SELECTION AND ENGINEERING OF ANTI-PANIE VHH SINGLE DOMAIN
ANTIBODIES AND THEIR FUSION TO CELLULOSE BINDING MODULES
A Thesis
Presented to
The Faculty of Graduate Studies
of
The University of Guelph
By TED INGEMAR JAKOB FJALLMAN
In partial fulfilment of requirements
for the degree of
Doctor of Philosophy
May, 2008
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SELECTION AND ENGINEERING OF ANTI-PANIE VHH SINGLE DOMAIN ANTIBODIES AND THEIR FUSION TO CELLULOSE BINDING MODULES
Ted Ingemar Jakob Fjallman Advisor: University of Guelph, 2008 Professor J. Christopher Hall
Plant pathogens from the genera Pythium, Phytophthora and Fusarium are responsible for 80% of root disease epidemics in hydroponic greenhouses worldwide. Many of these pathogens secrete virulence factors belonging to the Nepl-Like-Protein (NLP) family of necrosis-inducing proteins. This thesis describes the development of a platform, consisting of antibody-cellulose binding modules bound to cellulose, for combating epidemics of plants through the capture of PaNie (NLPPya) secreted by Pythium aphanidermatum. Four recombinant single domain antibodies (VHHS) with affinities to
PaNie ranging from 800 nM to 20.2 uM were developed by selection from a naive ribosome display library and a hyperimmune phage display library. Three of these antibodies bind to a synthesized peptide containing the highly conserved NPP1 domain of the NLP family. All four antibodies have varied complementarity determining regions and framework regions representing three of the four VHH subfamilies. The genes of the three highest affinity VHHS were fused to a cellulose binding module (CBM) with an intervening thrombin cleavage site. All VHH-CBM fusions were functional and the highest affinity binder P10-CBM was analysed in more detail. P10-CBM could be purified directly from culture using cellulose beads and cleaved with thrombin to yield fully functional P10 VHH. P10-CBM was bound to paper to create a bioactive filter paper for the capture of PaNie. The data obtained suggest that the affinity of paper-bound P10-
CBM is the same as for soluble P10-CBM. P10-CBM is also able to bind PaNie while attached to the cellulose surfaces of tobacco roots as shown by confocal laser scanning microscopy. Improvements of the affinity of the VHH-CBM-based platforms and the utility of the antibodies for the elucidation of NLP-associated disease are discussed.
Features of PaNie and the VnH-PaNie interaction suggest that NLPs may be pore forming toxins. In the future, VHH-CBMS could be produced in situ by non-pathogenic biocontrol agents and may be used to remove pathogenic toxins from hydroponic systems used in greenhouses and space-based advanced life support systems. ACKNOWLEDGEMENTS
"Science never ends, but our lives do. So, learn to pass on the torch"
This thesis is dedicated to all the people who never hesitated to pass part of their life's torch to me and brighten my path. Thank you for the deep thoughts, the laughs and above all the patience and honesty.
My development as a researcher would not have been so insightful were it not for Chris' trust and patience. I hope you realize what a gift you have given me, by letting me work things out my own way. Your impact on society through your teaching and guidance works on a deep level in the lives of everyone who has worked with you. You have taught me how to be an effective human being, not just a knowledgeable scholar.
My gratitude also extends to my advisors, examiners and teachers throughout these years.
Special thanks to Profs Michael D. McLean and John C. Sutton for giving expert advice for my laboratory and greenhouse work and for reading my thesis thoroughly. Mike, you always forced me to be humble about my science and I thank you for that. Drs Roger
MacKenzie and Mehdi Arbabi-Gharoudi; you made it possible for me to push myself to the limit and focus like I had never focused before. Of course none of this would have been possible without the funding from Flowers Canada, CresTech, NSERC NRC, CRC
Chair and Syngenta.
What got me through the rough parts was no doubt the sisterly and brotherly companionship of the 2002-2008 Hall lab crew. You all give me great hope for the
i future. Nina, you can slap me with bologni any day... seriously your humour kept me sane at times; Shokouh, your kindred spirit awakens my thirst for knowledge and my joy of sharing; Yongqing, your care and objectivity blend so harmoniously, I hope one day I will pick up some of that wisdom; Tsafrir, patience incarnate -just seeing you in the hallway had a soothing effect on me; Greg, your integrity is a role model for me; Adam, your honesty is so refreshing.
The last three years of my PhD were frustrating and even painful at times, but Cindy, you made me see that painful events only turn into suffering if you let them. In fact, you have given me so much renewed energy to tackle problems, by helping me redefine what a problem is. You renewed my interest in philosophy and at the same time made me a more pragmatic scientist - a combination I have struggled to attain all my life.
Last but not least, thank you to my family for a free upbringing that gave me the confidence and skills to be a freethinker. It is the fact that whenever we had discussions and got the encyclopedia out to settle disputes, which made me value objectivity as the one tool that can bridge the different perceptions in this world.
Thanks to all of you, I am now resolved that I can positively contribute to the future of humanity. I have grown so much, but I have not forgotten the creative child in me; thus, I want to say "Tack" (thanks in Swedish) with the following illustration. It is intended to show that a little part of everyone contributed to this thesis. Congratulations to you!
