant DNA technology and structurally well characterized, its binding partners for affinity chromatography for purification were not discovered. To do so cyclotide trypsin inhibitor II named MCoTI-II (from Momordica cochinchinensis plant) was used instead. This cyclotide was a powerful trypsin inhibitor isolated from the squash family. Its 3-D structure is also well determined by NMR spectroscopy which has confirmed their cyclic cystine knot CCK topology (Camarero et al., 2007). Biological synthesis of a completely functional MCoTI-II inside living E. coli strain BL21 (DE3) cells was the first successful story of producing cyclotide recombinantly. This was done by utilizing an intein-mediated cyclization approach, engineering E. coli cell with lot of mutations for glutathione reductases and thioredoxins to promote disulfide bridge formation which increased MCoTI-II yield. A higher intracellular concentrations that could be enough for screening in vivo, with trypsin inhibition activity (porcine pancreatic trypsin Ki= 25±5 pM) which resembles the naturally

6 purified cyclotide from plant. This permits modifications with introduction of specific biophysical or chemical probes. Moreover cyclotides can now be biosynthesized in bacterial cells like E. coli using modified protein splicing units. These characteristics of cyclotides makes it an ideal for the production of libraries based on the CCK cyclotide framework that are genetically-encoded (Jagadish et al., 2013). Cyclotides can now be screened for its ability to facilitate processes occuring inside living cells for drug development purposes (Camarero et al., 2007; Slazak et al., 2015). Different approaches can now be used for the production of cyclotides like for MCOTI-I using protein trans splicing PTS and expressed proteins ligations methods in E. coli as well as in eukaryotic systems like unicellular yeasts (Jagadish et al., 2013 and Jagadish et al., 2015). We have used both strategies in our molecular studies and also reported for the first time cyclotide MCOTI-I in Saccharomyces cerevisae (Jagadish et al., 2015).

Another recombinantly synthesized cyclotide approach was developed using “nonsense suppressing orthogonal tRNA/synthetase technology”. This technology has also allowed the genetic encoding and incorporation of a large multiplicity of unnatural amino acids (Uaas) in proteins (Liu and Schultz, 2010). Previously scientists have tried a lot the same thing to incorporate unnatural amino acids in vivo for site specific incorporation into proteins or peptides. This was done by evolving the E. coli orthogonal t-RNA synthatases for its specificities to add unnatural amino acids of our choice by generating that are not endogenous to the host. An orthogonal suppressor t-RNA/amino acyl t-RNA synthatase pair was used in E. coli that is specific for the t-RNAasp originally present in Saccharomyces cerevisae and its partner aspartyl-tRNA synthatases. Both these were used invitro and invivo and were well characterized (Pastrank et al., 2000).

1.8. Labeling cyclotide using unnatural amino acid Uaa

Biosynthesis of cyclotides with Uaas can be possible by using different intein-based methods like EPL (expressed protein ligation) and PTS (protein trans-splicing) but with different yields in both the cases (Jagadish et al., 2013). Jagadish and his coworkers (2013) reported the MCOTI-I, a trypsin inhibitory cyclotide, expression with p- methoxyphenylalanine (OmeF) and p-azidophenylalanaine non-natural amino acid

7 incorporated for site specific incorporation of coumarin fluorescent dye as probe and used it in cell production for natively folded cyclotides (MCoTI-AziF & MCoTI-OmeF) for the first time. The efficienct PTS-mediated cyclization was combined with nonsense suppressing orthogonal tRNA/synthetase technology made the in-cell cyclotides production containing Uaas possible. Of particular interest was the introduction of azido-containing Uaas p- azidophenylalanine (AziF) which reacted with DBCO-AMCA a fluorescent probe (dibenzo- cyclooctyne DBCO and amino-methyl-coumarin acetate AMCA) and produced in-cell fluorescently labeled cyclotides. This approach for in-cell production of fluorescent-labeled protein was not easily applicable to cyclotides previously due to their restricted backbone- cyclized topology and small size. Thus now cyclotides containing the Uaa AziF expressed in living bacterial cells and easily labeled with fluorescent DBCO-AMCA can be used to observe cyclotide-protein interactions. The interactions were estimated by fluorescence resonance energy transfer (FRET) with modified trypsin fused at N-terminus with green fluorescent protein (EGFP). This finding opens the possibility for in-vitro and potentially also in-cell screening of genetically-encoded libraries of cyclotides for the rapid selection of novel cyclotide sequences able to bind a specific bait proteins using high throughput cell- based optical screening approaches (Jagadish et al., 2013). Protein libraries can now be screened invivo as well as invitro for selecgtion of bioactive members that can be useful for treating different drug targets like proteins of toxins or pathogens or even cancers.

1.9. Research objectives Besides extensive research on cyclotides we are still lacking the knowledge of the bioactive potential of indigenous plants possessing cyclotides and can still find new genetic variants of genes of cyclotides and correlate them. We need to study cyclotides for optical studies to trace the path of cyclotides within cell or towards the target when epitopes are attached for specific purpose to it and thus better understand its mechanism of action within cells. Florescent labeling is one the best strategy to screen and understand cyclotide interactions with other agents like other labeled proteins by FRET. For this we needed a fluorescent dye with no overlapping with the spectrums of other interacting labeled member like EGFP for which we decided to label it with Texas Red that has a negligible background which can interfere in studying protein protein interaction both invitro and invivo and by

8 selection and screening through optical approaches. Moreover it will add new approaches towards peptide based drug development and targeting studies. Besides studies available on the recombinant peptides production from yeast cell there was no such studies available of cyclotide production and studies for eukaryotic cell drug targeting. Our present research will be important using bioactive cyclotide as tools for drug delivery and targeting that are bifunctional.

Figure. 1.2. Overview of Cyclotide labeling with Texas red dye. Cyclotide MCoTI-AziF labeled with Texas red-DBCO amine dye and its in cell folding using intein-mediated protein trans-splicing (PTS) technique combined with non-sense suppressing orthogonal t-RNA synthatase technology. A diagrammatic representation of an approach shown in Figure 1.2 as an overview of our research project related to cyclotide labeling with Texas red succinamyl ester with MCoTI-AziF. Keeping the above discussion in mind and gaps in the cyclotide research for better understanding towards cyclotides, the present study is therefore designed to focusing on the following objectives of our research;  To screen selectively cyclotide bearing plants on the basis of their bioactivities.  To isolate cyclotide genes and study its gene variants in screened and selected plants.  To label cyclotide, MCOT-I mutant with fluorescent dye and study protein-protein interaction  To express cyclotide MCOTI-I in Yeast cells, a unicellular eukaryotic model

Chapter 2 Review of literature

2.1. History of peptide

The term Peptide was first used by its originator Emil Fischer the founder of the field of protein/peptide chemistry (1852-1919). He was Nobel laureate and awarded in the field of chemistry in 1902 and reported the synthesis of the first dipeptide, glycylglycine. Name of peptide was derived from pepsis means peptones or digestion which means digestive products of protein. Whereas first medicinal use of peptides was by Dr Frederik Paulsen, Ferring’s founder choosed to develop Ferring’s first medicines by using peptide hormones. For the first time the production was done through extracting peptides from animal organs and then modifying those using natural hormones and ligands (Shinde et al., 2013). 2.2. Cyclic peptides as stable bioactive compounds Peptides are marcomolecules composed of a few amino acids starting from a dipeptide structure to a polypeptide with numerous residues. On the basis of the chemical structure of peptides, there are mainly two types of peptides, linear and cyclic. The linear peptides for clinical application have been limited by the intrinsic properties of peptides which include rapid degradation of peptides by peptidase enzymes, leading to metabolic instability which complicates oral delivery of peptides. Passage through blood brain barrier is an additional problem for linear peptides which acts on CNS. Fast removal from the circulation may also limit the therapeutic uses of linear peptides. In order to counteract these undesirable properties, numerous modifications of the peptide structure have been considered.

Figure 2.1. A general structural representation of a cyclic structure of cyclotide

One of the most significant modifications is the cyclization of the linear peptides (Figure 2.1), which reduces the flexibility of linear molecule and stabilizes secondary structure of peptides and increases its activity. Thus cyclization of the peptide structure which include (between ends of the peptide sequence or side chains): head to tail, end to side chain, N-backbone to N-Backbone, side chain to N-backbone, end to N-Backbone, side chain to side chain through disulfide lanthionine, dicarbahydrazine or lactam bridges is necessary in order to decrease the conformational flexibility of linear peptides, importantly increase their stability to proteolysis by endo and exopeptides and to reduce hydrogen bonding which enhance membrane permeability (Insanu et al., 2012).

Many cyclic peptide natural products are biosynthesized by non-ribosomal peptide synthetases (NRPSs), which unlike the ribosome often utilize both regular and non- proteogenic amino acids as substrates. NRPSs also often incorporate activities such as epimerization, heterocyclization and N-methylation, further increasing the numbers based on diversity of these natural products. The structural complexity of many natural products keeps them apart from the common synthetic drugs, because complexity in structures makes them accessible to a biological target space that may be apart of enzyme active sites and receptors targeted by conventional molecules/drug. These compounds penetrate in cells via passive diffusion whereas others like the clinically important drug cyclosporine A, are bioavailable orally (Bockus et al., 2013). On the basis of size and structural complexity the cyclic peptides have have been placed at a reasonable space in drug discovery as they provide useful scaffolds in order to modulate protein-protein interactions and allosteric binding sites.

Cyclization in peptides also eliminates charged termini, which can favor both membrane permeability and metabolic stability. Cyclization of the peptide molecule leads to the development of a number of medicinal compounds with potent biological activities. It is also thought to reduce conformational entropy losses when it binds target, although some studies have shown the positive impact of cyclization on this entropy (Bockus et al., 2013).

Cyclic peptides are formed into a ring like structure due to the presence of amide ester or disulfide bonds. Because there is no exposure of CN-terminal groups to exopeptides, that is why cyclic, peptides are stable enough to degradation by enzymes (Insanu et al.,

2012). Most of the cyclic peptides isolated from natural sources like plants and marine sources but as only minute quantities are obtained from these sources, attempts have been made towards the synthesis of these cyclic peptides and their derivatives by various methods (Shinde et al., 2013).

As compared to linear peptides, cyclopeptides exhibit stronger biological activities, mainly due to the stable configuration due to their cyclic structure (Craik et al., 2004). The cyclic peptides found in Violaceae, Cucurbitaceae and Rubiaceae families of plants have been shown to exhibit significant antitumor activities. The mechanisms of action of these peptides on tumors have variations due to recovery of many types of peptides (Craik et al., 2004).

2.3. Types of Cyclic peptides/proteins:

As a result of continuously increasing number of peptides that are cyclic a brief categorization of cyclopeptides is described as discussed here.

(1) Cyclic peptides are divided on the basis of linkages they have;

 Homodetic peptides, cyclic structure containing only peptide linkages such as 2, 5- piperazinediones.  Heterodetic peptides where cyclic structure contains both peptide bond and other linkages.

(2) Cyclopeptides can be grouped into two types on the basis of number of amino acids from terrestrial plants.

 One is cyclopeptides, in which the number of amino acids are less than 14 and there is no presence of any type of disulfide bonds. These types of cyclopeptides are present in the plant families of the Caryophyllaceae and Rhamnaceae. Some cyclic petides have shown powerful antitumor activities, such as, dianthins E a cyclic hexopeptide in Dianthus superbus; cherimolacyclopeptide C, a cycloheptapeptide from Annona cherimola seeds; RA-XVII, a bicyclic hexapeptide in Rubiaceae, longicalycinin A, a cyclic heptopeptidein Dianthus superbus.

 Another form of cyclopeptide is the recently discovered family of proteins, in which 28-37 amino acid residues are present. There is also the presence of disulfide bonds in these cyclotides. They have well-defined secondary structures and also show similarity to proteins due to the formation of compact three-dimensional folded structure (Craik et al., 2004).

(3) According to the mechanisms of action of peptides, bioactive peptides may be classified by Ovadia et al., (2010) as

 Antimicrobial  Antioxidative  Antithrombotic  Opioid  Antihypertensive  Immunomodulatory  Mineral binding (4) Cyclic peptides are divided into the following categories based on a broad survey of their structures and known biological functions:  Highly charged molecules that work by bringing changes in bacterial membranes and mostly used as antimicrobial agents.  Non-polar cyclic peptides that contain abundantly modifications to the amide backbone (e.g., N-methylation) and lipophilic side chains and penetrate within eukaryotic cells by passively.  Cyclic peptides which are amphiphilic, possess mixed polarity but are not limited to microbial targets.  Cyclotides and cysteine-knot proteins, which are small (2-8 kDa) proteins with unusual topologies that can have remarkable oral activity (Gould et al., 2011). (5) Cyclic peptides are of two types on the basis of their synthesis  Ribosomally and non-ribosomally synthesized peptides. The sirolimus, cyclosporine A (CsA) and tracrolimus are the examples of non-ribosomally synthesized peptides that play a significant role in interference of cytokine signaling. For the treatment of

autoimmune diseases and in order to prevent the rejection of a transplanted organ CsA, a cyclic peptide is widely used, which has a fungal origin from Tolypocladium inflatum.  Ribosomally synthesized are gene-encoded synthesized as precursors and post- translationally modified small peptides (RiPPs) also occur in different taxa. The several domains which are involved in these precursors have their own specific functions. The posttranslational modifications which are necessary for their biosynthesis are carried out by multifaceted machineries of enzymes (Thell et al., 2014). (6) Cyclic peptides drugs can be divided into two categories on the basis of their size as;

 Currently used drugs that can be defined as those types of drugs which have molecular weights of <500 Da and they also have the capability of oral bioavailability.  Much larger drugs that are known as biologics with molecular weights of >5000 Da and they are also needed to be delivered via injection and not orally bioavailable like insulin, growth hormone, antibodies etc.

The reduced target selectivity is the limitation of small molecule drugs that have side effects for human beings, whereas protein therapeutics tends to be specific for their targets. Now it is time to reinvestigate novel and new drugs that fits between the extremes of two molecular weights, with the goal of binding oppertunities of small molecules with those of proteins (Craik et al., 2013).

2.4. Sources of cyclic peptides

Cyclic peptides can be obtained from different sources which include;  Marine  Plant  Animals  Microorgnisms  Food

14 and other type of sources that entered preclinical and clinical trials. Violaceae, Cucurbitaceae and Rubiaceae and families of plants are the richest sources of peptides. The marine therapeutics comprises molecules with antiviral, antiphrastic, antibiotic, anticancer and analgesic activity. Various anticancer compounds have been isolated from marine sources with diverse modes of action, such as anti-microtubule, anti-proliferative and antioxidant. There are several types of side effects like gastrointestinal pain, fatigue and depression of immune system of traditional chemotherapeutic agents for which natural anticancer drug discovery is necessary. The reason behind the structural stability of these types of peptides indicate the presence of unusual amino acid residues. For the treatment and prevention of cancer the use of peptides that are obtained from marine sources are gaining importance and they also play a significant role in the research of anticancer drug. Marine cyanobacterial compounds show remarkable cytotoxic effects on tumor cell lines of human while the effects of some of these compounds will be at minimum range. For example, by the inhibition of cell cycle Apratoxin A, from Lyngbya majuscula possess cytotoxic effects on cultured Human HeLa cells of cervical carcinoma and it is a desipeptide in nature (Malaker and Ahmad, 2013). Marine plant peptides will also be mentioned in upcoming section also.

2.4.1. Terrestrial plants

Cyclopeptides in which the number of amino acid residues are less than 14 and disulfide bond is not found, are present in plants of the Caryophyllaceae and Rhamnaceae families. Some of those cyclopetides are reported to be strongly antitumorous. A cycloheptapeptide named cherimolacyclopeptide from Annona cherimola seeds; a cyclic hexopeptide named dianthins E present in Dianthus superbus; another cyclic heptopeptide known as longicalycinin A isolated from Dianthus superbus var. longicalycinus; a bicyclic hexapeptide called RA-XVII (in rheumatoid arthritis) inhabitant of plants of Rubiaceae family and a family of cyclopentapeptides named astia isolated from the roots of a medicinal plant Aster tataricus are the examples of cyclopeptides. The other type of cyclopeptide involves the discovered cyclotides of viola, poaceae, fabaceae and many more plant families with unique structural features and bioactivities (Hsieh et al., 2004).

2.4.2. Marine plant

Cytotoxic or antitumor peptides comprise of about 3.5% of the total population of the marine plants. Due to the presence of some types of modifications such as D-amino acids, some new α-and β-amino acids, hydroxyl acid, as well as oxazole and thiophene in the structures of antitumor peptides the peptides from marine plants are normally stable and have an efficient bioavailability. Nostoc sp. GSV224 cyanobacterium only, a class of cyclic depsipeptides comprising 25 analogues have been isolated from the. Some strongly cytotoxic peptides have also been isolated from Cyanobacterium Lyngbya majuscule (legunamides, Homodolastatin, Wewakpeptins and Malevamide D) and L. semiplena (algae) (Craik et al., 2004).

2.5. Microbial cyclic antimicrobial peptides Cyclic antimicrobial peptides derived from microbial sources bind stably with its target sites and have tolerance to hydrolysis when exposed to proteases as compared to its linear precursors due to the absence of carboxyl and amino terminals. They also posses favorable degradability property under field conditions thus preventing accumulation of cyclic compounds to a potentially harmful level, which makes these peptides an attractive substitution for use in agriculture as fungicides. Antimicrobial cyclic peptides are of different types based on bond types within the ring structures; homo or heterodetic and complex cyclic peptides, with diversity in their physicochemical features. Mostly cyclic antimicrobial peptides acts by affecting the cell envelope’s integrity through direct interaction or disturbance of the biosynthesis of components such as glucan, chitin and sphingolipid. Cyclic antimicrobial peptides are effective against a range or broad spectrum of plant pathogens e.g., Cochliobolus, Alternaria, Geotrichum, Botrytis, Penicillium, Fusarium species, Sclerotinia and M. grisea) which might be due to similarities present in fungal envelopes, where these peptides are active. Such moachanisms of action are believed to reduce the chance of resistance that can be developed in microbial populations. These features are ideal merits of using cyclic peptides as antimicrobials or fungicides in the control of diseases affecting plants. Despite merits, there are issues to consider that are associated with the practical applications of mentioned cyclic antimicrobial peptides e.g. the effectiveness under certain physiological conditions, the selective toxicity to pathogen over their hosts, and the synergistic effects with other fungicides needs to be resolved.

Studies focused on natural antimicrobial cyclic peptides in potentially rich sources like plants, bacteria and yeasts for controlling plant diseases that continuously needs diversifications in cyclic structures, validation is needed in structure-activity relationships, and optimize treatment methods and formulation may be an new effective tool to diversify and improve the current management strategies that usually & heavily relied on conventional fungicides for plant diseases (Lee and Kim, 2015).

Diseases of plants due to fungal infections are responsible for affecting the quality and safety of fresh and processed food and major yield losses during agricultural production. Until recently the control of fungal diseases relied much on chemical drugs or fungicides, however, concerns about the potential side effects on human environment and health, has stimulated research to develop new antimicrobial agents that meet current safety standards (Lee and Kim, 2015). Similarly evidences of cellular responses to oxidative stresses have been observed by proteomic analysis which shows that modifications occurs in the peroxiredoxins (proteinaceous enzymes destroying peroxides). Tendem mass spectrometry showed modified of proteins in vivo as a result of stress in T-cell lymphoma cells. More strengthening to this concept was achieved when active role of these proteins was observed in susceptibility or resistance to apoptosis that could result as a response of α-induced tumor necrosis factor. This explains important role of proteins in oxidative responses (Rabilloud et al., 2002).

2.6. Food derived peptides

Food besides providing proteins as energy source also provides bioactive peptides that can be of functionally more important as in its originally unmodified or altered native forms. Like lactoferrin, in milk has been shown to have inhibitory activities against the herpes simplex virus, HIV, human cytomegalovirus, rotavirus and poliovirus when tested in vitro as possible antiviral agents in food. Food proteins, like lactalbumin or β-lactoglobulin, after chemical modifications acquire antiviral properties. Hydrophobic sites alongwith the positively charged arginine and lysine residues of proteins modified by 3-hydroxyphthalic anhydride are important targets for modifications. Hexapeptide PAF19 Ac-RKTWFW-NH2 obtained through screening of combinatorial library of a synthetic peptide showed anti-fungal

17 activity that cause postharvest demage to fruits results were similar to that of the hemolytic 26-amino acid melittin that was not actually toxic for bacteria or yeast (García et al., 2002). Several peptide fragments after digestion by proteolytic enzymes in the gut possess antiviral properties giving food proteins an advantage over synthetic pharmacologic compounds as being non-toxic and well accepted by the consumers, future therapeutics (Pellegrini and Engels, 2005).

The antitumor effect of these peptides follows mechanisms like given below;  Interruption of cell microtubular activity Interruption in cellular microtubules processes can stop the process of division during cell mitosis, due to which the prolongation of the cell cycle occurs. Scytonema pseudohofmanni, is a microtubule dissociation-inducing compound. In vitro, it also has various types of inhibitory effects on murine leukemia, human KB cells and lung cancer cells.  Induction of cell apoptosis A new method of cancer therapy is the induction of programmed death (apoptosis) for tumor cell. A group of cyclopeptides called astis can perform its activity on KB cells in vitro and in vivo in mice on lymphocytic leukemia (Craik et al., 2004).  Cell cycle blockade Depsipeptide (FR901228, NSC 630176). NSC630167 is an inhibitor of histone

deacetylase and it causes the cell cycle to arrest in the G0/G1 phase by supressiing the expression of oncogene c-myc mRNA during the study in some human solid-tumor cell lines in vitro.  Anti-multidrug resistance The over expression of gene encoded P-glycoprotein is responsible for the synthesis of drug resistance via tumors. It reduces the toxicity of drug by changing the configuration and mechanism of action. Cryptophycin-1 is the effective peptide that play role in this regard (Craik et al., 2004). 2.6.1. Other examples and functions of cyclic peptides Applications of cyclic peptides are different in various fields which are summarized as;

Despite the clues of structure-function relationship, due to the rigid three-dimensional conformation cyclic peptides that are isolated from Euphorbiaceae are essential for bioactivity over lipid membranes. The interaction between cyclotide labaditin and their synthetic open chain analogs with lipid bilayers have been analyzed that showed that native labaditin had increased membrane insertion. The native labaditin reduced the viability in Gram +ve bacteria by changing their mechanism of interaction with the lipid membrane due to the presence of change in confirmation, peptide adsorption and internalization (Pestana- Calsa et al., 2010). Cyclosporin and vancomycin are the cyclic peptides which are composed of modified amino acids like D-modification of amino acids and have influence on the cure of severe diseases such as cancer. The presence of modified amino acids, such as N-methylation in vancomycin and cyclosporin make them enable for the treatments of various life-threatening diseases such as different cancers, HIV etc. The cyclic peptide systems which have multiple knotted side chains are the examples of vancomycin related antibiotics. It also play important role for binding of fragments of the cell-wall precursor that are crucial in disease causing bacteria (Liskamp et al., 2008).

Two different types of cyclic peptides were obtained from the latex of Jatropha curcas L. which are named as curcacycline A and B (CSA, CSB). By using paper disk diffusion method the antimicrobial activity of curcacycline A was tested against Staphyloccus aureus ATCC 6538, Bacillus subtilis ATCC 6633 , E. coli ATCC 8939, Candida albicans ATCC

1023 and Pseudomonas aeruginosa ATCC 9027. CS-A also inhibited the growth of Bacillus subtilis and P. aeruginosa with zone of inhibition between 6.5-10.3 mm. The cytotoxic test of this compound described that it reduced the level of OVCAR 3 cells (ovarium cancer) at a concentration of about 1 mg/mL, while there is no such type of effect was reported on human colon cancer cell (Colo205) (Insanu et al., 2012).

In rheumatoid arthritis (RA), to overcome this type of aggressive disease some types of antibodies like cyclic citrullinated peptide (CCP) are used as marker. In order to evaluate the role of second generation of anti-CCP antibodies (anti CCP-2) for the prediction of erosive disease are mainly used in patients with rheumatoid arthritis and in order to define their role in seronegative RA. The positivity for both rheumatoid factor (RF) and anti CCP-2

19 antibodies in patients is that both had a higher incidence of erosions as compared to patients who are negative for both or positive for only one antibody (Shankar et al., 2006).

On the basis of their applications in drug targeting Asn-Gly-Arg (NGR) peptides are of high interest due to recognition of CD13 receptors. Structural analysis of thioether bond- linked novel cyclic NGR peptides has shown that a proto-epitope containing S, R and F amino acids has the ability to regulate negatively TNF secretion via macrophages (Femling et al., 2006).

The interaction between VEGF (vascular endothelial growth factor) and VEGFRs (receptors of VEGF) that is actually inhibition is responsible for anti-cancer treatment. Blockage of the kinase activity on VEGFR indirectly with inhibitors of protein-protein interactions showed great interests in studying oncology. Recently a recombinant humanized monoclonal antibody, approved agent ramucirumab (Cyramza) that specifically binds to VEGFR2, another anti-VEGFR strategy and reported to have greatly increased VEGFR1 binding affinity by using the ELISA-based test (Wang et al., 2015).

Hexapeptide PAF19 Ac-RKTWFW-NH2 obtained through screening of combinatorial library of a synthetic peptide showed anti-fungal activity that causes postharvest decay in fruits. Further modifications by substituting 1 or 2 amino acids in this hexapeptide improved activities against target microbes Fusarium oxysporum, Botrytis cinerea, Penicillium expansum, and reduced against non targeted ones E. coli and Saccharomyces cerevisiae similar to that of the 26-amino acid hemolytic melittin that was not actually toxic for bacteria or yeast (García et al., 2002).

For detecting endogenous and exogenous threats of body the immune system plays important role. Failure of regulation in homeostasis leads to malfunctioning of T cell signaling. To suppress the over-reactive T lymphocytes, various types of medications are present. But mostly of the drugs produce severe types of side-effects. Peptides which are synthesized ribosomally gaining recognition due to their enhanced selectivity and decreased toxicity in the industry of pharmaceuticals. Such peptides includes cyclotides of plants inhibit the proliferation of T lymphocyte. Head-to-tail backbone cyclization and cysteine-knot motif

20 are responsible for stability of cyclic peptides in order to making them effective pharmaceutical tools (Thell et al., 2014).

Besides, a lot successful stories related to peptides and especially to cyclic peptides with unique structural configurations and bioactivities treasure are hiding. A group of highly stable and unique class of bioactive cyclic peptide named cyclotides is most versatile as compared to all mentioned and is current target of most researches related to peptide based drug targeting and drug development.

2.7. Cyclotides

A number of defensive antimicrobial peptides like lipid transfer proteins, thionins and defensins have been reported to express when exposed to large array of pathogens (Castro and Fontes, 2005). Cyclotides, the plant mini-proteins protective for plants from pest and pathogens posseses a cyclic cystiene knot like that makes it stable. Cyclotides discovered from plants being uterotonic which was derived after boiling to make a medicinal tea possessing circular backbone responsible for the stability was investigated (Barry et al., 2003).

2. 7. 1. Discovering Cyclotides

The discovery of cyclotides is attributed to ethnobotanical investigations and bioassay directed screenings of potentially therapeutic plants. In 1965, a professor of Pharmacognosy at Uppsala University, Dr. Finn Sandberg, reported his observations of an indigenous plant used in the Central African Republic (Gerlach and Mondal, 2012). A remedy from the plant “Wetegere” (Gbaya language), later identified as Oldenlandia affinis (Roem. & Schult.) DC (Rubiaceae), was administered to hasten uterine contractions. In the 1970s, the Norwegian physician Lorents Gran participated in a Red Cross Relief Mission which included harvesting medicinal plants in the Northern Congo of Africa. Dr. Gran observed Lulua tribe’s women (Tsjiluba language) harvesting the above ground tissues of a plant called “kalata kalata” which subsequently was taxonomically verified as O. affinis. Elder healers prepared an aqueous decoction (~1 part powdered aerial tissue to 1 part boiling water) and then ingested

21 the “tea” to induce labor. Use of the plant as an uterotonic was surrounded by a degree of secrecy among the women, and although the decoction produced rapid deliveries, in some cases severe spasms ensued and emergency caesarian sections were required (Gran,1973a ; Gran,1973b).

Upon returning to his native country, Dr. Gran isolated several polypeptides in samples of O. affinis extracts that exhibited remarkably strong uterotonic activity. With the aid of a protein chemist, Dr. Knut Sletten, the principal bioactive peptide, now named kalata B1, was identified and almost fully sequenced (Gustafson et al., 1994). This peptide was speculated to be a cyclic structure; however, it was exceptionally resistant to degradation and N-terminal amino acid sequencing, and at the time the available enzymatic tests were insufficient to provide conclusive evidence of the cyclic structure of kalata B1. Therefore, the complete sequence of the prototypic cyclotide, kalata B1, was not reported until the three dimensional solution structure was confirmed using distance restrained simulated annealing and two dimensional nuclear magnetic resonance (NMR) spectroscopy. At around this time, three independent research facilities reported the discovery of macrocyclic peptides with six cysteine residues isolated from violaceous and rubiaceous plants. During a screening for new saponins, the hemolytic violapeptide I (from Viola sp.; Violaceae) was isolated, and the finding was published in a German specialist trade journal. In 1994, the National Cancer Institute (USA) was evaluating a collection of plants for anti‑HIV activity; the cyclotides, circulin A and B, were characterized from extracts of the tropical tree Chassalia parvifolia K. Schum (Rubiaceae). Finally, Merck Laboratory Researchers (USA) identified cyclopsychotride A from extracts of Psychotria vellosiana Benth. (Rubiaceae) while testing natural products for neurotensin antagonistic activity. During the next decade, additional reports on the isolation of polypeptides with a circular nature and unique cyclic cystine knot (CCK) motif from violaceous, rubiaceous, and cucurbitaceous plants were reported which prompted the formal designation of the cyclotides as a plant protein family in 1999 (Gerlach et al., 2012).

Small disulfide-rich, cyclotide bioactives are prominent as protease inhibitors, toxins, hormones, and growth factors. Many of these peptides contain a three-disulfide knotted structure formed by two disulfide bonds making a cystine-knot (CK) motif, together with the

22 connecting backbones and forming an embedded ring through which the third bond penetrates. Of particular interest in drug development is the knottin family (CK peptides) contain 25–45 residues, and often possess protease inhibitory activities, from which the name was derived. Knottins also form compact and well defined structures with extensive internal hydrogen bonding, endowing them with resistance to demage by proteolytic endopeptidases and denaturation by chemicals or heat, as shown by extensive studies, including those using sequencing for determination of their primary structures. Certain CK peptides of the knottin family have further evolved as macrocyclics such as cyclotides, harboring cyclic CKs (CCKs) with no termini, a feature that has made them resistant to exopeptidases (Nguyen et al., 2014).

2.7.2. Structure of cyclotides

Cyclotides are a family of head-to-tail cyclized small proteins with quite a remarkable structure. They consist of 28-37 amino acids, and are characterized by a cyclic cystine knot (CCK) formed by the interactions of 6 cysteine residues. These cysteine residues are oxidized to form a cystine knot core, in which an embedded ring, formed by two disulfide bonds (Cys1-Cys4 and Cys2-Cys5), is threaded by the 3rd disulfide bond (Cys3-Cys6) (Fig 2.2). That knot, together with the head-to-tail cyclic amide backbone defines the cyclic cysteine knot motif (Craik et al., 2001). This CCK motif is shared by all cyclotides and gives an exceptional rigid and stable structure, which makes it extraordinarily resistant towards physical, chemical, and enzymatic degradation. Since the molecular core of this tightly bound knot is occupied by disulfide bonds, the propensity for amino acid side chains that are not associated with the cystine knot protrudes outwards, where they constitute the molecular surface. These amino acids when solvent exposed include a number of residues that form patches of hydrophobic nature in an otherwise hydrophilic surface, making them soluble in both organic and aqueous solvents. This surface-exposed patch of hydrophobic nature contributes to some of the major biophysical properties of the cyclotide, including their late elution in HPLC runs (Pedersen, 2012).

Figure 2.2. Structural scaffold and features of Cyclotide with loops presented in different ways by Pedesrson et al., (2012)

The amino acids not allied with the cystine knot, are arranged in six intercysteine loops, which are successively numbered 1-6, starting at cysteine 1 (Fig 2.2). Cyclotides exhibit extensive variations in the size and composition of their loops between the cysteine residues. Loop 1 is the least variable, containing typically three amino acids including a glutamic acid in the middle of the loop. Loop 4 is similarly highly conserved, always comprising just a single residue and almost invariably containing a single hydroxyl bearing residue (serine, threonine or lysine). These two loops constitute segments the backbone that together with their connecting disulfide bonds form the embedded ring that is threaded by the third disulfide bond. The high degree of conservation in these two loops quite possibly reflects the fact that they form a central core of cystine knot CK. By contrast there is much more variation in both size and sequence of the other loops. The most highly conserved element within the loops is an asparagine, or occasionally an aspartic acid residue in loop 6 which seems to play a functional role in the cyclization. Cyclotides feature the regular elements of secondary structure typically seen in larger proteins. Generally cyclotides are formed by a cystine knot reinforced β-hairpin, comprising loop 4, 5 and part of loop 6 (Wang et al., 2009). This β-hairpin is connected with a third more disordered β-strand that is constituted of parts of both loops 1 and 6. Together these structural elements make up a distorted β-sheet. The secondary structure is stabilized by extensive hydrogen bondings that is like the fold itself, is highly conserved and particularly so within different cyclotide subfamilies.

The structure of tricyclon A describes that the presence of a loop that is disordered in other cyclotides forms a protruding sheet out of the globular core. It was found that the cyclotide folding was amenable to an extent that a range of structural elements can be added to it without affecting the CCK. Tricyclon A, unlike other cyclotides, deprives a hydrophobic patch and has least hemolytic power that makes it attractive from pahrmacuetical point of view. A 22 kDa protein provided clues of being processed (Mulvenna et al., 2005).

2.7.3. The Möbius, bracelet and trypsin inhibitor cyclotides

Cyclotides are divided into three subfamilies. The two large subfamilies Möbius and bracelet, and a minor subfamily of trypsin inhibitors. The bracelet and Mobius subfamilies are distinguished by the presence (Mobius) or absence (bracelet) of a cis-Pro peptide bond in loop 5 (Burman et al., 2011). Although the presence of this cis-Pro amino acid residue is the defining feature of the two subfamilies, there also tend to be high sequence similarities for several loops within subfamilies (Aboye et al., 2012). Furthermore, the two subfamilies also differ in terms of distribution of surface-exposed hydrophobic amino acid residues and their overall net charge, with one of the main differences being the presence of a group of charged residues in loop 5 of bracelets (Burman et al., 2011). In addition to the general β-sheet, members of the bracelet family also feature a short α-helical segment in loop 3. Recently the division into these subfamilies has been blurred with the emergence of chimeric cyclotides which displays properties of both Möbius and bracelet families. Trypsin inhibitor cyclotides (Table 2.1) are the smallest of the three subfamilies. They are classified as cyclotides on the basis that they are cyclic and contain a CCK motif, and are otherwise quite distinct from the Möbius and bracelet subfamilies, both in terms of structure and sequence homology. In addition to the CCK motif, the two members of this subfamily are dominated by a series of well-defined turns, a turn of helix and a small beta-hairpin. As their name implies they are potent trypsin inhibitors (Aboye et al., 2012). Overall structure, characteristics and PDB references for the three subfamilies are summarized in Table 2.1.

Table 2.1. Structures, characteristics and references of 3 families of cyclotides modifications

Structure

kalata B1 Cycloviolacin O1 MCOTI-II Sub family Mobius Bracelet Trypsin inhibitor Special Cis pro peptide bond in Cluster of charged Series of well defined structural loop5 amino acids in loop5, turns , a turn of helix character turn of helix PDB 1nb1 1nbj 1ha9 reference Reference Rosengren et al., 2003 Rosengren et al., 2003 Felizmenio-Qumio et al., to 2001 prototypic structure of sub-family Source= Daly et al., 2009.

In between subfamilies, loop numbers 2, 3, 5, and 6 show extensive sequence variations in their composition and size. The variation in the residues in these loops, superimposed on an otherwise highly conserved framework, makes cyclotides an excellent template to examine structure-activity relationships (Pedersen, 2012).

2.7.4. Cyclotide isolations

A number of potential cyclotides each having different source and mechanism of action or synthesis is summarized in sub sections below;

2.7.4.1. kalata B1

The cyclotides are investigated to be involved in disruption of biological membranes in herbivorous pests, presenting cyclotides for its potential in various applications as pesticidal molecules (Craik et al., 2012). By examining the gut of the Lepidopteran species Helicoverpa armigera larvae of cyclotide Kalata B1 ingested through scanning, light, and transmission electron microscopy it was found to induce swelling, blebbing, ultimately rupture cells of the epithelia and disruption of microvilli. This histological reaction was comparable to that observed in H. armigera larvae to delta-endotoxin produced by Bacillus thuringiensis that controlled cotton crops pests (Barbeta et al., 2008). Similar findings were reported by Jennings and his co-workers (2001) about the isolation of knotted cyclotides from Rubiaceae and Violaceae families focusing on machainsm of synthesis of kalata B1 from Oldenlandia affinis (the African plant).

2.7.4.2. Labaditin

Cyclotide labaditin and its synthetic open chain analogs have the phenomenon of great membrane insertion. The mechanism of this is based on the initial interactions of hydrophobic nature with the lipid membrane followed by change in confirmation, peptide adsorption of peptide and internalization hence, native labaditin reduced the viability in Gram +ve bacteria (Malaker and Ahmad, 2013).

2.7.4.3. Cycloviolacin

Potentially stable (Chemically and biologically) cyclotides like varv A, varv F, and cycloviolacin O2 from Viola arvensis Murr and Viola odorata L., besides being antimicrobial were investigated for cytotoxity against tumor cell lines having defined type of cytotoxic drugs resistance and this activity was dose dependent when compared with normal lymphocytes. This reflected cyclotides potential as pharmacological agent with a unique mode of action (Lindholm et al., 2002). Viola tricolor extracts (hydroalcoholic, ethyl acetate (EtOAc), water & n-butanol fractions 0–800 g/mL) showed potential anticancerous property by inhibiting angiogenesis and inducing apoptosis. Results were confirmed after investigation in human (MCF-7) breast cancer cells and Neuro2a mouse neuroblastoma cells. Whereas anti-angiogenic activities were experimented on chicken chorioallantoic membrane

(CAM). Viola tricolor is also a family which is repository of cyclotides that also has the same properties (Hamid et al., 2014). Eight known & eight new cyclotides (Viphi A–H) were isolated from Viola philippica, a member of traditional Chinese medicines and family Violaceae. In addition, Mram 8 and Viba 17 were reported as mature peptides for the first time. Sequences were elucidated by reduction, enzymatic digestion and tandem mass spectroscopy sequencing. Cytotoxic activities were shown against the cancer cell lines MM96L, BGC-823 and HeLa (He et al., 2011).

Sixteen peptides from extracts of O. affinis, Viola hederaceae, and V. odorata were studied for the 3-D structure of a novel peptide, cycloviolacin O1, using NMR based spectroscopy (Craik et al., 1999). The structure elucidated by these studies showed a distorted triple-stranded cystine-knot arrangement of disulphide (S-S) bonds and ß-sheet. This structure is similar to kalata B1 and circulin A and available structure suggested that sequence variation exist all the way through the peptide family although folding pattern seems to be conserved. Sequence examination revealed that two subfamilies of cyclotides exist, one with a circularization of the backbone like bracelet containing a number residues of positive charge and second Moebius that may be taken as a Moebius strip shaped with a backbone twisting seen due to the presence of cis-Pro peptide bond which was absent in case of bracelet. Removal of 24−28 residues of amino acids in kalata B1 caused disruption of few structural properties related to stability and an overall loss in hemolytic property that was associated with linearization and backbone truncation (Barry et al., 2003). From Viola ignobilis 13 cyclotides sequences that were previously unknown characterized by means of an experimental approach, which was named sequence fragment assembly by MALDI- TOF/TOF (Hashempour et al., 2013).

Much less of cyclotides function and distribution as well as induction for expression is known. An investigation studies on Viola hederacea (Australian violet) and the sweet violet (Swedish), Viola odorata, as model plants was carried out, results of constant expression throughout the year in V. hederacea that reflects a basic armory of cyclotide expression along with some special “add ons”, cyclotide variants expression influenced by external factors only. 14 times more expression induced in warmer climate of Swedish violet showed why expression was variable due to greater climatic changes (Trabi et al., 2004).

2.7.4.4. Cyclotides from Oldenlandia affinis

Although cyclotides are believed to be one of the largest family of naturally occurring cyclic peptides yet cyclotide-encoding cDNAs when isolated from Viola odorata and compared with evolutionarily distinct plant Oldenlandia affinis showed that they posses one to three cyclotide domains preceded by highly conserved NTRs (N-terminal repeat regions) within species and differ between different species. Besides having no homology at peptide sequence level still they shared structurally conserved helical motif reflecting their possible role in processing, folding and detoxifying cyclotide domains from their precursor proteins (Dutton et al., 2004). The identification of a well conserved signal sequence in cyclotide precursors was an important base of isolation, this physicochemical amino acid substitution and tolerance to cystine knot framework can help in protein engineering (Gruber et al. 2008). The discovery of Kalata B8 from O. affinis reported that isomerization at the site of Asp-Gly sequence which was involved in cyclization and it showed flexibility in the core CCK. This finding was previously found in an unrelated knottins (cyclic) from Momordica cochinchinensi. Kalata B8 a hybrid between Mobius and bracelet classes showed structure activity relationship also as it showed anti-HIV activity with no haemolytic activity due to unusual hydrophilic nature (Daly et al., 2006). Moreover, kalata B8 also shows disordered loop 6 and isomerization in its cyclic peptide backbone which is similar to what was observed in MCoTI-II that is a completely different family of cyclotides named trypsin inhibitor cyclotides.

