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EXPRESSION, PURIFICATION and CRYSTALLIZATION TRIALS of SMALL RUBBER PARTICLE PROTEIN (SRPP) from Hevea Brasiliensis

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EXPRESSION, PURIFICATION and CRYSTALLIZATION TRIALS of SMALL RUBBER PARTICLE PROTEIN (SRPP) from Hevea Brasiliensis View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Repository@USM EXPRESSION, PURIFICATION AND CRYSTALLIZATION TRIALS OF SMALL RUBBER PARTICLE PROTEIN (SRPP) FROM Hevea brasiliensis SARANPAL SINGH A/L SATINDER SINGH UNIVERSITI SAINS MALAYSIA EXPRESSION, PURIFICATION AND CRYSTALLIZATION TRIALS OF SMALL RUBBER PARTICLE PROTEIN (SRPP) FROM Hevea brasiliensis by SARANPAL SINGH A/L SATINDER SINGH Thesis submitted in fulfillment of requirements for the degree of Master of Science February ACKNOWLEDGEMENT I owe my highest gratitude to Professor Dr. K. Sudesh Kumar and Dr. Teh Aik Hong for being my true mentors and for always being available when needed. My gratitude goes to them for their thoughtful insights, motivation, patience, professional rigour, and intellectual contributions. I could not have imagined having better advisors and mentors during the pursuit of my master’s degree. Their meticulous reading and critical comments on my drafts gave me the kind of feedback that always revitalized, encouraged, and propelled me forward with enthusiasm. It is an honor to have work with them. I am indebted and grateful for the encouragement and inspiration shared by my lab mates and post-docs at CCB: Chiam Nyet Cheng, Chung Corrine, Jess Loh Swee Cheng, Sam Ka Kei, Yue Keong Choon, Tengku Yasmin, Sim Pei Fang, Dr. Go Furusawa, Dr. Sheri-Ann Tan, Dr. Suganthi Appalasamy, Dr. Lau Nyok Sean, Dr.Farrukh Jamil, Dr. Abhilash Usharraj, and Dr. Gincy Paily Thottahil. I also appreciate the help of the administrative department of CCB for being helpful: Ms. Tengku Zalina Tengku Ahmad, Cik Nurul Farhana Che Hassan, and Ms. Nazira Zainal Abidin. Not forgetting, I would also like to convey my special thanks and appreciation to Ms. Azyyati Mohd Padzil from Malaysian Genome of Institute (MGI) for helping me to perform and analyze Circular dichroism (CD) analysis on my protein samples. ii Special appreciation is extended to Ministry of Higher Education of Malaysia for providing me with a scholarship through the Mybrain15 scheme. I am also thankful to Ministry of Science, Technology and Innovation (MOSTI) for their financial support to purchase research materials during the course of this research. I owe my loving thanks to my parents, especially my mom, sister, brother in- law, and my late father, without whom I would not be here today. They have been a constant source of unwavering support, love and encouragement for the past 2 years. Also, hundreds of apologies and sincere appreciation to all whom I failed to mention here, but had contributed to this project in any way. iii TABLE OF CONTENTS Page ACKNOWLEDGEMENT ii TABLE OF CONTENTS iv LIST OF TABLES vii LIST OF FIGURES viii LIST OF ABBREVIATIONS x ABSTRAK xii ABSTRACT xiv CHAPTER 1-INTRODUCTION 1 1.1 Background of Research 1 1.2 Research Objectives 2 CHAPTER 2-LITERATURE REVIEW 3 2.1 Scientific Classification, Cultivars and Historical Outline of 3 Hevea brasiliensis. 