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• «* O w Q - CD .£ ^ CD -~ S3 • rs 3 GO o - O EG rt -^ g ^ co :cd CO ^ f <-C>J a Ec o y & ^ I 2 g ° Z ^ c 5 ^ ffi CD ^ .r ST M CD » -PS -Si S3 O ? '^ 5t > C * r — 5b CO O > iii Table of Contents ACKNOWLEDGEMENTS i TABLE OF CONTENTS iv LIST OF TABLES vm LIST OF FIGURES ix LIST OF ABBREVIATIONS xin 1. GENERAL INTRODUCTION AND RATIONALE 1 2. LITERATURE REVIEW AND RESEARCH OBJECTIVES 5 2.1. Antibody technology 5 2.1.1. Conventional immunoglobulins 5 2.1.2. Recombinant antibodies 7 2.1.3. Recombinant antibody applications 11 2.1.4. Selection of recombinant antibodies from antibody libraries 14 2.1.5. Affinity maturation 19 2.1.6. Antibody expression systems 21 2.2. Cellulose-protein scaffolds 31 2.2.1. Structure of cellulose in plants 31 2.2.2. Enzymatic digestion of plant cellulose 33 2.2.3. Cellulose binding modules 35 2.3. Epidemiology of Pythium aphanidermatum 39 2.3.1. Physiology of Pythium aphanidermatum 39 2.3.2. Associated disease 41 2.3.3. Occurrence and pathogenicity 42 iv 2.3.4. Mechanism of action 45 2.3.5. Current detection and control strategies 50 2.4. Research objectives 54 3. SCREENING OF A NAIVE LLAMA RIBOSOME DISPLAY LIBRARY AGAINST AN NLP PEPTIDE 55 3.1. Introduction 55 3.2. Materials and methods 56 3.2.1. Peptide design & preparation 56 3.2.2. Selection of antibodies by ribosome display 57 3.2.3. Cloning and expression of VHH sequences 62 3.2.4. Cloning and expression of PaNie 63 3.2.5. Anti-peptide ELISA 65 3.2.6. Anti-PaNie inhibition ELISA 65 3.3. Results 66 3.3.1. NLPsg2 peptide characteristics 66 3.3.2. Ribosome display selection and sequence analysis 74 3.3.3. VHH protein expression 78 3.3.4. Cloning and expression of PaNie 79 3.3.5. Binding of VHH cl05 to NLPsg2 83 3.3.6. Determination of VHH cl05-PaNie binding kinetics by CI-ELISA 84 3.4. Discussion 86 4. CONSTRUCTION AND SCREENING OF A HYPERIMMUNE LLAMA PHAGE DISPLAY LIBRARY AGAINST PANIE 91 v 4.1. Introduction 91 4.2. Materials and methods 93 4.2.1. Immunisation and serum preparation 93 4.2.2. Serum titre ELISAs 94 4.2.3. Library construction 95 4.2.4. Selection by phage display 99 4.2.5. Monoclonal phage ELISA 102 4.2.6. Soluble expression of selected clones 103 4.2.7. Soluble ELISA and surface plasmon resonance 104 4.3. Results 105 4.3.1. Anti-PaNie polyclonal serum titres 105 4.3.2. Library construction 109 4.3.3. Panning titre results 110 4.3.4. Monoclonal phage ELISA 113 4.3.5. Sequence analysis of selected VHH antibodies 114 4.3.6. Soluble expression of selected anti-PaNie VHHS 117 4.3.7. Kinetic studies of selected anti-PaNie VHHs 118 4.4. Discussion 123 5. CONSTRUCTION OF VHH-CBM FUSIONS FOR CAPTURE OF PANIE ON CELLULOSE 128 5.1. Introduction 128 5.2. Materials And Methods 129 5.2.1. VHH-CBM construction 129 vi 5.2.2. VHH-CBM binding to cellulose and thrombin cleavage 132 5.2.3. Antibody functionality by ELISA 133 5.2.4. Surface plasmon resonance 134 5.2.5. Bioactive paper assay development 134 5.2.6. PaNie leaf injections and analysis of necrosis 135 5.2.7. PaNie root assays and root preparations 136 5.2.8. Production oiPythium aphanidermatum exudates 137 5.2.9. Confocal laser scanning microscopy 138 5.3. Results 138 5.3.1. Expression and purification of VHH-CBM constructs 138 5.3.2. Binding of VHH-CBM to cellulose and cleavage with thrombin 141 5.3.3. Verification of VHH and VHH-CBM functionality 143 5.3.4. Binding of VHH-CBM to NLPsg2 146 5.3.5. Functionality of P10-CBM while bound to cellulose paper 147 5.3.6. PaNie necrosis elicitation in tobacco 151 5.3.7. Confocal laser scanning microscopy of VHH-CBM and PaNie bound to roots 155 5.4. Discussion 159 6. OVERALL CONCLUSIONS AND FUTURE DIRECTIONS 163 REFERENCES 169 vii LIST OF TABLES Table 1. Sales by, and investment in, the Canadian greenhouse industry 1 Table 2. Comparison of sequence homology between PaNie and a selection of other NLPs 50 Table 3. Primers used in the ribosome display cycle 59 Table 4. Kyte & Doolittle hydrophobicity scores for six peptides of PaNie 73 Table 5. Growth and induction conditions for PaNie expressions 82 Table 6. Primer oligos used in library construction 96 Table 7. DNA sequences often random pre-panning clones from the anti-PaNie VHH library 110 Table 8. Polyclonal phage titres for two trials of panning against PaNie by phage display Ill Table 9. Protein parameters for the selected anti-PaNie VHHS 116 Table 10. Association (kass) and dissociation (kdiSS) constants for the interaction of PaNie and VHHS during surface plasmon resonance 122 Table 11. Comparison of affinity constants (KDs) for the anti-PaNie VHHS P10, P16 and P31 122 Table 12. Primers used in VHH-CBM cloning 130 Table 13. Response units of P10 binding to PaNie at steady state (Req) 144 Table 14. Comparison of four papers for use as bioactive filters 149 viii LIST OF FIGURES Figure 1. Thesis outline 4 Figure 2. Schematic representation of the five major classes of human antibodies 6 Figure 3. Antibody fragments produced by various enzymatic and chemical treatments of IgG 7 Figure 4. Human IgG and Camelid Heavy chain IgG (HcIgG) and their derivatives 8 Figure 5. The in vitro ribosome display selection cycle 18 Figure 6. Molecular breakdown of a dense cellulose microfibril 32 Figure 7. An example of a cell wall hemicellulose 33 Figure 8. Degradation of insoluble cellulose by hydrolysis of p-l,4-glycosidic bonds to single, soluble sugar units 34 Figure 9. Scanning electron micrograph of a Phytophthora zoospore showing the ventral groove into which the anterior and posterior flagella are inserted 40 Figure 10. Fertilisation of oogonium by two antheridia in Pythium 41 Figure 11. Extreme root rot of pepper caused by P. aphanidermatum compared to non- infected control 42 Figure 12. Release of zoospores from sporangium in P. undulatum 43 Figure 13. Zoospore cysts of P. aphanidermatum germinating behind the root tip of wheat 45 Figure 14. Effect of Pseudomonas chlororaphis biocontrol on root rot caused by P. aphanidermatum 52 Figure 15. Basic structure and function of the