Goransson et al. (2003) profiled cyclotide expression in 6 different violets by LC-MS and showed that single species expected to contain more than 50 cyclotides. Aminoethylation of cysteine residues overcomed their sequencing problems linked to core structure i.e, their joined ends disulfide knots, and enzymatic cleavage sites and clustered or low content of positively charged residues. Thus cleavage sites as well as charges were introduced at the most conserved regions of cyclotides sequence, the cysteines. Complete sequencing of novel cyclotides were determined by MS-MS of nanospray. This strategy for changing conserved cysteines to enzymatic cleavage sites might also be useful in studying of other peptides and proteins displaying similar structural issues related to mass spec analysis. The CCK motif presents some major difficulties in the identification of cyclotides by sequencing involving

29 different HPLC purifications and derivitizations before MS. As issues like, difficulty of separating such hydrophobic peptides, requirement of HPLC methods for purification and analysis & lack of softwares to handle these circular permutants Major progress in methodology included the use of improved LC-MS/MS conditions and development of cyclotide database where mature cyclotides were excised, replicated and appended thus opening a new episode for cyclotide sequencing (Colgrave et al., 2010). As a result of these better proteomics based strategies 11 cyclotide sequences as well as already known ones were identified from Oldenlandia affinis.

2.7.4.5. Chassatides and panitides

Nguyen et al., 2012a characaterized nine novel linear cyclotides from monocot plant Panicum laxum giving an evidence of their existence at the protein level in the Poaceae family. This gives an evidence of existence of Ancient linear analogs that could have been existed before the divergence of dicots and monocots. Similarly Nguyen et al., 2012b also discovered four more uncyclotides and 14 novel cyclotides from Chassalia chartacea. Precursors of these cyclotides were shortest of all known cyclotides at that time. Originally members of this family like Chassalia kolly (Schumach.) Hepper (family Rubiaceae) a medicinally important plant used in nigeria and west African countries for the treatment of diseases like typhoid and as insect repellent. Antifungal & antibacterial activities were observed against Candida albicans, Escherica coli, Salmonella typhii, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis by using agar cup plate diffusion technique using plant extract rich in alkaloids, glycosides and flavonoids. Brine shrimp lethality assay was used to evaluate cytotoxicity with a greater LD50 value (1000 µg/mL) showing less toxicity. Lemna bioassay to check phytotoxicity was used that showed moderate growth inhibitory effects. 40% insecticidal rate against Rhizopertha dominica at 1572.7 µg/cm2 was seen using contact toxicity method.

2.7.4.6. Cliotides

Many bioactive peptides including stable and versatile cyclotides have been isolated from Clitoria ternatea. The cyclotides concentration from C. ternatea is usually somewhat

30 higher than that regarding nearly all cyclotide containing plants from the Violacecae and Rubiaceae families (SEN et al., 2013). Cyclotides are ultrastable peptides with three disulfide bridges and cyclized backbone. Their bioactivities like uterotonic, antimicrobial, antivirus, hemolytic and resistance to extreme treatments make them promising for their use in drug discovery. Abundance of cyclotides (named Cliotides in Clitoria) have been produced from this medicinal plant.The enzyme named butelase 1 was isolated recently from Clitoria ternatea which is liable for the biosynhesis and cyclization of backbone of cliotides. Bunga Telang Ligase is the local name commonly used in Singapore. This enzyme has extraordinary efficiency of cyclization of peptides of plant and animal origin and the yield is greater than −1 −1 −1 95%. Kcat value for Butelase 1 is up to 17 s and its catalytic efficiency is 542,000 M s , therefore, it is known as the fastest peptide ligase. Butelase 1 is the first asparagine/aspartate (Asx) peptide ligase. The sequence similarity between Butelase 1 and legumain proteases is 71%. The only difference is that Butelase 1 does not hydrolyze the legumain protease substrate. As butelase 1 mostly targets the NH2 terminal amino acids of the peptide substrate, so Carboxy terminus specific intermolecular peptide ligations can be checked with the help of it (Nguyen et al., 2014). A lectin found in the seeds of Clitoria ternatea was reported its lectin is useful in cancer studies and efforts have been made to get high yield of Clitoria ternatea lectin (CTL) (Chauhan et al., 2012).

2.7.5. Cyclotides bioactive action and Mechanisms

Cyclotides proved to be versatile in their bioactivities besides being stable drug delivery tool. These peptides behave as excellent antimicrobial agents, antihelminthetic, hormone like behavior, regulate autoimmune responses, anti-HIV, anticancerous, nematocidal and many more.

2.7.5.1. Regulation of autoimmune responses

The immunological system is very crucial for detecting threats to the body and malfunctioning of which down-regulate homeostasis which may lead to autoimmunity frequently linked with malfunctioning T lymphocyte cells signaling. Therefore several medications target to suppress T lymphocytes that become over-reactive but many of them

31 have life-threatening and severe side-effects. Peptides that are ribosomally synthesized are gaining attention due to decreased toxicity effects and enhanced selectivities as compared to small molecules in pharmaceutical industry where particularly circular peptides are unique in these properties with better oral administration properties. Plant cyclotides experimentally proved effective option to inhibit T lymphocyte cells proliferation with a cystine-knot motif, which confers them highly stable small molecules, thus becoming an attractive pharmaceutical tool (Thell et al., 2014).

2.7.5.2. Role in signal transduction like plant hormones

Plant peptides or polypeptides also exist as members of plant signal transduction mechanism in contrary to the concept of classical phytohormones like cytokinins, gibberellins etc. Peptides involved in cell proliferation, wound signal transduction and in the regulation of water / salt homeostasis, i.e. enod40, systemins, phytosulfokines and natriuretic peptides, but still much more are likely to be discovered (Schaller, 2001). In a research study CVX15 based peptides were grafted on loop 6 of MCOTI, a novel peptide that was stable in human serum and inhibited the viral replication of HIV-1 by targeting cytokine receptor CXCR4. An active CXCR4 antagonistic and HIV-1-cell-entry blocker were produced in this study. CXCR4 a chemokine (GPCRs protein) as it behaves as a co-receptor involved in the HIV viral entry into cell and also its overexpression is associated with multiple types of cancers. Thus novel therapeutic peptide based drug design approach can be of great pharmaceutical importance (Aboye et al., 2012).

2.7.5.3. Anthelminthic activities

Cyclotides have anthelminthic activities against Trichostrongylus colubriformis and Haemonchus contortus, important sheep’s gastrointestinal nematodes. For this purpose to investigation was done to check the interaction of kalata B1 a prototypic cyclotide with the external surface of H. contortus adult worms and larvae. Cyclotides showed toxicity without being ingested by the worms rather they interact with the external surfaces alone to show toxicity. Evidence for this work came when this cyclotide was labeled. For which a mutant of kB1 with synthetic lysine at position 29 was replaced with asparagine. The inclusion

32 containing mutant kB1 labeled by probing with fluorescein-conjugated avidin with biotin using NHS chemistry of this cyclotide, which is a deficient primary amine. The nematocidal activity of labeled i.e. biotinylated [N29K]-kB1 using a larval development assay was determined which helped in the understanding one action of the cyclotides of being anthelminthic by being toxic without ingestion and by interacting with the surface of the worm’s lipid-rich epicuticle layer (Colgrave et al., 2010).

Kalata B1 is toxic to 2 important nematodes Haemonchus contortus and Trichostrongylus colubriformis of sheep. A lysine scan was conducted that incorporated positive charges in kalata framework. From 29 total residues each non-cysteine residue was changed to lysine that decreased that decreased hemolytic and nematocidal activities whereas those residues which resisted lysine incorporation when given positive charge increase the same bioactivities that was 13 folds more than the non-mutant form (Huang et al., 2010).

2.7.5.4. Targeting GPCRs

Cyclotides possessing CCK framework that provides it remarkable stability and highly bioactive protein still lacks a lot in explaining about mechanisms involved in its activities. More specifically no receptor for cyclotide native to plants has been reported yet. Recent studies on kalata B7 was reported to induce contractions in human uterine muscle cells. Further techniques like second messenger-based reporter assays and Radioligand displacement confirmed the receptors of vasopressin V1a and oxytocin as part of the family of G protein coupled receptors which were found to be molecular targets cyclotide Kalata B7. Moreover Koehbach and his coworkers generated an oxytocic nonapeptide with high affinity for the receptor of oxytocin that were cyclotide based that enhanced uterine contractions thus strengthening the concept for developing peptide ligands that are cyclotide- based. Since targeting GPCRs that are 30% target of phamacuetical companies and is >10% already marketed target of drugs opens the hidden potential of cyclotides and cyclotide based peptide grafted libraries to be screened for drugs development purposes (Koehbach et al., 2013).

2.7.5.5. Antimicrobial activity

Staphylococcus aureus is an important causative agent of most skin infections, occurring via surgical wounds. A lot of emphasis is on the importance of identifying new compounds with antimicrobial characters where cyclotides gained a lot of interest owing to its high multifunctional properties and stability. Both kalataB2 (KB2) and cycloviolacin 2 (CyO2) proved to be anti-staphylococcal at 25 mM for CyO2 and 50 mM for KB2 with no cytotoxicity against monocytes suggesting that an increase in the phagocytotic index in vivo may possibly be associated with anti-pathogenic behavior (Fensterseifer et al., 2015). Due to continuous threat of resistant pathogens around us stress on the development of antimicrobial agents from pharmaceutical point of view is emphasized. Cyclotides are versatile in this regard being antimicrobial in nature also but still targeted antimicrobial effects need to be explored. Therefore, semipurified (fractionation & SPE-C18 column chromatography) cyclotides from the Iranian plant Viola odorata were tested against plant and human pathogenic bacteria S. aureus, P. aeruginosa, Xanthomonas oryzae, alstonia solanacearum, E. coli, R. cicil and Bacillus sp. Methods like minimal inhibitory concentration (MIC), radial diffusion assays (RDAs) and minimal bactericidal concentration (MBC) was used for assessment of antimicrobial activities. MIC value of semipurified cyclotides was 16 mg/ mL S. Aureus (gram-negative). Studies showed that plant pathogens are more susceptible than human ones (Zarrabi et al., 2013).

2.7.5.6. Anti-cancerous activity

Studies on tumor cells usually make use of overexpression of two proteins Hdm2 and HdmX inorder to promote cell survival by inactivating the pathway of p53 tumor suppressor and targeting the interaction of these 2 proteins is a part of therapeutic strategy for treating cancers. Previously reports showed that small linear peptides linked at p53 protein’s N- terminal fragment proved to be potent antagonists of Hdm2/HdmX. The poor stability and bioavailability of linear variants has been resolved by engineering of stable cyclotide MCoTI-I (MCo-PMI) for antagonizing intracellular p53 degradation pathway by binding with very low nanomolar affinity with both HdmX and Hdm2 with cytotoxic to wild-type

34 p53 cancer cell lines and high stability in human serum. Thus cyclotide MCoTI-I becomes an optimized scaffold for studying protein-protein interactions within cells (Ji et al., 2013).

2.8. A bioinformatics based approach for cyclotides variants and features

Studies are now getting attractions at genes level due to highly increasing number of cyclotide gene variants and types as this gene encoded peptide family has shown to be among the largest number of genetically encoded family of plants. Thus there is continuously increasing data and information regarding conserved sequences and variations also that are directly related to structure-function relationships. Bioinformatics based studies on cyclotides are focus of bioinformaticians now, as there is a need to develop databases and tools to handle them for informative analysis.

An approach of bioinformatics based and combined expression analysis showed discovery of cyclotide-like sequences in Poaceae family such as Zea mays (maize), Oryza sativa (rice) and Triticum aestivum (wheat), whereas Hordeum vulgare (barley) showed tissue-specific expression (Mulvenna et al., 2006). Elevan species of Australian Hybanthus (Violaceae) were sampled, and twenty-six novel sequences were characterized alongwith 246 new cyclotides that added the number of cyclotides in the Violaceae to be >9000 (Simonsen et al., 2005). Recent investigations made on >200 Rubiaceae species screened cyclotides in only 22 species, moreover a few also reported cyclotides in the Apocynaceae. Precursor gene sequence analysis and phylogenetic studies predicted that cyclotides evolved independently in different plant families after divergence from Asterids and Rosids (~125 million years ago). This evidence was also supported by the ubiquitos proteolytic enzymes machinery involved in post-translational cyclization of cyclotides in all plants where found (Gruber et al., 2008).

Furthermore Nguyen and his co-workers (2011b) also found different evolutionary paterns (horizontal/ convergent) supported by the discovery of A1b (Albumin 1 chain b) domain alongwith cyclotide domains (cliotide T1–T12) that separate during displacement from precursor structures that were novel in fabaceae plant Clitoria ternatea that originally posses antimicrobial and cytotoxic properties. Another recent studies on fabaceae plant species which are one of the largest families and significantly playing role in our lives, a

35 study on Clitoria ternatea, the Cter M precursor gene transcript to be found in all plant parts, it also proved to be insecticidal to cotton budworm Helicoverpa armigera (Poth et al., 2011a). Latter 12 novel cyclotides from the seed extracts of C. ternatea were analyzed and characterized by utilizing nanospray and MALDI-TOF. Cyclotides with novel sequence motifs were discovered and their discovery helped to know more about cyclotide biosynthesis. MS analysis of these new cyclotides showed that at cyclization site, Asn to Asp variants are more common in contrast to those reported earlier (Poth et al., 2011b). In vitro cell-based assays proved that cyclotides possess sufficient cytotoxic activities through which cell membrane integrity can be disrupted. Whereas, in vivo, taking a xenograft model, no noteworthy anticancer effect has been seen even by the most potent cyclotide, cycloviolacin O2. Chemosensitization for treating cancer makes use of cyclotides from C. ternatea. The work of Gerlach et al. (2012) proved that when cyclotides are combined with other anticancer reagents, their chemosensitizing abilities become more pronounced (Sen et al., 2013). Cliotides belonging to the cyclotides family are also known to have strong antimicrobial activity against K. pneumonia, E. coli, P. aeruginosa and cytotoxicity against HeLa cells. The cliotide (a cyclotide from Clitoria), cT19 has shown significant antibacterial and immunomodulatory activities (Shamsi et al., 2014).

2.8.1. CyBase:

Increasing bioinformatics based informations about the one the largest genetically encoded peptides mainly cyclic ones urged to develop a database CyBase cyclic proteins that serves as repository of sequences/structures/functions. CyBase was redesigned with increased management to growing protein data and improved user-interactivities. This manages synthetic circular proteins data also providing database search and display capabilities for synthetic sequences, structures and their respective function that doubles the amount of data when compared to the initial version, hosts a novel suite of tools that are useful for the characterization, visualization, and engineering proteins. The tool ‘Diversity Wheel’ is helpful for analysing circular protein sequence variations and another tool ‘Predict Linker’ helps in the engineering of cyclic proteins from their linear targets (Wang et al., 2008). The tools include 2D representations of sequence/structure, a summary of mutational studies of synthetic analogues and about grafting, informations of N- to C-terminal distances of known

36 protein structures and a tool for structural modeling to predict that which linker length to cyclize a protein is the best. Cybase can help to accelerate the discovery of natural cyclic proteins and their engineering assist in drug development projects (http://research1t.imb.uq.edu.au/cybase).

2.9. Candidate Genes involved in cyclotide synthesis

Cyclotides are ribosomally synthesized proteins that are produced in its prepropeptide form which contains cyclotide domains which are enzymatically folded and cyclised into a final active shape for which they are well known.

2.9.1. Asparginyl Endopeptidases (AEP)

An asparaginyl-specific endopeptidase (AEP, a cysteine protease) is also named as legumain-like proteinase (LLP). It is a type of VPE (Vacuolar processing enzyme) and responsible for the post translational modification of different proteins. LLP with an apparent mass of 38.1 kDa was isolated firstly from the cotyledons of kidney bean (Phaseolus vulgaris L.) where it degardes phaseolin (Senyuk et al., 1998). AEP or legumain was firstly also discovered in the ectoparasitic blood-feeders, the ticks to retard disease transmission by disrupting the ability to digest host proteins (Sojka et al., 2007). AEP is restricted to substrate specifically at the C-terminal of asparagine residues. Some of them have been reported from sweet potatoes leaves (SPAE), Vicia sativa, Canavalia ensiformis, Phaseolus vulgaris and Vigna mungo with putative catalytic site at N-glycosylation site (332nd Asn). Their levels of specific expression changed on induction and are tissue specific also. The possible function of SPAE was its engagement with bulk protein mobilization and degradation during leaf senescence stage were also considered (Chen et al., 2004; Chen et al., 2008). Investigations proved the possible function of AEP in the processing of proglutelin in rice for appropriate protein crystallization, protein storage vacuoles (PSV) structure and compartmentalization of storage proteins (Kumamaru et al., 2010). AEP (asparaginyl endopeptidase) normally catalyse peptide bond formation and cleavage that occurs at a highly conserved Asp (sometimes Asn) at the C-terminal of the cyclotide domain in precursor protein. Studies on Nicotiana benthamiana confirmed the hypothesis that do not produce circular proteins but

37 found to change linear forms cyclic form in non-cyclotide-bearing plants. Studies also confirmed clear cut inhibition of cyclic proteins formation when AEP was suppressed (Saska et al., 2007, Conlan et al., 2010).

Vacuolar processing enzyme VPEs or AEP are inhabitants of the large plant valcuole present in vegetative parts whose major role is to degrade or immobilize protein by using different proteases and to recycle nutrients. This enzyme is also involved in the conversions of proproteins to their respective mature functional/structural forms. These VPE / AEPs can also serve us as a tool to regulate or alter different plant growth stages alongwith maturation of different plant peptides and proteins (Chen et al., 2008). Recently attractive reports showed another role of AEP in the processing of intracellular Toll-like receptors (TLRs) that play a role in regulating immune responses against the influenza virus (IAV) as its single- stranded RNA is sensed by receptors among which TLR7 is one. For these transgenic mice showed different reduced immunological responses with and without AEPs. Thus, it shows AEP has a critical role in opening new possibilities for the treatment of TLR7-dependent inflammatory responses and influenza disease (Maschalidi et al., 2012).

AEP catalyzes both cleavage and ligation peptide bond of cyclotides in a single processing event. This enzyme if transformed into plants can be useful in future to manipulate desired functions or phenotypic characters of the plants and vacuolar possessing peptides and proteins like cyclotides also where its naturally not present by making transgenics. When cDNA encoding the precursor of the cyclotide kalata B1 was expressed Nicotiana plant produced the mature cyclotide, together with linear forms not commonly present. Moreover when AEP gene expression was down regulated or inhibited by inhibitor specifically, the amount of cyclic cyclotide reduced a lot as compared linear peptides that increased significantly. These results suggest that an AEP is performing a major function for catalyzing both peptide bond cleavage and ligation of cyclotides in a single processing event (Saska et al., 2012) and making cyclotide proteins both ends meet to form cyclic structures.

2.9.2. Protein Disulphide Isomerases (PDI)

Protein disulfide isomerase (PDI) is a chaperone protein that catalyzes oxidative protein folding by assisting in folding process in endoplasmic reticulum (ER). A

38 bioinformatics based study showed that Oryza sativa consists about nineteen PDI-like genes. Although, their biological roles are not clearly known yet it is believed to be involved during seed development (Kim et al., 2012).

A number of PDIs are involved in formation of disulfide bonds and folding have been reported from soybean leaf, Arabidopsis thaliana and Oryza sativa with conserved exons and introns. It was also found to be invoved in isomerizations during protein folding in bread wheat possessing isozymes imparting conserved as well as diversified sequences when phylogenetically studied (Wadahama et al., 2008; d’Aloisi et al., 2011). Novel VPE genes were also isolated from Solanum lycopersicum called tomato (Ariizumi et al., 2011). Similarly PDI was also isolated in Oldenlandia affinis (OaPDI) where most cyclic stable proteins called cyclotides have been found coexpressing and interacting there (Gruber et al., 2007). The enzyme-dependent folding of plant insecticidal cyclotides was observed by comparing in the absence and presence of OaPDI, folding of kalata B1 derivatives resulted in dramatic enhancement of correct oxidative folding at physiological pH. Experiments were conducted to understand the mechanism of such enzyme-assisted folding of plant cyclotides comparing the folding of cyclotide kalata B1 derivatives in the absence and presence of O. affinis PDI (OaPDI). OaPDI significantly improved the correct oxidative folding of cyclotide at physiological pH. Investigations in detail of folding intermediates of kalata B1 suggested that S-S isomerization plays is a key role of plant PDI for the production of insecticidal cyclotides (Gruber et al,. 2007).

Protein disulphide isomerase (PDI) is an oxidoreductase enzyme produced in higher amounts in the endoplasmic reticulum. A number of reports of the molecular studies of these genes like PDI and PDI-like genes have been reported with success stories from plants like wheat and rice. However few reports on its promoter sequences are available like Aegilops speltoides, Triticum urartu and Aegilops tauschii. Analysis showed that there is much variation in the sequences when compared in all different species but within species it is conserved (Dhanapal, 2012).

PDI gene expression is also well studied in soya bean plant where it showed seed specific expression and found associated non-covalently with proglycinin (a precursor seed

39 storage protein) and with beta-conglycinin (in tunicamycin presence) suggesting its another role as molecular chaperone (Wadahama et al., 2008).

Both PDI and AEP are highly important regarding the synthesis of cyclotide precursors into mature folded and bioactive form. Plants can also be modified/made to prepare cyclotides that originally do not possess it or lack variants with desired bioactive behaviors by using transgenic plants and co-expressing the transgenes in host. Natural way of synthesis always flashes light towards the development of strategies of synthesis of high importance in vivo or in vitro.

2.10. Production strategies of cyclotide

Cyclotides are mostly ribosomally synthesized such as Kalata of B series in a number of violaceae plants like Viola uliginosa, are becoming scarce and endangered. So to preserve such a rich potential source in vitro culturing and direct or indirect organogenesis techniques are employed using different MS based culturing methods. When observed by AFLP, polymorphism of such tetraploid plant was significantly low but flow cytometry revealed that a lot is still shared between the 2 ploidies. Surprisingly the eleven different cyclotides that were originally reported in the diploid aerial maternal parts were found to be significantly in very high amounts in the tetraploid parts. Thus this opens another option of enhanced cyclotide production methods (Slazak et al., 2015).

The recombinant cyclotide KB1was first time biosynthesized in E. coli by Kimura and his co-workers (2005). Biosynthetic approach using PTS (protein trans-splicing) very efficiently generates natural KB1 as well as its several mutants that generated cyclotide- based libraries that could be screened for their bioactivities in vitro. Native chemical ligation method made use of adding N-termini Cys teine rsidue and methionine at the C-terimini of KB1 in the engineered plasmid with a modified VMA intein (vacuolar membrane ATPase). Thus a mechanism (Figure 2.3) has been elaborated for the in vivo biosynthesis of cyclotides in which cellular environments were not more reductive than E. coli’s cytoplasm (Kimura et al., 2005).

Figure 2.3: The in vivo biosynthesis of cyclotides in bacterial cell. Synthesis of cyclotide Kalata B1 into completely folded and cyclized functional form with reactive inteins that on cleavage causes cyclization and reduced environment in the cell causes folding by disulphide bonds formation. Method of Intein-mediated backbone cyclization as shown above in Figure 2.3 has also been used for making Bowman-Birk inhibitor, sunflower trypsin inhibitor 1 (SFTI-1) which was a cyclic peptide. This method is also used for the biosynthesis of other circular peptides like backbone-cyclized naturally occurring θ-defensins and α-defensins. In-vivo biosynthesis of wild-type SFTI-1 inside E. coli, a small library of the cyclic peptide containing multiple Ala mutants was synthesized and trypsin-binding was estimated. Trypsin binding now has been a property of cyclotide to inhibit trypsin by binding to it such as in case of MCOTI. Results demonstrates evolutionary technologies using the cyclic peptide like SFTI-1 as a molecular scaffold (Austin et al., 2010). Thus synthesis of cyclotides both in bacterial species and by plants is very important for sufficient production of completely folded and cyclized cyclotide for the understanding the hidden roles of these peptides within plants and explore the benefits that could help mankind.

2.10.1. Cyclotide expression in yeast cells:

Expressing recombinant bioactive and fully functional cyclotides in eukaryotic cells like Saccharomyces cerevisiae was a chellange. Yeast cells are good models to study different human based functions like many proteins important to understand human biology, signaling proteins, cell cycle proteins, and enzymes for protein-processing were all discovered initially by studying their homologs in Saccharomyces. For the first time an attempt of cyclotide production was successfully done inside yeast cells by using intracellular PTS (protein trans-splicing) technique in combination with a highly efficient split-intein. α- 41 syn (α-synuclein), a small human lipid-binding protein related to Parkinson’s disease. A cyclic peptide cyclo-CLATWAVG (CP4) previously reported to reduce cytotoxicity by α- synuclein-induction in a yeast synucleopathy model, was used with modifications using linear derivative where Cys residue was replaced with Ser, grafted on loop 6 of MCoTI-I. Natural cyclotide MCoTI-I and its engineered bioactive form MCoCP4 were both expressed to a yield of 50 µg/L to 60 µg/L and confirmed by Mass spec. We used MCoTI-I which is a trypsin inhibitor because these are interesting candidates for designing drugs since they can be used as natural scaffolds to create novel biological activities and possess low toxicities to mammalian cells. MCoCP4 showed reduction in the toxicity of α –synuclein (human origin) in live yeast cells. This attempt is successful beginning of using yeast for the screening of genetically encoded libraries of active cyclotides to understand cyclotide interactions and mechanisms in a eukaryotic system (Jagadish et al., 2015).

Moreover it was found that the expressed constructs of peptides also prevented dopaminergic loss of neuron in a nematode animal model Caenorhabditis elegans established as Parkinson’s model. This work was a step ahead to a previous phage display studies that revealed the possibility of utilizing cyclic peptides as protein ligands in general but had a problem that they can’t access proteins into the eukaryotic cells (Kritzer et al., 2009). This work on yeast cells will improve the efficiency of using libraries of cyclic peptides like cyclotides for forward chemical genetics in model organisms of human disease representing eukaryotic system.

2.10.2. Engineered scaffold of cyclotides with grafted epitopes for enhanced bioactive role

Cyclotide kalata B1 (KB1) can also provide engineered superior natural scaffold for active peptides that are active orally and are useful therapeutics. The Bradykinin (BK)-antagonist peptides DAK or DALK were grafted into the KB1 scaffold whose stability was enhanced. Intracellular levels of Ca2+ showed that antagonists designed are specific blockers of bradykinin B1 receptor, but not for B2. Abdominal constriction assay done in vivo revealed significantly pain inhibition response in the model animal. This advantage was observed only for cyclic analogues as compared to linear ones when administered orally as well as for the

42 antagonistic peptide alone with no response. The combined effect of cystine-knot and cyclization proved to be more stronger (Clarence et al., 2012).

The cyclotide scaffold that cross cell membranes through macropinocytosis and can be evolved or engineered using methods of molecular evolution to hinder protein-protein interactions involved in different diseases like cancer or in the designing of new antimicrobial. For example, development of inhibitors of the β-tryptase (serine protease) and human leukocyte elastase (HLE) using the CCK backbone of cyclotide MCoTI-II. β-Tryptase is associated with different inflammatory and allergic problems, and the HLE implicated in pulmonary and respiratory disorders. Moreover MCoTI-based peptides can cross cell membranes in breast cancer cell lines and macrophage through macropinocytosis. It was also seen that grafted helix region from the MCV (molluscum contagiosum virus) FLICE- inhibitory protein (FLIP) into MCoTI-I (loop 6) that triggered apoptosis of virally infected cells. Similarly squash trypsin inhibitor a homolog of cyclotide the RGD sequence was grafted into the 1st loop of EETI-II possessed platelet inhibitory activity. The engineered cyclotides showed more potential of avoiding platelet aggregation than linear peptides variants grafted. In another example, Cyclopsychotride (Cpt) A is natural cyclotide of Psychotropia longipes that was found to be neurotensin inhibitor. Cpt A inhibited neurotensin binding with its receptor to HT-29 cell membranes and increased intracellular levels of Ca2+ which no other neurotensin antagonists can block (Garcia and Camarero, 2010).

2.11. Labeling of Cyclotide

Besides studying cyclotides as a stable peptide based drug that is bioavalable orally as well and well studied from different perspectives there was also a need to study the cyclotide drug in a bifunctional manner. That is being itself a harbor of activities and unique features. It may also serve as a stable drug delivery tool with different grafted epitopes of medicinal or agricultural importances (Jagadish et al., 2013). For this purpose cyclotide have to be labeled in vitro as well as in vivo using different strategies.

2.11.1. Incorporation of non-natural amino acids by orthorgonal t-RNA in peptides:

Scientists have tried a lot to incorporate non-natural amino acids in vivo for site specific incorporation into proteins or peptides. This is done by evolving the E. coli orthogonal t-RNA synthatases for their specificities to add unnatural amino acids of once’s choice by generating that are not endogenous to the host. An orthogonal suppressor t- RNA/amino acyl t-RNA synthatase pair was used in E. coli that is specific for the t-RNAasp originally present in Saccharomyces cerevisae and its partner aspartyl-tRNA synthatases. Both these were used in vitro and in vivo and well characterized. Antimicrobial resistance was also investigated for using this pair (Pastrank et al., 2000).

Non-natural amino acids if site-specifically incorporated become a powerful tool to manipulate or alter proteins for functional and structural studies, or to produce proteins with different properties. This can be done by either labeling pre-synthesized proteins with probes at reactive side chains such as lysine or cysteine residues or by probes incorporation into newly developing proteins in the presence of an altered aminoacyl-tRNA (aa-tRNA). The first approach generally requires mutagenesis extensively. In contrary, using an mRNA with a unique cognate codon and modified aa-tRNAs allows incorporation by co-translational techniques, diverse probes will then be positioned at almost any site with minimal perturbation and high specificity. The second method usually uses an amber suppressor aa- tRNA that can recognize unique nonsense codons (like UAG). However, now a variety of engineered and synthetic aa-tRNAs have also been produced for this purpose, including those that can recognize unique four-base codons. For co-translational incorporation to happen, the non-native amino acid must be bonded to tRNA with high efficiency and specificity, by using an orthogonal aa-tRNA synthetase (aaRS) that is engineered to recognize both tRNA and modified amino acid (Gubbens et al., 2010).

Thus amber suppressor tRNAs are widely used to incorporate multiple probes using non-natural amino acids simultaneously incorporated at different locations within the same protein without being modified. Certain tRNACys derivatives by synthesis, aminoacylation, and modification are developed like amber, ochre and opal suppressor tRNAs originally derived from yeast and E. coli (Youngman et al., 2008). tRNACys can incorporate modified cysteine (chemical) residue selectively at the cognate UGA, UAG and UAA stop codons using an in-vitro translation system. These synthetic tRNAs are aminoacylated in vitro and

44 further bond was stabilized by attaching covalently a fluorescent dye to sulfhydryl group of cysteine. Read through efficiency in sequence is amber > opal >ochre which was further improved by inhibiting eRF1/eRF3 with an RNA aptamer, thus restricting in higher eukaryotic translation systems the intrinsic hierarchy in stop codon selection limited to UGA and UAA termination suppression. This approach significantly expands the chances for incorporating nonnatural amino acids for studying protein structure/function (Gubbens et al., 2010).

2.11.2. Click chemistry for proteins labeling

Those reactions that meet the necessary criteria of being high yielding, selective, and possesing good reaction kinetics are well known as Click reactions. Whereas a subclass of click chemistry reactions whose components are inert to the surrounding biological milieu is called bioorthogonal. Because of the added complications of biocompatibility it goes one step higher then the typical terminology of click reaction. Within the group of bioorthogonal reactions there are cycloadditions lacking exogenous metals as catalysts, so-called Cu-free click reactions. Exogenous metals can have mild to severe cytotoxic effects when used in biological systems and can thus interfere with the delicate metabolic balance of the systems being studied (Jewett & Bertozzi, 2010).

Mimicking nature in organic synthesis may help the invention of new pharmaceuticals given the large number of possible structures. Click chemistry in combination with combinatorial chemistry, building chemical libraries and high-throughput screening speeds up new drug discoveries by making every reaction during multistep synthesis faster, predictable and efficient. One of the most popular reactions is the azide alkyne known as Huisgen cycloaddition reaction using a Copper (Cu+2) catalyst at room temperature (Fig 2.4). Click chemistry has also been used for selective labeling of biomolecules within living systems. A Click reaction that is to be performed in a biological system must meet an even more precise set of criteria than in an in vitro reaction. It must be bioorthogonal, meaning the reagents used may not interact with the biological system in any way, nor may they be toxic to cell. The reaction must also occur at neutral pH or around physiological pH and at or around body

45 temperature. Most click reactions have high energy content and are irreversible and involve carbon-hetero atom bonding processes. An example of it is the Staudinger ligation of azides.

Figure 2.4. A Cu free click reaction between an azide and Alkyne: In the reaction above azide 2 reacts neatly with alkyne 1 to afford the triazole 3 as a mixture of 1,4-adduct and 1,5- adduct at 98 °C in 18 hours.

Click chemistry has diverse applications like in two-dimensional gel electrophoresis separation, modification of peptide function with triazoles, preparative organic synthesis of 1,4-substituted triazoles, drug discovery, modification of natural products and synthetic pharmaceuticals, natural product discovery, macrocyclizations using Cu(I) catalyzed triazole couplings, modification of nucleotides and DNA by triazole ligation, supramolecular chemistry: rotaxanes, calixarenes, and catenanes, dendrimer design, Polymers and Biopolymers, carbohydrate clusters and carbohydrate conjugation by Cu(1) catalyzed triazole ligation reactions, surfaces, material science, nanotechnology and bioconjugation e.g. azidocoumarin (Kolb, 2003).

Bioorthogonal chemical reactions are leading the way for new inventions in biology. These reactions possess extreme selectivity and biocompatibility in a way that reacting reagents can form covalent bonds within a rich functionalized biological system—in some cases, living organisms. Now Cu-free click chemistry has been tailored to be bioorthogonal by eliminating a cytotoxic copper catalyst, allowing reaction to proceed fastly and without live cell toxicity (Baskin et al., 2007). Cu-free click reaction has been applied within cultured cells, live zebrafish, and mice.

For example the binding of specific DNA or molecular activity may be retained for zinc finger proteins labeled through copper free click chemistry reaction (Urnov et al., 2005). Residue specific labeling of zinc finger proteins which can be useful conducting in vitro screens for assembled zinc finger proteins through FRET assays based on solution and microarray based hybridization. Copper catalyzed azide alkyne cycloaddition help to revealed reaction and chemistry between protein-DNA interaction and protein-protein binding with the use of surfaced based assay. Fluorescent labeling strategies provide a useful tool for the quantitative measure of dynamics in protein and transcriptional factor to visualized protein protein interaction and their specific orthogonal chemistries with the use of different fluorescent dyes (Wang et al., 2009).

Another example of such bioorthogonal reaction includes the incorporation of phenyl azide chemistry into proteins through the use of p-azidophenylalanine (azF). The phenyl azide moiety opens up different routes to non-natural Post-Translational Modifcations (nnPTM): photochemical transformations and Click chemistry adduct addition. Azide–alkyne cycloaddition is fast becoming a important method for orthogonal biomolecule conjugation but has been mostly used in a passive manner, for example, to label proteins. Baskin and his coworkers reported this type of reaction for labeling proteins with a core dibenzylcyclooctyne (DBCO) reactive handle that was chosen to have very distinct properties like it is an amine derivative of DBCO that has hydrogen donor and acceptor groups opening up the potential to form H-bonds between residues not normally close enough to each other in the protein structure secondly they made use of a rhodamine dye (Texas Red) which is a planar, large and hydrophobic dye that has proved valuable in labeling proteins for fluorescent imaging (Hartley et al ., 2015). A similar type of CU-free click chemistry logic between texas red succinamyl ester and DBCO amine an alkyne was used in our studies also.

2.11.3. Labeling of cyclotides

Labeling peptides was one of the major needs of science in earlier times for immune assays like an ultrasensitive chemiluminoenzyme immunoassay (CLEIA) where digoxigenin- labeled bradykinin was used as tracer for quantifying kinins. This assay was so sensitive that

47 it can detect bradykinin levels upto 0.1 fmol/mL in its lower limit with ED50 of 0.78 pmol/mL in extracts of carrageenan inflamed and normal tissues (Decarie et al., 1994). Peptides have also been labeled for ELISA based studies for chemiluminiscence detection. Practices of peptides labeling with the stable 15N isotope and NMR-active helps in NMR based studies that illustrates structural and dynamics studies and use it as tracers. Studies on the recombinant vascular endothelial growth factor (VEGF) biotinylated ones illustrates such examples that are involved in various cancer related studies when we inhibit its binding with others. Studies showed that when such labeled peptide was substitute by benzyl or 2 methylene groups and also one peptide with the substitution with coumarinyl group linked by methylene, the coumarinylated peptide had an advantage of being used in co-crystallizations and in biological studies related to imaging and advantage over the other types labeled. Such peptides and labeling methods were advantageous for cancer based studies (Wang et al., 2015).

However, labeled cyclotides being head-to-tail cyclized peptides are not amenable by conventional strategies using recombinant labeling. This limitation was overcomed by growing Oldenlandia affinis, a cyclotide-bearing plant on a medium containing nitrogen-free agar that was supplemented with 15N salts and got complete labeling with no detrimental effect to plant biomass. Kalata B1 and kalata B2 were labeled by this method for NMR studies (Mylne and Craik, 2008). The uptake of cyclotide MCoTI-I in live HeLa cells was also an application of labeling peptide with fluorescent probes which was monitored using real time confocal fluorescence microscopy imaging. Results showed that MCoTI-I was internalized in HeLa cells readily depending on temperature with easy access to general lysosomal / endosomal pathways (Contreas et al., 2011). In their studies they also used labeled markers for clathrin-mediated and cholesterol-dependent endocytosis of both EGFP and cholera toxin B respectively for such endocytotic studies.

Backbone thermodynamics studies were done on MCoTI-I when it binds to Trypsin as trypsin protese inhibitor. A competition experiment of labeled trypsin-[15N]-MCoTI-I with unlabeled MCoTI-I was used to indicate that the backbone structure of MCoTI-I remained unchanged on trypsin binding and chemical changes that resulted in loop1 & 6 helped to accommodate the increased flexibility of the binding loops and are part of entropic

48 penalties/adjustments. Such interesting results were already observed in other protein-protein interactions of high-affinity that involved protease inhibitors (Puttamadappa et al., 2010). Colgrave et al., (2010b) used mutant of kB1 based on synthetic lysine in which the residue asparagine at 29th position was replaced with lysine that enabled labeling with biotin utilizing the NHS chemistry of such cyclotide, which is normally not present in a primary amine. The biotinylated kB1 was also tested for its nematocidal activity.

Camarero et al. (2007) reported the biological synthesis of a natively folded cyclotide in E. coli, MCoTI-II, intracellular backbone cyclization of a linear uncyclotide was through intein-based fusion of precursor and then correctly folding of cyclotides permits the likelihood of generating cell-based combinatorial libraries that can be simply screened within living cells, for their ability to inhibit or modulate cellular processes. Jagadish and his colleagues in 2013 used Expressed protein ligation EPL for the in-cell generation of MCoTI- I-based cyclotides containing different Uaas using stop codon orthogonal t-RNA synthatase technology to incorporate two different non-natural amino acids in recombinant MCOTI-I for allowing site-specific incorporation of fluorescent probes into this scaffold inside the living cell using E. coli as host. In-cell production, however, is less efficient using EPL i.e. only 4 µg/L & 14 µg/L MCoTI-OmeF & MCoTI-AziF respectively. However PTS-mediated using the efficient Npu DnaE split-inteins of Nostoc puntiforme PCC73102 origin gave a production of around 7 times more efficient, thereby giving an attractive alternative for the production of these types of peptides and polypeptides. This could help using optical properties to screen or probe the cyclotides with Uaas with a fluorescent material like DBCO-AMCA a fluorescent probe with a dibenzo-cyclooctyne (DBCO)-derivative of the fluorescent dye amino-methyl-coumarin acetate (AMCA) to provide in-cell fluorescently labeled cyclotides. FRET can be used measuring distances between molecules and their dynamics especially among molecules such as proteins in a few nanometer ranges (Van der Meer et al., 1994; Levene et al., 2003). The labeled cyclotide was used in fluorescence resonance energy transfer (FRET) to visualize the interaction between modified trypsin that was fused at N-terminus with green fluorescent protein (EGFP). AMCA-labeled cyclotide MCoTI-AziF efficiently binds trypsin-S195AEGFP (KD of 1.8 ± 0.7 nM) in vitro, and cyclotide-protein interaction was monitored by intermolecular FRET shown by the simultaneous increase and decrease of the fluorescence signal at 445 and 515 nm from donor

49 to acceptor, respectively. This finding opens the possibility for in-vitro and potentially also in-cell screening of genetically-encoded libraries of cyclotides for the rapid selection of novel cyclotide sequences able to bind a specific bait proteins using high throughput cell-based optical screening approaches (Jagadish et al., 2013).

2.12. The Future of peptide-based drugs

For the development of peptide based drug there is a need to find out new sources which contain lead materials in the future. Disadvantages and advantages of peptides as drugs are summarized in Table 2.2.

Table 2. 2. Advantages and disadvantages of peptides as drugs (Vlieghe et al., 2010).

Advantages Disadvantages

 High potency  Poor metabolic stability  High selectivity  Poor membrane permeability  Broad range of targets  Poor oral bioavailability  Potentially lower toxicity  High production costs than small molecules  Low accumulation in tissues  Rapid clearance High chemical and biological  Sometimes poor solubility diversity

There are some limitations in the development of peptide based drugs, firstly there is a need of some type of reagents like resins and amino acids which are protected that are very expensive for the development of small peptides. For purification and production of these drugs there should be development of methods that are cheaper. By using chemical synthesis or molecular biology techniques this type of limitation can be overcome. Second type of modifications will be necessary to enhance the permeability of membranes. Due to increase in number of peptides in clinical applications there is a need for the development of methods in order to improve the delivery and transport.