2.2 Global Distribution of H. brasiliensis Natural Rubber and 4 its Role as an Indispensible Biopolymer. 2.3 Composition of Hevea Latex 5 2.3.1 Hevea Frey-Wyssling Complexes 7 2.3.2 Hevea C-Serum 8 2.3.3 Hevea Bottom Fraction 9 2.3.4 Hevea Rubber Particle 10 2.4 Structure of Natural Rubber and Rubber Biosynthesis 15 2.4.1 Structure of Natural Rubber 15 2.4.2 Rubber Biosynthesis 15 15 2.4.2.1 Biosynthesis of Isopentenyl Diphosphate (IPP): a precursor molecule 16 iv 2.4.2.2 Initiation 18 2.4.2.3 Elongation by cis-prenyltransferase (CPT) 20 2.5 Small Rubber Particle Protein and Rubber Elongation 22 Factor (SRPP & REF). 2.5.1 Putative Role of SRPP and REF in Rubber Biosynthesis 26 2.5.2 Role of SRPP and REF in Plant Stress 27 2.5.3 SRPP and REF Protein as a Major Allergen in the Hevea Latex 27 2.6 Determination of three-dimensional structure of SRPP protein 28 2.7 Protein Crystallization by X-ray crystallography 29 CHAPTER 3-MATERIALS AND METHODS 31 3.1 Materials 31 3.2 Methods 38 3.2.1 Transmembrane Prediction 38 3.2.2 Expression Screening of Codon Optimized SRPP_His Protein 38 3.2.3 Denaturation, IMAC Purification and Refolding of SRPP_His 40 3.2.4 Size-Exclusion Chromatography for Refolded SRPP_His 41 3.2.5 Solubility Optimization of SRPP_His using Detergents 42 3.2.6 Preparation of SRPP_His Protein for Circular Dichroism (CD) 43 Spectroscopy 3.2.7 Dynamic Light Scattering (DLS) for SRPP_His 43 3.2.8 Construction of NusA_SRPP Recombinant Plasmid 44 3.2.9 Crystallization Screening 47 CHAPTER 4- RESULTS AND DISCUSSION 49 4.1 Transmembrane Prediction for SRPP Protein 49 4.2 Protein Expression of Codon Optimized SRPP_His 50 v 4.3 Solubilization and Immobilized Metal Ion Affinity Chromatography 52 (IMAC) Purification of SRPP_His Inclusion Body under Denaturing Condition 4.4 Refolding of SRPP_His in the presence of 1 M Arginine 54 4.5 Size-Exclusion Chromatography of SRPP_His with Arginine 56 4.6 Screening of Detergents to Solubilize SRPP_His Inclusion Body 60 4.7 Purification with IMAC and Size-Exclusion Chromatography 64 of SRPP_His in 0.2% Sarkosyl 4.8 Cloning, Protein Expression and Purification of SRPP Protein 70 with NusA Solubility Tag 4.9 Protein Crystallization Screening 79 4.9.1 Crystallization Trial for SRPP_His Refolded with 79 Arginine hydrochloride 4.9.2 In surfo Protein Crystallization of SRPP_His using 0.2% Sarkosyl 81 4.10 Future Directions 85 CHAPTER 5- CONCLUSION 87 REFERENCES 89 APPENDICES 102 Appendix A 102 Appendix B 103 Appendix C 104 Appendix D 106 vi LIST OF TABLES Tables Page 2.1 Nine cultivars of Hevea and their respective characteristics 3 of latex 3.1 Detergents used to screen the solubility of SRPP_His 42 3.2 Primer used in PCR for NusA_SRPP fusion construct. 45 3.3 Reagents used in PCR for NusA_SRPP fusion construct 45 3.4 PCR reaction setup for NusA_SRPP fusion construct. 45 4.1 Detergents used to solubilize the SRPP_His cell pellet 61 4.2 Far-UV CD Spectroscopy analysis for SRPP_His in 0.2% sarkosyl 68 vii LIST OF FIGURES Figure Page 2.1 Global distribution of H. brasiliensis, in native and exotic regions 5 2.2 Fractions of Hevea’s latex 7 2.3 A Frey-Wyssling complex depicting the double 8 membrane envelope 2.4 Rubber particles from the latex of H. brasiliensis. 12 2.5 Schematic drawing of the rubber molecule surface. 13 2.6 Image depicting the microstructure of Hevea’s natural rubber. 