ribosome display library and its components 58 ix Figure 16. Jalview sequence alignment showing conservation and consensus of selected NLPs i Figure 17. Selected conserved peptides displayed within the PaNie amino acid sequence 70 Figure 18. Hydrophobicity plot of PaNie according to Kyte & Doolittle 71 Figure 19. Third round ribosome display results 74 Figure 20. Fourth round ribosome display results, when competition as well as EDTA elution was used 75 Figure 21. Fifth round ribosome display selection results 76 Figure 22. Amino acid alignment of the five full length anti-NLPsg2 VHH clones 77 Figure 23. SDS-PAGE (left) and Western (right) analysis of VHH clones from the fifth round ribosome display panning against NLPsg2 79 Figure 24. Genomic DNA preparation (A) and PaNie gene amplification (B) from Pythium aphanidermatum 80 Figure 25. PaNie expression under various temperature and induction regimes 81 Figure 26. Binding of NLPsg2 to VHH cl05 84 Figure 27. Competitive inhibition ELISA (CI-ELISA) of VHH cl05 vs. PaNie 85 Figure 28. pMEDl vector map 98 Figure 29. Titres of anti-PaNie antibodies in serum from immunized mice, rabbits and llama : 106 Figure 30. Purity and activity of HcIgG and ConvIgG fractions from the PaNie immunized llama 108 x Figure 31. Monoclonal phage ELISA from the three different panning rounds compared by signal to noise ratio 113 Figure 32. Amino acid alignment of the three anti-PaNie VRH clones and the anti- NLPsg2 VHH cl05 115 Figure 33. SDS-PAGE of VHH P10, P16 and P31 purified from E coli 117 Figure 34. Competitive inhibition ELISA (CI-ELISA) of VHHs P10 (A), P16 (B) and P31 (C)vs. PaNie 119 Figure 35. Surface plasmon resonance of immobilised VHH P10, P16 and P31 vs. 24 nM - 2.4 uM PaNie 121 Figure 36. Construction of an anti-PaNie VHH-CBM expression plasmid based on pPICZaA 131 Figure 37. Leaf injection system mounted on a micromanipulator 136 Figure 38. Hydroponic plant growth unit with a 6-week-old tobacco plant 137 Figure 39. SDS-PAGE showing crude purifications of VHH-CBM protein and BSA standards 139 Figure 40. Coomassie-stained SDS-PAGE of VHH PI0 and P10-CBM after purification from a large-scale P. pastoris culture 140 Figure 41. Western blot (left) and Coomassie stained gel (right) of P10 (a) and P10-CBM (b) binding to CP-102 cellulose beads 141 Figure 42. Western blots showing the thrombin cleavage of P10 from P10-CBM directly purified from culture using cellulose beads 142 Figure 43. Comparison of P10 and P10-CBM binding to PaNie as determined by ELISA 143 xi Figure 44. Surface Plasmon resonance of VHH P10 expressed in P. pastoris 144 Figure 45. Scatchard plot of VHH P10 from P. pastoris binding to PaNie 145 Figure 46. Binding of NLPsg2-biotin to P10-CBM and P16-CBM 146 Figure 47. Binding of PaNie to a bioactive paper filter 147 Figure 48. Anti-PaNie bioactive filter paper 149 Figure 49. Necrosis induced by PaNie injection into the leaf of tobacco 152 Figure 50. Necrotic effect due to a week-long 5 uM PaNie application to the roots of hydroponically growing tobacco 153 Figure 51. Growth and chlorophyll parameters of tobacco after a week-long 5 uM treatment with PaNie 154 Figure 52. Polyclonal serum neutralisation of PaNie necrosis in leaves of tobacco 155 Figure 53. Confocal laser scanning microscopy depicting the binding of P10-CBM and P10 (ctr) VHHS to the surface of tobacco roots 156 Figure 54. Confocal laser scanning microscopy depicting the binding of Alexa Fluor 594- labelled PaNie to VHH-CBM displayed on the surface of tobacco roots 158 xu LIST OF ABBREVIATIONS Abbreviation Explanation 4-HBA 4-hydroxybenzoic acid A0 Absorbance at 0 uM inhibition Ab Antibody Ag Antigen AMP Ampicillin AOX1 Alcohol oxidase 1 AP Alkaline phosphatase ARM complex Antibody-ribosome-mRNA complex BcNEP Botrytis cinerea necrosis and ethylene inducing protein BLAST Basic local alignment and search tool BMGY Buffered medium with glycerol for yeast (1% w/v yeast extract, 2% w/v peptone, 2% w/v glucose, 0.1 M sodium phosphate buffer, 1.0% glycerol, 0.04 mg/ml biotin; pH 6.0) BMMY Buffered medium with methanol for yeast (1% w/v yeast extract, 2% w/v peptone, 0.1 M sodium phosphate buffer, 1.0% (v/v) methanol, and 0.04 mg/ml biotin; pH 6.0) BSA Bovine serum albumin CARB Carbenicillin CBM Cellulose binding module CDR Complementarity determining region CH Constant domain of heavy chain of Ig xui Abbreviation Explanation CI-ELISA Competition/Inhibition ELISA CL Constant domain of light chain of Ig CLSM Confocal laser scanning microscopy CM5 Carboxymethylated dextran chip DMSO Dimethyl sulfoxide EDTA Ethylene diamine tetracetic acid ELISA Enzyme-linked immunosorbent assay ER Endoplasmic reticulum ExPASy Expert protein analysis system F(ab')2 Bivalent antigen binding fragment of immunoglobulins fl Strain of filamentous phage Fab Antigen binding fragment of immunoglobulins FACS Fluorescence activated cell sorting Fc Crystallizable fragment of immunoglobulins fd Strain of filamentous phage FITC Fluorescein isothiocyanate FPLC Fast protein liquid chromatography Fv Variable domain binding fragment of conventional immunoglobulins GHs Glycosidic hydrolases glllp Coat protein 3 of filamentous phage GRAVY Grand average hydropathicity GroESL E. coli chaperonin XIV Abbreviation Explanation HA Hemagglutinin HB Heparin block buffer (200 mg/ml heparin in 50 mM Tris-acetate, 150 mM NaCl, 50 mM Mg-acetate, 0.1% v/v Tween 20, pH 7.5) HcIgG Heavy chain IgG from camelid family HEPES 4-(2-hydroxyethyl)-l-piperazine-ethanesulfonic acid HIS Histidine HIV Human immunodeficiency virus HRP Horseradish peroxidase IC50 Inhibitor concentration required to reduce A0 by 50% IgG Immunoglobulin G Ig Immunoglobulin (e.g. IgG) IPTG Isopropyl P-D-1-thiogalactopyranoside Jalview Java alignment editor KAN Kanamycin kass Association rate constant KD Equilibrium dissociation constant kDa Kilodaltons kdiss Dissociation rate constant LB Luria-Bertani media / lysogeny broth (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, pH 7.5) M13 Strain of filamentous phage MCS Multiple