Recently, peptides and small molecule drugs are conjugated to antibodies (to improve targeting), to carbohydrates (to improve solubility, protection from degradation or conformational rearrangements) to PEGs and lipids (to improve uptake and permeability). For the delivery and development of peptide-based drugs there are more common methods that are used as alternative methods. For example, recent discovery, there is the discovery of some type of cyclic peptides that are stable structurally. These types of peptides are produced in larger amount and also reported to play important role in the peptide-based drug design as valuable scaffolds (as shown in Figure 2.5). A B C

Figure 2.5. Schematic illustration of the miniprotein scaffold approach to peptide-based drug design. Panel A highlights potential sources of target epitopes, from fragments of proteins, from bioactive peptides, or from phage display. Panel B schematically illustrates a range of disulfide rich frameworks, including SFTI-1, cyclotides and theta-defensins. Panel C shows the bioactive epitopes grafted into the stable frameworks (Mahon et al., 2010).

The peptide-based drugs may be produced to a greater extent by using plants as main source for their production. Different types of technologies which include sortase-mediated ligation, protein splicing and genetic code reprogramming also play a significant role for the development of cyclic peptide based drugs (Craik et al., 2013).

Moreover further studies are needed for opening the possibility for in vitro and potentially in-cell screening of genetically-encoded libraries of cyclotides. For rapid selection of novel cyclotide sequences able to bind a specific bait proteins that are fluorescently labeled using high throughput cell-based optical screening approaches is required to work on for peptide based drugs development and targeting studies.

Chapter 3 Materials and methods

The present project was split in three sections according to the research objectives. SECTION-I 3.1. Screening of plant extracts and cyclotides for bioactive potential It was planned to screen the bioactivities of cyclotide bearing Plants’ extracts from selected medicinal plants and to compare pure cyclotide MCOTI-I for comparison. All experiments of bioactivities were done in the protein molecular biochemistry lab (PMBL), department of Biochemistry, University of Agriculture Faisalabad, Pakistan. 3.1.1. Selection of plant material Plants with reported cyclotides of different families (table 3.1) were selectively collected from the local market and botanical gardens of the University of Agriculture Faisalabad, and taxonomically identified by a taxonomist in the Department of Botany, University of Agriculture, Faisalabad. Seeds were grown in gardens and in Petri plates under sterilized conditions. Fresh leaves were plucked and stored in liquid nitrogen or at -80 oC till further analysis. Table 3.1. Common and scientific names of different family plants used Sr Plant scientific names Common names Plant Family No. 1. Viola hybrid Rose shades or pansy Violaceae 2. Viola tricolor Wild pansy, heartsease Violaceae 3. Viola Odorata Sweet violet (Banafsha) Violaceae 4. Panicum laxum Lax panicgrass Poaceae 5. Panicum virgatum Switchgrass Poaceae 6. Panicum maximum Guinea grass, buffalo Poaceae grass 7. Clitoria ternatea Butterfly pea, blue Fabaceae pea, Cordofan pea, Asian pigeon wing 8. Hamelia patens Fire bush, scarlet bush, Rubiaceae humming bird bush, madder family 9. Viola F1 super hybrid Local Pansy Violaceae 10. Petunia × atkinsiana (P. × hybrida) Common Rock Jasmine, Solanaceae stalked wild petunia, pine barrens ruellia, Petunia mix

3.1.2. Preparation of plants extracts

The extract of fresh leaves and seeds of the selected medicinal plants were processed according to the following procedure in two different buffers systems 1) phosphate buffer (10 mM Na2HPO4, 100 mM NaH2PO4, 100 mM KCl and 2 mM EDTA) and 2) protein extraction buffer (10 mM Na2HPO4, 15 mM NaH2PO4,100 mM KCl, 2mM EDTA, 1.5% PVPP, 1mM PMSF and 2 mM Thiourea). Samples were made dirt free by washing with tap water and then with distilled water and dried to remove excess water moisture. The leaves and seeds were mixed with extraction buffer in a ratio of 1:2 (w/v) and ground at 4 oC. The material was centrifuged at 10,000 rpm, 4oC for 10 minutes and the residues were discarded. The supernatant was separated and filtered through sterilized Whatman filter paper No.7 to remove particles present in it. The filtrates were stored at 4 oC in sterilized Falcon tubes to be used for different bio assays.

3.1.3. Proteinase K treatment The crude extracts (2 mL) were treated with proteinase K (ThermoFischer Cat# AM2548) 20 mg/mL (Kamoun et al., 2005). Samples were incubated at 37 °C for 2 hours followed by incubation at 100 °C for 10 minutes to inactivate the enzyme proteinase K. The samples were then subjected to different bio assays with and without proteinase K treatment to observe the activities with and without protein content.

3.1.4. Protein estimation

Protein estimation of prepared extract was determined by method confirmed by Bradford assay. Prepared standard solutions for standard curve and noted the absorbance by spectrophotometer at 595 nm, bovine serum albumin (BSA) was used as standard. The concentration of samples was determined by using standard curve (Bradford, 1976).

3.2. Antioxidant studies

Antioxidant perspective of the plant extracts/cyclotide proteins was analyzed by performing different activities as given below.

3.2.1. Total Phenolic contents (TPC)

TPC was determined by using Folin-Ciocalteu reagent method as described by Ainsworth and Gillespie (2007). To 100 mL of sample 200 µL of F-C reagent was added and vortexed thoroughly. Added 800 µL of 700 mM Na2CO3 into each sample and incubated at room temperature for 2 h. Then 200 µL sample was transferred to a clear 96-well plate and absorbance of each well was noted at 765 nm. Amount of TPC was calculated using a calibration curve (Figure 3.1) for Gallic acid. The results were expressed as Gallic acid equivalent (GAE) per dry matter.

Absorbance 2 1.8 1.6 1.4 1.2 1 0.8 y = 0.0055x + 0.0987 2 0.6 R = 0.9968 0.4 0.2 0 0 50 100 150 200 250 300 350 Concentration (mg/g of GAE)

Fig. 3.1. Standard curve for total phenolic contents using gallic acid as standard where concentration is taken in mg/g of gallic acid.

3.2.2. Total Flavonoid content (TFC)

TFC was determined of the extracts by Chang et al. (2002). One mL of extract containing 0.01 mg/L of dry matter was placed in a 10 mL of volumetric flask, and then 5 mL of distilled water was added followed by 0.3 mL of NaNO2. After 5 min 0.6 mL of 10% AlCl3 was added. After another 5 min 2 mL of 1M NaOH was added and volume was made with distilled water. Absorbance was noted at 510 nm. TFC amount were expressed as a catechin equivalent (100- 1300 ppm) per dry matter (Figure 3.2).

Fig. 3.2. Standard curve for total flavonoid contents using catechin as standard

3.2.3 Reducing power assay The reducing power of extracts or cyclotide MCOTI-I was determined according to the procedure described by Yen et al. (2000). Various concentrations of selected plant extracts were mixed with 2.5 mL of 200 mmol/L sodium phosphate buﬀer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide. The mixture was incubated at 50 oC for 20 min. After 2.5 mL of 10% trichloroacetic acid (w/v) were added, the mixture was centrifuged at 650 rpm for 10 min. The upper layer (5 mL) was mixed with 5 mL deionised water and 1 mL of 0.1% of ferric chloride, and the absorbance was noted at 700 nm. Higher absorbance indicates higher reducing power. The assays were carried out in triplicate and the results are expressed as mean values ± standard deviations. The extract concentration providing 0.5 of absorbance (EC50) was calculated.

3.2.4. DPPH scavenging assay

The antioxidant activity of the selected plant crude extracts and their polar fractions was assessed by measuring their scavenging ability of 1,1-diphenyl-2- picrylhydrazyl stable radicals (DPPH). The DPPH assay was performed as described by Bozin et al., (2008) with slight modifications. Crude extract and its polar fractions were added at an equal volume in 500 μL ethanolic solution of DPPH (0.1mM). After 30 minutes incubation at room temperature the absorbance was recorded at 517 nm. The experiment was performed thrice. BHT was used as standard control. Inhibition of free radical by DPPH was

55 calculated as;

__ I (%) = 100 x (A blank A sample / A blank)

Where Ablank is the absorbance of the control reaction mixture excluding the test compounds, and Asample is the absorbance of the test compounds. Percentage scavenging can be calculated.

3.3. Determination of DNA damaging protection activity

3.3.1. Preparation of Phosphate (PO4 ) Buffer

Phosphate (PO4) buffer (50 mM) was prepared of physiological pH 7.4 by dissolving

0.18 g of NaH2PO4 and 0.55 g of Na2HPO4 in 100 mL distilled water.

3.3.2. Preparation of DNA solution

Calf thymus ct-DNA, 0.5 µg/μL was taken and it was diluted up to two folds as 0.5 µg/3µL with 50 mM phosphate buffer at pH 7.4. The reaction was carried out in microcentrifuge tubes.

3.3.3. Preparation of 30% H2O2

30% H2O2 was also prepared from 36% H2O2 of stock solution.

3.3.4. Reaction Mixture preparation

3 µL of diluted ctDNA was transferred to a microcentrifuge tube followed by addition of 20 µL of stock solution of the plant extract or protein in the final reaction mixture. 3 µL of TAE buffer and 4 µL of 30% H2O2 were added successively.

3.3.5. Control 1 preparation 1

Added 10 µL of diluted ctDNA and 12 µL of phosphate buffer in microcentrifuge tube.

3.3.6. Control preparation 2

Added 10 µL of diluted ctDNA and 12 µL of H2O2 in microcentrifuge tube.

3.3.7. Procedure:

An agarose gel of 1% w/v in 1X TAE buffer was prepared by dissolving 1 g agarose in 100 mL 1X TAE buffer and used ethidium bromide dye to staining agarose gel. The gel was photographed under UV light using gel document system (Syngene, UK) after running the samples. For each run, a molecular marker (Fermentas), a negative control, hydrogen peroxide and positive control were loaded, as well as various samples of DNA damage treatments (Terras et al., 1993).

3.4. Evaluation of antimicrobial activity crude extract and its polar fractions:

3.4.1 Bacterial strains i. Escherichia coli (ATCC 35218) ii. Staphylococcus aureus (ATCC 25923) iii. Bacillus subtilis (ATCC 6633) iv. Pasturela multoceda (Local isolate)

The above mentioned strains were characterized from the Institute of Microbiology, University of Agriculture Faisalabad. The pathogenic strains were used to determine the antimicrobial activity of the protein or plant extracts of selected medicinal plants.

3.4.2. Bacterial growth medium, cultures and inoculum preparation

Pure cultures were maintained on nutrient agar medium in the petri plates. For the inoculums preparation 13 g/L of nutrient broth (Oxoid) was suspended in distilled water, mixed well and distributed homogenously. The medium was autoclaved at 121oC for 15 min. Loopfull of pure culture of a bacterial strain was mixed in the medium and placed in shaker for 24 hours at 37 oC. The inocula were stored at 4 oC. The inocula with 1×108 spores/mL were used for further analysis.

3. 4 .3. Antibacterial assay by disc diffusion method

Antimicrobial activity of protein/plant extracts was determined by using disc diffusion method according to CLSI, 2007 (The Clinical and Laboratory Standards Institute,

2007). Nutrient agar (Oxoid, UK) 28 g/L was suspended in distilled water and mixed to homogeneity followed by autoclaving. The media was inoculated and solidified on petri plates. The inoculum was added in the medium. Small filter paper discs were placed on an aluminium file and 70 μL of the protein/ extract sample was dropped on it. It was allowed to dry and then laid flat on growth medium in the Petri plates, and incubated at 37oC for 24 hours. The extracts having antibacterial activity inhibited the growth of bacteria and clear zones were formed around them. The diameters of inhibition zones were measured in millimeters with the help of a zone reader. The results were compared with the similar plates with standard antimicrobial agent Rifampicin (Nwachukwu et al., 2010).

3.5: Formation and hydrolysis of biofilm potential

3.5.1. Biofilm inhibition assay by microtitre plate method

The biofilm formation was be accomplished by method reported by Anjum et al. (2014). The wells of a sterile 96-well flat bottomed plastic tissue culture plate were filled with nutrient broth (Oxoid, UK), testing sample and bacterial suspension inoculated. Negative control wells contained nutrient broth only. The plates were be covered and incubated aerobically for 24 hours at 37 °C. The content of each well was washed three times with of sterile phosphate buffer. The plates were shaked in order to remove all non-adherent bacteria. The remaining attached bacteria were fixed of 99% methanol per well, and after 15 min methanol was removed and plates were left to dry. Then, plates were stained for 5 min with 50% crystal violet per well. Excess stain was rinsed off by placing the plate under running tap water. After that plates were air dried, the dye bound to the adherent cells was resolublized with 33% (v/v) glacial acetic acid per well. The OD of each well was measure using microplate reader (BioTek, USA) (Afzal et al., 2014). All the tests were tested thrice against both selected bacterial strain and the results were averaged. The bacterial growth inhibition (INH%) was calculated as follows:

INH%=100 – (OD630 sample*100)/ OD630 control

3.5.2. Inhibition of biofilm

The qualitative assay for biofilm inhibition was performed according to the method described by Dheepa et al. (2012). The glass slides were poured with nutrient broth and inoculated with a loop full of a pure culture of microbial strain for overnight. After 48 h incubation at 37 °C, the content of each tube was decanted. The slides were stained with 2% crystal violet for 7 min. Then the slides were hold with distilled water for 5 min. A positive result was indicated by the presence of an adherent film of stained material on the surface of the glass slides. Slides containing nutrient broth and inoculums only were included in the test as negative controls. Similarly, slides containing nutrient broth, inoculum and standard antibiotic i.e. Rifampicin were included in the test as positive control to inhibit microbial biofilm inhibition. The protein extracts were seen for their inhibition effect on microbial biofilm with respect to positive and negative control under microscope.

3.6. Cytotoxicity of plant extracts/MCOTI-I by Hemolytic Activity

3.6.1. Assay procedure

The cytotoxicity studies of the protein extracts of different plants were analyzed by hemolytic activity after Powell et al., (2000). Added 3 mL of freshly obtained blood in heparinized tubes to avoid coagulation and gently mixed, poured into a sterile 15 mL falcon tube and centrifuged for 5 min at 850×g. The supernatant was poured off and RBCs were washed three times with 5 mL of chilled (4 oC) sterile isotonic phosphate buffer saline (PBS) solution, adjusted to pH 7.4. The washed RBCs were counted on heamacytometer. The RBCs’ count was maintained to 7.068 x 108 cells per mL for each assay. The 20 µL of plant extract and fractions were taken in 2mL microcentrifuge tubes then added 180 µL diluted blood cell suspension. The samples were incubated for 35 minutes at 37 oC. Agitated it after 10 minutes and after incubation, the tubes placed it on ice for 5 minutes and centrifuged for 5 minutes at 1310 x g. After centrifugation 100 µL supernatant was taken from the tubes and diluted with 900 mL chilled PBS. All microcentrifuge tubes were maintained on ice after dilution. After this 200 µL mixture from each microcentrifuge tube was added into 96 well plates. For each assay, 0.1% triton X-100 was taken as a positive control and phosphate buffer saline (PBS) was taken for each assay as a negative control. The absorbance was noted at 576 nm with a BioTeK, µ Quant (BioTek, Winooski, VT, USA) (Powell et al. 2000).

Triton-X 100 (0.1 %) was used as positive control for 100% lyses and PBS buffer as Negative control 0% lyses. The experiment was performed in triplicate and results were average.

Hemolytic inhibition% was calculated with the help of following formula:

Lysis of RBCs (%) = (Absorbance of sample –Absorbance of Negative control/Absorbance of Positive control) ×100 3.7. Evaluation of thrombolytic activity of protein/peptide extract

3. 7. 1. Specimen

Venous blood was drawn from healthy human volunteers (n = 20) without a history of oral contraceptive or anticoagulant therapy (using a protocol approved by our Institutional Ethics Committee). 500 µL of blood was transformed to each of the previously weighed micro centrifuged tubes to form clots.

3. 7. 2. Procedure

The experiment was carried out in following steps, venous blood drawn from healthy volunteers (n = 20) was transferred in different pre weighed sterile microcentrifuge tube (500 µl/tube). Incubated at 37 oC for 45 minutes. After clot formation, serum was completely removed (aspirated out without disturbing the clot formed) and each tube having clot was again weighed to determine the clot weight. Each micro centrifuge tube containing clot was properly labeled and 100 µL of crude extract and its polar fractions was added to the tubes. Water was also added to one of tube containing clot and this serves as a negative thrombolytic control and streptokinase was added in another tube as to serve as positive thrombolytic control. All the tubes were then incubated at 37 oC for 90 minutes and observed for clot lysis. After incubation, fluid obtained was removed and tubes were again weighed to observe the difference in weight after clot disruption. Difference obtained in weight taken before and after clot lysis was expressed as percentage of clot lysis (Kumar et al., 2010).

3.8. Ames test or mutagenecity test: Following steps to evaluate mutagenecity of our test samples were pursued.

3. 8. 1. Muta-Chromplate kit

A commercial test kit, the Muta-Chromplate, was used to evaluate the mutagenicity of the herbal extracts. The kit was purchased from Environmental Biodetection Products Incorporation (EBPI, Ontario, Canada). This test kit was based on the validated Ames bacterial reverse-mutation test (Ames et al., 1975) but was performed entirely in liquid culture (fluctuation test).

3.8.2. Test bacterial strains

Two mutant strains, S. typhimuriumTA98 and S. typhimuriumTA100 were provided by EBPI. The bacteria were maintained on nutrient agar at 3±1 ºC. The bacteria were inoculated in nutrient broth and incubated at 37 ºC for 18-24 h prior to the test.

3.8.3. Chemicals and solutions used in test

The chemicals from different well known companies used were Davis-Mingioli salt (5.5 times concentrated), D-glucose (40%, w/v), bromocresol purple (2 mg/mL), D-biotin (0.1 mg/mL), and L-histidine (0.1 mg/mL). Two sterile standard mutagens were sodium azide

(NaN3, 0.5 µg/100 µL) for S. typhimurium TA100 and K2Cr2O7 (30 µg/100µL) for S. typhimurium TA98. All chemicals were kept at 3±1ºC until used.

Table 3.2. Scheme followed for Ames test to evaluate mutagenecity, showing trearments given for each category of test sample and test strains with mixture preparations (in mL).

Sr. Treatment Standards Protein Reagent Deionized Test No. (Microtiter plate) extract mixture water strain 1. Blank ------2.5 17.5 ---- 2. Background I for S. ------2.5 17.5 0.005 typhymorium TA100 3. Background II for ------2.5 17.5 0.005 S. typhymorium

TA98 4. Standard mutagen 0.1 ---- 2.5 17.5 0.005 for S. typhymorium TA100 5. Standard mutagen 0.1 ---- 2.5 17.5 0.005 for S. typhymorium TA98 6. Test Sample 1 ---- 0.005 2.5 17.5 0.005 7. Test Sample 2 ---- 0.005 2.5 17.5 0.005

3.8.4. Procedure

The Ames test was performed in micro titer plate as reported by (Maron and Ames, 1983). The plates were sealed in plastic bags and incubated at 37 °C for 4 days. The blank plate was observed first and the rest of plates were read only when all wells in blank were colored purple indicating the assay was not contaminated. The background, standard, and test plates were scored visually and all yellow, partial yellow and turbid wells were scored as positive wells while purple wells were scored as negative. The extract or protein was considered toxic to the test strain if all wells in the plate showed purple coloration. For an extract to be mutagenic, the number of positive wells had to be more than twice the number of positive well in the background plate.

SECTION-II 3.9. Cyclotide genes isolation studies 3.9.1. DNA isolation From plant tissues that are selected on the basis of their potential to posses cyclotide (Local pansy Viola plant, Clitoria, Panicum plants etc), genomic DNA was isolated by the CTAB method described by Doyle and Doyle (1990). Leaf tissues upto 100 to 200 mg was ground to a fine powder in liquid nitrogen in a pestle and mortar. In a microcentrifuge tube the powdered tissue was mixed with 700 µL pre-warmed CTAB buffer [100 mM Tris-HCl (pH 8.0), 20 mM EDTA, 1.4 M NaCl, 2 (w/v) CTAB and 0.2 (v/v) β-mercaptethanol and incubated at 65 C for 30 minutes. After lowering the temperature of the sample to room temperature, an equal volume of chloroform: isoamyl alcohol (24:1) was added, mixed well and centrifuged at 11200×g for 10 min at room temperature. The upper DNA containing phase was transferred into a new microcentrifuge tube and mixed with 0.6 volume isopropanol to precipitate the DNA. DNA was pelleted by centrifugation at 16000×g for 10 minutes, washed with 70% ethanol and air dried. Finally the pellet was dissolved in sterile distilled water.

3.9.2. PCR amplification The amplification of cyclotide gene/s from the plants was done by using specifically designed primers of respected cyclotide gene/s. Different bioinformatics tools like Primer3.0v, Clustalw, BLASTn and Justbio.com hosted tools (http://www.justbio.com) were used to design the primers. First of all, such sequencing portions of the genes were selected that had start and stop codon at a reasonable distance needed for ORF. Such information was taken from already reported sequences from GenBank (NCBI). By using this software, initially cleaned the sequence from unwanted test marks (spaces/gaps) by using of CLEANER hosted tool. Then calculated primer parameter by OLIGOCALC. Through COMPLEMENTOR tool got complementary reverse sequence of DNA. By PRIMER ANEAL tool quickly search and visualize where the primers will hybridize on your DNA template sequence. By the use of CUTTER got the information about restriction enzymes which cut the sequence on appropriate size and help in cloning for sequencing.

3.9.2.1 Dilution of Primers 63

Minispin both forward and reverse primers tubes to collect powder at the bottom of the tube. Forward and Reverse primer were diluted by adding the volume of TE buffer [for 10X; 100 mM of Tris-Cl in 10 mM EDTA (each at pH 8.0)] to make 100 µM stock concentration of primers (Sambrook and Russel, 3rd Edition, 2001). The stocks were first diluted to 10 µM with sterile dH2O and then final working dilutions of primers in PCR mixtures were optimized in the range of 0.1-1 µM.

PCR reaction components of ThermoSCIENTIFCTM were used in the PCR reaction (Table 3.4). The reaction conditions for amplifications of each gene were optimized (Mushtaq and Jamil, 2012). Table 3.3. General PCR reaction mixture for amplification of cyclotide genes (optimized separately for each gene)

Volume Sr.No. Ingredients (For 50 µL of Reaction Final Concentration Mixture) 1. 10X Taq buffer 5 µL 1X

2. dNTPs, 5 µL 0.2 mM of each (2 mM each) 3. Forward primer 5µL (variable) 0.1-1 µM

4. Reverse primer 5 µL (variable) 0.1-1 µM

5. 25 mM MgCl2 Variable 1-6 mM

6. Template DNA Variable 100 ng-1 µg

7. Taq DNA 0.5 µL 1.25 U polymerease 8. Water Variable To make volume up (nucleases free) to 50 µL

Agarose gel (0.8% w/v) was prepared with ethidium bromide as dye and was run on agarose gel electrophoresis apparatus alongwith 6X loading dye added samples and marker DNA ladder. Results were documented for analysis.

3.9.3. PCR product extraction and purification

For column purification of DNA fragments after PCR, a kit (FavorPrepTM, Gel Purification Mini Kit Cat. No. FAGPK001) method was used.

User defined protocol was followed for the purification of the amplified products, eluted in smaller volumes and the results after purification was confirmed on 0.8% agarose gel electrophoresis and results were documented as mentioned earlier.

3.9.4. Plasmid DNA isolations

The plasmid DNA was isolated by using by following the user defined protocol of EZ Plasmid Miniprep Kit (CAT# M1000-50). The eluted plasmid DNA was collected in a collection tube and quantified using 260/280 rations by UV/VIS spectrophotometer (Agilent).

3. 9. 5. Sequencing and bioinformatics analysis of the sequences Sequencing of required and isolated genes was done by using Sanger’s method. Sequence was submitted in GenBank (NCBI). The sequence analysis was done by using suitable bioinformatics tools (Obregon et al., 2009). Sequences obtained after extensive PCR based screening and isolation of selected cyclotide genes were then analysed and trimmed by using different online tools and softwares. The sequence ready after processing was then submitted in the GenBank databases of nucleotide sequences submission. Alignments, BLASTn, phylogenetic analysis, domains finding alongwith different tools were applied to get maximum information related to selected gene/s of cyclotide.

SECTION-III (Part completed in School of Pharmacy, University of Southern California USC, Los Angeles, USA during IRSIP, HEC research scholarship)

3.10. Fluorescent labeling of cyclotide MCOTI-I proteins for studying protein-protein interactions

In order to study cyclotide based protein-protein interaction studies in vitro, the orthogonal tRNA synthetase technology was employed for the production of an MCOTI-I- AziF (mutant) with non-natural amino acid incorporated that binds to an amine fluorescent dye texas red through click chemistry reaction.

3.10.1. Instrumentation and materials used

Analytical HPLC was performed on a HP1100 series instrument with 220 and 280 nm detection using a Vydac C18 column (5 micron, 4.6 x 150 mm) at a flow rate of 1 mL/min. Semipreparative HPLC was performed on a Waters Delta Prep system fitted with a Waters 2487 Ultraviolet-visible (UV/Vis) detector using a Vydac C18 column (15-20 μm, 10 x 250 mm) at a flow rate of 5 mL/min. All runs used linear gradients of 0.1% aqueous trifluoroacetic acid (TFA, solvent A) vs. 0.1% TFA, 90% acetonitrile in H2O (solvent B).

UV/Visible spectroscopy was carried out on an Agilent 8453 diode array spectrophotometer. Electrospray mass spectrometry (ESMS) analysis was routinely applied to all cyclized peptides. ES-MS was performed on an Applied Biosystems API 3000 triple quadrupole electrospray mass spectrometer using Analyst 1.4.2. Calculated masses were obtained using Analyst 1.4.2.

DNA sequencing was performed by Retrogen DNA facility (San Diego,CA), and the sequence data were analyzed with DNA Star Lasergene v5.5.2. All chemicals were obtained from Aldrich (Milwaukee, WI) or Novabiochem (San Diego, CA) unless otherwise indicated. Restriction enzymes were purchased from New England Biolabs. Primers were ordered from IDT (Integrated DNA Technologies).

3.11. Synthesis of DBCO-AMCA

The synthesis of DBCO-TxRD was performed. The amine-reactive Texas Red®-X, succinimidyl ester was used to can be used to create bright red-fluorescent bioconjugates with excitation/emission maxima ~595/615 nm. This reactive dye contains an additional seven-atom aminohexanoyl spacer ("X") between the fluorophore and the succinimidyl ester group. Briefly, Texas Red®-X, succinimidyl ester (TxRD-X, ThermoFischer scientific) (2 mg, Cat# T-20175) was reacted with 5,6-dihydro-11,12-didehydrodibenzo-[b,f]-azocino-3- oxopropyl- 4-amine (DBCO-amine, Click Chemistry Tools, Bioconjugate Technology Company) (7 mg, 25.3 μmol) in DMF (100 μL) containing 5% di-isopropyl-ethylamine (DIEA) for 30 min at room temperature. The reaction was monitored by HPLC and was complete in 30 min. The product (DBCO-AMCA) was purified by reverse phase chromatography using a C18 Sepak cartridge 2 (Waters). The pure product was characterized by ES-MS.

3.12. Cloning of E. coli expression plasmids

All clones used in this study were gifted by the Dr. Julio Camrero lab, School of Pharmacy, University of Southern California (USC), Los Angeles California (Jagadish et al., 2013).

3. 12. 1. pERAzi Genes for argU, thrU, tyrU, glyT and thrT tRNAs were subcloned from the plasmid pRARE2 (Novagen), digested with Sph I and Nhe I, and ligated into plasmid pEvol-AziRS[1] pre-cut with the same restriction enzymes to afford pERAzi. pVLOmeRS The OmeRS gene was amplified by polymerase chain reaction (PCR) using plasmid pBKJY16 as template. The 5’- primer (5’-CT ATG ACT AGT GAC GAA TTT GAA ATG ATA AAG) encoded a Spe I restriction site. The 3’-primer (5’-GTG ATG AGA TCT TTA TAA TCT CTT TCT AAT TGG CTC) encoded a Bgl II. The PCR product was purified digested with Spe I and Bgl II, and ligated into a Spe I, Bgl I-treated pLei-tRNAOpt-STAT3 plasmid to give the plasmid pVLOmeRS.

3. 12. 2. pTXB1-MCoTI-I

The cloning of pTXB1-MCoTI-I was done according to the method described by Lacey et al 2011

3.12.3. pET28-TS-MCoTI-I

The gene fragment containing the DnaE IN Npu (residues 775-876, UniProtKB: B2J066) was amplified by PCR using plasmid pYY1-Npu-IN as template and the following primers: 5’-primer (5'-AAAAACATATGAAACGGAAATATTGAC) and 3’-primer (5ʹ-TTTTAAG CTTAATTCGGCAAATTATCAACCC-3’) which introduced a Nde I and Hind III restriction sites, respectively. The resulting DNA fragment was purified and digested with Sal I and Not I. 5’- Phosphorylated oligonucleotides coding for the DnaE IC Npu (residues 1- 36, UniProtKB: B2J821) were synthesized and PAGE purified by IDT DNA. Complementary strands were annealed in 20 mM sodium phosphate, 0.3 M NaCl buffer at pH 7.4 and the resulting double stranded DNA (dsDNA) was purified using Qiagen’s PCR Purification Kit. 5’- Phosphorylated oligonucleotides coding for MCoTI-I (Table S1, Jagadish et al., 2013) were synthesized and PAGE purified by IDT DNA. Complementary strands were annealed and purified as described above. The three DNA fragments were ligated using T4 DNA ligase (New England Biolabs). The 3 ligation product was then amplified by PCR using the following primers: 5’-primer (5’- AAA ACC ATG GGC AGC AGC CAT CAT CAT -3’) and 3’-primer (5’- TTT TAA GCT TAA TTC GGC AAA TTA TCA ACC C -3’), which introduced Nco I and Hind III restriction sites, respectively. The PCR product was purified, digested with Nco I and Hind III, and ligated into a Nco I, Hind III treated pET28a plasmid (Novagen) to give the plasmid pET28-TS-MCoTI-I.

3.12.4. pET28-TS-MCoTI-stop

The codon encoding the residue Asp15 in plasmid pET28-MCoTI was replaced by the stop codon (TAG) by site directed mutagenesis using the Quick Change Lightning Multi Site Directed Mutagenesis kit (Agilent technologies) and the primer (5’-GCGTTGCCGTCGT tag TCTGACTGCCC-3’). The resulting plasmid was sequenced to confirm the mutation introduced.

3.12.5. pET25-Tryp-EGFP

The gene fragment encoding EGFP was amplified by PCR using plasmid pEGFP-N1 (Clontech) as a template. The 5’-primer (5’- TCT AGA GGT GGT TCT GGT GGT TCT TCT GGT GGT GTC GAC AGC AAG GGC GAG GAG CTG TTC ACC GGG G -3’) introduced a Nco I restricition site and the flexible linker (Gly-Gly-Ser)3 in frame with EGFP. The 3’-primer (5’- A AGC TTA TTA GTG GTG ATG ATG GTG ATG AGA ACC ACC CTT GTA CAG CTC GTC CAT GCC GAG AGT G -3’) introduced a Hind III restriction site, a poly-His tag in frame with EGFP and a stop codon. The resulting DNA fragment was purified, double digested with Nco I and Hind III, and ligated onto a Nco I, Hind III-treated pET25b plasmid (Novagen) to give pET25- EGFP. Mature anionic rat trypsin gene was mutated at positions 16 (I16A) and 195 (S195A) using the plasmid pPicZalphaWTTg (generous gift from Dr. Teaster Baird Jr, SFSU) by site directed mutagenesis kit (Agilent biosystems) as per the manufacturer’s protocol using the mutagenic primers (5’- GGA GAT ATA CAT ATG gcc GTT GGA GGA TAC ACC -3’ and 5’-CAC AGG GCC ACC gcc GTC ACC CTG GCA GC -3’, respectively). The mutations were confirmed by DNA sequencing. Inactive mature anionic rat trypsin was amplified by PCR using the mutated plasmid pPicZalphaWTTg as template and the following primers: 5’- primer (5’- CAT ATG ATC GTT GGA GGA TAC ACC TGC C -3’) and 3’-primer (5’- CC A TG GCG TTG GCA GCA ATT GTG TCC TG -3’), which introduced a Nde I and Nco I restriction sites, respectively. 4 The resulting DNA fragment was purified, digested with Nde I and Nco I and ligated into Nde I, Nco I-treated pET25-EGFP plasmid to give the plasmid pET28-Tryp-EGFP (Jagadish et al., 2013).

3.13. Bacterial expression and purification

Expression and purification of intein precursor 1a E. coli BL21 (DE3) or Origami (DE3) cells (Novagen) were transformed with plasmid pTXB1-MCoTI (Puttamadappa et al., 2010). Expression was carried out in 2XYT medium (1 L) containing ampicillin (100 μg/L) at 30 °C for BL21 (DE3) or room temperature for Origami (DE3) cells. Briefly, 5 mL of an overnight starter culture derived from either a single clone were used to inoculate 1 L of 2XYT media. Cells were grown to an OD at 600 nm of ≈ 0.6 at 37 °C. Protein expression was induced by addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.3 mM at 30 °C for 4 h in BL21 (DE3) cells and room temperature for overnight in Origami (DE3)

69 cells. The cells were harvested by centrifugation. For fusion protein purification, the cells were resuspended in 30 mL of lysis buffer (0.1 mM EDTA, 1 mM PMSF, 50 mM sodium phosphate, 250 mM NaCl buffer at pH 7.2 containing 5% glycerol) in the presence or absence of 20 mM ICH2CONH2 and then lysed by sonication. The lysate was clarified by centrifugation at 15,000 rpm in a Sorval SS-34 rotor for 30 min. The clarified supernatant was incubated with chitin beads (1-3 mL beads/L cells, New England Biolabs), previously equilibrated with column buffer (0.1 mM EDTA, 50 mM sodium phosphate, 250 mM NaCl buffer at pH 7.2) at 4 °C for 1 h with gentle rocking. The beads were extensively washed with 50 bed-volumes of column buffer (50 mM sodium phosphate, 0.1 mM EDTA, 250 mM NaCl buffer at pH 7.2) containing 0.1% Triton X100 and then rinsed and equilibrated with 50 bed-volumes of column buffer. Quantification of the precursor intein was carried out spectrophotometrically using an extinction coefficient per chain at 280 nm of 38,150 M-1cm- 1. The proteins expressed by this method was also used in the different bioactivities performed in the section 3.2 to 3.8.

In vitro EPL-mediated cyclization of cyclotides MCoTI-I Purified intein-fusion proteins 1a, 1b and 1c adsorbed on chitin beads (1 mL) were cleaved in freshly degassed column buffer containing 100 mM GSH (total volume 1.5 mL). The cleavage/cyclization reaction was kept for 20 h at 250 °C with gentle rocking. Once the cleavage was complete the beads were filtered and analyzed by analytical HPLC. Folded cyclotide MCoTI-I was purified by semipreparative HPLC using a linear gradient of 10-30% solvent B over 30 min (Jagadish et al., 2013).

SDS-PAGE confirmation of inteins expressed at each time after expression was done by running gel for total proteins in cells, in supernatant, in pellets, and in Ni-NTA beads binded alongwith 10-180 kDa protein ladder.

3.14. Expression and purification of intein precursor 1b In-cell expression of cyclotide MCoTI-I using protein trans-splicing Origami (DE3) cells (Novagen) were co-transformed with pET28-TS-MCoTI. Precursor intein 2a was expressed as previously described for 1a in presence of kanamycin (25 μg/L) instead. Cells were harvested and lysed as described above. MCoTI-I was purified from the cell lysate

70 using sepharose-trypsin beads. Capture of in-cell generated MCoTI-cyclotides using trypsin- immobilized shepharose Beads Trypsin-immobilized agarose beads were prepared as previously described. Briefly, NHS activated sepharose was washed with 15 volumes of ice- cold 1 mM HCl. Each volume of beads was incubated with an equal volume of coupling buffer (200 mM sodium phosphate, 250 mM NaCl buffer at pH 6,) containing 2-4 mg of porcine pancreatic trypsin type IX-S (14,000 units/mg)/mL for 3 h with gentle rocking at room temperature. The beads were then rinsed with 10 volumes of coupling buffer, and incubated with excess coupling buffer containing 100 mM ethanolamine (Eastman Kodak) for 3 hours with gentle rocking at room temperature. Finally, the beads were washed with 50 volumes of washing buffer (200 mM sodium acetate, 250 mM NaCl buffer at pH 4.5) and stored at 4°C until use. The sepharose-trypsin beads are stable for a month under these conditions. Affinity purification of MCoTI-cyclotides was carried out as follows, 30 mL of clarified lysate was incubated with 500 μL of trypsin-sepharose for one hour at room temperature with gentle rocking, and centrifuged at 3000 rpm for 1 min. The beads were washed with 50 volumes of column buffer containing 0.1% Tween 20 and then rinsed with 50 volumes of column buffer without detergent. The sepharose beads were treated with 3 x 0.5 mL of 8 M GdmCl at room temperature for 15 min and then eluted by gravity. The eluted fractions were analyzed by HPLC and ES-MS. In-cell expression of cyclotide MCoTI-AziF using protein trans-splicing Origami (DE3) cells (Novagen) were transformed with pET28- TS-MCoTI-stop and pERAzi. Precursor intein 2c was expressed as previously described for 1°c but in presence of kanamycin (25 μg/L) and chloroamphenicol (35 μg/L). Cells were harvested and lysed as described above. MCoTI-AziF was purified from the cell lysate using sepharose-trypsin beads as described earlier and characterized by LC-MS.

Expression was carried out in 2XYT medium (1 L) in the presence of antibiotics at room temperature for Origami (DE3) cells. Cells were grown to an OD at 600 nm of ≈ 0.2 at 37 °C at which point 1 mM p-azido-phenylalanine (Chem-Impex International Inc., prepared in 0.2g/2 mL 1N NaOH) was added. Protein expression was induced with IPTG when the OD at 600 nm was ≈ 0.6. Arabinose (0.02 ) was added when the OD at 600 nm reached a value of 0.4. Cells were harvested and the intein precursor purified as described for precursor 1a. The expression level for intein precursor 1c was ≈ 7 mg/L. (Note: all steps involving proteins containing p-azido-phenylalanine needs to be carried out in complete darkness to avoid 71 photodecomposition of the aryl-azido group). Purified products were characterized as the desired product by ES-MS [MCoTI-I: expected averaged mass 3480.9 Da, found mass 3481.0 ± 0.9 Da; and MCoTI-AziF: expected averaged mass 3553.9 Da; mass found 3528.8 ± 0.3 Da, this mass corresponds to the photodecomposition product]. Quantification was carried out spectrophotometrically using an extinction coefficient per chain at 280 nm of 2,240 M-1cm-1 (MCoTI-I) and 3,730 M-1cm-1 (MCoTI-AziF).

3.15. In vitro labeling of Texas red Succinamyl ester with Lys-MCOTI-I

For the optimization studies of texas red behavior towards sepharose beads materials and product yield appearing in the HPLC experiments and method of detachment of labeled dye from the beads such in vitro studies was done. In the presence of alkaline DIEA solvent (D125806 sigma-aldrich >99% CAS 7087-68-5) reaction between the wild type MCOTI-I-

Lys-NH2 was carried out with texas red succinimyl ester. Minimum volume of DMF was used to dissolve a certain concentration of MCOTI-I-Lys this mixture was used to mix dye. 10 µg/µLit of Texas red succinimyl ester solution was prepared in DMF (degassed and dry by beads). Make a mixture of both solutions in 1:5 (i.e. 14.3 nM: 71.5nM) nanomolar ratios (using GraphPad, masses used MCOTI-I= 3481.9 amu, Texas Red succinimyl ester=816.94 amu). Both solutions were reacted in an alkaline medium of DIEA that should be 5% of the total volumes of both solutions. The reaction was carried at room temperature RT and for 10- 20 mins only. The reaction progress and products were analyzed on HPLC based monitoring till end point and the mass of product MCOTI-I-Lys-TxRD was checked. The original peak of wild type MCOTI-I should completey disappear from HPLC output if reaction completes. Each time before injection in HPLC the reaction mixture to be mixed with 10X more volume of 8M GdmHCl. The reaction can be stopped by lowering pH by adding pure acetic acid (5- 10% of total reaction mix volume). A mass spec analysis peak should be observed as 4182.91 amu for labeled MCOTI-I-Lys-TxRD.

Binding of the trypsin sepharose beads of different natures were checked by mixing the labeled MCOTI-I-Lys-TxRD with different volumes of beads and elutions with different buffers and flow throughs were tested with HPLC to check if it binds the hydrophobic sticky texas red dye.

3.16. In vitro labeling of MCoTI-I-AziF with DBCO-TxRD

Trypsin-sepharose immobilized MCoTI-AziF with mass expected 3554.9 Da for N3 form (500 μL, ≈2 μg, ≈0.48 nmol) was reacted with DBCO-TxRD (0.2 mg, 0.33μmol) in 500 μL PBS buffer (20 mM sodium phosphate, 100 mM NaCl buffer at pH 7.2) at 37° C for 30 mins. Once the labeling reaction was complete the excess of dye was removed by washing the trypsin-sepharose immobilized TxRD-labeled MCoTI-AziF with PBS (5 x 10 mL). TxRD-labeled MCoTI-AziF was eluted with 8 M GdmCl and analyzed by LC-MS and ES- MS (TxRD-labeled MCoTI-AziF; expected averaged mass 4532.9 Da, found mass 4532.0 ± 0.7).