15 2.7 Illustration of isoprenoid biosynthesis. 17 2.8 Image depicting the production of IPP molecule 19 by the mevalonate pathway. 2.9 Sequence homology of SRPP protein to REF from 23 H. brasiliensis 2.10 Phylogenetic analysis of SRPP and REF protein family 24 2.11 Proposed model of SRPP and REF protein attached 25 on the monolayer membrane of the rubber particle 2.12 Protein crystallization phase diagram 29 3.1 Crystallization plates 48 4.1 TMHMM prediction result of 50 transmembrane helices in SRPP. 4.2 Expression profile of SRPP_His on 15% SDS-PAGE. 51 4.3 Elution profile of SRPP_His purified using immobilized 53 nickel ion affinity column under denaturing conditions. 4.4 SDS-PAGE of SRPP_His derived from immobilized 54 nickel ion affinity under denaturing condition 4.5 Elution profile of SRPP_His using size-exclusion chromatography 56 viii 4.6 DLS analysis performed on SRPP_His refolded in 58 arginine hydrochloride buffer 4.7 CD spectrum of SRPP_His dissolved in 1xPBS, 250 mM 59 arginine hydrochloride. 4.8 Anionic detergent (0.1-1.0% sarkosyl) that was screened 62 for the solubilization of SRPP_His. 4.9 Elution profile of SRPP_His purified using immobilized 65 nickel ion affinity column with 0.2% (w/v) sarkosyl. 4.10 Elution profile of SRPP_His with 0.2% (w/v) sarkosyl 66 using size-exclusion chromatography. 4.11 Circular Dichroism (CD) spectrum of SRPP_His protein 67 solubilized with 0.2% (w/v) of sarkosyl. 4.12 DNA band of the amplified product of SRPP (625bp) 71 for pET43.1a. 4.13 Expression profile of NusA_SRPP fusion protein 72 induced with 0.1 mM IPTG at 25°C on 15% SDS-PAGE. 4.14 Elution profile of NusA_SRPP purified using immobilized 73 nickel ion affinity column. 4.15 SDS-PAGE gel image of NusA_SRPP after first 74 run of immobilized nickel ion affinity chromatography. 4.16 Elution profile of NusA_SRPP purified using immobilized 75 nickel ion affinity column for the second run 4.17 SDS-PAGE analysis of NusA_SRPP fusion protein from second 76 run using immobilized nickel ion affinity column purification. 4.18 Elution profile of NusA_SRPP fusion protein using 77 size-exclusion chromatography. 4.19 SDS-PAGE analysis of NusA_SRPP fusion protein 78 resulting from size-exclusion chromatography. 4.20 Images of crystallization trials performed using 80 5 and 10 mg/mL of SRPP_His solubilized in arginine hydrochloride. 4.21 Needle-like crystals of SRPP_His protein solubilized 81 in 0.2% (w/v) of sarkosyl formed at 20°C using sitting-drop vapour diffusion method. ix 4.22 Needle-like crystal formations of SRPP_His 83 protein solubilized in 0.2% (w/v) of sarkosyl in the optimization conditions. LIST OF ABBREVIATIONS Acetyl-CoA Acetyl Coenzyme A CPT Cis-prenyl tranferase CD Circular dichroism DMAPP Dimethylallyl diphosphate DXS 1-deoxy-D-xylulose 5- phosphate synthase DLS Dynamic light scattering EDTA Ethylenediaminetetraacetic acid FPP Farnesyl pyrophosphate GGPP Geranyl-geranyl pyrophosphate GPP Geranyl pyrophosphate HLL Hevea latex lectin-like protein HMG-CoA 3-hydroxy-3-methyglutaryl Coenzyme A IPP Isopentenyl diphosphate IMAC Immobilized metal affinity chromatography IPTG Isopropyl-β-D- thiogalactopyranoside LB Luria-bertani LRP Large rubber particle MEP Methylerythritol 4-phosphate
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