cloning site xv Abbreviation Explanation MT Melting temperature NEB New England Biolabs Nepl Necrosis and ethylene inducing protein 1 of Fusarium oxysporum NHS/EDC N-hydroxysulfosuccinimide/l-Ethyl-3-[3- dimethylaminopropyl]carbodiimide hydrochloride Nip Necrosis-inducing protein of Erwinia carotovora NLP Nepl-like protein NLPpya NLP produced by Pythium aphanidermatum (= PaNie) NLPsg2 NLPpya (PaNie) peptide GHRHDWENVWWLDNSGSGK-biotin NPP1 Necrosis-inducing Phytophthora protein 1 NPQ Non-photochemical quenching NTA Nitrilo-triacetic acid NZW New Zealand White o/n Overnight; 12-16 h OD Optical density PaNie Pythium aphanidermatum Necrosis inducing elicitor (=NLPpya) PBS Phosphate buffered saline (137 mM NaCl, 10 raM Na2HP04, 1.8 raM KH2P04,2.7 mM KCl, pH 7.4) PBST Phosphate buffered saline + 0.05% v/v Tween 20 PCR Polymerase chain reaction PEG Polyethylene glycol xvi Abbreviation Explanation Pfu Plaque forming unit PsojNIP Phytophthora sojae necrosis inducing protein RBS Ribosome binding site Req Response at steady state during surface plasmon resonance Ri Root inducing RIA Radioimmunoassay R-max Maximum response defined as saturation of surface plasmon resonance RT Room temperature; 20-25°C RT-PCR Reverse transcription followed by PCR RU Resonance unit (in surface plasmon resonance) ScFv Single chain Fv SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis SOC medium Super optimal catabolite repression medium (20 g/L tryptone, 5 g/L yeast extract, 10 mM NaCl, 2.5 mM KC1, 10 raM MgS04, 10 raM MgCl2, 20 mM glucose, pH 7.0) Ta Annealing temperature T-DNA Agrobacterium-transferred DNA TEAE Triethylamine elution Ti Tumour inducing TMB 3,3',5,5'-tetramethyl benzidine TMV Tobacco mosaic virus Tris 2-Amino-2-hydroxymethyl-propane-l,3-diol xvii Abbreviation Explanation TRITC Tetramethyl rhodamine isothiocyanate TSP Total soluble protein Tween 20 Polyoxyethylene (20) sorbitan monolaurate VH Variable domain of heavy chain of Ig VHH Variable heavy domain from HcIgG VL Variable domain of light chain of Ig w/v Weight/volume (% expressed in g/ml) YPD 10 g/L yeast extract, 20 g/L peptone & 2% w/v dextrose YT 5 g/L yeast extract, 8 g/L tryptone, 2.5 g/L NaCl, pH 7.2 xvm 1. GENERAL INTRODUCTION AND RATIONALE The greenhouse industry suffers from considerable revenue loss due to crop destruction caused by pathogenic forms of the following three genera: Pythium, Phytophthora and Fusarium. These genera are responsible for 80% of root disease epidemics in hydroponic systems (Schuerger, 1998). Commercial nutrient disposal from hydroponic systems has a negative effect on the environment and legislation is becoming stricter, effectively prohibiting such discharge in the future. The greenhouse industry, which is expanding both in terms of sales and investments (see Table 1), will have to recirculate the nutrient solution if nutrient leaching is to be prevented. However, recirculation will increase incidence and severity of diseases due to Pythium, Phytophthora and Fusarium. Thus, there is a need to find new environmentally compatible tools for fighting disease in greenhouse-based hydroponic systems. Table 1. Sales by, and investment in, the Canadian greenhouse industry. Data obtained from Statistics Canada (2006). Canadian Greenhouse industry 2004* 2005* 2006* Ornamental Plant Sales 1.44 1.42 1.52 Vegetables Sales 0.72 0.73 0.83 Total Greenhouse Sales 2.17 2.15 2.34 Total Investment 3.07 3.30 3.70 * In billions of Canadian dollars. 1 Single domain antibody fragments, which are very stable under adverse conditions such as heat and extreme pH (Muyldermans, 2001), could be used to specifically capture and quantify pathogens and their toxins in hydroponic systems. Binding these antibodies to filters would provide a biotechnology that is bio-compatible and simple to implement for hydroponic greenhouse growers. This thesis describes the selection and engineering of recombinant antibodies and their potential uses as antibody-based filters for the capture of soluble pathogenic toxins in hydroponic systems. Soluble toxins represent simpler targets than whole-cell pathogens or filamentous fungi and are important because they cause many of the deleterious effects of plant pathogens. The toxin chosen in this study is the Pythium aphanidermatum Necrosis inducing elicitor (PaNie). Root rot due P. aphanidermatum is one of the most common diseases of hydroponic systems in Ontario (Zheng et ah, 2000) and is a major problem because it thrives in recirculating nutrient solutions (Sutton et ah, 2006). PaNie shares up to 71% amino acid sequence identity with elicitors from species of Phytophthora and Fusarium and the continued discovery of more PaNie-related elicitors has led researches to coin a collective family name, the NLP family (Nepl like proteins), named after one of the most well-characterised elicitors from Fusarium oxysporum (Gijzen and Nurnberger, 2006). The requirements of antibodies for the capture of PaNie on filters are high affinity, high specificity, low cost of production, and simple methods for attachment to a filter matrix. The requirements for a filter matrix are biocompatibility, low cost, and adaptability. 2 Cellulose fibres constitute the most abundant renewable resource on the planet; in Canada alone, approx. 