Later the reaction was carried out in a buffered 6M GdmCl (mixing 750 µL of 8M

GdmCl+ 250 µL 1M Na2HPO4 pH 9.0+ DMF i.e. 10% v/v of total volume of mixture). Reaction between 5-azido pentanoic acid (712256-250mg Sigma-Aldrich CAS # 79583-98-5, mass =143.14 amu) and DBCO-amine (761540-10 mg Sigma-Aldrich CAS # 1255942-06-3, 276.33 amu expected mass) was also done in optimized buffered 6M GdmCl of pH 7.0 to check if it works for click chemistry reactions. The reaction was tested for completion with equimolar concentrations 1:1 and also with 1:2 and 1:5. HPLC based monitoring of click reaction completion was seen and product mass was confirmed by mass spectrometer. Quantification of TxRD-labeled MCoTI-I-AziF by LCMS using built in softwares by analyzing peak areas after comparison with freshly prepared standards.

3.17. Expression of trypsin-S195A-EGFP

Origami (DE3) cells (Novagen) were transformed with plasmid pET25-Tryp-EGFP. Cells were grown in LB media containing ampicillin (100 μg/L) to an OD at 600 nm of ≈ 0.6 at 37° C. Protein expression was induced with 0.3 mM IPTG for 6 h at 30° C. The cells were harvested by centrifugation, resuspended in 30 mL of lysis buffer (0.1 mM PMSF, 10 mM imidazole, 25 mM sodium phosphate, 150 mM NaCl buffer at pH 8.0 containing 5% glycerol) and lysed by sonication. The lysate was clarified by centrifugation at 15,000 rpm in a Sorval SS-34 rotor for 30 minutes. The clarified supernatant was incubated with 1 mL of Ni-NTA agarose beads (Qiagen) previously equilibrated with column buffer (20 mM

73 imidazole, 50 mM sodium phosphate, 300 mM NaCl buffer at pH 8.0) at 4°C for 1 hour with gentle rocking. The Ni-NTA agarose beads were washed sequentially with column buffer containing (100 mL) followed by column buffer containing 20 mM imidazole (100 mL). The fusion protein was eluted with 2 mL of column buffer containing 100 mM EDTA. The Protein was characterized as the desired product by ES-MS.

Quantification of Typsin-S195A-EGFP was carried out spectrophotometrically using an extinction coefficient per chain at 484 nm of 56000 M-1cm-1(Shaner et al 2005).

Expression level of soluble protein was estimated ≈ 0.7 mg/L (Jagadish et al., 2013). Measurement of affinity constant between trypsin and TxRD-labeled MCoTI-I-AziF The dissociation constant between trypsin and TxRD-labeled MCoTI-I-AziF was measured by fluorescence polarization anisotropy at 25 °C using a Jobin Yvon/Spex Fluorolog 3 spectrofluorometer with the excitation bandwidth set at 5 nm and emission at 5 nm. The excitation wavelength for TxRD was set at 489 nm and emission was monitored at 613 nm (in this experimental system EGFP is the donor and TxRD acts as acceptor which then emits the detectable signals with only 1.8% background). The equilibrium dissociation constant (KD) for the interaction was obtained by titrating a fixed concentration of TxRD-labeled MCoTI-AziF (2 nM) with increasing concentrations of trypsin in 0.5 mM EDTA, 50 mM sodium phosphate, 150 mM NaCl at pH 7 by assuming formation of a 1:1 complex. The KD value was further calculated in ± nM.

3.18. Measurement of affinity constant between trypsin and TxRD-labeled MCoTI-I-AziF

The dissociation constant between trypsin-S195A-EGFP and TxRD-labeled MCoTI-I-AziF was measured by fluorescence resonance emission transfer (FRET) at 25° C using a Jobin Yvon/Spex Fluorolog 3 spectrofluorometer with the excitation bandwidth set at 5 nm and emission at 5 nm as described before.

3.19. Western blot based optimization induction conditions of inteins expression for MCOTI-I production

MCOTI-I cyclotides induction conditions were optimized again inorder to reduce the time of cyclotide production with incorporation of non-natural amino acid to unwanted sites in peptide products produced that usually create erroneous background in labeling results. Origami (DE3) cells (Novagen) were transformed by pET28-TS-MCoTI-I in the same way as described in section 3.12.3. Regular 0.3 mM IPTG induction overnight at room temperature was given and inteins were captured by Ni-beads as describes before in extraction methods. In another experiment 1 mM IPTG induction was given after 0.6 O.D at 37 oC incubation on shaker and equal number of cells were collected after every 1 hour i.e. 1hrs, 2hrs, 3hrs and 4hrs. Last 4 hrs sample was taken twice and incubated overnight in 1x M9 (sterile 10X M9=

60 g anhydrous Na2HPO4+30 g KH2PO4+10 g NaCl + 1mL 100 mM CaCl2autoclave and to 100 mL add 900 mL dH2O alongwith 1mL 1M MgSO4 + 10 mL 20% glucose / dextrose+5 mL 20% NH4Cl+100 µL 0.5% Vitamin B12this makes 1X M9) media at RT for proper folding followed by five times washes after 30 mins shaking pellets with 1X M9 media containing relevant antibiotic kanamycin also. Western blotting of the same amount of cells and using standard amounts of inteins running seperately in SDS-PAGE was used alongwith protein marker (10-180 kDa tricolor protein ladder).

1 x 108 /mL cells in each condition of inductions were taken. Samples were boiled in SDS loading dye (2-mercepto ethanol amine added) before running the SDS PAGE for western blot. PAGE run at 130 volts for 50 mins. Separate the gel from the castings and separately activate the PVDF membrane by methanol washes (20 sec). Place it for adsorption on the gel. First of all remove air bubbles by gentle pressing and soak all things in tank and wash with western transfer buffer. Placed by pressing it in the X-cell II, blotting module (invitrogen) apparatus and placed it again into the vertical SDS-PAGE apparatus and pressed casting locks. Apparatus was filled with western transfer buffer in the centre and sides were filled with ddH2O as coolant and run on 25 volts for 90 mins. Bands of proteins were transferred to the PVDF membrane. Then added blocker (milk powder 5% in 1X TBST buffer) approximately 10 ml in bath tank. After 30 minutes and washed with 1 X TBST buffer (from 10x TBST=Tris Base and NaCl + tween 20 pH7.4) and transferred antibody of anti-histidines (6X histidine epitope tag antibody in a ratio of 1:5000 dilution in 1X TBST buffer) then placed over night at 4 °C and discarded the mixture of primary antibody. Washed 3 times with 1X TBST buffer and added anti-mouse (mammalian) Horse Raddish 75

Peroxidase HRP secondary antibody (in 1:5000 in 1xTBST, for 1.5 hours). After washing three times with 1X TBST buffer, added substrate from westernblot ECl2 substrate system containing 50 µL chemiluminescent solution per 2 mL of substrate solution. Shaked it for 5 mins and immediately placed PVDF membrane (wash by rinsing covered it with aluminium foil) it in the STORM850 Rinsing scanner by inverting the side. Software based fluorescence, scanning and documentation of western blot results was done (Jagadish et al., 2013).

3.20. Expression of cyclotide MCOTI-I in yeast cells (Already Published, Jagadish et al., 2015).

All chemicals were obtained from Aldrich (Milwaukee, WI) or Novabiochem (San Diego, CA) unless otherwise indicated. Restriction enzymes were purchased from New England Biolabs. Primers were ordered from Integrated DNA Technologies. Sacchraomyces cerevisiae strains INVSc1 and W303-1a, and plasmids pYES2/NT, pYC2/NT, were purchased from Invitrogen. Plasmid p426GPD was purchased from ATCC (ATCC 87361). Plasmid propagations were done in Escherichia coli DH5α cells (Invitrogen) using LB Miller medium and the appropriate antibiotic.

3.20.1. Cloning of S. cerevisiae expression plasmids

A) pYES2/NT-TS-MCoTI-I

The gene fragment encoding the Npu DnaE IC and IN (residues 775-876, UniProtKB: B2J066) intein fragments fused the N- and C-terminus of cyclotide MCoTI-I, respectively, was cloned as previously described. Codon optimization of IC-MCoTI-I-IN for S. cerevisiae was done by Genscript USA. The resulting gene was subcloned into a pUC57-Kan to provide the plasmid pUC57-TS-MCoTI-I. The DNA region encoding IC-MCoTI-I-IN was amplified by PCR using plasmid pUC57-TS-MCoTI-I as a template. The 5’-primer (5’- AAA AAA AGC TTA TGG TTC TTC TCA CCA CCA CCA C -3’) and 3’-primer (5ʹ- AAA AAG GAT CCT TAT TAG TTT GGC AAG TTA TCA ACG CGC -3’) introduced a Hind III and BamH I restricition sites respectively. The PCR product was purified, double digested with Hind III and BamH I, and ligated onto a Hind III and BamH I-treated pYES2/NT plasmid to give pYES2/NT-TS-MCoTI-I.

B) pYC2/NT-TS-MCoTI-I

The DNA region encoding IC-MCoTI-I-IN was amplified by PCR and digested with Hind III and BamH I as described above for pYES2/NT-TS-MCoTI-I. The digested amplicon was ligated onto a Hind III and BamH I-treated pYC2/NT plasmid to give pYC2/NT-TS-MCoTI-I (Jagadish et al., 2015).

C) p426GPD-TS-MCoTI-I

The gene encoding IC-MCoTI-I-IN was amplified by PCR using plasmid pUC57-TS-MCoTI-I as a template. The 5’-primer (5’- AAA AAG GAT CCA TGG TTC TTC TCA CCA CCA CCA C -3’) and 3’-primer (5ʹ- AAA AAA AGC TTT TAT TAG TTT GGC AAG TTA TCA ACG CGC -3’) introduced a BamH I and Hind III restricition sites respectively.

3.20.2. S. cerevisiae transformation and selection

Expression plasmids derived from plasmids pYC2/NT and pYES2/NT, and p426GPD were transformed into S. cerevisiae strains INVSc1 and W303-1a, respectively, by electroporation. Briefly, cells were first treated with filter sterilized LiAc/DTT/TE buffer (1 mM EDTA, 10 mM DTT, 10 mM Tris•HCl and 100 mM LiAcO) at pH 7.5. 10-100 ng of DNA in 1-5 μL of water was placed in a sterile screw cap microfuge tube. Electrocompetent cells (40 μL) were added and mixed. The mixture was transferred to a pre-chilled 0.2 mm Biorad electroporation cuvette. Electroporation was performed with a Gene Pulser Xcell electroporator (Bio-Rad) set immediately diluted with 1 mL of ice-cold sterile 1 M sorbitol and returned to ice. Aliquots of the transformation mix were either plated directly or diluted as needed with additional 1 M sorbitol and spread onto synthetic complete media lacking uracil (SC-U) agar plates (Jagadish et al., 2015).

3.20.3. Expression of MCoTI-I in S. cerevisiae

Expression using an inducible promoter (GAL1) was carried out in INVSc1 cells (Invitrogen) at 30° C in SC-U media containing 2 % galactose. Single colonies were grown into 15 mL of SC-U medium containing 2% glucose and allowed to grow overnight at 30 °C with shaking.

Cells were diluted to an optical density at 600 nm (OD600) of 0.4 into SC-U medium

77 containing 2% galactose for 48 h at 30 °C. Expression using a constitutive promoter (GPD) was performed using W303-1a cells at 30 °C in SC-U media containing 2% glucose for 48 h. In both cases cells were harvested by centrifugation, resuspended in 30 mL of lysis buffer (1 mM PMSF, 50 mM Tris•HCl, 100 mM protease cocktail mixture (Thermo Scientific), 150 mM NaCl) at pH 7.4 containing 1% nonidet P-40. After sonication, the lysate was clarified by centrifugation at 15,000 rpm in a sorval SS-34 rotor for 30 min. The clarified supernatant was incubated with Ni-NTA beads (Thermo Scientific), previously equilibrated with lysis buffer at pH 7.4 containing 10 mM imidazole, for 1 h at 4°C with gentle rocking. The beads were extensively washed with 50 bed-volumes of washing buffer (20 mM imidazole, 50 mM Tris•HCl, 150 mM NaCl) at pH 7.4. Quantification of the split intein fragments was performed by SDS-PAGE gel. Folded cyclotide MCoTI-I was identified and quantified by LC-MS/MS (Jagadish et al., 2015)

Chapter 4 Results and Discussion

Plants have been used throughout the history of mankind in traditional medication system. Up till now about two-third to three quarters of the world population have been relying on traditional ethno pharmaceutical products for the treatment of many ailments and diseases. That’s why there is an increased interest to study the phytochemicals for their biological and pharmacological effects. From plants many bioactive agents like phytochemicals have been reported for their bioactivities and some of these are isolated and purified for their specific biological activities (Alviano and Alviano, 2009). For this a number of agents like secondary metabolites, proteins and peptides of biological importance and their bioactive genes are of much interest now in terms of research and pharmaceutical importance.

In the present research work molecular level studies related to cyclotide gene/s isolation from indigenous plants (names mentioned below) and gene sequence analysis was done (section-II). Moreover cyclotide MCOTI-I (Jagadish et al., 2103) was also expressed and purified for different studies like biological in vitro assays including antimicrobial activity, biofilm formation inhibition and biofilm hydrolysis/breakdown, antioxidant potential, mutagenecity assay, thrombolytic assay and toxicological studies were performed to confirm the bioactivities of translated cyclotide MCOTI-I protein (section-I). Different experiments were done to optimize the molecular studies related to MCOTI-I expression in yeast cell for the first time (Published , Jagadish et al., 2015), for expression conditions optimizations in E.coli host and molecular studies for the labeling of MCOTI-I (wild type) and mutant MCOTI-I-AziF (with non-natural amino acid) with fluorescent dye Texas Red (TxRd) were done for studying protein-protein interaction (sections III, project done in USC lab, USA as a part of IRSIP HEC Scholarship).

Section –I

Bioactive potential of indigenous cyclotide bearing plants along with MCOTI-I In section-I, in order to confirm bioactive potential of cyclotide bearing plants of indigenous origin, crude proteins /peptides were extracted from medicinal plants bearing the

79 cyclotide proteins (from literature survey) and the plants’ bioactive potential of extracts with and without proteins from plant were also estimated. Two different buffer systems were employed to segregate the effect of bioactive secondary metabolites from bioactive proteins/peptides, present in selected medicinal plants. The main focus of the current study in section-I was to performed comprehensive antimicrobial, antioxidant and toxicological potential of the following selected medicinal plants in comparison with MCOTI-I cyclotide also; 1. Viola odorata (Banafsha) leaves 6. Pansy F1 seeds

2. Viola Hybrida leaves 7. Panicum vigatum leaves

3. Viola tricolor leaves 8. Panicum laxum leaves

4. Clitoria ternatea seeds 9. Panicum maximum leaves

5. Ptunia mix leaves 10. Hamelia patens seeds

The bioactive potentials were evaluated by conducting the following stepwise discussed experiments;

4.1. Extraction of plants extracts with proteins and other phytochemicals from indigenous cyclotide bearing plants using different buffer systems

Combined effect of bioactive secondary metabolites and bioactive peptides/proteins present in selected plants was estimated simultaneously using a buffer system which had the ability to dissolve hydrophilic bioactive secondary metabolites and proteins in it. Phosphate buffer saline (PBS) pH=7.4 was employed (see section 3.1.4) to extract secondary metabolites as a major portion with proteins as minor part. Biological effect of extracts prepared in PBS were analyzed discretely to see the combined biological effect of extracted proteins and other bioactive secondary metabolites like phenolics, flavonoides, alkaloids, glycosides, tannins and other such phytochemicals present in plants (Ebrahimzadeh et al., 2015). In order to evaluate purely the bioactivities of bioactive proteins and peptides as major bioactive component, another buffer system was used to extract only proteins/peptides from selected plants. Then extracted proteins/peptides were analyzed for their bioactivities parallel to the plant extracts prepared in PBS. Plants have many bioactive proteins/peptides present in them

80 including our desired protein/peptide family of circular proteins called “Cyclotides”. The cyclotides in contrast with the other circular proteins have highly defined three-dimensional structures and, despite their small size, may be regarded as mini proteins. This attribute arises primarily from the knotted network of disulfide bonds that stabilizes the structures. The well- defined structures are associated with a range of biological activities. Indeed, the cyclotides were originally discovered either from screening programs or from anecdotal reports of their biological activity in traditional medicines (Cameron et al., 2008).

Gene encoding the desired cyclotide protein i.e. MCOTI-I was isolated and expressed using suitable expression vectors pASK and pTXB1 (shown in section-III) and translated protein was purified by affinity chromatography technique. After purification of MCOTI-I cyclotide protein, bioactivities of purified protein were checked, each bioactivity using MCOTI-I was done in PBS buffer.

Finally, different results of bioactivities performed by using different extracts of same plant were analyzed and correlated to prove that the bioactivities of MCOTI-I possessed the same potential and that of naturally occurring in plants extracts bearing proteins were identical while excluding the effects of other bioactive secondary metabolic compounds of selected plants.

For extraction, freshly plucked leaflets were taken from botanical garden of university of Agriculture Faisalabad and lab artificial grown plants. Seeds were purchased from local market and both seeds and fresh leaflets were grounded to fine powder and suspended the powdered plant material in Phosphate buffer saline (PBS) to prepare extracts (1g powdered plant / 5 mL of PBS) and similarly seeds and leaves were ground to fine powder using liquid nitrogen to protect their proteins and peptides components being damaged/ degraded. To extract the proteins from powdered plant samples (1 g powdered plant/5 mL of protein extraction buffer), a specific buffer system named protein extraction buffer was used. In which phenyl methyl sulfonyl floride (PMSF) was used to inhibit proteases, polyvinyl poly pyrrolidone (PVPP) and EDTA were used to scavenge secondary metabolites present in plants and thiourea was used to concentrate the protein contents in supernatant (Rabilloud et al., 2002). The extract compositions are mentioned in chapter 3 in detail (see section 3.1.4). Extracts were dialyzed to remove ions and other hydrophilic bioactive components. All these

81 techniques and different components of buffer were used to maximize the yield of proteins in aqueous medium so as to analyze the biological effects of naturally occurring proteins in plants (Jamil et al., 2007). Pure cyclotide MCOTI-I (obtained from section-III) was dissolved in PBS (at a concentration of 40 µg/mL of PBS and 50 µlit was used from this in most bioassays performed like antimicrobial activity) to use it for further bioactive studies in order to explore cyclotide MCOTI-I bioactive potential and for comparison with protein extracts. In this way biochemical effects of secondary metabolites present in selected plants, naturally occurring proteins of selected plants and gene amplified and purified protein products were correlated by performing different in-vitro bioassays at Protein Molecular Biochemistry Laboratory (PMBL), Department of Biochemistry, University of Agriculture Faisalabad Pakistan.

4.1.1 Protein estimation by Bradford method

Above mentioned protein extraction buffer and PBS were employed for the extraction of total soluble proteins from leaves and seeds of selected medicinal plants. Protein concentration was determined by Bradford analysis using BSA calibration curve as standard. The highest concentration of protein was recorded in seed samples of selected medicinal plants while using protein extraction buffer contrary to PBS and protein concentration in leaves of respected plant was low when compared with seeds (Fig. 4.1). In plant leaves maximum concentration of protein was detected in Ptunia mix leaves (3.21 mg/g of powdered plant sample) while using protein extraction buffer PEB and using PBS (1.74 mg/g). Minimum protein concentration was determined in seeds of Pansy F1 (0.56 mg/g in PEB & 0.52 mg/g using PBS). Extracts prepared in PEB showed more protein concentrations on quantification as compared to the same sample extracted in PBS.

For the extraction many factors are important such as pH (acidic, basic), ionic composition of the system, temperature, solvent volume, time for extraction, sequence of the solvents used, types of polymers (protein/peptide) and their molecular weight, concentration of the targeted protein/ peptide, partitioning behavior and many more parameters influence the extraction and stability of proteins (Abugoch et al., 2003; Platis and Labrou, 2006; and Zhao et al., 2006). Hence, no single buffer is appropriate for use as a universal extraction buffer for the extraction of all the targeted proteins.

3.5

3.21 2.89 3 Protein Extraction Buffer 2.5 2.25 PBS Buffer 1.95 2 1.71 1.76 1.37 1.5 1.33 0.86 0.98 0.96 0.86 0.93 1 0.72 0.8 0.56 0.52 0.47 0.36

0.5 0.32 Proteinconcentration mg/g 0

Figure 4.1. Protein estimation by Bradford assay of plant extracts prepared in two different buffers (A graphical representation). The extractable proteins (mg soluble protein/g of seeds weight and fresh leaves weight of selected plants, mean ± SD, n = 3) profile of selected medicinal plant samples using protein extraction buffer (pH 7) and PBS (pH 7.4). Proteins were quantified by Bradford method. The samples 1-10 correspond to the seeds and leaves of selected medicinal plants as mentioned before in the list above. Each bar represents data from at least three independent experiments with error bars showing standard deviation (Westphal et al., 2004; Chinnasamy and Rampitsch, 2006). The figure 4.1 clearly shows that extracts prepared in protein extraction buffer (PEB) extracted more concentrations of proteins/peptides as compared to the PBS buffer extracts.

4.2. In vitro antioxidant potential of selected medicinal plants

Antioxidant activities of extracts prepared from leaves and seeds of selected plants in two different buffer systems were performed/evaluated by different methods.

4.2.1. Total phenolic contents (TPC)

Selected plant extracts were prepared in PBS and protein extraction buffer as mentioned before. Plants extracts were analyzed for their total phenolic components by Folin– Ciocalteau method. It is well known that plant have polyphenolic components and polyphenols are widely distributed in the plant kingdom. These are sometimes present in surprisingly high concentrations (Katalinicc et al., 2006). Results of total phenolic components (TPC) are documented in the figure 4.2 in graphical way using PEB and PBS.

25 22.13 19.22 20 16.05 14.82 14.77 14.78 13.80 13.74 15 13.77 12.34 13.03 10.97 9.67 9.61 10.76 10 8.48 8.03 8.06 7.08 6.45 5

0 Total phenolic GAE) (mg/g contents phenolic Total Viola Viola Viola Clitoria Ptunia Pansy F1 Panicum Panicum Panicum Hamelia odorata Hybrida tricolor ternatea mix vigatum laxum maximum patens (Banafsha)

TPC(mg/g GAE) by using PBS

TPC(mg/g GAE) using protein extraction buffer Figure 4.2. Total phenolic contents of selected plants using two different buffer system The extractable phenolic compounds (mg of soluble phenolic compounds/g of seeds weight and fresh leaves weight of selected plants, mean ± SD, n = 3) as mentioned in section 3.2.1. Results showed that maximum phenolic compounds were present in plant extracts prepared in PBS and concentration of phenolic compounds were lowered in respective plant extracts which were prepared in protein extraction buffer. Reason behind this phenomenon would be the composition of protein extraction buffer which would maximize the protein concentration in extract while reduced the amount of secondary metabolites present in powdered plant samples. This is the property of protein extraction buffer. Results showed that highest TPC contents are in viola hybrid plant using PBS extracts whereas lowest TPC contents were present in panicum vigatum i.e. 8.06 % using same buffer. There was a significant difference of TPC contents of all plants seeds and leaf parts when used different buffer like PEB (Fig. 4.2; Appendix-I). Statistical analysis of the data produced in each

84 activity was analysed for validation and comparisons by SPSS 21.0v statistics and MiniTab 13.0v (shown in appendixes I-V).

The phenolic compounds are the largest and mainly ubiquitous groups of plant’s secondary metabolites that acquire an aromatic ring having one or more hydroxyl moieties. Current interest in these metabolites are aroused from their antioxidant, anticarcinogenic, anti-inflammatory and antimutagenic activities (Atoui et al., 2005; Geetha et al., 2005). Phenols and polyphenols wield their defensive effects through varied mechanism like preventing the creation of carcinogens from precursor substances by behaving as blocking agents (Claudine et al., 2004). The studies carried out on Acacia species have confirmed that extracts bearing phenols and polyphenols have strong antioxidant and antimutagenic activities (Singh et al., 2007).

Total phenolic components and total flavonoids components are responsible for antioxidant activities shown by different plant extracts. Methanolic extracts have much more amount of phenolic compounds. Polarity of methanol is relatively efficient to extract phenolic components while aqueous solvents/ buffers have low tendency to extract phenolic components relatively (Arabshahi-Delouee and Urooj, 2007).

4.2.2. Total flavonoids contents (TFC)

Colorimetric aluminum chloride method was used for flavonoid determination (Chang et al., 2002) as mentioned in 3.2.2. It has been recognized that results for flavonoid content determination of different extracts of all selected plants showed that the extracts prepared in PBS were exhibited higher contents of flavonoids as compared to protein extracts prepared in protein extraction buffer (Fig. 4.3). Least flavonoid components were observed in case of protein extracts. Maximum TFC flavanoids were seen in the Panicum vigatum extracts prepared in PBS whereas lowest seen in the Hamelia seeds prepared in the same buffer. Whereas extracts of PEB viola tricolor leaves with showed maximum TFC whereas Hamelia seeds showed least which is in agreement with the PBS extracts’ data.

40 36.99

30 26.76 21.67 18.91 20 16.87 16.69 13.18 10.28 8.55 7.97 10 6.14 6.65 6.83 CATECHIN eQ. CATECHIN 5.35 5.55 3.91 2.81 3.06 3.60 2.75 0 Viola Viola Viola Clitoria Ptunia Pansy F1 Panicum Panicum Panicum Hamelia

TOTAL FLaVONOID FLaVONOID CONTENTS(MG/G TOTAL odorata Hybrida tricolor ternatea mix vigatum laxum maximum patens (Banafsha) TFC(mg/g Catechin Eq.) using PBS TFC(mg/g catechin Eq.) using protein extraction buffer Figure 4.3. Total flavonoid contents of selected plants using two different buffer system: The extractable flavonoid compounds/contents (mg of soluble flavonoid compounds/g of seeds weight and fresh leaves weight of selected plants equivalent to catechin, mean ± SD, n = 3). Epidemiological studies suggest that the ingestion of foods that are rich in flavonoid provide protection against human diseases that are related with oxidative stress, like coronary heart disease and cancer (Jaouad et al. 2006). Antioxidant activity and their effects on human nutrition and health are considerable. The mechanisms of action of flavonoids are through scavenging or chelating process (Pourmorad et al. 2006).

Verma et al. (2009) explored the different extracts of M. oleifera leaves to study in vitro and in vivo antioxidant potential of selected plant. Oxidative DNA nicking protective activity was performed to evaluate peroxide scavenging potential of different plant fractions. Polyphenolics having antioxidant potential were investigated by HPLC analysis, which indicated the presence of phenolic acids i.e. gallic acid, chlorogenic, ellagic and ferulic acid. Presence of antioxidant flavonoids were also confirmed by HPLC analysis i.e. kaempferol, quercetin and rutin (Mata et al., 2007). So flavonoids also seem to be important factor to contribute in the antioxidant potential of the plants.

4.2.3. Reducing power assay

The reducing potential of the protein extracts of selected plants were estimated following the method by Yen et al. (2000). Ferric ion (Fe+3) reduction to ferrous ion (Fe+2) is employed as an indicator of electron donating activity of phenolic/flavonoids compounds

86 present in plants, which is a central mechanism of antioxidant activity (Nabavi et al., 2008). The occurrence of antioxidants in the selected plant samples would result in decreasing the oxidation of Fe3+ to Fe2+ by providing an electron. Formation of Fe+3 to Fe+2 complex in the presence of potassium ferricyanide and ferric chloride, can be monitored by measuring the formation of Perl's Prussian blue at 700 nm. Increase in absorbance at 700 nm specifies an increase in reductive ability of plant extracts. Figure 4.4 shows the reducing powers of the plant extracts prepared in different buffer system and purified cyclotide MCOTI-I to evaluate if MCOTI-I cyclotide protein have the potential of reducing power. There was an observed concentration dependent behavior of plant extracts in reducing power activity. All extracts had shown good reducing power that was comparable with vitamin C and using the value of ascorbic acid, results were calculated in percentage numeric values.

20 18 16.17

16.02 16 14 13.02 12.94 11.82 11.97 12 10.39 9.77 9.45 9.81 9.98 10 8.5 8.72 8.38 8.18 7.74 7.83 8.32 8.33 8 6.92 7.21 7 6.25 6.33 6.2 6.54 5.995.54 6 5.83 5.06 4.11

4 3.65 %age Reducing activity power Reducing %age 2 0 Viola Viola Viola Clitoria Ptunia Pansy F1 Panicum Panicum Panicum Hamelia Pure odorata Hybrida tricolor ternatea mix vigatum laxum maximum patens cyclotide (Banafsha) MCOT-I %age reducing power(PBS)

%age reducing power without PK (protein extraction buffer) %age reducing power (protein extraction buffer) with proteinase K treament

Figure 4.4. Percentage Reducing Power activity of selected plant extracts using two different buffer system. The reducing power of selected plant extracts prepared in PBS (1g powdered plant leaves or seeds/5 mL of PBS) and protein extraction buffer (1g powdered plant leaves or seeds/5ml of Protein extraction buffer) in comparison with ascorbic acid, mean ± SD, n = 3. Proteinase K treatments showed if there is possible role of some peptide/proteins that could potentially be candidate of reducing nature.

Ptunia mix leaves (16.17%) and Hamelia patens seeds (16.02%) showed maximum reducing power activity in their PBS extracts while Panicum vigatum leaves showed minimum reducing power activity i.e. 5.06%. Results revealed that all plant extracts prepared in PBS showed maximum reducing power activities while respective plant extracts prepared in protein extraction buffer showed decrease in reducing power activities significantly, for example, 9.98% in contrast to 16.17% observed in its PBS extract. Reason of these marked differences in reducing power activities is that PBS has more capacity to dissolve hydrophilic soluble secondary metabolites while in protein extraction buffer a component of PVPP had adsorbed the secondary metabolites and render these secondary metabolites inaccessible to aqueous layer. Proteins in aqueous layer of protein extraction buffer showed the reducing power activities that is confirmed by treatment of protein extracts with proteinase K (see section 3.1.5). The decrease in reducing power activities confirmed that intact proteins showed reducing power activities. In case of some plants e.g. Hamelia patens protein extraction buffer fraction showed slight decrease i.e. from 11.97% to 10.39% in reducing power activity after treatment of proteinase K and this may be due to possibly two reasons, one the unsuccessful activity of proteinase K during handling when enzyme kept denatured or second is the yielding of more reduced bioactivity due to proteolysis of peptides after protein lysis by proteinase K (Li et al., 2014). There’s a little possibility that active phenolic contents still appeared in the PEB extracts i.e. 10.76 mg/g but reducing power may not be due to flavonoids as very little concentration was seen. Cyclotides are one of the most intact proteins that resist most damages that could be a reason of such an active behavior. However cyclotide MCOTI-I showed a considerable reducing power activity i.e. from 8.38 to 6.54% (without and with proteinase K digestion respectively). Amino acids in the cyclotide backbone may be responsible for reducing power activity of MCOTI-I. It also demonstrated that MCOTI-I also has a slight reducing power.

4.2.4. DPPH radical scavenging assay

Plants samples were assessed for their potential to scavenge free radicals by DPPH radical scavenging assay. Butylated hydroxyl toluene BHT (a synthetic antioxidant) and ascorbic acid as a standard antioxidant were used to compare the results of antioxidant potential of selected plant extracts alongwith MCOTI-I protein. All plants’ extracts showed

88 considerable DPPH scavenging activities and surprisingly purified MCOTI-I showed high DPPH scavenging activity in both the buffers. Plant extract prepared in PBS showed maximum DPPH scavenging activities among all other plant species (fig. 4.5). Hamelia patens seeds PBS extract showed maximum activity (80.15%) while PBS solution of purified MCOTI-I showed minimum DPPH scavenging activity 37.32 and 38.42 % in PBS and PEB which were close but still significant without other plant constituents. In PEB extract of Panicum maximum it was maximum (72.85%).

90 80.15 75.01 73.86 73.48 74.35 77.38 80 71.27 68.34 72.22 72.66 72.85 70.81 64.76 65.34 70.31 66.87 67.54 63.26 70 58.83 63.78 58.43 60 58.84 52.54 46.44 48.94 50 39.65 41.43 39.51 41.44 38.42 40 32.56 37.32 30 21.23 20 10 %age scavenging%age activity 0

%age DPPH scavening (PBS)

%age DPPH scavenging(protein extraction buffer)

Figure 4.5. Percentage DPPH scavenging activity of selected plants using two different buffer systems. The percentage DPPH free radical scavenging activity of selected plant extracts prepared in PBS (1g powdered plant leaves or seeds/5 mL of PBS) and protein extraction buffer (1g powdered plant leaves or seeds/5 mL of Protein extraction buffer) in comparison with ascorbic acid and butylated hydroxyl toluene (BHT) mean ± SD, n = 3. Radical scavenging activity of MCOTI-I was also evaluated in both buffers. With treatment of Proteinase K enzyme to protein extracts DPPH scavenging activity was markedly decreased in Hamelia PBS (upto 58.43%) whearas PEB extract of Panicum decreased to 63.26% and MCOTI-I in PBS decreased upto 21.23%. This may be due to proteolysis of bioactive proteins present in protein extracts. Stable MCOTI-I cyclotide, besides being digested, still showed antioxidant behavior as well as in the protein extraction buffer extracts.

It was observed that the water fraction of the methanol extract obtained by decreasing solvent polarity exhibited maximum free radical-scavenging activity (74.1%) at

89 concentration of 100 µg/mL. The potential of L-ascorbic acid to scavenge DPPH radical activity became almost stable after 50 µg/mL and there was no increase in scavenging activity from the concentrations 50 µg/mL to 100 µg/mL. The IC50 value for the water fraction (increasing polarity) was observed to be 35.4 µg/ mL. The activity of the extracts in the DPPH assay indicates their hydrogen-donating ability as the free radicals are known to cause auto-oxidation of the unsaturated lipids (Kim et al., 2002).

4.2.5. DNA damage protection assay

Reactive oxygen species can cause damage to cellular biomolecules like DNA, RNA, enzymes, lipids, carbohydrates and consequently may adversely affect immune functions. Oxidation of bases in DNA, deoxyribose lesions and strand breaks may lead to mutagenic changes and a variety of diseases (Apati et al., 2003). The cellular damage resulting from hydroxyl radicals is the strongest among free radicals. Hydroxyl radicals can be generated by biochemical reactions. Superoxide radical is converted by superoxide dismutase to hydrogen peroxide, which subsequently can produce extremely reactive hydroxyl radicals in the presence of transition metal ions, such as iron and copper or by UV photolysis. Hydroxyl radicals can attack DNA to cause strand scission. Evans et al. (2004) reported that oxidative attack by OH- radical on the deoxyribose moiety may lead to the release of free bases from DNA; generating strand breaks with various sugar modifications and simple abasic (AP) sites. In fact, one of the major types of damage generated by ROS is AP site, a site where a DNA base is lost.

Free radical induced plasmid pBR322 break system is used to evaluate the pro oxidant and antioxidant effects of a compound on DNA (Fig. 4.6). This assay is based on the principle of Fenton reaction (Tian and Hua, 2004). In a Fenton reaction, Fe+2 reacts with

H2O2, resulting in the production of hydroxyl radical, which is considered to be the most harmful radical to biomolecules, Fe+2 are oxidized to Fe+3 in the Fenton reaction. With the attack of •OH generated from the Fenton reaction, supercoiled plasmid DNA is broken into three forms; supercoiled (SC), open circular (OC) and linear form.

The degree of antioxidant or pro oxidant effect of the test samples is presented by the ratio of SC percentage of test samples to that of the control, in an agarose gel electrophoresis assay (Tian and Hua, 2004).

1KB Ladder

Uncoiled

3000 bp unprotectant

1500 bp Coiled protectant

1000 bp

Figure 4.6. DNA damage protection assay for selected plants using pBR322 plasmid

DNA. Lane 1= 1kb ladder (Fermentas). Lane 2= plasmid pBR322 only. Lane 3=H2O2 + FeSO4 + plasmid pBR322. Lane 4= Viola Hybrida leaves + H2O2+ FeSO4 + plasmid pBR322. Lane 5= Viola tricolor leaves+ H2O2+ FeSO4 + plasmid pBR322. Lane 6= Clitoria ternatea seeds + H2O2+ FeSO4 + plasmid pBR322. Lane 7= Ptunia mix leaves + H2O2+ FeSO4 + plasmid pBR322. Lane 8= Purified Cyclotide MCOTI-I + H2O2+ FeSO4 + plasmid pBR322

DNA damaging protection assay of protein extracts of Viola tricolor leaves, Clitoria ternatea seeds and Ptunia mix leaves showed minimum protection against oxidative DNA damage as the results showed open circular (OC) bands in agarose gel while protein extract of Viola Hybrida leaves showed maximum protection against oxidative DNA damage as supercoiled structure of plasmid remained intact (Fig. 4.6). In lane 3 plasmid DNA was completely destroyed or somewhat linearized. Similar results were found for pure cyclotide MCOTI-I which showed minimum DNA damage protection potential of cyclotide MCOTI-I. All results shown here were in PBS buffer.

Another experiment was conducted by using calf thymus (CT) DNA to check the oxidative DNA damage protection potential of selected plant extracts prepared in protein extraction buffer. In this assay protein extracts of selected plants along with proteinase K enzyme treated fractions of protein extracts were engaged to qualify the potential of plant extracted proteins against oxidative DNA damage.

Figure 4.7. DNA damage protection assay for selected plants using ctDNA(calf thymus

DNA) Lane 1= cyclotide protein MCOTI-I + H2O2+ FeSO4 + ctDNA (calf thymus DNA). Lane 2= cyclotide protein MCOTI-I treated with proteinase K + H2O2+ FeSO4 + ctDNA(calf thymus DNA). Lane 3=Viola odorata + H2O2+ FeSO4 + ctDNA (calf thymus DNA). Lane 4=Viola Odorata treated with proteinase K + H2O2+ FeSO4 + ctDNA (calf thymus DNA). Lane 5=Viola Hybrida + H2O2+ FeSO4 + ctDNA(calf thymus DNA). Lane 6=Viola Hybrida treated with proteinase K + H2O2+ FeSO4 + ctDNA (calf thymus DNA). Lane 7=Viola tricolor leaves+ H2O2+ FeSO4 + ctDNA (calf thymus DNA). Lane 8=Viola tricolor leaves treated with proteinase K + H2O2+ FeSO4 + ctDNA (calf thymus DNA). Lane 9=Clitoria seeds + H2O2+ FeSO4 + ctDNA (calf thymus DNA). Lane 10=Clitoria seeds treated with proteinase K + H2O2+ FeSO4 + ctDNA (calf thymus DNA). Lane 11=Petunia mix seeds + H2O2+ FeSO4 + ctDNA(calf thymus DNA). Lane 12=Petunia mix seeds treated with proteinase K + H2O2+ FeSO4 + ctDNA (calf thymus DNA). Lane 13=Pansy F1 seeds + H2O2+ FeSO4 + ctDNA (calf thymus DNA). Lane 14=Pansy F1 seeds treated with proteinase K + H2O2+ FeSO4 + ctDNA (calf thymus DNA). Lane 15= Hamelia patens seeds+ H2O2+ FeSO4 + ctDNA (calf thymus DNA). Lane 16= Hamelia patens seeds treated with proteinase K + H2O2+ FeSO4 + ctDNA (calf thymus DNA). Lane 17= Pure ctDNA (calf thymus DNA) as control. Lane 18= Pure ctDNA (calf thymus DNA) as control. Lane 19= Pure ctDNA (calf thymus DNA) + H2O2 as control 1. Lane 20= Pure ctDNA (calf thymus DNA) + H2O2 + FeSO4 as control 2.

Results of DNA damage protection assay performed by using ctDNA revealed that pure cyclotide with and without proteinase K enzyme treatment MCOTI-I did not protect the DNA damage caused by reactive hydroxyl radicals produced in reaction mixture by Fenton

92 reaction (Fig. 4.7). The protein extract of Viola odorata without pretreatment of proteinase K enzyme showed some protection against oxidative DNA damage while in the protein extract of Viola odorata with pretreatment of proteinase K had less DNA damage protection. This may be due to bioactive protein lysis by proteinase K leading to generation of reactive peptides by products that damage DNA itself. All protein extracts with or without pretreatment of Proteinase K showed probably the similar results. The DNA treated with

H2O2 and FeSO4 was used to detect the oxidative DNA damage alone in the absence of any plant extract. Results were evaluated by intact undamaged ctDNA (calf thymus) band in contrast with damaged DNA smear formation as showed in figure 4.7. Calf thymus DNA has previously been used in many such DNA protection studies, for example, chitosan depolymerised products had protection effects from free radicals (Prashanth et al., 2007).

Three cyclotides, cyo2, kB1 and vaby D were subjected to DNA protection assay by Wang et al. (2008) in some earlier studies where he found no protection.

The inhibition of permanent (transmissible) damage of DNA in living organisms, known as anti-mutagenesis, encompasses several enzymatic and non-enzymatic mechanisms (De Flora and Ramel, 1988) to counteract the modification of DNA components such as nitrogen bases and sugars by electrophiles. Free radicals released during oxidative stress are among the most widespread intracellular DNA modifiers (Møller et al., 1998). Their involvement in carcinogenesis, inflammation, diabetes, atherosclerosis, brain and heart ischemia, aging etc has been intensely addressed during the last years.

The data for antioxidant perspectives evaluated for cyclotide MCOTI-I and cyclotide bearing plant extract prepared in two different buffers PBS and PEB is given in appendix-I.

4.3. Antimicrobial activity of selected medicinal plants

The crude extracts of selected medicinal plants were prepared in phosphate buffer saline (PBS) and screened for antimicrobial activity by disc diffusion method. The zones of inhibition were measured and the results were represented by plus/minus signs (Table 4.1). The results indicate that some plant extracts had a broad spectrum activity by forming clear zones of inhibition while others had negligible zones of inhibition and had very poor activity against the selected bacterial strains (Fig. 4.9 and 4.10). Negative results of some plants

93 indicate that the plants had no active compound, or if present it was either in very low concentration or had lost its activity.