68 M m3 of lumber and 20 M tonnes of dry pulp chips were produced during 2007 (Statcan, 2007). In addition, cellulose fibres can be processed into a plethora of different products, from filter papers to car parts (Mohanty et al, 2002). Cellulose binding modules (CBMs) are protein domains that have evolved as part of cellulose degrading enzymes and bind cellulose almost irreversibly (Tomme et al, 1998), but do not degrade it. Because they are proteins, their coding DNA can be directly fused to antibody genes, allowing the resultant antibody-CBM fusion proteins to bind to cellulose. The major objectives of this thesis were to 1) create PaNie-specific antibodies, 2) fuse these antibodies to a CBM using recombinant DNA technology, and 3) use the antibody- CBM fusions to capture PaNie on paper and the surface of roots. Figure 1 outlines the structure of the thesis. Chapter 2, i.e. the literature review, describes where antibody fragments come from and how they are produced; cellulose as a matrix and how CBMs attach to it; and the epidemiology of Pythium root rot and how capture of PaNie may help ameliorate disease symptoms. At the end of chapter 2 the specific research objectives of the thesis are outlined. Chapter 3 presents the work done to design a common NLP peptide and the development of an antibody against this NLP peptide. Chapter 4 presents research on developing antibodies to the full length PaNie, with higher affinity than that of chapter 3. And finally chapter 5 presents the research done to fuse the anti-PaNie antibodies to CBMs and to formulate different biotechnological applications for the capture of PaNie. Chapter 6 provides overall conclusions including a synopsis of in- chapter discussions and an outlook for future research directions. 3 Selection and engineering «f anti-PaNie ">"nll single domain antibodies and their fusion to culfatosc binding modules General introduction and rationale I X "A ;Iviterawre*Wigw a0 Screening i»f a naive llama ( t r truUNni ,*nd suit,! nv~ < i Construction oPVnH-('BM ri^osmpc display litynry hvpu«nmuB«, it in 1 pit ^ fu£iun#- fwr ciifitaro nf njawKi an NF P peptide- di pi* t bnn a tin t I* iNic ltiNftc on cellulose Antibody technology Peptide Design Production of antisera Construction, expression & Cellulose binding modules Ribosome display panning Library construction purification of VHH-CBMs Epidemiology of Pythium Expression of VHHs Phage display panning Bioactive paper development aphanidermatum Cloning & expression of PaNie Expression of VHHs PaNie necrosis characterization Research objectives Antibody binding kinetics Antibody binding kinetics Root coating with VHH-CBMs Overall conclusions and future directions Chapter 2 Chapter 3 Chapter 4 Chapter 5 Figure 1. Thesis outline. In the interest of brevity the chapter summaries do not include the exact in-chapter headings. 2. LITERATURE REVIEW AND RESEARCH OBJECTIVES 2.1. Antibody technology To understand the development of antibodies as biotechnological tools, this section describes the basic nature of immunoglobulins, how they are applied for human use, how antibody fragments can be engineered for diverse applications and how they can be produced in large quantities. 2.1.1. Conventional immunoglobulins All antibodies and antibody fragments are derived from immunoglobulins (Igs). There are five classes of conventional immunoglobulins: IgG, IgD, IgE, IgA and IgM (see Figure 2). Of these five classes of antibodies, IgGs are most commonly used, especially for diagnostic applications. IgG is a tetramer consisting of four polypeptide chains: two identical heavy chains (ca. 450 amino acids each) and two identical light chains (ca. 250 amino acids each) (Maynard and Georgiou, 2000). The N-terminal domains of the heavy and light chains are called variable domains. They comprise the paratope, which is responsible for binding to the epitope of the antigen. Thus, each IgG molecule presents two identical paratopes that can bind to two epitopes of two antigens or to two repeating epitopes on the same antigen (Kuby, 1997). 5 Figure 2. Schematic representation of the five major classes of human antibodies. Light chains are shown in shades of pink while heavy chains are shown in shades of blue, green, yellow, brown and purple. Disulphide bonds are indicated by thick black lines. Source: Immunology 4th Edition by Janis Kuby. Conventional IgGs can be broken into smaller fragments, by either enzymatic digestion or reducing conditions. Papain digestion produces two antigen binding fragments (Fab) and a crystallisable fragment (Fc), whereas pepsin digestion produces a bivalent F(ab')2 fragment and two truncated Fc fragments (see Figure 3). Under certain conditions, pepsin 6 digestion can yield Fv fragments, which consist only of the variable regions of the heavy and light chains (Seddas-Dozolme et ah, 1999). Figure 3. Antibody fragments produced by various enzymatic and chemical treatments of IgG. Light chains are in grey and heavy chains in blue. Source: Immunology 4th Edition by Janis Kuby. 2.1.2. Recombinant antibodies Using recombinant DNA technology, antibodies are easily obtained by expressing the required gene in a bacterial, yeast or plant host as well as mammalian or insect cell culture. The technology also enables novel fragments (see Figure 4), such as single chain 7 variable fragments (scFv) and camelid heavy chain variable domain fragments (VHH), to be produced, which can be matured to bind haptens, proteins, microorganisms etc. Heavy chain Light chain -\ r~ cHi-| * ~2 c :H2-{ ,* ,1 IgG scF„ c :H3{ 0 V H-[ V„H H ^ CH2-| HcIgG * i CH3-[ Figure 4. Human IgG and Camelid Heavy chain IgG (HcIgG) and their derivatives: Antigen binding fragment (Fab), variable fragment (Fv), single chain variable fragment (scFv) and HcIgG variable domain fragment (VRH). The labels on IgG denote the three heavy chain constant domains (CH1-3), the light chain constant domain (CL), and the two variable domains (VH, VL and VHH for the HcIgG). Both the heavy and light chain variable domains exhibit certain complementarity determining regions (CDRs) shown as blue striations. 8 2.1.2.1. Single chain variable fragment (scFv) One VL and one VH are fused into a single chain using a linker peptide to make a 26-27 kDa scFv (see Figure 4). Expression levels of Fvs in prokaryotes are low, because the two chains often do not anneal in the reductive prokaryotic cytoplasm, but because scFvs are encoded by one gene, genetic manipulations to improve expression levels are simplified. Although scFvs are designed to be monovalent, they often form dimers or higher molecular weight species, because of the pairing of the VH chain of one scFv polypeptide with the VL chain of another (Maynard and Georgiou, 2000). If the stability of a scFv is poor, engineering the linker peptide may improve the function. Many different linkers have been designed, based on structural and other considerations (Pantoliano et al, 1991; Turner et al, 1997; Robinson and Sauer, 1998). The two variable domains are connected either Vn-linker-VL or VL-linker-VH. The order affects expression, stability and dimerisation and the former is more commonly used (Merk et al, 1999). There is a growing number of reports, in which scFvs have replaced immunogenic polyclonal sera or monoclonal antibodies as antidotes. One of the earliest reports came from Mousli et al. (1999), who produced a scFv, which neutralises toxin II from the snake Androctonus australis. 2.1.2.2. Camelid heavy chain variable domain fragment (VHH) The Camelidae family (camels, llamas and alpacas) contain, in addition to conventional IgG antibodies which form from association of two heavy and two light chains, heavy- chain IgGs (HcIgG) formed from the association of only two heavy chains 9 (Muyldermans, 2001). VHH stands for the VH of the HcIgGs (see Figure 4). It is the smallest available antigen binding protein (15 kDa) and is often referred to as a single domain antibody. Despite absence of VH-VL combinatorial diversity, HcIgGs exhibit a broad range of antigen binding by having enlarged hypervariable regions. In general, VHHS are well expressed, with expression levels being, in some cases, ten times greater than those of conventional antibodies. VHHS are highly soluble, heat stable (MT <72°C) and can bind target antigens with an affinity in the low nM range (Muyldermans, 2001). HcIgGs do not have the CHI domain due to a mutation in the donor splice site flanking CHI exon. Unlike conventional antibodies, HcIgGs used as enzyme inhibitors can be displaced from active sites by small competitive inhibitors such as acarbose and dorzolamide (Lauwereys et al, 1998), suggesting that the HcIgGs bind in an analogous fashion to these inhibitors by mimicking substrate rather than just blocking substrate access. The complete VHH is coded from one original exon. This makes cloning and library synthesis much easier. Conventional antibody libraries require many clones to make sure the original VH-VL pairs are recovered. In contrast, VHH libraries can be relatively small, due to their monomeric nature. In addition, it is relatively simple to generate diverse synthetic VHH libraries, because amino acids in the long CDR3 can be altered or added without changing conformation (Muyldermans, 2001). In conventional libraries the alteration of the CDR3 is more complex due to the pairing of two variable domains. The high stability and refolding capability of VHHS allows repeated denaturation and renaturation without loss of function. In conventional Abs, the antigen binding surface is 10 flat or concave, but the long CDR3s of VHHS can form loops which can fit into active sites of enzymes and can thus act as powerful modulators of biological activities. However, a long CDR3 is not a prerequisite for enzyme inhibition. The CDR3 accounts for 50-100% of antigen binding and so is often the starting point for affinity maturation by site directed mutagenesis (Thompson et al, 1996). The CDR3 domain has even been used to create peptide drugs mimicking target receptors. VHHS are suitable as human therapeutics, because of their high degree of similarity to the human VH, for example making humanisation more straightforward than from murine antibodies. The relatively small size of VHHS (15 kDa) also encourages the development of VnH-based therapies, because VHHS may evade the patients own immune system. Mouse studies have shown that VHHS injected into the blood stream of mice result in no anti-VnH Igs and no T-cell response, even after repeated doses (Muyldermans, 2001). The relatively small size of VHHS would be an advantage for the development of immunomodulators within plant tissues as well. Muyldermans mentions that a VHH has been cloned in tobacco plants to expression levels of 0.1% of TSP. 2.1.3. Recombinant antibody applications In the late 1980s efficient cloning and expression of antibody genes in bacteria allowed the production of antibodies without the need to continually immunise and boost animals (Winter and Milstein, 1991). The mRNA used for cloning is isolated from hybridoma, spleen or lymph cells, reverse transcribed to cDNA and antibody genes are amplified using the Polymerase Chain Reaction (PCR). This technology enabled the production of 11 recombinant antibodies or fragments thereof, which because of their simplicity and small size enable new therapeutic and diagnostic uses. The invention of hybridoma technology significantly boosted the share of monoclonal antibody-based assays in the diagnostics industry (Maynard and Georgiou, 2000) and led to many biomedical applications of monoclonal antibodies such as neutralisation of toxins in vivo, passive immunisation, delivery of radionuclides for imaging, immunosuppression and cancer therapy (Whittum-Hudson et al, 1996). In 2006, four of the 'blockbuster' mAb-based drugs, Herceptin, Remicade, Avastatin, and Humira, brought in more than $11 B in sales (Lawrence, 2007). Environmental applications include detection of small molecules like herbicides in river water, soil and plant extracts at concentrations of 1 ng/ml (Deschamps and Hall, 1990). Recombinant antibodies can augment and even replace monoclonal antibodies in many of these applications, especially where Fc effector functions are not required and small size is an advantage. For example, recombinant antibodies can be used as environmental biosensors with detection limits of 20 to 10000 ng/ml of organic pesticide (Tout et al, 2001). In addition, recombinant antibodies can be used as agents of bioremediation to remove microbes and organic pollutants from contaminated water or soil (Harris, 1999). Furthermore, it has been suggested that recombinant antibodies and degradative enzymes could be fused to phage, allowing these fusion phages to bind as well as degrade pollutants in the environment (Hall et al, 1997). Even very small pollutants can be bound by recombinant antibodies as demonstrated by Tout et al (2001), who produced a scFv against picloram, a small auxinic herbicide (MW 241.5). Small plant hormones can be bound by 12 recombinant antibodies, thus providing a means to modulate plant metabolism in a process termed immunomodulation. 