The antibacterial activity was also assessed for proteins/peptide extracts prepared in PEB (Figure 4.8 & 4.10). To confirm that the antibacterial activity was due to isolated proteins and peptides, the protein extracts were treated with Proteinase-K for proteolytic activity. Prior to antibacterial assay, Proteinase-K was denatured by heat treatment. The antibacterial activity was declined in the proteinase K treated samples as compared to the untreated proteins extracts. The decline in antibacterial activity explained that the antibacterial activity was due to bioactive proteins and peptides isolated from selected medicinal plants (Powell et al., 2000). There was no significant decline in antibacterial activity of some plant extracts after treatment of Proteinase-K enzyme and this may be due to some compounds other than peptides (Figures 4.8-4.10).

In order to validate our results of antibacterial activity due to bioactive protein and peptides, a family of bioactive cyclotide proteins was focused. Cyclotide proteins were reported in Rubiaceae and Violaceae family (Pelegrini et al., 2007) and selected medicinal plants belong to aforesaid families i.e. viola species (Chen et al., 2005). MCOTI-I was expressed and purified by using affinity chromatography technique as shown in section-III. Purified protein was also ensured for its antibacterial activity along with extracted proteins from selected plants. The purified MCOTI-I protein had a broad-spectrum antibacterial activity against gram positive and gram negative bacteria (Pranting et al., 2010). Zarrabi and his co-workers also evaluated antibacterial activity of semipurified cyclotides from viola odorata plants against plant and human pathogenic bacteria like Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Xanthomonas oryzae, Ralstonia solanacearum, R. cicil and Bacillus species (Zarrabi et al., 2013).

Antibacterial activity of protein extracts and purified MCOTI-I protein with and without Proteinase-K treatment are mentioned (appendix-II) against four different gram positive and gram negative bacterial strains i.e. Pasturella multocida, Escherichia coli, Bacillus subtilis and Staphylococcus aureus.

Table 4.1. Grading of results for antimicrobial activity of different plant extracts by disc/well diffusion method.

Sr. No. Activity Code Zone size (mm) Interpretation

1 - 0-1 No or poor activity

2 + 2-11 Activity present

3 ++ 12-20 Moderate activity

4 +++ 21-30 Strong activity

5 ++++ 31-40 Highly strong activity

5 (zone of ihibition in mm) in ihibitionof (zone 0

-5 Antibacterial activity of selected plants ofselected activity Antibacterial

Staphylococcus aureus Bacillus subtilis Escherichia coli Pasturella multocida Figure 4.8. Antibacterial activity of different plants extracts prepared in Protein extraction buffer PEB and purified Cyclotide MCOTI-I protein with and without proteinase K treatment against Gram positive (Staphylococcus aureus and Bacillus subtilis strains) and Gram negative (Escherichia coli and Pasturella multocida strain) bacteria. “P” in each case shows plant extract treated with proteinase K. Each data bar shows the data (n) of atleast 3 times taken with mean value ± SD. Data was also statistically analysed for significance and comparison between buffers.

45 40

30 25 20 15

10 inhibition in mm) in inhibition 5

Antibactarial activity (zone of (zone activity Antibactarial 0 -5

Staphylococcus aureus Bacillus subtilis Escherichia coli Pasturella multocida Figure 4.9. Antibacterial activity of different plants extracts prepared in PBS and Purified Cyclotide MCOTI-I protein with and without proteinase K treatment against Gram positive (Staphylococcus aureus and Bacillus subtilis strains.) and Gram negative (Escherichia coli and Pasturella multocida strains) bacteria. . “P” in each case shows plant extract treated with proteinase K. Each data bar shows the data (n) of atleast 3 times taken with mean value ± SD. Data was also statistically analysed for significance and comparison between buffers. Maximum antibacterial activities were shown by the protein extract isolated from Clitoria ternatea seeds and minimum activity is shown by Pansy F1 seeds. Panicum plants in general showed no or neglible antibacterial response towards the microbes used (Appendix- II, Figure 4.9 and 4.10). Purified cyclotide protein MCOTI-I showed broad spectrum antibacterial activity and maximum antibacterial activity was recorded against Pasteurella multocida. Various types of peptides and proteins are produced by plants and these peptides have marked inhibitory effect on growth and development of pathogens. In order to produce pathogenic resistant plant varieties, there is a need to incorporate the genes of such bioactive proteins and peptides into plant varieties to enhance production of crops (Castro and Fontes, 2005). Antimicrobial peptide kills bacteria by blocking both DNA and protein synthesis or even metabolism (Park et al., 1998). Peptides like cyclotides find applications associated with inherent bioactivities, particularly anti-HIV, anti-microbial and anti-tumor (Gerlach et al., 2010; Lindholm et al., 2002). In non-therapeutic areas, one approach is to employ them as natural pesticides utilizing their host defense function.

Zone of Inhibition of bacterial growth measured in millimeter

Figure 4.10. Antibacterial activity or zones of inhibition of different plants extracts: Plant extracts prepared in Protein extraction buffer (with and without pretreatment of Proteinase K enzyme), Phosphate buffer saline PBS and purified Cyclotide MCoTI-I protein (with and without pretreatment of Proteinase K enzyme) against Gram positive

(Staphylococcus aureus and Bacillus subtilis strains) and gram negative (Escherichia coli and Pasturella multocida strains). Their reversible and non-toxic anti-fouling effect makes them potential candidates for inhibiting bacterial or fungal growth in fields or on surfaces (Göransson et al., 2004). An effort to insert the cyclotide gene to crops so as to improve their defense has also been already realized (Gillon et al., 2008).

4.4. Biofilm formation inhibition/hydrolysis

In the current study the bacterial biofilm inhibition/hydrolysis assay was performed by Petri plate method and microtiter plate assay (Salman et al., 2014).

Biofilm is the involvement of microorganisms which is considered by attachment of the microbial cells to solid surfaces. It has the capacity to show wide-ranging microbial resistance contrary to antimicrobial compounds. Microbial cells are frequently surrounded in suspicious matrix which is made up of extracellular polysaccharides with entrenched proteins and DNA. Biofilm can be formed mutually by prokaryotes (bacteria, archaea) and eukaryotes (algae, fungi) (Bryers, 2008). Biofilm can be developed naturally, medically and industrially. National Institutes of Health assessed that biofilms are associated with 80% of all bacterial infections. By recent evaluation, biofilm-dependent diseases are responsible for 19 million infections per annum in the US (Hall-Stoodley et al., 2009; Wolcot et al., 2010).

In quantitative biofilm formation inhibition assay, the protein extracts isolated from selected plants and pure MCOTI-I cyclotide were assayed for their biofilm formation inhibition by micro titer plate method and results were quantified in percentage inhibition of biofilm formation contrary to standard antibiotic Rifampicin. Maximum biofilm formation inhibition activity (84.21%) was found in purified MCOTI-I cyclotide (Figure 4.11 & 4.12). A marked decrease (57.44%) in the activity was observed when MCOTI-I was treated with proteinase K. The decrease in bioactivity might be due to partial loss of integrity of cyclotide protein. Cyclotides are resistant to heat lysis and proteolysis (Nawrot et al., 2011) that is why it still shows its behavior after resisting proteolysis. Clitoria ternatea had maximum biofilm inhibition activity. Clitoria ternatea also showed maximum antibacterial activity. When protein extract of Clitoria ternatea was treated with proteinase K enzyme, its activity was

98 decreased due to proteolysis of bioactive proteins/peptides. Minimum activity was shown by Panicum species and Pansy F1 plants. Thus results suggest that Clitoria possessed maximum antibacterial and biofilm inhibitory bioactive peptide or proteins contents as compared to all other plant samples. Maximum bacterial growth Minimum bacterial growth bacterial growth supperesed by plant sample

Figure 4.11. Biofilm formation inhibition of protein extracts prepared in protein extraction buffer and purified MCOTI-I cyclotide by petri plate method with slight modification

100 90 84.21 80.81 80 72.96 70 61.96 54.41 57.44 60 %age Biofilm formation 50 43.44 inhibition 31.43 36.4 40 28.51 28.12 26.28 30 17.55 27.42 10.83 15.46 13.80 20 13.72 15.12 12.26 14.55 11.56 10 9.26

0 Percentage Biofilm formation inhibition formation Biofilm Percentage

Plant name

Figure 4.12. Percentage Biofilm formation inhibition of protein extracts prepared in protein extraction buffer and purified MCOTI-I cyclotide by microtiter plate assay method. . “P/Pk” in each case shows plant extract treated with proteinase K. Each data bar shows the data (n) of atleast 3 times taken with mean value ± SD. Data was also statistically analysed for significance and comparison between buffers.

4.5. In-vitro cytotoxicity assay Hemolytic activity of selected plant extracts prepared in phosphate buffer saline and protein extraction buffer along with purified cyclotide MCOTI-I with and without treatment of proteinase K enzyme, were assayed by the method of Powell et al., 2000. Maximum hemolytic activity (48.50%) was observed in Ptunia mix in phosphate buffer and 30.21% in protein extraction buffer PEB. Upon treatment of proteinase K a decrease in activity was observed. In some plant extracts with treatment of proteinase K enzyme showed increased hemolytic activity, that may be due to production of more cytotoxic peptide fragments. Increase in hemolytic activity may also be due to proteinase K enzyme catalysis of RBCs membrane. In case of pure cyclotide MCOTI-I percentage hemolytic activity was recorded to be 50.42% (Appendix-III) and upon treatment of proteinase K enzyme the activity was decreased (32.15%) as shown in Figure 4.13.

100

120

100

Percent haemolytic activity haemolytic Percent 20

Heamolytic activity(PBS buffer)

Hemolytic activity(Protein extraction buffer)

Hemolytic activity(Protein extraction buffer) with Proteinase K treatment Figure 4.13. Percent hemolytic activity of selected plants prepared in PBS and protein extraction buffer with and without treatment of proteinase K enzyme. Each data bar shows the data (n) of atleast 3 times taken with mean value ± SD. Data was also statistically analysed for significance and comparison between buffers. Cyclotides are plant-derived proteins that naturally exhibit various biological activities and whose unique cyclic structure makes them unusually stable and resistant to denaturation or degradation (Contreras et al., 2011). Hemolytic activity, i.e., the ability to cause lysis of erythrocytes, was one of the first activities reported for cyclotides of viola peptide-I (Gustafson et al., 2004). Overall, cyclotides are only mildly hemolytic, with median hemolytic doses (Daly et al., 2000) which are weak compared to the potent hemolytic agent. Interestingly, hemolytic activity is lost on linearization of cyclotides (Barry et al., 2003), showing that the intact cyclic backbone is important for this activity, as also appears to be the case for a number of other cyclotide activities, including anti-HIV activity. So far there have been no reports describing a functional role for this hemolytic activity in a specific host defense interaction. Most investigations of the hemolytic activity of cyclotides have been more concerned with engineering out this activity for cases where a cyclotide framework is targeted for use as a pharmaceutical template. For example, an alanine scan identified a

101 region of the kalata B1 surface in which substitution of any one of nine resides with Ala significantly reduced the hemolytic activity.

Independent studies showed that the membrane binding was functional, in that cyclotides were able to induce leakage of contents from phospholipid vesicles and form large pores in lipid bilayers (Haung et al., 2009), and that membrane binding modulated cytotoxicity. Surface plasmon resonance studies (Kamimori, et al., 2005) also established a specific interaction between cyclotides and phospholipid bilayers. These studies have provided details on the selectivity of particular cyclotides for particular lipid subtypes. Overall, cyclotides appear to have a preference for phosphatidyl-ethanolamine (PE) as opposed to phosphatidyl-choline lipids (PC). Furthermore, for membranes of a given PE content kalata B1 has a higher affinity for membranes in a liquid disordered phase i.e. more rigid membranes rich in cholesterol (Chol) and sphingomyelin (SM) that are raft-like domains (Craik, 2012).

Host defense proteins (HDPs) are oncolytic agents that overcome the limits of current drugs. Antimicrobial proteins/peptides (AMPs) are cationic, amphipathic, which enables the peptides to interact with and disrupt lipid membranes. The anticancer effect of peptides is based on disrupting the cytoplasmic and mitochondrial membranes (Mai et al., 2001; Ellerby et al., 1999; Shai, 2002).

4.6. Mutagenicity assay by Ames test

Mutagenicity assay of the selected plant’s PBS and protein extraction buffer extracts were performed by Ames test (Ames et al., 1975). EBPI's Muta-ChromoPlateTM kit was used for the detection of mutagenic activity of different plant extracts prepared in PBS and protein extraction buffer.

The test employs two mutant strains, TA98 and TA100 of Salmonella typhimurium, carrying mutation(s) in the operon coding for histidine biosynthesis. When these bacteria are exposed to mutagenic agents, reverse mutation from amino acid (histidine) auxotrophy to prototrophy occurs. Traditionally, reverse-mutation assays have been performed using agar plates, known as ‘pour plate’, ‘agar-overlay’ or “plate-incorporation” assays. An alternate assay performed

102 entirely in liquid culture is the Fluctuation test, originally devised by Luria and Delbruck (1943) and was modified by Hubbard in 1984.

Plant extracts prepared in phosphate buffer saline and protein extraction buffer were screened for their mutagenic activity. All plant extracts showed no or slight mutagenicity (Figure 4.14 and 4.15, appendix-IV) towards bacterial test strains especially. The extracts prepared in phosphate buffer saline showed some mutagenicity that may be due to presence of some secondary metabolites while the same plant extract when prepared in protein extraction buffer in which it was assumed that minimum secondary metabolites concentration, showed no mutagenicity. Some plants showed slight mutagenicity in their protein extracts and after treating their protein extracts with proteinase K enzyme followed by heat treatment, showed no mutagenic activity. The decrease in mutagenic activity may be due to some mutagenic peptides present in protein extracts of respective plants, which the enzymatic proteolysis and heat denaturation showed no mutagenic activity in test strains (appendix-IV).

Figure 4.14. Percent mutagenicity by Ames test of selected plant extracts prepared in PBS and protein extraction buffer (PEB) with and without treatment of proteinase K enzyme. . “P” in each case shows plant extract treated with proteinase K. Each data bar

103 shows the data (n) of atleast 3 times taken with mean value ± SD. Data was also statistically analysed for significance and comparison between buffers.

Figure 4.15 Representation of Ames test of mutagenicity at selected plant extracts prepared in PBS and protein extraction buffer (PEB) with and without treatment of proteinase K enzyme (all results of all plates not shown here due to high numbers). Studies on other peptides showed that a number of HIV-1 proteins have cytotoxic effect, therefore it was expected that the anticancer effect of HIV-1 p24 protein with these methods. After the prediction, mutagenicity of two anticancer peptides and two non- anticancer peptides was studied by Ames test. Our results show that, the accuracy and the specificity of local alignment kernel based method were 89.7% and 92.68%, respectively. The accuracy and specificity of PseAAC-based method were 83.82% and 85.36%, respectively. By computational analysis, out of 22 peptides of p24 protein, four peptides are anticancer and 18 were non-anticancer. In the Ames test results, it is clear that anticancer peptides (ARP788.8 and ARP788.21) are not mutagenic. Therefore, the results demonstrate that the described computation methods are useful to identify potential anticancer peptides, which are worthy of further experimental validation and 2 peptides (ARP788.8 and ARP788.21) of HIV-1 p24 protein can be used as new anticancer candidates without mutagenicity (Hajisharifi et al., 2014).

104

Similarly genetic activity of peptides isolated from cell-free culture filterate of dimorphic yeast Candida tropicalis (strain CBS94) was investigated in Salmonella/microsome reversion assay. The peptides induced either base-pair substitutions or fiame-shift mutagenesis and one of this promoted both kind of mutations. A peptide also inhibited Mu induced lysis of Escherichia coli, indicating that the peptide could interfere with the growth of a bacterial virus (Tashmeem et al., 1991). Thus research studies in reference to previously reported ones strengthens our results and findings that peptides or proteins can be mutagenic.

4.8. Thrombolytic activity

Thrombosis is the formation of blood clot within the vascular system. Vascular thrombosis is the major clinical problem in developed western countries. Many thrombolytic drugs such as streptokinase, urokinase and tissue plasminogen activator (t-PA) are reported yet. These thrombolytic agents activate the protein plasminogen into plasmin, which dissolve arterial thrombi particularly in cardiac blood vessels (Vaidya et al., 2012).

In-vitro thrombolytic activity of protein extracts prepared in protein extraction buffer was tested by following the method of Kumar et al. (2010). Protein extracts and purified cyclotide MCOTI-I were treated with proteinase K enzyme followed by degradation of enzyme to confirm the thrombolytic activity of proteins. Significant decrease in percentage thrombolytic activity results (Figure 4.16, 4.17 & Appendix-V) were recorded after treating the protein extracts with proteinase K enzyme which confirmed that the thrombolytic activity corresponding to extracted proteins from different selected plants. Purified cyclotide MCOTI-I also showed a significant thrombolysis which is confirmed by decrease in percentage thrombolytic activity when degrading the cyclotide with proteolysis by proteinase K enzyme followed by heat degradation of enzyme to exclude the possible thrombolytic effect of treated enzyme.

Maximum thrombolytic activity was shown by Viola species, the thrombolytic activity of protein extracts of Viola species plants was also markedly decreased when protein extracts were analyzed after treatment of proteinase K enzyme.

105

Purified cyclotide MCOTI-I showed a significant thrombolytic activity (50.86%±2.49) that was reported for the first time in case of cyclotide MCOTI-I. A marked decrease in thrombolytic activity was noted when MCOTI-I solution was treated with proteinase K enzyme (17.46% ±3.54). This decrease in activity may be due to degradation or linearization of cyclic structure of MCOTI-I upon proteolysis catalyzed by proteinase K enzyme.

90 80 70 60 50 40 30 20 10

of selected selected plants of Percentage thrombolytic activity thrombolytic Percentage

%age thrombolytic activity

Figure 4.16 Percentage thrombolytic activity of selected plants and MCOTI-I prepared in protein extraction buffer with and without treatment of proteinase K enzyme. Thrombolysis Slight thrombolysis

No activity

Figure 4.17. General representation of thrombolytic activity of selected plants and MCOTI-I prepared in protein extraction buffer with and without treatment of proteinase K enzyme.

106

Thrombolytics play an important role in several diseases, including deep vein thrombosis (DVT), ST-segment elevation myocardial infarction (STEMI), ischemic stroke, peripheral arterial disease and pulmonary embolism (PE). Campbell and Hilleman (2010) represented Recombinant thrombolytic peptides which were in forms of tissue plasminogen activator (t-PA). Tissue plasminogen activator is an enzyme which catalyzes the conversion of plasminogen into active plasmin and further plasmin helps to dissolve clots. Recombinant thrombolytic peptides were structurally modified to increase their half-life and fibrin specificity. Three clinically related recombinant thrombolytic peptides were employed in study named alteplase (t-PA), reteplase (r-PA), and tenecteplase (TNK). All of these peptides showed significant thrombolysis both in-vitro and in-vivo thrombolysis models. Kumar et al. (2010) determined thrombolytic activities by using in-vitro model assay method of some plants available in Bangladesh, namely Tamarindus indica (Fabaceae), Lawsonia inermis (Lythraceae), and spices, namely Nigella sativa (Ranunculaceae) , Cinnamomum tamala (Lauraceae). For negative control they used distilled water and streptokinase as a positive control. The clot lysis activity in term of percentage of weight loss of in vitro formed clots were conducted. The analysis shows that L. inermis and C. longa have anti-clotting property that could lyse blood clots by using in-vitro model assay method.

Ghosh et al. (2012) studied the comprehensive explanation of new generation streptokinase base thrombolytic agents, their mode of action, structural modification and present situation of the drugs. They also worked on the development of new mutant and hybrid by genetic engineering. Streptokinase is broadly used as a thrombolytic drug for the cure of myocardial infarction. Ren et al., (2008) reported the dual-action of vasodilative and thrombolytic activities of H-Pro-Ala-Lys (PAK, 3a) and five novel analogs H-Pro-Ala-AA (2b−f, AA = Val, Phe, Ser, Glu, and His) were coordinated with Cu (II) to form Cu(II)−Pro- Ala-AA [(3a−f)−Cu(II) . The coordination chemistry was established by the d−d transition followed in their UV, circular dichroism (CD) spectra and the molecular ion in their electrospray ionization mass spectrometry (ESI-MS) spectra. The particle size of powders in solution revealed that the synchronization generally resulted at nanoscale. The bioassays inferred that comparing to the PAK peptides themselves and CuCl2, the coordination led to a 3000-fold increase of the in vitro thrombolytic activity, a 10-fold increase of the in vivo 107 thrombolytic activity, and an additional vasodilation effect was noted. Thus Cu (II) −peptide coordination really is a way for thrombolytic peptide design.

Section -II 4.9. Isolation of Cyclotide genes from selected local plants Potentially cyclotide bearing local plants (reported from literature) were screened for the presence of cyclotide gene or genes by Polymerase Chain Reaction (PCR). The selectivity and specificity of gene isolation was made possible by using specifically designed primers using different bioinformatics tools like primer 3.0v, online tools on justbio.com etc. Primers were designed against each plant family and target gene to screen if genes present and to check how much genetic variation is present among them as compared to our local flora and reported ones. Different primers were designed from the data available as sequences in Genbank (NCBI). Genomic DNA of selected plants was isolated by CTAB method (Doyle and Doyle, 1990) was used for template in PCR reaction. Primers for Violaceae plants. VBF: 5’-AAGTTATAACATCCTTCTTAATTAGCC VBR: 5’-CTCTGCAAGTGGTAGATCTTGGAAC

M 1 2 1 2 A B

300 & 400bp PCR 500 bp products of cyclotide precursor 2 gene 250 bp

Figure 4.18. PCR amplification of cyclotide genes from Viola plants. A) Shows the genomic DNA isolated from the viola plants, Viola odorata and Viola hybrida by CTAB method as shown in Lane 1 & 2 respectively. M represents 1 kbp Fermentas DNA ladder. B) Shows the PCR amplicons of V. odorata and V. hybrid having isoforms of approximately 300 and 400 bp sizes amplicons.

108

Cyclotide genes isolated by specific primers VBF/VBR (for violaceae family) at concentrations of 1X Taq PCR buffer, 0.2 mM dNTPs, 1 µM forward and reverse primers,

2.5 mM MgCl2, 5 µg of genomic DNA and 1.25u of Taq DNA polymerases (thermo scientific #EP0402) in a total 50 µL reaction mixture. Thermal cycling conditions of 30 cycles of repeated denaturation at 94 oC for 30 secs, annealing at 50 oC for 40 secs (Tm of primers = 57 & 62 oC respectively) and polymerization of 72 oC for 50 secs from viola plants V. odorata , viola tricolor and V. hybrida showing the presence of two different isoforms of a single gene that may be with and without intronic regions. Amplicon of 400 bp showed the expected gene CDS amplified from DNA whereas 300 bp amplicon had either to another specifically amplified isoform of same cyclotide sequence or may be a non-specific band. The primers VBF / VBR were designed from the sequence reported for the 342 bp CDS of precursor-2-mRNA sequence of cyclotide genes from viola baoshanesis (accession number DQ851861), having no introns in the RNA of 676 bp and an expected size of amplicon was 400 bp. The partial sequence obtained after DNA sequencing of the ~400 bp amplicon (Figure 4.18) of cyclotide precursor 2 gene from viola species was similar to that reported earlier (Zhang et al., 2009).

Similarly for isolation from panicum species of poaceae family, primers for panitides from panicum species were designed from the Steinchisma laxum/panicum laxum panitide L4 gene, complete CDS (GenBank: KC182529.1) with an expected size of amplicon was around 250 bp product (target) of CDS from 489 bp intronless DNA fragment. The designed primers are shown below;

PanLF (forward) 5’-ACATGGGGAAACACAAGCAG-3’

PanLR (Reverse) 5’-GAAGCACACATCCAACAGTCC-3’

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1 2 A B ~400bp

1000bp

750bp

500bp

Figure 4.19. Genomic DNA isolation and Isolation of Panitide gene from panicum plant. A) DNA of panicum plant (Panicum laxum) shown in lane 1 and from Clitoria ternatea shown in Lane 2. B) Approximately 400 bp panitide gene amplified by specific primers alongwith 1 kbp marker.

Genomic DNA was isolated and 4 µg of DNA was subjected to PCR amplification at thermal cycling conditions of 32 cycles of repeated denaturation at 94 oC for 40 secs, annealing at 46 oC for 30 secs and polymerization of 72 oC for 45 secs. The optimized reaction mixture was almost same as tried for Viola plants except the MgCl2 was 2 mM. The panitide gene (Figure 4.19) screened from panicum laxum, panicum vigatum and P. maximum were confirmed on gel. The only isolated gene was purified by following protocol of Favorprep PCR cleanup mini kit (Favorgen, cat# FAPCK001) and was sequenced. The sequence of Panitides from Panicum laxum (258 bp) DNA fragment had similarities 100% to that from already reported one showing that panitides besides of different origin showed maximum conservation in genetic sequences.

Cyclotide genes were similarly isolated from the fabaceae plant family also and for this we selected local Clitoria ternatea plant and the selected primers were designed by using 584 bp mRNA CterB (accession number JF931998) sequence from Genbank databases (Nguyen et al., 2011) with 408 bp CDS. 50 ng of genomic DNA was used as template DNA for PCR reaction with MgCl2 concentration optimized at 2 mM and the forward and reverse primers used were of 5 µM each. Other PCR reagent concentrations were kept fixed. The thermal cycling conditions optimized were for 32 cycles of repeated initial denaturation at 94

110 oC (1 min), annealing 46 oC (30 sec) and final extension at 72 oC (45 sec). Following primers sequences were used for amplification process;

Primers for Clitoria ternatea of Fabaceae (for cliotides)

CterBF 5-ACTCATAGCAGAACCTCTAACGAAT CterBR 5-GGGTCTGATTTTTATTTAAGATAGTTG The results were documented in gel documentation system as shown in figure 4.20.

M 1 2 3

683 bp Cliotide gene amplicon 750 bp

500 bp

Figure 4.20. Isolation of Cliotide gene i.e. cyclotide genes from Clitoria plants. Lane 1-3 shows cliotide amplicons isolated from genomic DNA of indigenous Clitoria plant alongwith M marker of 1 kbp.

The amplicons after confirmation by agarose gel electrophoresis were purified by using user defined protocol given by Favorprep PCR cleanup mini kit (Favorgen, cat# FAPCK001) and was sequenced from CAMB, Lahore, Pakistan. The sequences obtained were used to generate a contig of all and after trimming of unusual sequences or basepairs the full sequence obtained from the genomic DNA was submitted through BankIT in Genbank database og NCBI which is now online availble accession number KP889219 as shown below in figure 4.21;

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A) B)

TGTTTACTTAATAGGAGGCAGACCTCTAACGAATATCAGTAATCAAATAATGGGTACTATTGCAAGGTATTATGCTCATGTTGTGCTCTT CTTGGTTGCCACTTCTGGTATGTAGCATTTATTTTCTTTTCTTCATGATTTCTATTTTAGAAGTACAAGAAATTAGAATATACGTGGGTG CACTTGTTCTAATTACTTTGGCATTTTGATTTAGTTATCTTTACCGTGAAGAAGACAGAAGCTGGTGTTCCTTGTGCAGAATCTTGTGTG TGGATTCCATGTACTGTGACAGCACTTCTTGGTTGTTCTTGTAAGGATAAAGTTTGTTATTTAAATCATGTCATTGCATCTGAGGCAAAG ACAATGGATGAACATCACCTGTTATGTCAATCTCATGAAGATTGCTACAAAAAAGGAAGTGGAAACTTTTGCGCTCCTTTTCTTAATCAT GATGTTAAATATGGTTGGTGTTTCCGTGCTGAATCTGAAGGATATCTACTGAAAGACTTCTTGAAGATGCAGCCCAGAGACATCTTGAAG ATATCTAAAGCAATCGCTAAGTAAAAACAGTCATGTAACATAGTATCAACTATCTTAAATAAATACAAGACCCACTGATCCTCTGTAGTG AATGTAATGTAATTTACATTTCCCCAGAATAATATATATCTTTTGGGATATGA Figure 4.21. Cliotide gene of 683bp submitted in Genbank (accession number KP889219): Cliotide gene from local Clitoria ternatea was sequenced and submitted in Genbank databases (NCBI). Non-coding regions are highlighted in yellow and regions of CDS are underlined.

A 35% GC content was observed in the 683 bp complete gene and comprises intronic sequence of 107 bp between two exonic sequences i.e. exon1 from 50-107 bp location and exon2 is from 215-564th bp location in total sequence reported. The other regions are not coding for the protein domains here that are shown in the 683 bp full length genetic sequence of Cliotide isolated from the genomic DNA (Figure 4.20 & 4.21). The query and subject sequences were also analyzed by different bioinformatics tools and compared as;

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Figure 4.22. CDS of Cliotide gene with protein Translation and its comparison: 408 bp CDS sequences alongwith translated precursor protein sequence of both Cliotide precursor gene from native Clitoria ternatea (our submission accession number KP889219) represented in Black “” arrows (right side of figure) and the other without arrows shows precursor CterB (accession number JF931998) sequence for comparison (primers designed from this) and studying gene sequence variations.

Figure 4.22 shows compared sequence of our isolated cliotide gene with accession number KP889219 obtained after sequencing with sequence from where primers originally designed i.e. JF931998. The CDS translated protein sequence shows its various domains present in the 135 amino acids premature uncyclised peptide. Domains include cyclotide domain followed by an Albumin-1 domain and a sequence resembling transcriptional regulator NarL; Provisional, seen in a descending way shown in two colored boxes Yellow and Blue respectively (Figure 4.22). Presence of a signal peptide sequence is seen and cleavage site between position 29 and 30 i.e. “TEA-GV” with values of D=0.718 and D-cutoff=0.450 (Networks=SignalP-noTM). Further the albumin-1 domain was also separately analysed for the presence of signal peptide but found no signal peptide (Figure 4.23). SignalP 4.1 server predicts the presence and location of signal peptide cleavage sites in amino acid sequences from different organisms: Gram-positive prokaryotes, Gram-negative prokaryotes, and eukaryotes. The method incorporates a prediction of cleavage sites and a signal peptide/non-

113 signal peptide prediction based on a combination of several artificial neural networks (Petersen et al., 2011 & Nielsen et al., 1997).

A) B)

Figure 4.23. Prediction of signal peptide with the CDS by signal-4.1 Prediction (euk networks) sequence: A) figure shows the graphical display of the result of signal peptide prediction in albumin-1 domain, showing no signal peptide present. Whereas B) shows the the graphical display of a signal peptide sequence present in the cyclotide domain with cleavage site between position 29 and 30 i.e. “TEA-GV” with values of D=0.718 and D- cutoff=0.450

Cyclotide domain of 29 residues consists of “PCAESCVWIPCTVTALLGCSCKDKVCYLN” possessing absolutely conserved protein sequences that proves cyclotides besides being isolated from different sources of different region (Pakistan) still remains conserved. The figure 4.22 also shows 62 residues’ albumin-1 domain in a chimeric form with this sequence was seen as “AKTMDEHHLLCQSHEDCYKKGSGNFCAPFLNHDVKYGWCFRAESEGYLLKDFLK MQPRDILK” within which amino acid residue at 96th position “F” was replaced to “L” by codon change from “TTT” to “CTT”. Between the 2 domains another sequence variation was found to be from residue at position 65th “F” to “S” by codon change from “TTT” to “TCT”. Within the albumin-1 chimera another domain was present comprising transcriptional

114 regulator sequence of NarL provisional (23 a.a), as “CFRAESEGYLLKDFLKMQPRDILK” and within this sequence variantion was seen from aminoacid “F” to “S” (position 110th) due to genetic sequence variation from “TTT” to “TCT”. Similarity and variations were compared by the sequences of cliotides analysed by Nguyen and his co-workers (2011). Graphical representation of domains conserved in the precursor sequence is shown in figure 4.24 ;

Figure 4.24. Results of conserved domains search from NCBI. (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi?RID=F9VXW60H01R&mode=all)

The superfamily /Non-specific hit with e-value = 5.26e-19 belongs to pfam08027 or superfamily cl06866, Albumin I. The albumin I protein is a hormone-like peptide that stimulates kinase activity upon binding a membrane bound 43 kDa receptor. The structure of this domain reveals a knottin like fold, comprise of three beta strands. Similarly superfamily and specific hit e-value = 4.88e-09 belonging to pfam03784 and cl04259 is Cyclotide family. This family contains a set of cyclic peptides with a variety of activities. PRK10651 is the transcriptional regulator NarL; Provisional from interval 85-114 with E-value 7.74e-03 (Live blast search RID = F9VXW60H01R).

Transmembrane domains or sequences (TMHs) present in my protein are predicted by using online tools at www.cbs.dtu.dk/services/TMHMM. For the total 135 residues CDS the

115 number of TMHs=1 whereas the expected residues in number were 19.78918 in first 60 amino acids. The probability of 0.81211 was for N-terminal sequence of its presence. The transmembrane inside sequence was from residues 1-6 and the helix position were from 7-24 and outside that transmembrane the peptide left was from position 25-135th as shown in figure 4.25.

Figure 4.25. Probabilities of Trans membrane domains (http://www.cbs.dtu.dk/services/TMHMM) General characteristics of our protein sequence were also studied using different online tools as shown below in Appendix-VI. The percentage of amino acids present in our Cliotide CDS that comprised different domains along with composition of only cyclotide domain (cliotide) is given below in the Appendix- VII. Further through analysis the protein statistics were also shown highlighting the certain groups of amino acids classified according to their biochemical properties.

For motifs present in my Cliotide gene sequence of complete CDS i.e. 408 bp fragment was analysed by MAST (motif alignment and search engine tool) by online tool available at http://meme.nbcr.net as shown in figure 4.26 no possible roles of motifs found in the sequence (Bodén and Bailey, 2008).

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Figure 4.26. Possible Motifs present in the cliotide sequence.

Analysis of the sequence and sequence variabilities studied by us will add a new member to the already report cyclotide members and add the knowledge of these genes too.

Section – III (NOTE=The experiments conducted in the present section were performed in the University of Southern California, School of pharmacy, Dr. Julio Camerero Lab, California, USA)

4.10. Labeling of MCOTI-I cyclotide with florescent dye Texas Red for studying protein-protein interaction studies

Labeling of bioactive peptides has always been a challenge as well as a practice of all times due to a number of applications that can be used according to the chemistry of labeling strategies and giving us useful informations that has solved many mysteries in understanding peptide based interactions machanisms. Labeling peptides was one of the major needs of

117 science in earlier times for immune assays like an ultrasensitive chemiluminoenzyme immunoassay (CLEIA) where digoxigenin-labeled bradykinin was used as tracer for quantifying kinins (Décarie et al., 1994). Examples include copper catalyzed azide alkyne cycloaddition help to revealed reaction and chemistry between protein-DNA interaction and protein-protein binding with the use of surfaced based assay. Biomolecules such as proteins and DNA linked to specific surfaces using special chemical attachment and introduced in to solution with specific binding domains. Many surface based assays such as biotin avidin linkage chemistry is used with specific biomolecules which provide help in binding of fluorescent labeling of specific biomolecules and motivate molecules for the addition of orthogonal chemistry. Fluorescent labeling strategies provide a useful tool for the quantitative measure of dynamics in protein and transcriptional factors to visualize protein- protein interaction and their specific orthogonal chemistries with the use of different fluorescent dyes (Wang et al., 2009).

Similarly practices of peptides labeling with the stable 15N isotope and NMR-active helps in NMR based studies that illustrates structural and dynamics studies. However, labeled cyclotides being head-to-tail cyclized peptides are not amenable to conventional strategies by using recombinant labeling (Mylne and Craik, 2008). Moreover, the uptake of cyclotide MCoTI-I in live HeLa cells was also an application of labeling peptides with fluorescent probes which was monitored using real time confocal fluorescence microscopy imaging. Results showed that MCoTI-I was internalized in HeLa cells readily depending on temperature with easy access to general lysosomal / endosomal pathways (Contreas et al., 2011). Research practices showed that backbone thermodynamics studies were done on MCoTI-I when it binds to Trypsin as trypsin protease inhibitor. A competition experiment of labeled trypsin-[15N]-MCoTI-I with unlabeled MCoTI-I was used to indicate that the backbone structure of MCoTI-I remained unchanged on trypsin binding and chemical changes that resulted in loop1 & 6 helped to accommodate the increased flexibility of the binding loops and are part of entropic penalties/adjustments. Such interesting results were already observed in other protein-protein interactions of high-affinity that involved protease inhibitors (Puttamadappa et al., 2010).

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Proteins like cyclotide labeling can help increase the possibility of in-vitro and potentially also in-cell screening of genetically-encoded libraries of cyclotides for the rapid selection of novel cyclotide sequences able to bind a specific bait proteins using high throughput cell-based optical screening approaches (Jagadish et al., 2013). For this purpose following the strategy defined by Jagadish and his co-workers (2013) may be used with high efficiency of PTS-mediated cyclization combined with nonsense suppressing orthogonal tRNA/synthetase technology and can make the in-cell production of cyclotides containing Uaas possible. Of particular interest is the introduction azido-containing Uaas that is p- azidophenylalanine (AziF), which can react with Texas red- DBCO-amine a fluorescent probe with a dibenzo-cyclooctyne (DBCO)-derivative of the fluorescent dye texas red succinimyl ester (previously used amino-methyl-coumarin acetate AMCA) to provide in-cell fluorescently labeled cyclotides. The classical approach for in-cell production of fluorescent-labeled proteins by fusing a fluorescent protein to the target protein is not applicable to cyclotides due to their small size and restricted backbone-cyclized topology. So in case of cyclotides containing the Uaa AziF can be expressed in live bacterial cells Origami (DE3) and easily labeled with DBCO-TxRd to monitor cyclotide-protein interactions. The labeled cyclotide in our studies was used in fluorescence resonance energy transfer (FRET) to visualize the interaction between modified trypsin that was fused at N-terminus with green fluorescent protein (EGFP). Texas Red- labeled cyclotide MCoTI-AziF efficiently binds trypsin-S195AEGFP (KD of 29.7 ± 1.08 nM in comparison to the coumarin labeled i.e. 1.8 ± 0.7 nM) in vitro, and cyclotide-protein interaction was monitored by intermolecular FRET shown by the simultaneous decrease and increase of the fluorescence signal at 489 and 613 nm with least background of each other in comparison to the Coumarin labeled MCOTI-I and EGFP that was 445 and 515 nm from donor to acceptor, respectively.

The Experiments conducted for protein interaction studies using texas red fluorescent dye were as under;

 Preparation and HPLC analysis of pure Texas Red and DBCO amine product (Flourescent Dye)

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 Mass spectrometric analysis of Texas Red DBCO amine product purified (Lyophilized)  Cloning and expression of MCOTI-I Wild type (as controls) & MCOTI-I- stop codon-mutant (Phe-Ala-azide).

 In vitro Labeling of MCOTI-I wild type (Lys-NH2 in Loop 2) with texas red succinamyl ester (AS CONTROL FOR OPTIMIZATIONS)  In vitro labeling of mutant MCOTI-I-Azi with dye and LC/MS based analysis of Reaction progress  HPLC analysis and purification of MCOTI-I-Azide-TexasRed-DBCO and mass spectrometric confirmation of product.  Expression of trypsin-EGFP for flourimetry.  Titration of MCOTI-I-Azi-TexasRed-DBCO with trypsin-EGFP (FRET) and

calculation of KD for binding efficiency

4.10.1. HPLC analysis of Cu++ free click chemistry Reaction of Texas red and DBCO amine and Texas Red DBCO amine

Before starting the labeling reaction between MCOTI-I-AziF and the flourescent dye, the first step was to prepare the dye in its alkyne form so that it reacts with N3 form of MCOTI- I-AziF. For this purpose the pure Texas Red succinimyl ester (Life technologies) was first analysed on HPLC at 220 nm signal dector value of UV/VIS detector at 30-100% gradient RP-HPLC on C-18 columns. Texas red future spectrophotometric estmations were calculted -1 -1 using molar extinction co-efficeint = 80,000 cm M at A595 nm. 20 µL (1 µg) was injected and analyed on HPLC (Figure 4.27) without reaction with alkyne containing DBCO amine.

The introduction of azido-containing Uaas that is p-azidophenylalanine (AziF), which can react with DBCO-AMCA a fluorescent probe with a dibenzo-cyclooctyne (DBCO)- derivative of the fluorescent dye amino-methyl-coumarin acetate (AMCA) to provide in-cell fluorescently labeled cyclotides has also been practiced before (Jagadish et al., 2013). Similar experiments coumarin-DBCO was prepared and we are using TxRd-DBCO to prepare and label.

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Figure 4.27. HPLC analysis of pure texas red succinimyl ester. 30-100% HPLC analysis was done to see the Texas Red purity and retention time of reactant before adding the DBCO amine . TxRd appears at 15 mins Retention time and the product was collected as analysed by mass spec as shown in figure 4.29.

Similarly purity and retention time of alkyne group containing compound DBCO amine was checked on HPLC (Figure 4.28) before starting the click chemistry reaction between TxRd and DBCO at room temoerature and using DMF as solvent medium for the reaction to occur.

MWD1 A, Sig=220,16 Ref=800,100 (JAGS\061814000020.D)

Norm. 1.903

700

600

500

400

300

200 4.960

100 1.712 0 0 5 10 15 20 25 30 min

Figure 4.28. HPLC analysis of DBCO-Amine pure: Pure DBCO amine before reaction at 0 mins of TxRd addition and retention time appears at 21 mins for the product of TxRd- DBCO and and 5 min Rt for pure unreacted DBCO was seen using gradient HPLC 30-100%.

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The figure 4.28 shows 0 mins of TxRd addition HPLC results with only DBCO amine appeaering at 5 mins Rt and product will start appearing after equimolar (1:1) mixing of both Texas red succinimyl ester and DBCO amine in DMF at RT. The product and progress of the reaction appeared in figure 4.29 (B-D) by the appearance of product TxRd-DBCO and by the disappearance of DBCO amine only since it was consumed to the reaction progress.

A MWD1 B, Sig=595,16 Ref=800,100 (JAGS\061814000024.D) Norm. 21.086 Reaction after 5 mins B

500

400

300 Unreacted TxRd with disappeared 200 DBCOUnreacted amine TxRd

15.314 100

0 0 5 10 15 20 25 30 min

C MWD1 B, Sig=595,16 Ref=800,100 (JAGS\061814000021.D) D Norm.