2.1.3.1. Intracellular antibodies Antibodies that are expressed intracellularly and manipulate cellular pathways including pathogen induced pathways, by e.g. inhibiting the function of key enzymes or hormones, are termed intrabodies (Cattaneo and Biocca, 1999). Intrabodies expressed in plants are often referred to as plantibodies, although the term is used loosely for all antibodies produced in plants. Plantibodies are very useful for studying phytohormone activity and are generally more suitable in such studies than knock-outs, because the whole pathway is preserved, blocking the activity of the product, rather than its production (De Jaeger et al, 2000). Thus, plantibody production may offer a sensitive approach to modulate protein activity and for studying cellular processes. In addition, plants could act as bioreactors for the large-scale production of antibodies. In contrast to most microbial bioreactors, plants are not limited to producing only antibody fragments but can even produce multimeric antibodies (Ma and Hein, 1995). Various antibodies, including recombinant scFv and Fab fragments, have been produced in the cytosol, apoplast and endoplasmic reticulum (ER) of plants. The accumulation of antibodies greatly depends on the plant species, as was shown by differences in expression of Fab between Arabidopsis and tobacco. ScFv fragments tend to accumulate at low levels, because of instability or inefficient synthesis or folding of the proteins (De Wilde et al, 1999). For most efficient expression of full-size antibodies in plants, N- 13 terminal signal sequences are introduced for targeting of the individual chains to the ER. C-terminal ER retention signals like KDEL can also be introduced to protect antibodies from cytosolic proteinases and facilitate optimal purification. Expression levels of up to 1% of total soluble protein (TSP) have been achieved using this method (Artsaenko et al, 1995; De Jaeger et al, 1999). In 2002, total global production of antibodies was approx. 700 kg at a cost of $1000 per gram. Expression levels of 1% TSP equal approx. 0.2-0.4 g of antibody per tobacco plant or -50 kg of antibody per hectare have been achieved (Turpen et al, 1995). Negating losses due to processing, the 2002 global production of antibodies would thus only require 15 hectares of tobacco. The expression of antibodies in seeds of plants will simplify the storage and purification procedures and indeed it has been reported that a scFv expressed in seeds of transgenic tobacco accumulated to 0.67% of TSP (Fiedler and Conrad, 1995). A separate report shows that intact scFvs could be stored in seeds at room temperature (RT) for at least one year (De Wilde et al, 1999). 2.1.4. Selection of recombinant antibodies from antibody libraries An antibody library refers to a repertoire of antibodies, where the antibodies are displayed on a vehicle, such as a ribosome, phage or cell, and can be selected by panning. Screening refers to the process of selecting the desired binders after panning. One of the earliest methods used to pan a library involves cloning the antibody gene repertoire into a "k bacteriophage expression vector. After infection of bacterial cells, grown on agar plates, clear zones (plaques) are formed, containing antibodies from lysed cells. The plate is overlayed with a filter membrane to bind proteins and reacted with a 14 radiolabeled antigen and spots containing desired antibodies can be visualised using autoradiography. A single bacteriophage clone associated with each plaque is retrieved from the plate and amplified in fresh cells after which the DNA corresponding to single antibody clone can be obtained and used (Huse et al, 1989). The advent of naive libraries or non-immunised libraries and high-throughput screening means there is no longer a strict requirement for animal immunisations (Churchill et al, 2002). High-throughput screening is made possible by establishing a direct physical link between a gene, the protein it encodes and the molecule recognised by the protein. Three main display technologies exist to create this link; phage display, cell surface display and ribosome display. 2.1.4.1. Phage display Phage display offers an efficient and flexible way for the generation of antibodies by creating large and diverse libraries of antibody variable domains, which can be screened in vitro using phage display technology (Hall et al, 1997). Various antibody fragments, including VHHS, are presented on the phage coat protein surface, by expression from a phagemid vector, thus creating a genotype-phenotype linkage. This linkage is exploited to select and enrich for antibodies with high specific affinities for a given antigen and to co-select the concomitant gene. Therefore, screening is significantly reduced in comparison to hybridoma technology and large repertoires of antibodies can be screened within weeks. Phage display is most advantageous with regard to its simplicity and the stability of the phage particles. However, because protein synthesis and assembly of 15 phage particles takes place in E. coli, the library size is restricted by the DNA transformation efficiency. Thus, in contrast to e.g. ribosome display, phage display is more suited to smaller immune libraries than to larger naive libraries. For a detailed review of phage display see Dufner et ah (2006) and Yau et ah (2003). The most common filamentous phages used are Ml3, fd and fl. The antibody genes are fused to the bacteriophage genes encoding surface-exposed proteins, such as glllp. Upon infection, the altered phage uses the host's biosynthetic machinery to reproduce itself with the antibodies displayed on its surface. An