After 1 hour 700 Purified TxRd- 600 DBCO amine

500 TxRd-DBCO 21.141

product 400 (fluorescent alkyne dye) 300

200

100

0 0 5 10 15 20 25 30 min

Figure 4.29. HPLC analysis results of the click chemistry reaction between Texas Red and DBCO amine: The figures A-D above shows the 30-100% HPLC results of the reaction between texas red succinimyl ester and DBCO amine reaction after 2 mins (A), 5 mins (B) and 60 mins (C) of incubations at room temperature RT and at each time 1 µLit from the reaction mixture of 500 µg of each from 500 µlit reaction mixtures was injected. DBCO that is getting reduced in the reaction progress and the product TxRD-DBCO-amine is appearing

122 in the progress at approximately 21 minutes Retention time. Later (D) the product of the reaction was purifed by HPLC and lyophilised for future use before labeling peptide.

Figure 4.29 A-D sections shows HPLC of flourescent dye i.e. TxRD-DBCO-amine formation after DBCO complete consumption and injecting to pool out pure TxRD-DBCO-amine product only at 595nm absorbance i.e. lambda max for texas red dye. Later both the products and reactants were confirmed by m/s analysis (Figures 4.30).

A B

Figure 4.30. M/S of both DBCO amines and TxRd-DBCO amines. A) shows mass of pure DBCO-amine and B) shows mass of HPLC purified and Lyophilized Texas red-DBCO amine dye . Expected mass was 978.27 amu whereas mass obtained is 978.8±0.38 amu.

The purified and lyophilized alkyne flourescent dye TxRd-DBCO will be furher used to label MCOTI-I-AziF i.e. in Alkyne and azide based copper free click chemistry reaction for studying protein-protein interactions in further parts of this section. But before it was mendatory to express and optimize the expression condition for MCOTI-I and MCOTI-I- AziF (with p-azido phenylalanine, a non natural amino acid)

4.11. Cloning and expression of wild type MCOTI-I

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For cloning MCOTI-I (wild type, constructs from already reported Jagadish et al., 2013) different constructs like pTXB1 (pTXB1 6706 bp were used and expressed in Bl-21 E. coli host) and pASK (pASK IBA 3-3.34 kb, expressed in origami DE3 strains). These were used for different intein-based expression and folding methods like Expressed protein ligation (EPL) and protein trans-splicing (PTS) (Figure 4.31).

Both constructs are used to express MCOTI-I that natively folds the peptide within cell (pASK) whereas the other (pTXB1) folds in vitro, both have different yields of the peptide amount reported and product can be confirmed by mass spectrometry (Jagadish et al., 2013).

Figure 4.31. Constructs pASK and pTXB1. A) pASK construct contains tetracycline promoter (200 µg/mL for induction of polymerases)/chloramphenicol resistant/ 6xHis tags. B) pTXB1 which is IPTG inducible/ possess Ampicillin resistant/ Chitin Binding domain CBD/ thiols based intein cleavage.

Plasmids were extracted through miniprep following the user defined protocols amplification and PCR amplification of MCOTI-I gene was done and confirmed on agarose gel (0.8%). Only PCR of MCOTI-I gene was done using plasmid as template DNA, with 100 bp ladder (purified). Same reaction mixture concentrations were used as in viola plant’s PCR (in section 4.9) except that plasmid DNA used as template was 0.25 ng, forward/reverse primers were 0.25 µM each, MgCl2 2 mM.

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The plasmid DNAs for expression were both restricted alongwith amplicons and confirmed by gel electrophoresis before ligation and transformation. The pTXB1 was restricted by nde I and spe-I digested plasmids. Figure 4.32 show the constructs/ plasmids restricted and extracted ones alongwith an insert or amplicon with MCOTI-I + inteins sequences.

A) B) C)

D) E) F)

Figure 4.32. PCR amplification, plasmid DNA isolation, restriction and ligation of MCOTI-I in pTXB1 and pASK vectors: A) PCR product of about 200 bp representing MCOTI-I alongwith N-/C inteins (MCOTI-I +IC+IN= ~200bp) with 100 bp DNA ladder (BioLabs, N3231S). B) PCR product and plasmid restriction with 1kbp DNA ladder (BioLabs, N3232S). C) Purified restricted insert for ligation. D) Plasmid DNA isolation by user defined protocol of EZ gene alongwith hindIII ladder. E) Digestion before ligation of

125 pASK with ndeI and speI (biolabs, R3133S). F) Digestion before ligation of pTXB1 with ndeI and speI alongwith 1 kbp DNA ladder.

Plasmid and inserts after ligations were expressed and induced at conditions given i.e. 0.3 mM IPTG induction at RT overnight along with antibiotics. The protein expressed was run in 1X SDS buffer at 135V for 50 mins and stained in coomessie brilliant blue before documentation (Figure 4.33). SDS-PAGE gels (PAGEr precasting gels, Lonza company) and X-cell sure electrophoresis apparatus.

B Tp S P B M

~11 KDa

Cleaved IN fragment

~6 KDa

Figure 4.33. SDS-PAGE confirmation of inteins cleaved during MCOTI-I expression and cyclization: Polyacrylamide gel showing inteins (Ic = approx 6.0 kDa and IN= 11 kDa) completely cleaved PTS based method of expression of MCOTI-I and cyclization within strain origami DE3 (Novagen) competent cells using pASK construct alongwith 10-180 kDa prestained Tricolor protein ladder (Morganville scientific, PM0100). TP=total protein after sonification, S=supernant obtained from lysis, P=pellets of lysed cells, B= Ni-beads captured Ic inteins and M = protein ladder.

The yield of the PTS after trypsin capture in current findings was 68 µg/L MCOTI-I and approximately 32 mg/L Inteins production. The protein quantity was estimated by UV/VIS spectrophotometry at 280 nm and by calculating the peak areas of protein peaks and comparing it with known amounts injected. These are the same proteins purified (Figure 4.34) and lyophilized and then used in the bioactivities based studies (sections 4.1 to 4.9).

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Figure 4.34. HPLC of pASK product MCOTI-I after trypsin capture: 0-70% of buffer B for 30 mins at 280/220 nm UV/Vis detector wavelength each time when analyzing MCOTI-I. The protein peak of MCOTI-I appeared at 14-15 min retention time each time the exact Rt changes as acidic conditions of experiment varies. The Pure MCOTI is usually dissolved in Buffer A or acidified phosphate buffer before injecting as cationic peptides separate better like this. Peaks at 22-23mins shows trypsin degradation products sometimes.

Another way of expressing MCOTI-I by EPL method using pTXB1 construct and GSH based cyclization after chitin based beads capture (Figure 4.35) of inteins (CBD tagged in construct) observed yield upto 112 µg/L. In vitro folding by GSH was continuously monitored exact mass was obtained. Reaction was immediately stopped by acidifying the medium with glacial acetic acid 5% v/v of mixture, and then the proteins were captured by using affinity chromatography using trypsin specificity for MCOTI-I capture. By using an intein-mediated cyclization approach the cyclized cyclotide peptide folds spontaneously within the cytoplasm of bacterial cell to give the fully functional cyclotide. Previous findings using similar expression strategy i.e. EPL was done for MCOTI-II (Momordica cochinchinensis trypsin inhibitor II, original) showed that E. coli cell was engineered with some mutations in the glutathione reductases and thioredoxins to promote disulfide bridge formation which increased MCoTI-II yield (Camerero et al., 2007). Under these conditions,

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it was possible to reach intracellular concentrations high enough for in vivo screening experiments, i.e. up to 5 µM of natively folded cyclotide. The trypsin inhibition activity calculated Ki for porcine pancreatic trypsin for this recombinant cyclotide was 25±5 pM, which resembles to the value reported for the naturally purified cylotide from plant.

M TP S CB CB2

MCOTI gyrase intein ~ 34 KDa

Approx 27 gyrase intein KDa

Figure 4.35. SDS-PAGE results showing EPL based expression of MCOTI-I: TP is the total insoluble proteins and cells contents, S is the supernantant/ soluble fraction after cell lysis, CB is the chitin binded or captured, CB2 is chitin binding of inteins after completely folded by GSH reaction.

pTXB1 construct cleaved MCOTI-I (WT) on treatment with GSH and cleaved and uncleaved inteins checked on SDS PAGE if uncleaved left tagged by CBD. Quantification was carried out spectrophotometrically using an extinction coefficient per chain at 280 nm of 2,240 M- 1cm-1 (MCoTI-I) and for future experiments 3,730 M-1cm-1 (MCoTI-AziF) in labeling reaction of mutant MCOTI-I with unnatural amino acid p-Azido-phenylalanine.

Many samples were run on HPLC were run to check the progress of folding events induced by GSH treatments and to monitor if folded product formed. Reactions were acidified or stopped each time before injecting in HPLC. Equal volumes were used each time from total mixture (Figures 4.36 a & b). 128

A B

MWD1 A, Sig=220,16 Ref=800,100 (JAGS\092014000001.D) MWD1 A, Sig=220,16 Ref=800,100 (JAGS\091814000002.D)

Norm. Norm.

2.016 2.145 6.605 6.896

1.946 2.214 6.896

24.793 25.235

3.764 3.107

300 300 14.719

250 250

200 200

31.880 14.776

150 150

23.952 24.636

100 15.320 100

24.329

14.554

13.752

8.057

13.819 8.209

50 50

7.659 11.107

0 0 0 5 10 15 20 25 30 min 0 5 10 15 20 25 30 min

MWD1 A, Sig=220,16 Ref=800,100 (JAGS\092514000004.D)

Norm.

2.131 4.246 4.969 8.608 14.835

600 6.788

500 C

400

300 20.655

200

14.237

30.088

7.908

12.805

8.383

19.827

19.195

13.064

20.506

10.639

17.578

21.099

13.262 10.547

100 12.574

9.115 9.263

20.197

18.479 18.225

0 5 10 15 20 25 30 min

Figure 4.36. Analytical HPLC showing progress for GSH based (in vitro) cyclization/folding of intein-fusion linear precursors A to C. The reaction was induced by addition of reduced glutathione (GSH) and analyzed after 1 day at room temperature and shaking. A = after 1 h of GSH addition. B= Checking the folding done after 1 day incubation. C= HPLC showing 500 µL mix injection volume for purification after reductive biosynthesis and oxidative folding is complete. Star sign indicates folded MCOTI-I

129

The mixture after confirmation of complete product formation was then used to harvest all folded MCOTI formed by using modified trypsin immobilized on sepharose beads (that only binds but do not cleave proteins). The trypsin beads were then used to capture specifically using affinity chromatography type technique to harvest all product formed and was washed and then eluted out by using 8 M guanidinium HCl that denatures the proteins complex and trypsin sets free our MCOTI-I and do not unfold or cleave the bonds in cyclotide. The medium was first neutralized to pH 7 of GSH and protein mixture so that trypsin beads could work in capturing method by 1 M NaH2PO4 The trypsin capture continued till no MCOTI remains behind in the mixture as shown in the HPLC figure below in 4.37.

MWD1 A, Sig=220,16 Ref=800,100 (JAGS\092314000005.D) MWD1 A, Sig=220,16 Ref=800,100 (JAGS\092414000001.D)

Norm. Norm.

2.000 2.534 4.660 8.698

1.920 2.118 2.312 4.709 8.687

300 300

250 250

200 200

150 150

14.854

8.354 8.253

100 100 14.936

50 50 8.942

21.128

20.043

19.064

8.932

12.774

7.887

17.392

12.761

19.244

10.704

7.909 10.567

0 0 0 5 10 15 20 25 30 min 0 5 10 15 20 25 30 min

MWD1 A, Sig=220,16 Ref=800,100 (JAGS\092414000008.D)

Norm.

2.164 21.479

600 6.828

500 23.707

400 21.158

300 23.294

200 4.347

30.226

13.124

9.313

13.862

12.075

14.646

13.655

12.714

24.708 20.803

100 8.148

18.062

10.234

3.403 10.762

0 5 10 15 20 25 30 min Figure 4.37. HPLC of captured MCOTI-I for checking trypsin elutions after GdmHCl to detach till no protein remains behind. Sections A-C shows the progressive removal of

130

MCOTI-I by trypsin capturing from the reaction mixture till we see no MCOTI-I product of reductive biosynthesis and oxidative folding at 14 to 15 min peaks. No unbound proteins were seen. Star sign shows product MCOTI-I which is not seen in the last. MWD1 A, Sig=220,16 Ref=800,100 (JAGS\092314000001.D)

Norm.

2.022 2.180 4.497 8.570 1.871 A

300

250 1.738

200

150

100

14.710 8.273

0 0 5 10 15 20 25 30 min

Figure 4.38. Mass of purified MCOTI-I from EPL method: A= HPLC of 50 µL of Pure MCOTI-I, B= mass spec anlaysis results showing charged states of MCOTI-I calculations shows 3481.9 ±0.64 SD obtained mass (871.3 and 1161.3 amu 4th and 3rd charged states,

131

expected mass = 3481). u ,

amu

The charge states were calculated (for m/s results in Figure 4.38) following the formulas below for both 3rd and 4th states. A protein can have four different charge states on being cleaved during m/s analysis.

4.12. Labeling of MCOTI-I-AziF with Texas Red for protein-protein interaction studies

Molecular technique using orthogonal tRNA synthastases technology (having evolved tRNA not competent with endogenous tRNAs alter amino acid incorporations and uses orthogonal tRNA synthatases to incorporate non-natural amino acids using ATP) to incorporate a non- natural amino acid named p-azido-phenylalanine (with reactive azide N3) was utilized to label the cyclotide MCOTI-I with a florescent dye Texas red. The same technique was also used to label cyclotide MCOTI with coumarin dye (Jagadish et al., 2013). Coumarin (donor) being excited at 350 nm and EGFP (acceptor of energy) at 450 nm both had close wavelengths so showed more backgrounds in FRET based analysis or FACS based cell sorting techniques. Considering this in mind the current project was designed to change the dye with a fluorescent dye Texas Red (610nm) as acceptor of energy and kept EGFP as donor and here the difference of energies was more that would decrease the background as at the same time when one fluorescent protein is excited the other protein labeled with another dye should not be excited at the same time. Excitations and emissions were checked from the Chroma Tech. graph/spectra viewer and life technologies websites of both dyes.

Non-natural amino acids, if site-specifically incorporated, become a powerful tool to manipulate proteins for functional and structural studies, or to create proteins with different properties. This can be done by either labeling pre-synthesized proteins with probes at reactive side chains such as lysine or cysteine residues, or by probes incorporation into newly developing proteins in the presence of an altered aminoacyl-tRNA (aa-tRNA). This method usually uses an amber suppressor aa-tRNA that can recognize unique nonsense codons (like UAG). However, a variety of synthetic and engineered aa-tRNAs have also been developed for this purpose, including those that recognize four-base codons. For co-translational incorporation to happen the non-native amino acid must be bonded to tRNA with high

132 efficiency, either by using an orthogonal aa-tRNA synthetase (aaRS) that is engineered to recognize both tRNA and modified amino acid (Gubbens et al., 2010). Similarly for producing a cyclotide MCOTI-I by co-translational methodology two constructs were used (figure 4.39).

Figure 4.39. pVLOmeRS and pET28a constructs used in expression of MCOTI-I-AziF: The construct pET28a with MCOTI insert and intein sequences with mutation for stop codon modified for unnatural amino acid incorporation. Construct pVLOmeRS possessing gene for tRNA synthatase for orthogonal tRNA that incorporate unnatural amino acid p-azido phenyalanine (amber codon TAG) at the stop codon of MCOTI-I where our synthesized dye Texas Red-DBCO amine will attach by Cu++ free click chemistry reaction.

Expression Plasmids (Figure 4.39) i.e. pET28 is IPTG inducible and pVLOmeRS is arabinose inducible whereas unnatural amino acid can be added in the cell’s culture in the log phases so that enough can incorporate within cell before inducing the expression plasmids. Construct pVLOmeRS for incorporating the non-natural amino acid phenylalanine azide into the MCOTI-I framework by suppressing orthogonal stop codon tRNA synthatase technology. Production of MCOTI-AziF in low concentrations in 2 liters of 2XYT media was seen in figure 4.40 below and induction conditions will be mentioned in upcoming segments.

133

Figure 4.40. LC/MS analysis of expressed mutant MCOT-I-AziF captured on trypsin beads: Analysis shows that the cells were used to extract MCOTI-I-aziF by trypsin capture without adding TxRd-DBCO the dye for labeling reaction. Since minute amounts of products are expected therefore a very little amounts are injected that highly sensitive LC/MS can detect. Figure shows the unreacted form of MCOTI-AziF i.e. N3 form represented by its 3rd and 4th charged states 1185.7 amu and 889.4 amu respectively. Where as no labelled product was dected at this stage shown at its charged states 1134.2 amu and 1511.9 amu.

The unreacted form of MCOTI-AziF i.e. N3 form posses its 3rd charged state as 1185.7 amu 889.4 amu and its 4th charged state of MCOTI-I-AziF (N3 form). Whereas 882.9 amu for the NH reacted form of MCOTI-AziF protein when exposed in air (sensitive in light). We therefore avoided exposure to light and air and perform all the reaction and extraction procedures in red lamp (dark room). 1511.9 amu and 1134.2 amu are 4th and 3rd charged states of MCOTI-I-AziF-TxRD i.e. labelled product of Texas Red-DBCO amine (TxRd- DBCO) with MCOTI-AziF. No product formation was found as alkyne florescent dye was not added yet (Figure 4.40). Latter addition of dye in different ratios and reaction progress was monitored by Lc/Ms analysis (Figure 4.41).

134

A B

D C

Figure 4.41. LC/MS analysis results showing reaction of Texas Red-DBCO amine dye with MCOTI-I-AziF: A) Shows that 1:2 molar ratios of mixing MCOTI-I-AziF : TxRd- DBCO. MCOTI-I-AziF utilizes its N3 form for reacting with dye in 5% DMF solution as reaction medium. LC/MS observation was seen one hour after incubation. B) 1:50 molar ratios reaction proceeded by adding more of the alkyne dye, slight appearance of the MCOTI-I-AziF-TxRd-DBCO labelled cyclotide was seen with gradually decrease of the reactive N3 for of MCOTI-I-AziF and the NH form which is the reacted one is also increasing that some of protein is not reacting with small amounts of dye added rather getting reduced when exposed. Sections C) to D) shows the effect of increase of reaction ratios from 1:100 to 1:150 times and was left overnight at RT in dark but still found much of the

135 unreacted N3 and increase in the self reacting NH form of MCOTI-I-AziF wheras very low intensity of MCOTI-I-AziF-TxRd-DBCO.

The mass of MCOTI-I-AziF-TxRD was expected to be 4533.1 amu and was observed as 4532.1 ± 0.707 S.D amu from 3554.0 + 978.2 (MCOTI-I-AziF protein and TxRd-DBCO dye respectively). Products were judged according to the 3rd and 4th charge states (Figure 4.41).

Besides increasing the concentration of TxRd-DBCO dye from 1:2 times to 1:150 in terms of moles still we found much less concentrations of the MCOTI-I-AziF were found and still some concentrations of unreacted N3 formed that depleted only a little. Wheras self reacted NH form that was unable to react with the alkyne dye remained with slight increase. Besides using a very high concentration of TxRd-DBCO and incubating it overnight in 5% DMF overnight yet only small amounts of MCOTI-I-AziF-TxRD product appeared showing the reaction was slow in either the medium provided or due to the dye used. A possibility of low production of MCOTI-I-AziF in N3 form was the reason of slow reaction or product or may be the trypsin sepharose beads captured complex couldn’t be a favourable support for the reaction to occur.

Besides 150 times more dye TxRd-DBCO was added to let all reactants to form product labelled peptide MCOTI-I-AziF-TxRD. The possible reason for the dye or labeled peptide for not appearing in the LCMS (AB1-300 APSIS) results might be that the whole reaction invitro was carried on the trypsin beads that had already captured MCOTI-I-AziF, there might be the reason that this fluorescent dye might be sticking on the sepahrose beads material which is in accordance to the hyrophobic nature of dye also.

The expression of MCOTI-I-AziF was also confirmed on the SDS PAGE along with the total protein insoluble fraction of lysed cells and soluble fraction i.e. supernantant with intein captured His-pur Ni-NTA beads (as IC 6 kDa is tagged for Ni beads capture) shown in figure 4.42. This could confirm if expression and folding process was completed or not, and if any protein still left within the cell pellets or supernant. Results showed the proper folding of captured protein following expression.

136

Figure 4.42. SDS-PAGE results of inteins expressed for MCOTI-I-AziF: The figure shows approximately 6 kDa IC intein expressed confirming about cleavage of inteins and protein folded after cleavage. No uncleaved product appeared in the Total proteins (TP) i.e. whole cell, ‘P’ pellets (lysed cells insoluble) and supernatant (Sp) along with Ni beads on which tagged inteins are captured. No uncleaved or unfolded IN seen here.

Besides LC/MS confirmation of reaction progress and conditions that needed to be optimized in further experiments and confirmation of expression of folded MCOTI-I-AziF, it was tried to separate or purify the labeled MCOTI-I-AziF-TxRd (although in small amount) through HPLC. The whole volume eluted from the trypsin beads by guanidinium HCl washes was injected and each peak was collected from the fraction collector for further mass spectrometer based confirmation of the product possible peaks (Figure 4.43). Purified labeled peptide had to be used for further protein-protein interaction studies.

Figure 4.43. HPLC based attempt to pool out all the MCOTI-I-AziF-TxRD: All the washes and flow throughs by Guanidinium HCl wash of trypsin beads that captured MCOTI-

137

I-AziF-TxRD were injected in the high pressure chromatographic separation system (GdmHCl in 5-10% v/v DMF elutions of trypsin beads) and collected each peak appearing at the 595 nm, detector set point for Texas Red dye.

It was generally expected that if hydrophobic dye was attached to the MCOTI-I-AziF the labeled product may appear after the retention time of MCOTI-I. Still with care each peak of the eluent that appeared at 595 nm was analysed. Analysis of each peak on M/S was done but no significant results of exact product mass (4532.1 amu) was observed. This shows that the protein is needed to be hyper produced/expressed to be observed on HPLC. Moreover, the labeling reaction conditions with Texas red-DBCO amine may needed to be further optimized. Further the low conc protein MCOTI-I-AziF-TxRD being hydrophobic in nature after dye addition, might have stuck with beads or somewhere in the column material of HPLC that causes difficulty in labeling, seperation and purification.

The production of mutant MCOTI-I-Azide was attempted with non-natural reactive amino acid p-Azido-F incorporated and labeled it with Texas Red-DBCO amine. Besides minute amount seen on LCMS there was a failure to harvest from trypsin sepharose beads and also failure to see/purify on HPLC as its in less amount. Harvesting method had to be optimized. The trypsin captured MCOTI-I-AziF was eluted and pooled. Then the labeling experiment was done in a buffered medium (needed to be optimized too) for maximum labeled product. HPLC analysis and hydrophobic interactions with sepharose and column material with that of labeled peptide will be optimized using another labeled peptide at loop 2 Lysine on MCOTI-I (WT) to label (wild type WT i.e. without unnatural amino acid, used in section 4.11) with Texas Red succinimyl ester only and produce on large scale to purify as a control reaction.

4.13. Labeling of wild type (WT) MCOTI-I-Lys-NH2 with Texas Red succinimyl ester

For futher optimization studies to get better yields of labeled cyclotide the Texas Red succinamyl ester was reacted with known amount of MCOTI-I-Lys-NH2 (50µg) in the presence of DIEA as catalyst or base (organic amine, non-nucleophilic base in alkylations) that gives a labeled peptide product of WT as MCOTI-I-Lys-NH-TxRd (with loss in the ability to bind trypsin) as loop 2 lysine was involved in binding also. the reaction mixture was incubated at room temperature as its too reactive will form labeled peptide in an hour

138 completely, further reaction was stopped by acidifying the medium with 5% v/v of glacial acetic acid. The strategy of labeling is in figure 4.44;

Figure 4.44. Schematic representation of ester dye with amino group of a peptide

The labeled WT MCOTI-I-Lys-NH-TxRd was further analysed or purified by HPLC using same gradient modes at 0-70% buffer B and for 30 minutes. Here again the retention time was expected to appear after the time required for WT MCOTI-I to elute out or appear i.e. 14-15 min as its hydrophobic in nature now (Figure 4.45). Each time 5 µg of MCOTI-I-Lys-

TxRd in PO4 buffer and in the presence of 5% (v/v) DMF was injected into HPLC as it will be the same type of mixture that was used in the labeling reaction of MCOTI-I-AziF.

MWD1 B, Sig=595,16 Ref=800,100 (JAGS\100414000005.D)

Norm. 20.043

300 20.740 250

200

MCOTI-I-Lys-TxRD 150

100 18.649

50 21.901 19.761

0 5 10 15 20 25 30 min Figure 4.45. HPLC of MCOTI-I-Lys-TxRD labeled and mixed in phosphate (-PO4) buffer and in the presence of DMF. Results of MCOTI-I-Lys-TxRD eluted at 18.6 mins with other non reacted texas red dye isoforms on HPLC using 595 nm of TxRd max absorbed wavelength value for detector and 0-70% gradient for 30 min.

139

The MCOTI-I-Lys-TxRD peak appeared at 18-19 mins as expected due to hydrophobic groups of dye attached alongwith the isoforms of unreacted texas red succinimyl ester that appeared around 20-21 mins. The peaks may appear in some experiments slightly displaced in time of elution due the acidifying conditions variation in handlings. The peaks were confirmed for the presence of labeled MCOTI-I-Lys-TxRD by mass spectrometric analysis of each peak. The 3rd and 4th charge states of the peptide were seen at 1047.0 ± 0.205 amu and 1395.4 ± 0.467 amu respectively (Figure 4.46)

Figure 4.46. Mass spec results of MCOTI-I-Lys-TxRD: the figure shows the mass analysed for labeled MCOTI-I-Lys-TxRD. 3481.99 (MCOTI-I) + 703.2 (TxRD) = 4185.2 (MCOTI-I-Lys-TxRD) mass obtained with 3rd and 4th charge states seen at 1047.0 ± 0.205 amu and 1395.4 ± 0.467 amu respectively for observed 4184.6 amu (1396.06=3rd & 1047.29=4th expected masses of charged states).

140

Results here also show that besides injecting 5 µg of the MCOTI-I-Lys-TxRD in to HPLC system that was expected to show a high concentration with intensity sharp peak, gave in actual a short peak of low intensity that showed that the hydrophobic sticky nature of the texas red dye made it difficult to elute out in the HPLC column. Thus to elute or purify MCOTI-I-AziF-TxRD higher amounts of peptide had to be injected by hyperproducing in large scale.

Furthermore, in order to optimize labeling issues on trypsin-sepharose beads (as complications appeared in labeling on beads), we planned to first use fresh trypsin sepharose beads, check its loading capacity and then its interaction with the reaction mixture containing MCOTI-I-Lys-TxRD.

4.13.1. Preparation of trypsin-sepharose beads for capturing MCOTI-I or MCOTI-I- AziF

The 50 µL of trypsin sepharose freshly prepapred beads (section 3.14.1) after activation were used to see with 1.5 µg of MCOTI-I to check if the capturing ability was working. The result of trypsin capture is shown in figure 4.47;

MWD1 A, Sig=220,16 Ref=800,100 (JAGS\101314000003.D)

Norm.

1.982 2.244 4.653

175 8.373

150

1.694 1.764 125

100 15.105

50 8.077

0 5 10 15 20 25 30 min Figure 4.47. HPLC chromatogram of trypsin capture tested using MCOTI-I: 1.5 µg of purified MCOTI-I from pASK construct was eluted by in GdmHCL for checking trypsin beads.

141

Further the Optimization of loading or binding capacity of trypsin-sepharose beads by its known volumes were judged using fixed concentration of WT MCOTI-I i.e. 5 µg and different volumes of trypsin sepharose beads 50-100 µL were used. More than 86% MCOTI- I was bound to 50 µL of beads that was enough to use 200 µL of beads to capture 8 µg of MCOTI-I-AziF from 4 litres of cell culture expected (Figure 4.48 A-D). This was done to capture maximum amounts of MCOTI-I-AziF with minimum volume of trypsin sepharose beads as smaller volumes capture maximum of smaller concentrations.

MWD1 A, Sig=220,16 Ref=800,100 (JAGS\093114000002.D) MWD1 A, Sig=220,16 Ref=800,100 (JAGS\093114000001.D)

Norm. Norm.

1.984 2.268

2.015 2.470 4.602 8.407 8.775 1.801 A B 140 140

120 120

100 100

80 80

60 15.449 60

40 19.047 40

10.562 7.810

20 18.630 20

0 0

-20 -20 0 5 10 15 20 25 30 min 0 5 10 15 20 25 30 min

MWD1 A, Sig=220,16 Ref=800,100 (JAGS\100414000004.D) MWD1 A, Sig=220,16 Ref=800,100 (JAGS\100414000003.D)

Norm. Norm. 2.246 C 1.970 D 140 140

120 120

100 100

80 80 14.877

60 60

40 40

20 20

0 0

-20 -20 0 5 10 15 20 25 30 min 0 5 10 15 20 25 30 min

Figure 4.48. HPLC results of different volumes of trypsin beads capturing same amount of MCOTI-I: Loading capacity of trypsin beads was estimated using same amount i.e. 5 µg of MCOTI-I when allowed to be captured by diferent volumes of trypsin beads i.e. 50 µL (section A) and 100 µL (section C). And at each time after incubation or capturing event the washes of beads by buffer A or PO4 buffers were also analysed in HPLC to see if something left unbound (shown in sections B + D).

142

In both volumes capture was almost the same, only a little more in 50 µL trypsin beads whereas a little less that was not captured by 100 µL beads appeared in the washes. This shows that smaller amount of trypsin beads can capture more the MCOTI-I amounts as maximum chances of binding and hitting occurs in smaller volumes and maximum comes out when eluted with the same volumes of GdmHCl since more washes with GdmHCl for elutions removes completely without leaving behind the traces of MCOTI-I. Flow throughs of phosphate buffer washes were also checked in each case. Thus for maximum capture of MCOTI-I-AziF (that could yeild ~2 µg from 1 Litres) minimum volume i.e. 50 µL of trypsin sepharose beads were used that had the ability to bind up to 5 µg of pure MCOTI-I originally. As 2-3 µg of MCOTI-I-AziF was expected, therefore, all MCOTI-I-AziF would have been captured and purified by this amount.

Furthermore after several experiments and HPLC analysis it was found that 5 µg MCOTI-I Wild Type, when captured by trypsin-sepharose beads and eluted by 100% buffer- B, had 50% product recovery as observed on HPLC analysis. Moreover it was found by several experiments and HPLCs that 76% product was recovered after eluting with GdmHCl using 100 µL of trypsin-beads. 92% was seen when used 50 µL beads of beads to capture 5 µg of MCOTI-I whereas only 8-9% of injection loss was seen.

Futher different buffers or solvent systems that could best elute maximum yield of MCOTI-I from beads were also checked, and analysed through HPLC results. It could possibly suggest that which solvent could further be best used to elute trypsin captured MCOTI-I labeled and non-labeled products. Five micrograms of MCOTI-I in each experiment was used to be captured by 50 µL of trypsin sepharose beads (beads gel agarose, few residual charged groups pH = 4-9).

Fifty percent buffer B elutes of MCOTI-I were captured on trypsin beads, showing very minute amount eluted. Futher 100% buffer B was tested that eluted a good yield of MCOTI-I captured on trypsin beads but alongwith many other unwanted products bound to beads that were eluted. A peak at 14-15 mins (at 220 nm) was confirmed by Mass spectrophotometric analysis at each time (Figure 4.49). Each time when buffer B was used for eluting MCOTI-I captured on trypsin beads the mixture was first lyophilized to remove

143 buffer B as everything would be eluted in the start of HPLC process if Buffer B solutions was used directly for injection.

One hundred percent buffer B elute of MCOTI-I that was mixed with trp-sep-GdmHCl beads (at 220nm) showed that treatment with already GdmHCl to trypsin beads, carried out maximum failure of capturing MCOTI-I. However, a little that was bound was eluted and seen with a minor peak (Figure 4.49). Isopropanol washes for elutions were also analysed on HPLC followed by lyophilization and then again solubilizing in Buffer A before injection and was found to show very dirty noisy peaks (Figure 4.49).

Similarly sepharose GSH beads with no ability of capturing MCOTI-I when washed or eluted with 100% Buffer B showed no significant MCOTI-I presence.

MWD1 A, Sig=220,16 Ref=800,100 (JAGS\100814000002.D) MWD1 A, Sig=220,16 Ref=800,100 (JAGS\100814000001.D)

Norm. Norm. 1.946

A 2.012 B

300 16.830

80 17.955

250

60 16.974

17.351 200 MCOTI-I 2.649

MCOTI-I

16.349

17.719 18.113 40 150

100 14.079 15.324

20 16.565

50 17.631 10.829

0 2.524

0 5 10 15 20 25 30 min 0 5 10 15 20 25 30 min MWD1 A, Sig=220,16 Ref=800,100 (JAGS\100614000001.D) Norm. C D

250 1.965 17.377

MCOTI-I 200

150 15.976

100

15.780 15.565 50

0 5 10 15 20 25 30 min Figure 4.49. HPLC Analysis showing different solvents and beads types optimized for maximum elutions of captured peptides. A) Shows 10% of MCOTI-I eluted after elutions using 50% buffer B. B) HPLC result shows through peak area calculation that 55% of captured MCOTI-I eluted when 100% buffer B was used. C) Shows 15% MCOTI-I eluted by 100% buffer B when MCOTI-I was mixed with trypsin-sep-GdmHCl beads (original trypsin beads washed before capture with GdmHCl 8 M solution). D) Result of isoprpanol based wash for eluting MCOTI-I gave dirty noisy peaks and mass.

144

Further it was decided to check if the TxRd labeled peptide MCOTI-I-Lys-NH-TxRd had the ability to bind with sepharose-GSH (usually used in recombinant proteins purifications tagged with GSH transferase) or sepharose-Trypsin-GdmHCl-washed (before capture) beads that already had no or lost trypsin based MCOTI-I capturing ability. Although lysine based labeling made the MCOTI-I-Lys-NH-TxRd peptide a mutant with no ability to bind with tryspin sepharose beads it was tested if the dye made it possible to stick. Further if this could happen a strategy to break the dye interactions with the bead material was tried. For this HPLC analysis at 595 nm (Max. wavelength for Texas Red) of MCOTI-I-Lys-TxRD was done after lyophilization and dissolving in buffer A or phosphate buffer then mixed/incubated with trypsin-sepharose beads 50 µL that were already washed with Gdm- HCl (8 M) and after that the beads were washed with DMF. The product peak was appeared at 17.92 min (Figure 4.50) that was confirmed by mass spectrophotometer. In another experiment DMF flow throughs and washes of MCOTI-I-Lys-TxRD incubated with sepharose-GSH (glutathione beads) only i.e. without trypsin immobilized on beads. HPLC analysis showed that the sticky dye Texas Red also sticks to the sepharose-GSH beads material although its trypsin binding was omitted. Washes with DMF still eluted the MCOTI- I-Lys-TxRD. Moreover, this was also confirmed by looking the washes of phophate buffers that are always obtained after MCOTI-I-Lys-TxRD incubations with beads and beads were further washed before being eluted by different solvents. Nothing was found in washes of the product MCOTI-I-Lys-TxRD that showed the labeled products was either stuck to the beads material or to the column (Figure 4.50).

Experimental results by HPLC analysis of flow throughs and washes of DMF solvent on sepharose-trypsin (50 µL) beads showed that the product MCOTI-I-Lys-TxRD still appeared, although the trypsin specificity for binding was already lost. This shows that the dye was sticking somewhere with the material of beads of sepharose only. Nothing was seen in the washes also as shown in the picture at top side (small image) in Figure 4.50. So this proves that Texas Red-DBCO labeling on the beads (sepharose-trypsin) with MCOTI-I-Azide- PheAla was not a better strategy to label in vitro, rather it was needed to elute the MCOTI-I- AziF from the beads first after completely capturing and then do labeling by Texas Red- DBCO by CU++ free click chemistry reaction.

145

MWD1 B, Sig=595,16 Ref=800,100 (JAGS\100814000007.D) Norm. A 175

150

125 20.089

100

19.418 21.169 75 MCOTI-I-Lys-TxRD

25 17.922

0 0 5 10 15 20 25 30 min

MWD1 B, Sig=595,16 Ref=800,100 (JAGS\100814000006.D) MWD1 B, Sig=595,16 Ref=800,100 (JAGS\100414000002.D)

Norm. Norm. 20.142 19.541 B C 350

200

300 20.314 19.793

250 150

200

100 150 NO MCOTI-I-Lys- 100 MCOTI-I-Lys- 50 TxRD Peak

TxRD 21.225 17.687

50 19.137 19.567

0 2.029

0 2.026 0 5 10 15 20 25 30 min 0 5 10 15 20 25 30 min

MWD1 B, Sig=595,16 Ref=800,100 (JAGS\100814000005.D)

MWD1 B, Sig=595,16 Ref=800,100 (JAGS\100414000001.D)

Norm. Norm. 20.549 200 No product

350 D 19.929 in washes 150

300

100

250 50 20.024 0 200 MCOTI-I-Lys-TxRD 0 5 10 15 20 25 30 min

150

100

18.895 21.505

50 19.803

0 2.058 0 5 10 15 20 25 30 min MWD1 E, Sig=280,16 Ref=800,100 (JAGS\100614000003.D) MWD1 C, Sig=595,16 Ref=800,100 (JAGS\100614000001.D) Norm. Norm. 140 E 140 F 120 120

100 100

80 80

60 60

40 40

21.650 17.214

20 21.120 20 1.968

0 0

-20 -20 0 5 10 15 20 25 30 min 0 5 10 15 20 25 30 min Figure 4.50. HPLC results of MCOTI-I-Lys-NH-TxRd interactions. A) shows labeled WT product appearing after DMF washes on sepharose-trypsin (GdmHCl washed) beads.

146

Sections B) and C) shows DMF washed sepharose-GSH beads (incubated with MCOTI-I- Lys-NH-TxRd) products and the PO4 buffer washes after DMF respectively, showing sticking of peptide only beads and elutions by DMF not by phosphate buffers. D) shows product appearing after DMF wash from trypsin sepharose beads and small pic shows nothing in flow throughs i.e. all sticking to beads. E) and F) Showing no labeled peptide appearing after 100% and 50% Buffer B washes from sepharose-GSH beads. Each time peaks were checked by mass spec for product confirmations. Moreoever HPLC analysis done on pure MCOTI-I-Lys- TxRd (w/o beads) showed only 18% recovery of product as compared to the amounts injected. Sections E and F of figure 4.50 also showed 100% Buffer B and 50% were also tested to elute MCOTI-I-Lys-TxRD from sepharose-GSH beads but no product peak appeared, showing that all the labeled protein bound to bead’s material. But similar elution buffer when used for GdmHCL treated trypsin-sepharose beads showed some MCOTI-I-Lys-TxRD coming out in elutions that shows that GdmHCl treatment before sticking step of MCOTI-I- Lys-TxRD makes the surface area less acceptable to dye binding or dye interactions remains there that becomes weaker when eluted by 100% Buffer B. The yields or results of MCOTI- I-Lys-TxRD products appearing from HPLC analysis using different sepharose beads and using different solvents for the product elutions are given in Table 4.2. Products were also checked in the elutes that were collected after reaction mixture incubations (5 µg) and those that could appear in flow throughs by phosphate buffer washes after incubations to clean the beads (Table 4.2).

Table 4.2. HPLC yields of MCOTI-I-Lys-TxRd using different sepharose beads and solvents for elutions.

SR# Sepharose Product Washes DMF Buffer B 100% Buffer B beads type elutes+FT with (ACN, 0.1% 50% GdmHCl TFA, H2O)

1. Trypsin-sep 2.5% 16% 2.6% 12.4% 11% (NHS active) comes out comes out only

2. Trypsin- 25.8% 6.8% 3.2% 6% 12% sep(Gdm.HCl washed)

3. Sepharose- 1% 5.8% 1% 5.3% 4.32% GSH

147

Complications were observed due to the highly sticky hydrophobic nature of Texas Red dye and maximum binding of it was seen with carbon skeleton of Beads material and column. Attempts of different solvents failed to completely elute maximum yields of the dye or dye binded peptide. Product recovery by HPLC was much less then injected. The reaction of labeling was too slow in the DMF medium for MCOTI-I-AziF, that also needed to be optimized in a buffered medium. This will be done first on azides and alkyne groups other than those involved in our peptide based labeling reactions to optimize first. The results suggest the avoidance of trypsin beads during the labeling reaction can give maximum yields when used 50 µL of trypsin sepharose beads. So labeling can be performed in a new buffered medium after detaching or eluting from the trypsin beads (in vitro). For captured MCOTI-I GdmHCl it seemed to be the best eluting solvent yet. Quantification of MCOTI-I-AziF- TxRD-DBCO was done by LC/MS based analysis. Furthermore, inteins induction and expression studies through western blot analysis was also conducted. We expressed the MCOTI-I in yeasts cells for the first time also (Published data, Jagadish 2015).

Considering the above based results and conclusions it was planned further to detach or harvest MCoTI-I-AziF from trypsin-sepharose beads to quantify the mutant protein and label it in vitro. In vitro labeling in buffered GdmHCl was optimized and then used, where click reaction of azide (protein) and alkyne (Dye) was possible. For this, a buffer preparation was needed that harvested/eluted as well as label in same medium. 4.14.1 Optimization and HPLC Monitoring of Cu++ free click reaction in buffered Guanidium-HCl It was decided to use 5-azido pentanoic acid (as azido) to be used in the optimization of Cu++ free click chemistry reaction with an alkyne group named DBCO amine. Both the reactants and the buffered GdmHCl were analysed on HPLC first and then the reactants was mixed in the buffer at room temperature as click reactions can work even faster at such conditions at neutral pH. For this the RP-HPLC conditions were set to 1 mL/min, 0-70% gradient of Buffer B for 30 mins at 220 nm in each experiment. Figure 4.51 shows the HPLC results showing buffered GdmHCl without reactants and then DBCO amine.

148

MWD1 A, Sig=220,16 Ref=800,100 (JAGS\101514000003.D)

Norm.

2.363

3.835 2.105

1750 4.685

1500

1250

1000

750 6.676

500

250 8.719

1.882

1.801

8.361 9.002 0 0 5 10 15 20 25 30 min MWD1 A, Sig=220,16 Ref=800,100 (JAGS\101514000005.D)

Norm.

2.122 2.389 3.896

700 6.679 DBCO amine 600 1.25ug

500 19.703

400

300

200

22.972 8.718

100

24.790

1.923

8.369

12.961

14.627 1.821 0 0 5 10 15 20 25 30 min

Figure 4.51. HPLC of Buffered GdmHCl and DBCO amine: The figure in the top shows buffered GdmHCl (6 M) diluted by 1 M Na2HPO4 solution with DMF added to 10% v/v of the total mixture (blank for the reaction). The figure at the bottom shows pure DBCO amine presented by 2 isomeric peaks at approximately 20 min and 23 min when used 1.25 µg

The buffer was made by diluting Guanidinium-HCL 8 M  6 M using 1 M Na2HPO4 and adding 10% DMF (v/v) and kept at room temperature (pH=7.0). The buffered GdmHCl is now believed to be ideal for both eluting the trypsin captured MCOTI-I-AziF and also to carry out the click chemistry reaction in a neutral environment pH=7. The DMF was added to let Texas Red or any other insoluble precipitates to become solubilize. Before running HPLC using buffered GdmHCl diluted the mixture in Buffer A or with 5% (v/v) of acetic acid to acidify medium before running the separation of cationic cyclotide.

Further reaction was started by the addition of 5-azido pentanoic acid to the mixture already containing DBCO amine in GdmHCl (6 M). The molar ratios were optimized from 1:1 to 1:10 for a complete reaction and obtained 5-Azidopentanoic acid-DBCO product of click reaction as shown in figure 4.52 below;

149

MWD1 A, Sig=220,16 Ref=800,100 (JAGS\101914000006.D)

Norm. A

17.188 19.651

700 17.534

600

500

400

300 23.023

18.312 18.567

200 24.858 12.912

100 10.540

0 0 5 10 15 20 25 30 min

150

MWD1 A, Sig=220,16 Ref=800,100 (JAGS\102114000003.D) Norm. B

5-azidopentanoic acid- 17.135

DBCO-Product isomers 17.477 700 DBCO-amine isomers

600

19.605

500

400 5-azidopentanoic acid 18.294

300

18.550

23.015 24.863

200

10.710

12.847 10.502

100

0 0 5 10 15 20 25 30 min MWD1 A, Sig=220,16 Ref=800,100 (JAGS\101514000002.D)

Norm. Isomeric forms of 5-azido

2.249 3.622 6.871 2.022 C pentanoic acid-DBCO amine 700 click reaction product

600

500

400 17.022

5-azidopentanoic acid 17.390 300

200

8.439

18.052 18.311

100

12.211

23.558

8.110

24.933

1.789

12.910

14.508

1.700 3.287 0 0 5 10 15 20 25 30 min Figure 4.52. Reaction of 5-azido pentanoic acid with DBCO amine in buffered GdmHCl. A) Shows the reaction after 1 h at RT after addition of 5-azidopentanoic acid (12 mins peaks) in a mixture of DBCO amine present in buffered GdmHCl and appearance of product seen at 17 mins, confirmed by mass spec analysis. B) 1:10 ratios of 5-azido pentanoic acid:DBCO amine (5 h), after sometime DBCO peaks are reducing since used in making products. C) Isomeric Products of 5-azidopentanoic acid-DBCO recovered after dilution in buffer A.

4.14.2. Mass spectrophotometric analysis of each reactant and product of azido and alkyne reaction for optimization of buffered GdmHCl in future labeling MCOTI-I-AziF

Mass spectrometric analysis of both DBCO amine (Only DBCO-amine from 20 min peak) and 5- azido pentanoic acid (12 min peak) collected by HPLC fraction collector were both analysed on the mass spectrometer. Product of reaction 5-azidopentanoic-DBCO was also analysed for both peaks of 17 min and were found to be similar as expected. Results in Figure 4.53 show agreement with expected masses of DBCO amine = 276 M.W and 5-azido

151 pentanoic acid = 148 amu and product was expected to be 5-azido pentanoic acid-DBCO = 424 amu.

5-azido pentanoic acid

DBCO-amine

152

Product of click reaction = 5- azidopentanoic acid-DBCO-amine

Figure 4.53. Results of mass spectrophotometric analysis of reactants and products of click reaction optimized. The figures shows the masses obtained for DBCO amine = 277.2 ± 0.84 amu (expected=276.3), 5-azido pentanoic acid = 148.9 ± 0.63 amu (expected=143 amu) and product of reaction (5-azidopentanoic acid-DBCO) = 420.1 ± 2.76 amu (expected close to 430 amu)

4.14.3. Expression and quantification of MCOTI-I-AziF at large scale for labeling reaction after optimization steps

After optimization of the Buffered GdmHCl for click reaction between Texas red- DBCO amine (TxRd-DBCO) and MCOTI-I-AziF for peptide labeling with flourecscent dye and confirming that the labeling have to be done in vitro after detaching from trypsin beads. It was decided to further express the MCOTI-I-AziF using same constructs as shown in figure 4.39 i.e. both pVLOmeRS and pET28a constructs with slight changes in induction conditions. For this schematic plan shown in figure 4.54 was followed to complete the labeling process for studying protein-protein interactions based on FRET analysis when checked with trypsin-EGFP protein as trypsin would specifically bind with MCOTI-I-AziF and TxRd and EGFP would serve as donor acceptors for FRET signals. KD values was calculated in the end.

153

Figure 4.54. Summarized view of Intein mediated protein-trans splicing, in cell folding of mutant cyclotide and its labeling with TxRd for interactions with Trypsin-EGFP.

This time the expression was done for four litres of culture conditions to get a higher yield of MCOTI-I-AziF for maximum labeling and purification by HPLC since much of the labeled peptide sticks.

Constructs for the said purpose used were pET28a vector with mutant MCoTI-I-Azide gene (Kan resistant/ IPTG inducible) and pERAzi (pVLOmeRS) vector with orthogonal tRNA synthetases gene (Chloramphenicol resistant/ Arabinose inducible) that has the ability to bind suppressor tRNA with anti-codon specificity to bind/incorporate p-azido phenyalanine at stop codon. Both constructs were co-transformed in the chemically competent host cells E. Coli Origami DE3 strain by heat shock method. The induction conditions after cells grown on LB agar plates with antibiotics kanamycin (50 mg/mL, each added 50 µL/50 mL) and chloramphenicol (75 mg/mL) were;

Induction conditions per litre (37oC)

• At 0.2 O.D growth = added 0.2 g/2 mL p- azido Phenyl alanine i.e. 1 mM • 0.4 OD = stock is 20 % arabinose 5 mL/L and added till 0.4 % previous was 0.02 % • 0.6 OD = 0.5 mM IPTG (previous was 0.3 mM) • Over night at RT, keep in dark or Red light after azide addition as its photoreactive in an orbital shaker at 220 rpm. We used terrific broth as growth medium this time as compared 2XYT media previously. 154

Expression and in cell folding was judged by SDS-PAGE analysis showing expressed inteins

(IC tagged and captured by Ni-NTA beads) (Figure 4.55).

Sp P B M

IN cleaved (Intein)

Figure 4.55. Results of SDS PAGE of the MCOTI-I-Azide expressed: SDS-PAGE results inteins expressed for MCOTI-I-AziF folding, lanes showing P is the pellets (insoluble cell lyate after sonication), Sp is the supernatant (soluble fraction after cell lysis), B is the Ni- NTA beads that captured inteins showing IC Amino terminal 6XHis-tagged-inteins with uncompletely cleaved showing maximum protein folding still needed. Very little Ic is shown below that which shows still uncleaved is there that should be incubated more for proper complete folding.

Once it was confirmed by SDS PAGE analysis that the complete folding and expression of

MCOTI-I-AziF has occurred and later when the inteins when quantified by A280 that its expression level was almost 13.8 mg/L of inteins (Ic tagged with His, detached from beads by 0.5 M EDTA)(1 OD=1.12 mg/mL) which is in agreement with the already reported expression level during coumarin labeling (Jagadish et al., 2013), then the next step was to inject for LC/MS a known volume 20 µL of the expressed MCOTI-I-AziF to quantify the recombinant protein. Once the trypsin sepharose beads 50 µL had captured maximal amounts of MCOTI-I-AziF then the protein interactions with the trypsin beads was set free by buffered GdmHCl (6 M diluted by Na2HPO4 and DMF solutions) as optimized in section 4.14.1. Significant amounts of MCOTI-I-AziF was found to be extracted from 4 litres of

155 culture under induction conditions as described above and was confirmed by LC/MS results (Figure 4.56). Charged states were similar used to differentiate between the NH form already reacted and N3 azide reactive forms as described previously in section 4.12 and figure 4.40 where earlier labeling was not much successful and after optimizations under new conditions the same is succesfully expressed for labeling (in sufficient amount then expected) as mentioned in this section.

Unreacted N3 form of MCOTI-I-AziF

Already Reacted NH form of MCOTI-I-AziF

Figure 4.56. LC/MS analysis of MCOTI-I-AziF expressed showing NH and N3 forms: LC/MS results of MCOTI-I-AziF expressed showing NH and N3 peaks clearly as much of the reactive forms N3 are there with non-natural amino acid p-azidophenyl alanine present invitro (datached from beads after capture by trypsin sepharose). At this stage no labeling was done as it shows no labeled product.

Production of MCOTI-I-AziF was found to be 2.29 µg/L after following the optimized induction conditions for its expression. Fixed volume and fix concentrations of WT MCOTI- I were injected in the LCMS system for analysis as standards i.e. 0.5 µg, 1 µg and 1.5 µg. Each time volume injected was 20 µL. The quantification was done by using the peak areas corresponding to known standard concentrations of MCOTI-I and the estimation of the

156 mutant cyclotide MCOTI-I-AziF was done when compared with the peak areas of known concentrations of MCoTI-I (0.5 µg, 1 µg and 1.5 µg) (Figure 4.57). Built in software of the LC/MS system was used for calclulation of peak areas and its concentrations. Different method of quantification was used here as very little amounts of MCOTI-I-AziF was expressed from large scale induction conditions also and already very small volumes are there i.e. only 500 µL and we cannot expose our N3 form of MCOTI-I-AziF before labeling reaction as its self reactive when exposed to light or air much.

1.5 ug MCOTI-I

Selected peak areas for quantification of charged 1.0 ug states

1.5ug

0.5ug

Figure 4.57. LCMS based quantification using standard WT-MCoTI-I represented by 3rd charged state of peaks area selected (871.6 amu) each time. 3rd charge states 871.6 amu were used to estimate the peak areas corresponding to known concentrations of MCOTI- I i.e. 0.5 µg, 1 µg and 1.5 µg and with fixed volume each time injected. These expreriments

157 were done freshly as readings may change with different reaction conditions used each time and results may vary.

Similarly a known volume i.e. 20 µL of MCOTI-I-AziF shown in Figure 4.54 were quantified by LC/MS software (Figure 4.58) and peak areas of standards and knowns were compared to quantify for 3rd charge stae here also.

Figure 4.58. LC/MS based quantification of MCOTI-I-AziF by peak areas: A) N3 only without labeling 3rd charge (889.4 amu). B) NH only i.e. self reacted from N3 to NH form not participating in labeling reaction (882.9 amu). C) 3rd charge state of MCoT-I-AziF-TxRd (1134.2 amu) showing no labeled peptide peaks that produced after reaction completion after

158

TxRD-DBCO additions since no reaction was started yet. Corresponding peak areas are shown in the tables.

4.14.4. Labeling reaction of MCOTI-I-AziF: Texas Red-DBCO

After expression and quantification of MCOTI-I-AziF i.e. 2.29 µg/L (by LC/MS based quantifiaction) the next step was to calculate the molar concentrations of the reactants by the help of molarity calclator online tool availble at graphpad (http://www.graphpad.com/quickcalcs/moleform/) using molecular mass and volumes present i.e. dye and peptide. Later the buffered GdmHCl (6 M) with pH 7 and 10% v/v DMF

(dimethyl formamide, miscible with polar H2O and organic solvents with low evaporations) was used to react at room temperature for labeling. The different molar concentrations ratios used were 1:5, 1:25 and 1:100. 2.045 µM concentration was used for MCOTI-I-AziF and respectivey 5 times, 25 times and 100 times more molar concentrations corresponding to some fixed volumes of TxRd-DBCO (powdered dye may be dissolved in DMF) were added after each hour of incubations at each time and continous shaking. Each time a fixed volume representing 5% of total volume of reaction mixture was taken out for analysis of labeled product MCOTI-I-AziF-TxRd-DBCO through LC/MS till the reaction was complete. Actual labeling step is a slower one and required much higher concentrations then actually expected to label the cylotide peptide with texad red DBCO amine.

The reaction progress and LC/MS results of the stepwise results of MCOTI-I-AziF labeling reaction with TxRd-DBCO (Figure 4.59);

159

N3 1:25 1:5 (uM) i.e. 2.045uM of MCOTI- (uM) I-AziF : 10.229 uM TxRd-DBCO

1:100 (uM) completed labeling

MCOTI-I-AziF-TxRd Unreacted NH Labeled (1.48ug/L after completion)

Figure 4.59. LC/MS of labeling reaction completing between MCOTI-I-AziF and TxRd- DBCO: Results showing the progress of the reaction at different molar ratios 1:5 to 1:100 till completely all reactive N3 form is converted to MCOTI-I-AziF-TxRd-DBCO and only NH

160 unreacted remains behind in buffered GdmHCl (6 M). charge states of each labeled and unlabeled MCOTI-I-AziF were given in LC/MS operating system. Bold Arrows ↓ shows product appearing. 4.14.5. HPLC Purification and M/S confirmation of labelled peptide MCOTI-I-Azide- TxRD Once the labeling reaction of the cyclotide peptide was completed (Figure 4.59) the next step was to purify the labeled cyclotide (1.43 µg/L left, quantified by LC/MS) from the reaction mixtures and for which the only best choice to separate out this hydrophobically labeled peptide was through HPLC. As volume and concentration was too small so using preparative HPLC was not a good idea. For this 10-60% gradient HPLC and 0-70% gradient were both run seperately at 595 nm for 30 minutes and each peak appearing in the HPLC run was collected seperately and analysed on mass spectrometer to get the labeled peptide. Figure 4.60 shows HPLC results of labeled peptide for collection and identification.

10-60% HPLC

0-70% HPLC

MCOTI-I-AziF-TxRD-DBCO

Figure 4.60. HPLC purification of MCOTI-I-AziF-TxRd-DBCO. Figure above shows two types of gradients run for purification, 10-60% RP-HPLC using C-18 columns at 595 nm

161 detector (max wavelength for Texas Red dye) and the other was 0-70%. The peak representing 24.89 min and 23.95 min respectively retention time represents our labeled MCOTI-I-AziF-TxRd-DBCO confirmed by m/s.

Peaks apearing in the 10-60% gradient RP-HPLC i.e. 24.89 min and in 0-70% gradient at 23.95 min were both collected and analysed on m/s and both exactly showed successful labeled masses i.e. 4532 ± 0.7 amu as closest to expected one with 3rd and 4th charge states 1133.9 and 1511.1 amu respectively. The results of mass spec analysis are shown in figure 4.61.

3rd & 4th charged states (1133.9 & 1511.1)

Obtained= 4532 ± 0.7 amu

Figure 4.61. M/S of fluorescently labeled MCOTI-I-AziF-TxRd-DBCO. 4532 ± 0.7 amu mass of labeled MCOTI-I-AziF-TxRd-DBCO was obtained with 3rd and 4th charged states as 1133.9 and 1511.1 amu.

4.14.6. Protein-protein interaction studies based on FRET analysis

FRET is an advanced method for probing, measuring distances between molecules and their dynamics especially among biochemical molecules such as protein and nucleotides that are present in a few nanometer ranges and more sensitive like cellular machine. Where in FRET fluorescent molecules are present in the condensed state that has a very limited number of

162 excitation and emission cycles, these molecule can strikes with the other molecules chemically (Meer et al., 1994; Levene et al., 2003).

The labeled cyclotide was used in fluorescence resonance energy transfer (FRET) to visualize the interaction between modified trypsin that was fused at N-terminus with green fluorescent protein (EGFP) and cyclotide-protein interaction was monitored by intermolecular FRET shown by the simultaneous decrease and increase of the fluorescence signal from donor to acceptor, respectively (Jagadish et al., 2013).

Mass spectrophotometric results shown in figure 4.61 represents the labeled peptide with a molar concentration of 20.1 µM which was in purified form and was enough to be used in the Protein-protein interaction studies based on FRET analysis on a fluorimeter after expressing Trypsin-EGFP protein using pET25 expression system. This system was based on a donor acceptor system where EGFP behaves as a donor and TxRd labeled MCOTI-I-AziF as an acceptor. Whereas trypsin is modified for no cleavage activity rather it still binds MCOTI-I- AziF-TxRd-DBCO as an inhibitor protein specifically and this interaction results in proteins interaction studies giving FRET as signal as result of excitation and emission that varies as distance between both or interaction varies.

For this purpose the trypsin-EGFP was first expressed using pET25b expression system in E.coli Bl21 strain using IPTG as inducer and kanamycin as an antibiotic marker. For spectrophotometric quantification the extinction coefficient value of EGFP protein at 484 nm was 56,000 cm-1M-1. The figure 4.62 shows Trypsin-EGFP expressed in form of results of SDS PAGE and mass spectrophotometeric output results showing different peaks. The recombinant green frourescent protein was captured by affinity chromatography using Ni- NTA beads and the interaction or bonding was relieved by washing it with 0.5 M EDTA.

163

Figure 4.62. Expression of Trypsin EGFP : SDS PAGE and m/s results of trypsin EGFP expressed in Bl21 and using 0.3 mM IPTG induced Over night, Kanamycin resistant and pET25 construct.

For flourimetry and FRET analysis the Trypsin-EGFP was excited at 489 nm and after energy transfer the emission was obtained at 613 nm with only 1.8% background of EGFP. 1X PBS was used as a buffer for the FRET analysis reaction. 10 nM from 20.2 µM stock MCOTI-I-AziF-TxRd-DBCO was used as fixed amount of protein to interact with different concentrations/dilutions of trypsin-EGFP proteins (stock=21.5 µM) i.e 5 nM, 10, 25, 50, 100, 250, 500 and 1000 nM concentrations and the total reaction mixture was in 1 mL.

The absorption spectra of each concentration of trypsin-EGFP was taken at 508 nm on the fluorimeter (Figure 4.63) and before that instrument working was first checked by distilled sterilized and Millipore filtered water then buffer (blank). These all steps were done without labeled peptide.

164

1X PBS d.H2O

14000000

12000000 buffer blank 10000000 5nm EGFP & buf 10nm EGFP 8000000 25nm EGFP

6000000 50nm EGFP 100nm EGFP 4000000 250nm EGFP 500nm EGFP 2000000 1000nm EGFP

500 507 514 521 528 535 542 549 556 563 570 577 584 591 598 605 612 619 626

Figure 4.63. Absorption spectra for Trypsin-EGFP of different concentrations. Absorption spectra taken from fluorimeter at 508 nm without labeled MCOTI-I-AziF. The instrument check was done with dH2O and by blank i.e. buffer 1X PBS as shown above in figure.

For FRET analysis studies i.e. fluorimetry, titration between 10 nM MCOTI-I-AziF-TxRd- DBCO and different concentrations of Trypsin-EGFP (from 5 nM-1000 nM) was done in 1X PBS buffer. To the buffer first labeled peptide was added whose spectra is shown in figure 4.64 and then trypsin EGFP stepwise in an ascending concentrations. The energy transfer by the interaction between two labeled proteins helps us in understanding the protein interaction studies that be useful in drug development and targeting studies. The maximum FRET based

165 energy transfer was seen at 500 nM (saturation point) of trypsin EGFP and then binding effieciency was calculated by the calculation of KD values.

MCOTI-I-AziF- TxRd-DBCO

40000 MCOTI-I-Azide-TxRd- DBCO= 10 nM constant 35000 throughout titration 30000 5nm EGFP & buf

25000 10nm EGFP

20000 25nm EGFP 15000 50nm EGFP 10000 100nm EGFP 5000 250nm EGFP

500nm EGFP

607 619 567 571 575 579 583 587 591 595 599 603 611 615 623 627 631 635 639 643 647 563

Figure 4.64. FRET analysis shows MCOTI-I-AziF-TxRD-DBCO and Trypsin-EGFP interactions. Above figures shows a absorption spectrum of 10 nM MCOTI-I-AziF-TxRd- DBCO and also protein interaction studies in which excitation at 489 nm and Emission at 613 nm (b/w 500 to 650 nm) was set and readings were graphically expressed showing clear energy transfers by interactions of EGFP labeled trypsin.

166

10 nM of MCOTI-I-Azi-TxRD fixed concentration was used to observe the transfer of energy from Trypsin-EGFP binding which excited the labeled MCOTI-I-AziF by binding with trypsin and this binding saturation was obtained at or near 250 nM trypsin-EGFP and maximum at 500 nM concentration above which no further change was observed by increasing further the trypsin-EGFP concentration (Figure 4.64). Texas red excitation was taken at 489 nm and emission at 613 nm and saturation of TxRD-MCOTI-I-Azi for FRET at 500 nM concentration of trypsin-EGFP. Measurement of affinity constant between trypsin-

EGFP and MCoTI-I-AziF-TxRD-DBCO was calculated by graphpad software i.e. KD the dissociation constant between 2 labeled proteins was measured by fluorescence polarization anisotropy at 25 °C using a Jobin Yvon/Spex Fluorolog 3 spectrofluorometer with the excitation bandwidth set at 5 nm and emission at 5 nm. The excitation wavelength for TxRD was set at 489 nm and emission was monitored at 613 nm (in this experimental system EGFP is the donor and TxRD acts as acceptor which then emits the detectable signals with only

1.8% background of each other’s spectra that interfere in results). The KD value was further calculated to be 29.7 ± 1.08 nM (graphpad prism software) as shown in figure 4.65. Studies of labeling MCOTI-AziF with coumarin i.e. AMCA-labeled cyclotide MCoTI-AziF efficiently binds trypsin-S195AEGFP (KD of 1.8 ± 0.7 nM) in vitro which is much less as compared to our results. Labeling with texas red is a better alternative approach for studying protein interactions using cyclotides but a tougher approach to purify in bulk for different studies.

29.7±1.08 nM

Figure 4.65. Calculation of Binding efficiency KD. Figure shows affinity constant or binding dissociation constant value of MCOTI-I-AziF-TxRD-DBCO with trypsin-EGFP and Calculation of KD value by GraphPad was 29.7 ± 1.08 nM which is higher and better.

167

4.15. Expression optimization of Wild Type MCOTI-I production (Wester blot analysis).

Besides the labeling of cyclotides with flourescent dye for studying proteins interactions, were also optimized the MCOTI-I expression conditions for maximum or equivalent production of MCOTI-I by estimating inteins in much lesser time and with much less non specific products that could probably interfere where interactions were affected by unwanted products. Both overnight incubations and expressions in 3-4 h gave equivalent amounts of MCOTI-I productions.

Standard condition for producing MCOTI-I in E.coli cells as host was at 0.6 O.D of cells the induction overnight at room temperature (RT) was carried out at 0.3 mM IPTG using pASK construct i.e. by PTS system. Later after series of experimemnts we optimized its production using conditions i.e. at 0.6 O.D of cells induced by 1 mM IPTG and collected same number of cells after each hour during 1-4 hrs time of induction or incubation at 37 oC followed by each time 1X M9 buffer washes and over night incubation at RT for expression optimizations and complete folding of proteins to mature forms. Each time the equal number of cells (1 x 108 cells/mL) were used as sample in the Western blot analysis (VWR, western blotting scanner system-chemilumicent storm-860, Molecular Dyanamics) alongwith standard Ni captured inteins of different concentrations i.e. 10, 25 and 50 ng. Results of western blot analysis after folowing the standard protocol in 3.19 section (using PVDF nitrocellulose membranes, 1xTBST buffers, 5% milk as blockers, anti-Histidines primary antibodies as intein-C tagged with His & mouse HRP secondary antibody before scanning) shows that approximately 50 ng inteins produced at 1 mM IPTG inductions for 4 hrs at 37 oC & 1X M9 washes whereas same production was seen the previous standard optimization condition with 0.3 mM IPTG inductions over night, as inteins are result of folded cyclotides produced. This study may be helpful considering the MCOTI-I-AziF production for labeling purposes also for invivo labeling and removal of unwanted amino acids (like p-azidophenylalanine) that incorporate in cellular proteins when incubated overnight and are responsible for the background produced during analysis of results during FACS analysis for cell sorting. Figure 4.66 shows the western blot results of expression studies in optimized inductions. Equal

168 amounts of cells 9.995 x 1011 cells/different volumes of each induced condition 0.3 & 1 mM IPTG induction was used to extract MCOTI-I and captured by equal volumes of trypsin sepharose beads 50 µL and quantified to be almost equal when observed through HPLC.

Figure 4.66. Western blot analysis of inteins production at 1 mM IPTG inductions. The figure shows the in cell production of inteins produced as a result of MCOTI-I cyclization and folding in PTS system optimized under conditions including 1 mM IPTG, 3-4 hrs incubation time and at 37 oC. After primary and secondary antibody application for HRP the cells after 4 hours incubation and those cells of 4 hour incubation plus 1X M9 overnight incubations and washes both showed same amounts of cleaved Inteins i.e. 50 ng which resulted by MCOTI-I folding. A tricolor protein ladder in along with our samples.

4.16. Expression of Cyclotide MCOTI-I in yeast cells (Publised, Jagadish et al., 2015).

To express cyclotide MCoTI-I inside living yeast cells (Saccharomyces cerevisiae) protein trans-splicing (PTS) system was used to facilitate the intracellular backbone cyclization. This process has been previously used to express small cyclic peptides (Kritzer et al., 2009, Young et al., 2011) and more recently cyclotides (Jagadish et al., 2013) in bacterial expression systems but never used earlier in a eukaryotic expression system to express large disulfide-containing cyclic proteins such as cyclotides. PTS is a post-translational modification similar to protein splicing with the difference that the intein self-processing domain is split into two inactive fragments IN (N-terminal) and IC (C-terminal). When the two intein fragments bind to each other under appropriate conditions, they form an active protein splicing or intein domain in trans (Jagadish et al,. 2013, Kwon et al., 2006). PTS-mediated

169 backbone cyclization can be accomplished by rearranging the order of the intein fragments, i.e. by fusing the IN and IC fragments to the C- and N-termini of the linear polypeptide precursor to be cyclized. To boost the intracellular expression of folded cyclotide MCoTI-I in yeast we used the Nostoc puntiforme PCC73102 (Npu) DnaE split-intein. This DnaE intein has the highest reported rate of protein trans-splicing (1/2 ≈ 60 s) and has a high splicing yield ( Iwai et al., 2006, Zettler et al., 2009).

Accordingly, we designed the split-intein construct, where the MCoTI-I linear precursor was fused directly to the N- and C-termini of the Npu DnaE IC and IN polypeptides, respectively. To facilitate backbone cyclization we used the native Cys residue located at the N-terminal position of loop 6. A His-tag was also added at the N-terminus of the construct to facilitate identification of the precursor and intein-containing byproducts of the cellular cyclization process.

Expression in yeast S. cerevisiae of cyclotide MCoTI-I using PTS was tested by employing two different high-copy 2 episomal expression plasmids, pYES2/NT (under the control of a GAL1 inducible promoter) and p426GPD (under the control of a GPD constitutive promoter). Expression plasmids encoding the split-intein precursor derived from plasmids pYC2/NT and p426GPD were transformed into S. cerevisiae strains INVSc1 and W303-1a, respectively, by electroporation (eppendrof electroporator 2510 model). Expression of the MCoTI-precursor split-intein was accomplished for 48 h at 30° C in media containing either 2% galactose (inducible GAL1 promoter) or 2% glucose (constitutive GPD promoter). Under these conditions the precursor was expressed at relatively high levels in both cases, ≈ 10 mg/L (GAL1 promoter) and ≈ 7 mg/L (GPD promoter). In both cases the precursor was completely cleaved (Fig. 4.67), indicating the intrinsic high reactivity of the split-intein construct to undergo protein trans-splicing. Next, we quantified the amount of natively folded MCoTI-I generated in-cell by LC-MS analysis using pure MCoTI-I as standard as shown before in our previous experiments of producing MCOTI-I-AziF. Correctly folded MCoTI- cyclotides are able to bind trypsin with high affinity (Ki ≈ 20-30 pM). Therefore, this step can be used for affinity purification and to test the biological activity of the recombinant cyclotides. By either using the whole cell lysate or the fraction purified with trypsin- immobilized sepharose beads the LC-MS analysis revealed in both fractions the presence of a

170 major peak that had the expected mass of the natively folded MCoTI-I cyclotide (Figs. 4.68). Quantification of the amount of cyclotide gave similar yields in both fractions ≈ 50 µg/L (GAL1 promoter) and ≈ 60 µg/L (GPD promoter), which correspond approximately to an intracellular concentration of approximately 450 nM and 660 nM, respectively. These results indicate that in cell produced cyclotide MCoTI-I is biologically active and therefore adopts a native cyclotide fold (Data published, Jagadish et al., 2015).

Figure 4.67. In-cell expression of MCOTI-I based cyclotides in S. cerevisiae cells using Npu DnaE intein-mediated PTS. A. SDS-PAGE analysis of the recombinant expression of cyclotide precursors using a high-copy µ episomal plasmid under the control of the GAL1 (inducible, left) or GPD (constitutive, right) promoters. B. Analytical HPLC-MS/MS traces of the soluble cell extract (left) or trypsin pull-down (right) fraction from S. cerevisiae cells expressing precursor under the control of a GAL1 inducible promoter. The peak marked with an asterisk corresponds to folded cyclotide MCoTI-I.

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Figure 4.68. Mass spectrum of in-cell generated (yeast cell) cyclotide MCoTI-I. Precursor ion scan was performed using the ion (m/z = 871.6) in Q3. PUBLISHED (Jagadish et al., 2015) Final Conclusions

Protein extraction buffer is a better substitute than phosphate buffer for extraction of bioactive components, when we want target proteins as major bioactive component responsible in a plant metabolite mixtures. Experiments related to bioactive roles of plant extracts with and without protein content, proved that plant extracts with protein content showed significant bioactivities and their absence marks a significant decrease in most bioactivities. Isolated Genes (selected) including cliotides, panitides and from violaceae from local plants for the first time and these local isolates’ genes showed least variations in their cyclotide domains. Purified Wild type MCOTI-I after expression and natively folding by PTS or EPL methods showed significant antimicrobial, hemolytic, reducing and thrombolytic activities. But MCOTI-I showed no protection to DNA demage & mutagenicity responses. Control experiments were conducted on WT-MCoTI-I-Lys-TexasRed for checking the differences in elution times when hyphobic sticky dye is attached to the MCOTI-I that can roughly predict where mutant with trypsin binding ability in HPLC. Moreover elution yields and interactions with trypsin-sepharose or sepharose beads will also be estimated. Polymers of carbon like sepharose column beads and cellular contents strongly stick to the Dye due to its nature (Hydrophobic). This made it very hard to purify by HPLC columns also (C-18). It was found that only invitro labeling possible of the mutant MCOTI-I-AziF using buffered

172

GdmHCl (optimized) and once the protein is detached from the beads they are then labeled with Texas Red-DBCO amine dye in the buffer in a eppendrof microcentrifuge tube. NOTE: This click reaction was optimized Using 5-azido pentanoic acid and DBCO-amine (costs less) in buffered GdmHCl before doing the MCOTI-I-azide reaction with dye. MCOTI-I-Azi- Phe labeled Invitro with Texas red-DBCO amine at 1:100 molar ratios after several tries with different increasing ratios of protein and dye.

Lower amounts of product (MCOTI-I-Azi-TxRD-DBCO) was purified by HPLC and then was confirmed by M/S after analyzing all expected peak elutions & quantified by LCMS. Trypsin-EGFP produced separately was then titrated for flourimetry with concentrations without binding to TxRD product and FRET analysis against TxRD-DBCO- MCOTI-I-Azi were seen. After attaining saturation regarding labeled protein cyclotide binding to EGFP-trypsin the value of KD was calculated (binding efficiency).

Western blot based Optimization studies was also done on induction conditions of MCOTI-I expression at 1 mM IPTG induction for 4 h at 37 oC giving same amount of inteins as recently produced by 0.3 mM IPTG induction overnight at RT using Origami DE3 cells. Attempt for the first (published) producing natively folded active MCOTI-I in the yeast cells was successfully done and confirmed by m/s analysis. This can now open opportunies towards understanding the drug development and targeting studies from bacterial system to eukaryotic system, which may be a successful ladder towards studying cyclotides interaction in human cells that resembles eukaryotic system.

Better extraction methods are used in our studies when we consider proteins for bioactive studies. Further bioactive roles of proteins in plants are highlighted by our studies. Local potential of Cyclotide bearing plants are explored emphasizing on their pharmacuetical importances or potential to focus more in future. We proved a better molecular understanding towards stable or conserved cyclotide genes like in clitoria even isolates of distant areas analysed through bioinformatics. Current studies emphasized on the better understanding to protein labeling strategies and drug targeting and protein interactions for diseases control studies. This is a new emerging domain of cyclotide based research. Through this study can monitor inhibitory responses with diseases causing agents when grafted proteins on trypsin or

173 cyclotides and drug targeting studies may be mentioned. Here we also optimized induction conditions or method for maximum production of MCOTI-I or same production reducing time and background of unwanted materials which incorporate during overnight incubations giving already reacted products like NH derivative of p-azidophenyalanine incorporated in MCOTI-I framework. Inductions within 3-4 hours can now give the same amounts of MCOTI-I besides overnight productions. Eukaryotic systems are exposed for the different studies done on cyclotides that was never done before.

Acknowledgement

I am highly thankful to the higher education commission Islamabad Pakistan for providing support in all aspects from finance to infrastructural and technological facilities provided in our studies. I acknowledge the support of Molecular biochemistry lab and Plant Molecular biochemistry lab for providing the facilities of gene isolation and bioactivity based studies. I also highly appreciate Dr. Julio A Camerero, School of Pharmacy, University of Southern California, USA for providing all the facilities and support to work with cyclotide MCOTI-I during expression, labeling and first time production studies in Yeast cells.

174

SUMMARY

Humans have been exposed to a number of challenges related to health, food and agricultural issues including a number of pathogens and pests. Many ways have been adopted to combat theses targets. Among those successful remedies plants are one of the safest source of natural drugs and agents that can be a source to combat these targeted agents without any side effects. The bioactive agents that can compete with the pests and pathogens include a number of bioactive secondary metabolites such as phenolics and alkaloids and bioactive peptides etc. Bioactive peptides, specifically cyclic peptides, are one of the most attractive choice to address our problems related to human health and food issues. Cyclic peptides including “cyclotides” have been proven to be stable and versatile active agents with capability of being bioactive. It has also proven to be an excellent tool for the delivery of drugs to the requisite target areas under the influence of signal molecules attached or by affinity matches along with bioactive epitopes attached. Cyclotides comprise about 30 amino acids and are stable peptides rich in cysteine, having three absolutely conserved disulphide linkages with head to tail cyclized backbone. In the present studies bioactive potential of cyclotide bearing indigenous plants plants including Panicum, Clitoria, Viola, Ptunia and Hamelia were explored. It was found that bioactive potential of the plant extracts was due to peptides that had antimicrobial, antioxidant and thrombolytic activities. Cyclotide MCOTI-I was used in pure form for comparison purpose in each bioactivity assay as a standard. Out of the screened plants Clitoria had highest activities.

In the next section of studies we focused on the screening and isolation of cyclotide gene/s from the selected cyclotide series of plants using specifically designed primers. Clitoria DNA with 683 bp cliotides had the most unique chimeric arrangement. The sequence was submitted in GenBank (Accession number KP889219) and analysed using different online bioinformatics tools. A conserved domain of cyclotide chimerically arranged with Albumin-1 was also conserved with variation in the intronic regions other than CDS.

Further studies were done on the labeling of cyclotide MCOTI-I protein to check the protein-protein interactions at molecular level for cancer based or other inhibitory based protein interaction studies. For this purpose the dye was prepared in its alkyne form by

175 reacting with Texas Red succinimyl ester (TxRd) with DBCO amine using click chemistry reaction. The prepared dye TxRd-DBCO was purified and was further used to label the cyclotide MCOTI-I-AziF with unnatural amino acid (p-azido phenylalanine AziF) having reactive N3 azide group. The cyclotide MCOTI-I-AziF was expressed in origami DE3 cells and captured by trypsin sepharose beads. The trypsin capture volumes of 50 µL were optimized to be enough for 5 µg. MCOTI-I was also used as a model to get labeled with

Texas red succinimyl ester in the presence of DEAE base to label at the NH2 group lysine present in loop 2. The MCOTI-I-NH-Lys-TxRd was modified due to labeling with loss of trypsin binding but still showed sticky or binding in the C-18 material of the HPLC column as well as on the sepharose material since texas red itself is quite sticky and hydrophobic in nature. This nature of fluorescent dye makes it impossible to label on the beads material and purify the MCOTI-I-AziF-TxRd-DBCO through HPLC. Next the MCOTI-I-AziF was expressed in larger quantity from 4 litres of culture and captured by trypsin beads. Before next step buffered GdmHCl 6 M was used to optimize reactions between 5-azidopentanoic acid and DBCO amine as controls or standards. The Gdm-HCl was used to detach all the MCOTI-I-AziF and quantified by LC/MS analysis. Two different vectors were employed for expression of MCOTI-I-AziF by orthogonal tRNA synthatase technology using pVLOmeRS and pET28 constructs under IPTG/arabinose induction along with 2 g/2 mL NaOH solution of p-AziF was added in each litre of culture of origami 2 DE3. The expressed MCOTI-I-AziF was then reacted in the buffered GdmHCl 6 M (pH 7.0) alongwith DMF with TxRd-DBCO dye and the product and progress of the reaction was monitored by LC/MS for the MCOTI-I- AziF-TxRd-DBCO. Each time HPLC based analysis and mass spectrophotometric analysis was done for the confirmation of the products. Once the MCOTI-I-AziF-TxRd-DBCO was produced in a 20.2 µM concentration then was used to interact trypsin-EGFP proteins separately expressed using pET25 system. Using 10 nM MCOTI-I-AziF-TxRd-DBCO was used to get binding saturation with 500 nM trypsin-EGFP was achieved using fluorescent assays like using fluorimeter for FRET based analysis that indicated the protein interaction.

KD value for checking the binding efficiency was calculated to be KD of 29.7 nM.

Expression of MCOTI-I was optimized for the same quantity by IPTG induction from overnight incubation to 3-4 hours. Cyclotide MCOTI-I was also expressed for the first

176 time in the eukaryotic system i.e yeast cells which was folded natively and confirmed by mass spectrophotometric analysis.

The current research will help us understand the bioactive role of cyclotide bearing indigenous plants. Moreover current studies assist to understand the fluorescent labeling approaches of cycotides such as MCOTI-I in general for in cell screening through optical approaches and screening of bioactive peptide libraries that can inhibit various pathogenic or toxic proteins or groups. Our findings will also help in using better induction conditions for MCOTI-I production and using a eukaryotic system (yeast) for studying cyclotides in future studies like cancer based studies or drug development and targeting.

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Appendixes

Appendix-I

Different antioxidant Activities of leaf and seed extracts of selected medicinal plants prepared in PBS and protein extraction buffer with and without proteinase K treatment.

TFC (mg/g) Reducing power DPPH TPC (mg/g) Gallic Catechin (%) scavenging DNA damage protection Sr.No Plant names acid equivalent equivalent activity (%) assay Means ± S.D Means ± S.D Means ±S.D Means ±S.D Plasmid Different buffers PBS PEB PBS PEB PBS PEB PBS PEB ctDNA DNA 16.05± 13.77±1 21.67±2 16.87±2 9.77±0 8.18 58.81± 58.84± Slight 1 Viola odorata *** 1.6 .2 .1 .5 .6 ±1.6 6.0 1.4 Protectant 6.92 46.44± Non 2 Viola odorata PK *** *** *** *** *** *** *** ±1.2 0.7 Protectant 22.13± 14.82±2 10.28±0 8.55 7.74±0 6.24 71.27± 64.76± Non Slight 3 Viola Hybrida 1.8 .3 .3 ±0.5 .6 ±0.9 0.9 2.4 Protectant Protectant 5.83 48.94± Non 4 Viola Hybrida PK *** *** *** *** *** *** *** ±0.8 0.9 Protectant 9.66 8.48 26.76±3 18.92±1 9.44±0 7.83 75.00± 68.34± Non Slight 5 Viola tricolor ±2.0 ±1.3 .6 .6 .4 ±1.2 1.3 1.2 Protectant Protectant 7.21 52.54± Non 6 Viola tricolor PK *** *** *** *** *** *** *** ±0.9 0.7 Protectant 14.76± 13.80±0 7.96 5.34 13.01± 9.81 73.85± 65.34± Non Slight 7 Clitoria ternatea 1.1 .5 ±2.9 ±1.4 0.6 ±0.8 2.6 2.1 Protectant Protectant Clitoria ternatea 6.33 39.65± Non 8 *** *** *** *** *** *** *** PK ±0.5 0.8 Protectant 9.61 7.07 6.14 2.81 16.17± 9.98 73.47± 70.31± Non Slight 9 Ptunia mix ±1.3 ±0.8 ±1.5 ±0.5 1.0 ±0.4 0.8 2.7 Protectant Protectant 8.32 32.56± Non 10 Ptunia mix PK *** *** *** *** *** *** *** ±0.7 0.1 Protectant 10.96± 8.03 6.65 3.05 8.5 7.41 72.22± 66.87± Slight 11 Pansy F1 *** 0.3 ±1.4 ±1.8 ±0.5 ±0.7 ±0.5 2.2 1.1 Protectant 6.2 41.43± Non 12 Pansy F1 PK *** *** *** *** *** *** *** ±0.5 1.4 Protectant 8.06 6.45 36.98±2 16.69±3 5.06±0 4.11 74.34± 67.54± Slight 13 Panicum vigatum ±0.6 ±0.8 .4 .7 .3 ±0.7 0.5 0.6 Protectant Panicum vigatum 3.65 39.51± Non 14 *** *** *** *** *** *** *** PK ±0.6 2.0 Protectant 13.74± 12.34±0 13.18±2 6.83 11.81± 5.98 72.65± 63.78± Slight 15 Panicum laxum *** 0.4 .1 .6 ±0.9 1.3 ±0.7 1.9 0.6 Protectant 5.54 41.44± Non 16 Panicum laxum PK *** *** *** *** *** *** *** ±0.4 0.8 Protectant Panicum 19.21± 14.78±0 5.55 3.60 12.93± 8.72 77.38± 72.85± Slight 17 *** maximum 1.6 .4 ±1.3 ±1.7 0.5 ±0.6 0.9 0.5 Protectant Panicum 8.33 63.26± Non 18 *** *** *** *** *** *** *** maximum PK ±0.5 0.7 Protectant 13.03± 10.76±0 3.91 2.75 16.02± 11.97± 80.15± 70.81± Slight 19 Hamelia patens *** 0.1 .2 ±1.5 ±0.9 0.7 0.3 3.0 0.9 Protectant 20 Hamelia patens *** *** *** *** *** 10.39± *** 58.43± *** Non

201

PK 0.4 0.4 Protectant

8.38 37.32± 38.42± Non Non 21 cyclotide MCOTI-I *** *** *** *** ±0.1 *** 0.1 0.1 Protectant Protectant

6.54±0 21.23± cyclotide MCOTI-I Non Non 22 *** *** *** *** .3 *** 0.1 *** PK Protectant Protectant

PBS = phosphate buffer saline, PEB = Protein extraction buffer, S.D = standard deviation PK = Proteinase K enzyme treated fraction of protein extract of selected plants, *** = Test not performed.

202

Statistical analysis of different antioxidant Activities of leaf and seed extracts of selected medicinal plants prepared in PBS and protein extraction buffer (PEB) with and without proteinase K treatment

Plant TPC (PBS) TPC (PEB) DPPH (PBS) DPPH (PEB) TFC (PBS) TFC (PEB) Reducing Power (PBS) Reducing Power (PEB) 01-Viola odorata (Banafsha) 16.05±0.94c 13.78±0.73ab 58.83±3.47d 60.08±0.84d 21.68±1.21c 20.27±1.46a 9.77±0.40c 8.18±0.97a 02-Viola Hybrida 22.13±1.06a 14.82±1.33a 71.27±0.58c 64.71±1.41c 10.28±0.21de 11.28±0.32c 7.74±0.36e 6.25±0.56ab 03-Viola tricolor 9.67±1.16fg 11.49±0.75c 75.00±0.79bc 70.13±0.71ab 26.77±2.11b 18.92±0.96ab 9.45±0.26cd 7.83±0.70bc 04-Clitoria ternatea 14.77±0.68cd 13.80±0.30ab 73.86±1.55bc 66.90±1.23c 7.97±1.68ef 5.35±0.81de 13.01±0.40b 9.81±0.49bcd 05-Ptunia mix 9.61±0.77fg 7.08±0.48d 73.48±0.49bc 70.85±1.61ab 6.14±0.88fg 2.81±0.32e 16.17±0.60a 9.99±0.24b-e 06-Pansy F1 10.97±0.19ef 12.03±0.86bc 72.22±1.30c 71.14±0.69ab 6.65±1.07efg 3.06±0.34e 8.50±0.42de 7.00±0.30cde 07-Panicum vigatum 8.06±0.36g 7.83±0.47d 74.35±0.32bc 66.85±0.35c 41.58±1.42a 16.70±2.19b 5.06±0.18f 4.11±0.44cde 08-Panicum laxum 13.74±0.24d 13.68±0.08ab 72.66±1.15c 64.25±0.37c 13.18±1.52d 6.83±0.52d 11.82±0.80b 5.99±0.41c-f 09-Panicum maximum 19.22±0.93b 14.78±0.28a 77.38±0.55ab 72.72±0.32a 5.55±0.76fg 3.60±0.99e 12.94±0.31b 8.72±0.38c-g 10-Hamelia patens 13.03±0.10de 12.62±0.13bc 80.15±1.76a 70.02±0.57b 3.91±0.87g 2.75±0.53e 16.02±0.46a 11.97±0.20c-g 11-Viola odorata (Banafsha)P 45.56±0.46gh 7.65±0.59d-g 12-Viola Hybrida P 48.20±0.88fg 6.92±0.78d-g 13-Viola tricolor P 50.09±1.25f 8.93±0.87e-h 14-Clitoria ternatea P 38.63±0.53j 8.07±0.90e-i 15-Ptunia mix P 31.07±0.75k 8.28±0.86e-i 16-Pansy F1 P 42.51±0.61i 7.76±0.93f-j 17-Panicum vigatum P 38.50±0.60j 5.33±0.94g-j 18-Panicum laxum P 43.65±1.28hi 5.70±0.34hij 19-Panicum maximum P 65.18±1.22c 9.42±0.62ij 20-Hamelia patens P 57.26±0.74e 10.94±0.31jk 21-Pure cyclotide MCOT-I 45.58±0.37e 44.96±0.11hi 8.38±0.11de 8.52±0.30jk 22-Pure cyclotide MCOT-I P 24.21±1.79l 8.81±0.64k Mean 13.72±0.81 12.19±0.51 70.43±1.70 54.90±1.80 14.40±2.20 9.16±1.30 10.81±0.60 8.01±.25 t-value (PBS vs PEB) 1.60NS 6.34** 2.08* 4.30**

Mean (untreated) - - - 67.77±0.73 - - - 7.98±0.42 Mean (treated with PK) - - - 46.06±1.70 - - - 7.90±0.35 t-value - - - 11.47** - - - 0.15NS

NS = Non-significant (P>0.05); * = Significant (P<0.05); ** = Highly significant (P<0.01)

Means sharing similar letter in a column are statistically non-significant (P>0.05)

203

Appendix-II

Antibacterial and biofilm formation inhibition activity of different plants extracts prepared in Protein extraction buffer PEB (with and without pretreatment of Proteinase K enzyme), Phosphate buffer saline PBS and purified Cyclotide MCOTI-I protein (with and without pretreatment of Proteinase K enzyme) against Gram positive (Staphylococcus aureus and Bacillus subtilis strains) and gram negative (Escherichia coli and Pasturella multocida strains).

%age Biofilm Antibacterial Antibacterial Antibacterial Antibacterial Sr.N Plant names activity(B.S) activity(S.A) activity(E.C) activity(P.M) formation o. Mean ± S.D Mean ± S.D Mean ± S.D Mean ± S.D inhibition

Different buffers PBS PEB PBS PEB PBS PEB PBS PEB PEB

Viola 15.0++ 12.33++ 13.33++ 13.67++ 9.33+ 10.67+ 6.33+ 1 8.67+ ±1.5 31.44 ±1.3 odorata ±2.0 ±0.5 ±1.5 ±3.8 ±2.5 ±1.2 ±1.2 Viola 3.33+ 1.33- 2 6.66+ ±2.0 5.0+ ±3.0 5.67+ ±0.5 1.67- ±1.5 5.67+ ±4.0 3.0+ ±2.6 28.12±0.5 odorata PK ±1.5 ±0.6 22.66+++ 23.0+++ 16.33++ 15.0++ 15.0++ 17.33++ 16.67++ 17.33++±2. 3 Viola Hybrida 54.41±1.5 ±2.5 ±3.6 ±2.0 ±3.0 ±3.0 ±2.5 ±2.5 1 Viola Hybrida 5.33+ 12.33++ 3.67+ 7.67+ 3.33+ 4 4.0+ ±1.0 5.33+ ±3.5 4.67+ ±3.1 36.40±1.5 PK ±2.0 ±0.5 ±1.5 ±0+.6 ±1.5 20.0++ 19.0++ 14.67++ 15.66++ 14.67++ 7.33+ 5 Viola tricolor 1.33- ±1.2 5.33+ ±2.1 43.44±0.7 ±2.0 ±3.0 ±1.5 ±0.6 ±2.3 ±2.1 Viola tricolor 2.33+ 6 6.66+ ±2.0 3.33+ ±1.5 5.66+ ±0.5 1.0- ±1.0 0.0- ±0.0 1.0- ±1.0 2.33+ ±1.5 26.28±0.5 PK ±0.6 Clitoria 27.66+++ 31.33++++ 26.33+++ 27.66+++±0 18.0++ 26.33+++ 25.0+++ 26.67+++±2 7 72.97±1.3 ternatea ±3.0 ±2.5 ±1.5 .6 ±3.6 ±2.5 ±2.0 .5 Clitoria 16.0++ 4.33+ 11.33+ 10.67+ 5.33+ 10.67+ 8 7.66+ ±1.5 6.0+ ±3.6 61.96±1.5 ternatea PK ±3.0 ±3.5 ±3.2 ±2.5 ±2.1 ±3.2 4.0+ 5.33+ 12.33++ 13.33++ 9 Ptunia mix 1.67- ±0.5 3.66+ ±0.6 9.33+ ±3.2 9.0+ ±1.0 17.55±0.5 ±2.6 ±1.5 ±3.1 ±2.1 Ptunia mix 1.67- 9.61+ 10 0.33- ±0.5 0.66 - ±0.5 2.0+ ±1.0 1.33- ±0.6 8.33+ ±3.1 4.0+ ±2.0 15.13±0.9 PK ±1.2 ±4.0 10.33++ 5.33+ 1.33- 6.33+ 11 Pansy F1 2.0+ ±1.0 3.0+ ±1.0 4.33+ ±1.5 1.0- ±1.0 10.84±2.1 ±1.5 ±1.2 ±1.5 ±2.1 2.33+ 1.67- 12 Pansy F1 PK 2.33+ ±2.5 0.33- ±0.5 1.67- ±1.1 1.0- ±1.0 1.0- ±1.0 1.00- ±1.7 12.27±0.5 ±1.5 ±1.5 Panicum 0.67- 0.67- 13 2.66+ ±1.5 1.33- ±0.5 2.33+ ±0.5 1.0- ±1.0 0.0- ±0.0 0.0- ±0.0 15.47±2.0 vigatum ±1.0 ±0.6 Panicum 2.66+ 1.33- 1.67- 14 0.33- ±0.5 0.66- ±0.5 0.33- ±0.6 1.67- ±1.5 2.33+ ±2.5 14.56±0.5 vigatum PK ±2.0 ±1.2 ±1.5 Panicum 0.0- ± 15 0.66- ±0.5 1.33- ±0.5 0.66- ±1.2 0.00- ±0.0 0.0-±0.0 0.0- ±0.0 0.0- ±0.0 9.26±0.4 laxum 0.0 Panicum 0.67- 1.67- 16 1.0- ±1.0 1.33- ±1.5 0.33- ±0.5 0.33- ±0.6 0.67- ±1.2 1.0- ±1.7 13.73±1.3 laxum PK ±1.2 ±1.5 Panicum 4.33+ 17 1.66- ±0.5 1.0- ±1.0 2.0+ ±0.0 1.00- ±1.0 1.0- ±1.0 0.67- ±0.6 1.0- ±1.0 13.80±0.9 maximum ±0.5 Panicum 0.67- 18 0.33- ±0.5 0.33- ±0.5 0.33-±0.5 0.34- ±0.6 0.67- ±1.2 2.67+ ±1.5 1.0- ±1.0 11.56 ±0.5 maximum PK ±1.2 Hamelia 13.67++ 4.67+ 4.33+ 19 5.33+ ±2.0 4.0+ ±1.0 9.67+ ±2.1 1.67- ±0.6 3.00+ ±1.7 28.52±0.9 patens ±2.5 ±1.5 ±0.6 Hamelia 1.33- 20 1.00- ±1.0 2.0+ ±1.1 0.33- ±0.5 0.33- ±0.6 1.33- ±1.5 1.0- ±1.0 1.33- ±1.5 27.43±0.3 patens PK ±1.5

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cyclotide 37.02++++ 37.33++++ 34.0++++ 33.66++++ 25.67+++ 27.67+++ 39.33++++ 37.0++++ 21 84.21±1.5 MCOTI-I ±1.7 ±0.5 ±3.6 ±1.5 ±2.5 ±1.5 ±2.5 ±1.7 cyclotide 13.66++ 11.34+ 12.67+ 13.33++ 8.33+ 22 11.0+ ±5.2 9.0+ ±2.6 10.33±4.0 57.44±2.0 MCOTI-I PK ±6.6 ±6.1 ±6.4 ±4.0 ±2.1 29.33+++ 30.33+++ 25.33+++ 24.34+++±1 27.0+++ 26.67+++ 31.0++++ 23 Rifampicin 30.67±1.2 80.81± 2.0 ±2.0 ±1.3 ±2.0 .5 ±2.6 ±1.5 ±1.0 S.A = Staphylococcus aureus, B.S = Bacillus subtilis, E.C = Escherichia coli, P.M = Pasturella multocida, PBS = Phosphate buffer saline, PEB = Protein extraction buffer, S.D = standard deviation.

205

Statistical analysis of antibacterial and biofilm formation inhibition activity of different plants extracts prepared in Protein extraction buffer PEB (with and without pretreatment of Proteinase K enzyme), Phosphate buffer saline PBS and purified Cyclotide MCOTI-I protein (with and without pretreatment of Proteinase K enzyme) against Gram positive (Staphylococcus aureus and Bacillus subtilis strains) and gram negative (Escherichia coli and Pasturella multocida strains) Plant SA (PBS) SA ( PEB) BS (PBS) BS (PEB) EC (PBS) EC (PEB) PM(PBS) PM (PEB) Biofilm BS (PEB) 01-Viola odorata 13.33±0.88de 8.67±0.88e 15.00±1.15d 12.33±0.33e 13.67±2.19cd 9.33±1.45d 10.67±0.67e 6.33±0.67fg 31.44±1.63g (Banafsha) 02-Viola Hybrida 16.33±1.20c 15.00±1.73d 22.67±1.45c 23.00±2.08c 15.00±1.73bc 17.33±1.45b 16.67±1.45d 17.33±1.20d 54.41±2.65d 03-Viola tricolor 14.67±0.88cd 15.67±0.33d 20.00±1.15c 19.00±1.73d 1.33±0.67jk 14.67±1.33bc 5.33±1.20fg 7.33±1.20f 43.44±1.08e 04-Clitoria ternatea 26.33±0.88b 27.67±0.33b 27.67±1.76b 31.33±1.45b 18.00±2.08b 26.33±1.45a 25.00±1.15c 26.67±1.45c 72.97±2.22b 05-Ptunia mix 1.67±0.33ghi 3.67±0.33ghi 4.00±1.53efg 5.33±0.88f 9.33±1.86efg 12.33±1.76cd 9.00±0.58e 13.33±1.20e 17.55±1.17i 06-Pansy F1 3.00±0.58fgh 4.33±0.88gh 2.00±0.58fg 10.33±0.88e 1.00±0.58jk 5.33±0.67e 1.33±0.88h 6.33±1.20fg 10.84±1.50jk 07-Panicum vigatum 2.33±0.33ghi 1.00±0.58j 2.67±0.88fg 1.33±0.33ghi 0.00±0.00k 0.67±0.33gh 0.00±0.00h 0.67±0.33kl 15.47±1.07ij 08-Panicum laxum 0.00±0.00i 0.67±0.67j 0.67±0.33g 1.33±0.33ghi 0.00±0.00k 0.00±0.00h 0.00±0.00h 0.00±0.00l 9.26±0.55k 09-Panicum maximum 1.00±0.58hi 2.00±0.00hij 1.67±0.33fg 4.33±0.33fg 1.00±0.58jk 1.00±0.58gh 0.67±0.33h 1.00±0.58kl 13.80±0.88ijk 10-Hamelia patens 4.00±0.58fg 9.67±1.20e 5.33±1.20ef 13.67±1.45e 1.67±0.33ijk 4.67±0.88ef 3.00±1.00gh 4.33±0.33ghi 28.52±0.57gh 11-Viola odorata 5.67±0.33f 1.67±0.88ij 6.67±1.20e 5.00±1.73f 5.67±2.33ghi 3.33±0.88efg 3.00±1.53gh 1.33±0.33jkl 28.12±1.42gh (Banafsha)P 12-Viola Hybrida P 12.33±0.33de 5.33±2.03fg 5.33±1.20ef 4.00±0.58fgh 4.67±1.76hij 3.67±0.88efg 7.67±0.33ef 3.33±0.88h-k 36.40±2.70f 13-Viola tricolor P 5.67±0.33f 1.00±0.58j 6.67±1.20e 3.33±0.88fghi 0.00±0.00k 1.00±0.58gh 2.33±0.88gh 2.33±0.33i-l 26.28±0.97h 14-Clitoria ternatea P 11.33±1.86e 7.67±0.88ef 16.00±1.73d 4.33±2.03fg 10.67±1.45def 5.33±1.20e 10.67±1.86e 6.00±2.08fgh 61.96±2.27c 15-Ptunia mix P 2.00±0.58ghi 1.33±0.33ij 16.00±1.73d 0.67±0.33hi 8.33±1.76fgh 1.67±0.67fgh 9.67±2.33e 4.00±1.15g-j 15.13±0.83ij 16-Pansy F1 P 1.67±0.67ghi 1.00±0.58j 0.33±0.33g 0.33±0.33i 1.00±0.58jk 2.33±0.88efgh 1.00±1.00h 1.67±0.88i-l 12.27±1.03jk 17-Panicum vigatum P 0.67±0.33hi 0.33±0.33j 2.33±1.45fg 2.67±1.20fghi 1.67±0.88ijk 1.33±0.67gh 2.33±1.45gh 1.67±0.88i-l 14.56±0.45ij 18-Panicum laxum P 0.33±0.33hi 0.33±0.33j 0.33±0.33g 1.33±0.88ghi 0.67±0.67jk 0.67±0.67gh 1.00±1.00h 1.67±0.88i-l 13.73±1.20ijk 19-Panicum maximum P 0.33±0.33hi 0.33±0.33j 1.00±0.58g 0.33±0.33i 0.67±0.67jk 0.67±0.67gh 2.67±0.88gh 1.00±0.58kl 11.56±0.65jk 20-Hamelia patens P 0.33±0.33hi 0.33±0.33j 0.33±0.33g 0.67±0.67hi 1.33±0.88jk 1.00±0.58gh 1.33±0.88h 1.33±0.88jkl 27.43±0.60gh 21-Pure cyclotide MCOT-I 34.00±2.08a 33.67±0.88a 37.00±1.00a 37.33±0.33a 25.67±1.45a 27.67±0.88a 39.33±1.45a 37.00±1.00a 84.21±3.09a 22-Pure cyclotide MCOT-I 11.00±3.06e 9.00±1.53e 13.67±3.84d 11.33±3.53e 12.67±3.71cde 13.33±2.33c 10.33±2.33e 8.33±1.20f 57.44±3.65cd P 23-Rifampicin 25.33±1.20b 24.33±0.88c 29.33±1.20b 30.33±0.88b 27.00±1.53a 26.67±0.88a 30.67±0.67b 31.00±0.58b 80.81±1.89a Mean 8.41±1.20 7.59±1.10 10.30±1.30 9.70±1.30 7.00±1.00 7.84±1.10 8.40±1.30 8.00±1.20 t-value (PBS vs PEB) 0.50NS 0.30NS 0.57NS 0.24NS

Mean (untreated) 8.27±1.60 8.83±1.50 10.17±1.80 12.20±1.80 6.10±1.30 9.17±1.50 7.17±1.50 8.33±1.50 29.80±3.80 Mean (treated with PK) 4.03±0.83 1.93±0.49 5.50±1.10 2.27±0.42 3.47±0.73 2.10±0.34 4.17±0.74 2.43±0.38 24.70±2.80 t-value 2.39* 4.30** 2.19* 5.46** 1.76NS 4.49** 1.81NS 3.77** 1.08NS NS = Non-significant (P>0.05); * = Significant (P<0.05); ** = Highly significant (P<0.01) Means sharing similar letter in a column are statistically non-significant (P>0.05)

206

Appendix-III.

Hemolytic activity (Percentage) of selected plants prepared in PBS and protein extraction buffer with and without treatment of proteinase K enzyme Percent hemolytic activity using Protein Percent hemolytic activity using extraction buffer (without treatment of Phosphate buffer saline(PBS) Sr. No. Sample Names Proteinase K enzyme)

Mean ± S.D Mean ± S.D 1 Viola odorata 16.22±3.20 8.03±2.54 2 Viola odorata PK *** 5.98±1.54 3 Viola Hybrida 23.10±3.91 15.34±1.71 4 Viola Hybrida PK *** 10.89±2.32 5 Viola tricolor 22.03±3.26 22.89±0.58 6 Viola tricolor PK *** 16.45±1.32 7 Clitoria ternatea 23.84±5.50 21.54±1.57 Clitoria ternatea 8 *** 13.22±0.84 PK 9 Ptunia mix 48.50±8.05 30.21±2.94 10 Ptunia mix PK *** 19.76±2.54 11 Pansy F1 20.38±2.14 17.42±1.60 12 Pansy F1 PK *** 9.65±1.60 13 Panicum vigatum 44.16±5.54 43.34±1.62 Panicum vigatum 14 *** 28.37±1.94 PK 15 Panicum laxum 25.67±3.63 20.54±3.65 Panicum laxum 16 *** 25.9±1.45 PK Panicum 17 20.72±2.01 19.08±1.74 maximum Panicum 18 *** 12.59±2.43 maximum PK 19 Hamelia patens 24.97±4.72 10.32±1.27 Hamelia patens 20 *** 6.45±2.31 PK cyclotide 21 50.42±1.79 *** MCOTI-I cyclotide 22 32.15±1.34 *** MCOTI-I PK Positive control 23 96.58±1.36 96.45±0.65 Triton-X100 Negative control 24 1.33±0.28 1.39±0.22 PBS *** = activity not performed

207

Statistical analysis of Hemolytic activity (Percentage) of selected plants prepared in PBS and PEB with and without treatment of proteinase K enzyme Plant Hemolysis (PBS) Hemolysis (PEB) 01-Viola odorata (Banafsha) 16.22±1.85d 9.65±1.47k 02-Viola Hybrida 23.10±2.26c 17.99±1.09f-j 03-Viola tricolor 20.04±1.89cd 23.10±0.34fg 04-Clitoria ternatea 23.84±3.18c 20.32±0.91f-i 05-Ptunia mix 48.51±4.65b 33.47±1.70e 06-Pansy F1 20.39±1.24cd 15.87±0.92h-k 07-Panicum vigatum 44.16±3.20b 41.52±0.93cd 08-Panicum laxum 25.67±2.10c 24.63±2.11f 09-Panicum maximum 20.73±1.16cd 19.07±1.00f-i 10-Hamelia patens 24.98±2.73c 11.78±0.73jk 11-Viola odorata (Banafsha)P - 11.32±2.50jk 12-Viola Hybrida P - 20.32±1.43f-i 13-Viola tricolor P - 21.29±0.84f-i 14-Clitoria ternatea P - 22.40±0.72fgh 15-Ptunia mix P -- 36.20±6.15de 16-Pansy F1 P - 15.82±3.20h-k 17-Panicum vigatum P - 42.32±1.45cd 18-Panicum laxum P - 24.04±1.75fg 19-Panicum maximum P - 17.47±2.48g-j 20-Hamelia patens P - 15.17±4.91ijk 21-Pure cyclotide MCOT-I 50.42±1.04b 50.42±1.04b 22-Pure cyclotide MCOT-I P - 43.85±5.86bc 23-Rifampicin - - 24-PBS 1.33±0.16e 1.34±0.12l 25-TritonX100 96.58±0.79a 96.46±0.38a Mean 32.0±3.70 26.50±2.30 t-value (PBS vs PEB) 1.26NS

Mean (untreated) - 21.74±1.70 Mean (treated with PK) - 22.60±1.90 t-value - 0.35NS

NS = Non-significant (P>0.05)

Means sharing similar letter in a column are statistically non-significant (P>0.05)

208

Appendix-IV

Percentage mutagenicity by Ames test of selected plant extracts prepared in PBS and protein extraction buffer (PEB) with and without treatment of proteinase K enzyme

Sr. %age mutagenicity (TA98) %age mutagenicity (TA100) Plant names No. Mean ± S.D Mean ± S.D No. No. No. No.o %age %age No. No. No. No. %age %age of of of f +ve mutageni mutageni of of of of mutageni mutageni +ve +ve +ve wells city PBS city PEB +ve +ve +ve +ve city PBS city PEB Different buffers well wells well in Mean Mean wells wells wells wells Mean Mean s(K2 (K2C s in samp ±S.D ±S.D (Sod- (Sod- in in ±S.D ±S.D Cr2 r2O7 ) sam les Azid Azid samp samp O7 ) PEB ples PEB e) e) les les PBS PBS PBS PEB PBS PEB 22.97NM Viola 24.36SM± 15.71NM 17.14NM 1 76 70 19 11 74 70 17 12 ±4.9 odorata 1.5 ±1.4 ±2.0

Viola 19.23NM 12.86NM 20.27NM 12.86NM 2 76 70 15 9 74 70 15 9 odorata PK ±1.3 ±2.5 ±4.8 ±2.5 Viola 15.38NM 11.43NM 13.51NM 10.00NM 3 76 70 12 8 74 70 10 7 Hybrida ±2.6 ±1.8 ±3.0 ±1.2 Viola 12.82NM 10.00NM 17.57NM 8.57NM 4 76 70 10 7 74 70 13 6 Hybrida PK ±1.6 ±1.6 ±3.1 ±2.3 Viola 17.95NM 14.29NM 20.27NM 15.71NM 5 76 70 14 10 74 70 15 11 tricolor ±0.9 ±3.2 ±1.8 ±3.1 Viola 10.26NM 10.00NM 12.16NM 10.00NM 6 76 70 8 7 74 70 9 7 tricolor PK ±2.0 ±0.9 ±2.3 ±0.5 Clitoria 28.21M± 28.57M± 31.08M 27.14SM 7 76 70 22 20 74 70 23 19 ternatea 2.0 1.8 ±3.4 ±0.6 Clitoria 24.36SM± 25.71SM± 25.68SM 24.29 SM 8 76 70 19 18 74 70 19 17 ternatea PK 2.0 1.4 ±2.0 ± 1.3 19.23NM 19.71NM 21.62NM 17.14NM 9 Ptunia mix 76 70 18 18 74 70 16 12 ±0.5 ±2.6 ±3.2 ±1.8 Ptunia mix 24.36SM± 17.14NM 21.62NM 15.71NM 10 76 70 19 12 74 70 16 11 PK 3.6 ±1.2 ±3.2 ±1.6 21.79SM± 20.00NM 25.68 SM 12.86NM 11 Pansy F1 76 70 17 14 74 70 19 9 0.9 ±4.1 ±2.0 ±2.6 Pansy F1 25.64SM± 15.71NM 20.27NM 10.00NM 12 76 70 20 11 74 70 15 7 PK 1.5 ±3.3 ±4.5 ±2.6 Panicum 24.36SM± 14.29NM 25.68SM 15.71NM 13 76 70 19 10 74 70 19 11 vigatum 3.8 ±4.6 ±3.3 ±1.3 Panicum 12.82NM 17.14NM 20.27NM 12.86NM 14 76 70 10 12 74 70 15 9 vigatum PK ±6.9 ±2.5 ±1.5 ±2.6 Panicum 15.38NM 18.57NM 12.16NM 12.86NM 15 76 70 12 13 74 70 9 9 laxum ±2.4 ±1.2 ±3.1 ±1.2 Panicum 12.82NM 21.43SM± 17.57NM 8.57NM 16 76 70 10 15 74 70 13 6 laxum PK ±1.6 3.4 ±2.1 ±1.6 Panicum 7.69NM 8.57NM 10.81NM 7.14NM 17 76 70 6 6 74 70 8 5 maximum ±1.5 ±0.9 ±0.6 ±2.0 Panicum 6.41NM 7.14NM 6.76NM 4.29NM 18 76 70 5 5 74 70 5 3 maximum PK ±2.3 ±2.4 ±1.4 ±3.0 Hamelia 12.82NM 10.00NM 10.81NM 12.86NM 19 76 70 10 7 74 70 8 9 patens ±0.6 ±3.8 ±3.9 ±2.8 Hamelia 11.54NM 12.86NM 13.51NM 10.00NM 20 76 70 9 9 74 70 10 7 patens PK ±3.5 ±3.1 ±0.5 ±2.1

209

cyclotide 19.23NM 22.86SM± 21.62NM 20.00NM 21 76 70 15 16 74 70 16 14 MCOTI-I ±1.6 1.3 ±1.2 ±2.5 cyclotide 17.95NM 18.57NM 18.92NM 20.00NM 22 76 70 14 13 74 70 14 14 MCOTI-I PK ±3.7 ±3.1 ±1.9 ±1.5 Background 23 plate for TA *** *** *** *** *** *** 74 *** 9 *** 12.16 *** 100(PBS) Background 24 plate for TA *** *** *** *** *** *** *** 70 *** 8 *** 11.42 100(PEB) Background 8 25 plate for TA 76 *** *** 10.52 *** *** *** *** *** *** ***

98(PBS) Background 7 26 plate for TA *** 70 *** *** 10.00 *** *** *** *** *** ***

98(PEB)

PBS = Phosphate buffer saline, PEB = Protein extraction buffer, S.D = standard deviation. *** = Test not performed, M=Mutagenic, SM= slight mutagenic, NM=non mutagenic, PK=proteinase K enzyme

210

Statistical analysis of Percentage mutagenicity by Ames test of selected plant extracts prepared in PBS and protein extraction buffer (PEB) with and without treatment of proteinase K enzyme.

Plant AMES TA100 AMES TA100 AMES TA98 (PBS) AMES TA98 (PEB) (PBS) (PEB) 01-Viola odorata (Banafsha) 26.30±2.83c 17.11±1.17efg 22.69±0.89cde 16.10±0.81ef 02-Viola Hybrida 13.62±1.73hi 11.36±0.70lm 15.71±1.48ghi 12.69±1.02fgh 03-Viola tricolor 20.39±1.03def 12.85±1.78h-m 18.79±0.52efg 17.60±1.83e 04-Clitoria ternatea 31.27±1.94b 26.55±0.32b 29.26±1.16b 29.28±1.04b 05-Ptunia mix 21.76±1.83cde 18.51±1.06ef 19.21±0.31efg 23.39±1.50cd 06-Pansy F1 24.91±1.14cd 14.74±1.48g-k 22.71±0.55cde 24.44±2.34c 07-Panicum vigatum 25.84±1.88c 16.14±0.78fgh 24.00±2.21cd 19.58±2.65de 08-Panicum laxum 14.52±1.80ghi 13.30±0.72h-l 13.08±1.41ijk 17.55±0.71e 09-Panicum maximum 11.31±0.36ij 6.59±1.13o 9.19±0.86kl 9.27±0.52hi 10-Hamelia patens 13.62±2.24hi 11.44±1.61klm 12.66±0.34ijk 9.77±2.18hi 11-Viola odorata (Banafsha)P 25.81±2.79c 15.62±1.46f-j 18.77±0.73efg 15.64±1.44efg 12-Viola Hybrida P 16.78±1.81fgh 10.92±1.35lm 13.98±0.92hij 11.73±0.94ghi 13-Viola tricolor P 11.79±1.33ij 10.42±0.28lmn 10.92±1.15jkl 10.74±0.53hi 14-Clitoria ternatea P 24.91±1.14cd 24.62±0.74bc 26.66±1.16bc 26.34±0.81bc 15-Ptunia mix P 20.85±1.85def 16.08±0.91f-i 20.48±2.06def 18.55±0.71e 16-Pansy F1 P 24.92±2.58cd 12.77±1.48i-m 24.00±0.87cd 19.07±1.93e 17-Panicum vigatum P 19.01±0.88efg 12.39±1.50j-m 18.82±3.97efg 19.53±1.42de 18-Panicum laxum P 15.39±1.23ghi 9.97±0.94mn 13.98±0.92hij 18.51±1.99e 19-Panicum maximum P 7.68±0.83j 7.14±1.74no 7.87±1.33l 7.82±1.36i 20-Hamelia patens P 13.11±0.31hi 11.33±1.19lm 12.25±1.99ijk 9.22±1.82hi 21-Pure cyclotide MCOT-I 22.61±0.71cde 22.31±1.46cd 17.45±0.95fgh 23.92±0.75c 22-Pure cyclotide MCOT-I P 20.81±1.08def 20.41±0.86de 13.93±2.14hij 19.05±1.81e 23-Rifampicin - - - - 24-PBS - - - - 25-TritonX100 - - - - 26-Pot. DI Chrom TA 98(PBS) 100.00±0.00a - - - 27-Pot. DI Chrom TA 98(PEB) - 100.00±0.00a - - 28-Sod. Azid TA100(PBS) - - 100.00±0.00a - 29-Sod. Azid TA100(PEB) - - - 100.00±0.00a 30-Negative TA100(PBS) 12.23±1.60hij - - - 31-Negative TA100(PEB) - 10.86±1.05lm - - 32-Negative TA98(PBS) - - 10.92±1.15jkl - 33-Negative TA98(PBS) - - - 10.23±1.67hi 34-Streptikinase - - - - Mean 22.5±2.10 18.10±2.10 20.70±2.10 21.50±2.30 t-value (PBS vs PEB) 1.49NS 0.25NS

Mean (untreated) 20.35±1.30 14.86±0.99 18.73±1.10 17.97±1.20 Mean (treated with PK) 18.03±1.20 13.13±0.91 16.77±1.20 15.72±.10 t-value 1.34NS 1.29NS 1.22NS 1.38NS

NS = Non-significant (P>0.05); * = Significant (P<0.05); ** = Highly significant (P<0.01)

Means sharing similar letter in a column are statistically non-significant (P>0.05)

211

Appendix-V

Percentage thrombolytic activity of selected plants and MCOTI-I prepared in protein extraction buffer with and without treatment of proteinase K enzyme.

Percentage(%age) Thrombolytic activity Serial Number. Plant Names Mean ± S.D

1 Viola odorata 55.54±4.28 2 Viola odorata PK 35.20±1.43 3 Viola Hybrida 61.60±1.46 4 Viola Hybrida PK 12.24±1.32 5 Viola tricolor 64.12±3.66 6 Viola tricolor PK 40.76±1.45 7 Clitoria ternatea 52.25±2.00 8 Clitoria ternatea PK 42.14±1.89 9 Ptunia mix 61.64±0.63 10 Ptunia mix PK 55.33±4.64 11 Pansy F1 56.81±1.41 12 Pansy F1 PK 33.07±3.42 13 Panicum vigatum 52.88±0.69 14 Panicum vigatum PK 38.23±1.00 15 Panicum laxum 48.22±0.28 16 Panicum laxum PK 41.50±2.32 17 Panicum maximum 42.78±2.42 18 Panicum maximum PK 26.13±1.54 19 Hamelia patens 53.33±0.74 20 Hamelia patens PK 41.82±3.74 21 cyclotide MCOTI-I 50.86±2.49 22 cyclotide MCOTI-I PK 17.46±3.54 23 Streptokinase enzyme as positive control 78.64±1.01 24 Phosphate buffer saline(PBS) as negative control 1.89±0.34

212

Statistical analysis of percentage thrombolytic activity of selected plants and MCOTI-I prepared in protein extraction buffer with and without treatment of proteinase K enzyme Plant Thrombolysis (PEB) 01-Viola odorata (Banafsha) 55.54±2.47cd 02-Viola Hybrida 61.60±0.84b 03-Viola tricolor 64.12±2.11b 04-Clitoria ternatea 52.25±1.16de 05-Ptunia mix 61.64±0.36b 06-Pansy F1 56.81±0.82c 07-Panicum vigatum 52.88±0.40cde 08-Panicum laxum 48.22±0.16f 09-Panicum maximum 62.78±1.40b 10-Hamelia patens 53.33±0.43cde 11-Viola odorata (Banafsha)P 35.20±0.83hi 12-Viola Hybrida P 12.24±0.76l 13-Viola tricolor P 40.76±0.83g 14-Clitoria ternatea P 42.14±1.09g 15-Ptunia mix P 55.33±2.68cd 16-Pansy F1 P 33.07±1.97i 17-Panicum vigatum P 38.23±0.58gh 18-Panicum laxum P 41.50±1.34g 19-Panicum maximum P 26.13±0.89j 20-Hamelia patens P 41.82±2.16g 21-Pure cyclotide MCOT-I 50.86±1.44ef 22-Pure cyclotide MCOT-I P 17.46±2.05k 23-Rifampicin - 24-PBS - 25-TritonX100 - 26-Pot. DI Chrom TA 98(PBS) - 27-Pot. DI Chrom TA 98(PEB) - 28-Sod. Azid TA100(PBS) - 29-Sod. Azid TA100(PEB) - 30-Negative TA100(PBS) - 31-Negative TA100(PEB) - 32-Negative TA98(PBS) - 33-Negative TA98(PBS) - 34-Streptikinase 78.65±0.57a Mean (untreated) 56.92±1.00 Mean (treated with PK) 36.60±2.00 t-value 8.90** NS = Non-significant (P>0.05); * = Significant (P<0.05); ** = Highly significant (P<0.01) Means sharing similar letter in a column are statistically non-significant (P>0.05)

213

Appendix-VI The characters predicted through different bioinformatics tools of Cliotide sequence

Character Results Details of observations Tools used studied 1)Hydrophobi P C A E S C V WI P C T V T A L L GC S C KD KRedV C Y L N =IVL (strong), (http://www.justbio.com/in c pattern (only cyclotide domain) yellow=FCMA (high), dex.php?page=seqpainter) green=GTSWYP(medium) blue=HNQDEKR(Low) (for complete CDS protein)

2)Molecular 15113.85 (for complete CDS protein) ExPasy/protparam weight 3120.78 (only cyclotide domain) 3)Theoratical 8.31 (for complete CDS protein) ExPasy/protparam pI

6.3 (only cyclotide domain) 4)Molar A280 19060 and 1.261 Extinction data was observed for ExPasy/protparam & A280/cm (1 (for complete CDS protein) assuming the protein in the 6M Guanidine mg/ml) all cys are reduced and if we assume HCl / 20mM at pH 6.5 alloxidised (half) then; 20260 & 1.340 6970 & 2.233 (only cyclotide domain)Cys (reduced) 7690 & 2.464cys (oxidised) 5)Protein 1) MYRISTYL, PATTERN 1) G-{EDRKHPFYW}-x(2)- PROSITE™ database patterns 2) CK2_PHOSPHO_SITE, [STAGCN]-{P} http://www.justbio.com/ind PATTERN. ex.php?page=patsearch 3) PKC_PHOSPHO_SITE, 2) [ST]-x(2)-[DE] PATTERN. 3) [ST]-x-[RK] 4) PROKAR_LIPOPROTEIN, 4) {DERK}(6)- RULE. [LIVMFWSTAG](2)- 5) CAMP_PHOSPHO_SITE, [LIVMFYSTAGCQ]- PATTERN. [AGS]-C 5) [RK](2)-x-[ST] (Within whole CDS) 6) Asp + Glu 13 & 16 negatively and positively charged http://web.expasy.org/cgi- & Arg + Lys residues bin/protparam/protparam

7)Formula C679H1060N176O188S13 For 135 a.a. CDS ttp://web.expasy.org/cgi- and atoms & 2116 bin/protparam/protparam 8)Estimated  30 hours  mammalian reticulocytes, ttp://web.expasy.org/cgi- half-life  >20 hours in vitro bin/protparam/protparam  >10 hours  yeast, in vivo  Escherichia coli, in vivo 9)Instability  The instability index (II) =27.16 This classifies the protein as http://web.expasy.org/cgi- index  Aliphatic index= 87.41 stable. bin/protparam/protparam  Grand average of hydropathicity (GRAVY)= 0.118

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Appendix-VII a) Composition of amino acids with number and molecular percentage (135 amino acids) for complete CDS vs cyclotide domain only (29 residues) for Cliotide A 12 vs 2 8.89% vs 6.9 C 10 vs 6 7.41% vs 20.60 D 6 vs 1 4.44% vs 3.45 E 7 vs 1 5.19% vs 3.45 F 6 vs 0 4.44% vs 0 G 7 vs 1 5.19% vs 3.45 H 6 vs 0 4.44% vs 0 I 7 vs 1 5.19% vs 3.45 K 13 vs 2 9.63% vs 6.9 L 12 vs 3 8.89% vs 10.34 M 3 vs 0 2.22% vs 0 N 3 vs 1 2.22% vs 3.45 P 4 vs 2 2.96% vs 6.9 Q 2 vs 0 1.48% vs 0 R 3 vs 0 2.22% vs 0 S 8 vs 2 5.93% vs 6.9 T 7 vs 2 5.19% vs 6.9 V 11 vs 3 8.15% vs 10.34 W 2 vs 1 1.48% vs 3.45 Y 6 vs 1 4.44% vs 3.45 b)Protein Stats giving the number of occurrences of certain groups of residues with number and %age of occurrence for cyclotide domain only (www.sequence manipulation suite/proteinstat) Aliphatic G,A,V,L,I 12 38.71% Aromatic F,W,Y 2 6.45% Sulphur C,M 6 19.35% Basic K,R,H 2 6.45% Acidic B,D,E,N,Q,Z 3 9.68% Aliphatic hydroxyl S,T 4 12.90% tRNA synthetase class I 17 54.84% Z,E,Q,R,C,M,V,I,L,Y,W tRNA synthetase class II 14 45.16% B,G,A,P,S,T,H,D,N,K,F

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