infected E. coli cell can produce over a thousand identical phage particles (Smith, 1988). The exposed antibody is selected by binding to immobilised antigen during panning. The phage expressing this antibody is later desorbed and can be used for re-infection of E. coli cells (Maynard, 2000). Usually 3-6 rounds of panning are sufficient to eliminate the majority of non-binders and in so doing enrich the chances of selecting specific antibodies. The murine and human immune systems are capable of selecting antibodies from B-cell repertoires of 5 x 108 and 1012 cells, respectively (Rolink and Melchers, 1993). Phage selection seems to be at least as powerful as immune selection and repertoires of 2 x 107 clones are common (Winter et ah, 1994). Antibodies with affinities of 10"n M, at least as high as those of hybridomas, have been found by phage display (Katz, 1997). In addition, some phage-display libraries have been constructed to select proteins with catalytic activity, e.g. hen egg-white lysozyme, P-lactamase, subtilisin, alkaline 16 phosphatase, muclease and P-galactosidase (Maenaka et al, 1996; O'Neil and Hoess, 1995). Functional peptides have also been displayed on other viruses such as tobacco mosaic virus (Turpen et al, 1995), baculovirus (Ernst et al, 2000) and herpes simplex virus (Spear et al, 2003). 2.1.4.2. Cell surface display Microbial cells such as E. coli and S. cerevisiae are used to display multiple copies of an antibody. To screen such a library, the cells are incubated with a fiuorescently tagged ligand and thus become labelled if they display the correct antibodies. Fluorescence Activated Cell Sorting (FACS) is used to isolate the labelled cells. This system is simple and enables affinity maturation, due to the ease of optimisation of the ligand concentration and the time for dissociation of antibody:ligand complexes. However, there is a bias for well-expressed antibodies, because the cell sorter will detect only cells with a minimum expression of 1000 antibodies per cell. In addition, the FACS method can only screen 5xl08 clones, thus limiting library complexity (Boder and Wittrup, 1997). 2.1.4.3. Ribosome display Rather than being expressed on phage particles or on cells, the antibodies are displayed on ribosome complexes. This means that transcription, translation and selection can occur in vitro. This enables the major advantage of using ribosome display, namely the ability for antibodies to be affinity matured between each round of panning, without subcloning. The DNA and subsequent mRNA construct is designed with a spacer, usually glllp, and no stop codon, so that when translated from the mRNA, the protein remains bound to the 17 ribosome and the mRNA, creating a complex, known as the antibody-ribosome-mRNA (ARM) complex (see Figure 5). It is through this genotype-phenotype linkage that one can screen for the genes associated with a functional protein (in this case an antibody). Library amplification Cloning and expression i ^W\y/^ Reverse In vitro transcription transcription &PCR & translation Soluble selection Figure 5. The in vitro ribosome display selection cycle. After library amplification the dsDNA is transcribed to mRNA. The ribosomes attach to the ribosome binding site and translate the produced mRNA into proteins. All molecules remain attached to each other, because there is no stop codon on the mRNA, and thus an antibody- ribosome-mRNA (ARM) complex is formed. In this example, selection is performed using streptavidin coated magnetic beads which bind biotinylated antigen. The dsDNA, which is used for further panning or for cloning and expression, is produced by reverse transcription PCR. 18 He et at. (1997) were first to describe the ribosome display method. In their paper they point out that the major advantage of ribosome display lies in the ability to perform RT- PCR on the mRNA, while it is still attached to the ARM complex. Another advantage is that in contrast to the phage display method, the library size is not limited by transformation efficiency and the library does not exhibit a bias against bacteriotoxic proteins. Potentially, a library size of 1015 different sequences can be made, enabling development of technologies that mimic the mammalian immune response, which may allow high affinity antibodies to be produced without animal immunisation (Roberts, 1999). 2.1.5. Affinity maturation Antibodies isolated either from animals or from repertoire libraries commonly exhibit 6 9 affinities (KD) in the range of 10' and 10" M (Maynard and Georgiou, 2000). Affinities of antibodies can be obtained using competition ELIS A, surface plasmon resonance and fluorescence polarisation. The surface plasmon resonance technique is the most sensitive and involves immobilising the antigen on a dextran matrix sensor chip; the antibody flows over the matrix and binding is detected by a decrease in light scattering intensity at a given incidence angle (Maynard and Georgiou, 2000). To obtain affinities in the picomolar or even subpicomolar range, some form of maturation usually has to take place. These techniques are usually applied to the CDR3, because it contributes most of the antigen-binding contacts, and include site-specific, random and/or combinatorial mutagenesis, chain shuffling, artificial extension of the 19 CDR3 and recombination of preferred mutations from any of the above methods (Maynard and Georgiou, 2000). Furthermore, in vitro mutagenesis can broaden the range of specificity of an antibody. For example, in vitro mutagenesis of the CDR1 and CDR3 regions of an anti-HIV antibody resulted in improved binding to a broader range of human immunodeficiency virus (HIV) type 1 variants (Barbas et al, 1994). Combinatorial mutagenesis works on the principle that, using degenerate primers, libraries of mutant antibodies are created by randomising CDR3 residues. If a crystal structure of the Ab-Ag complex is available the selection of residues can be narrowed. The crystal structure also enables more rational structure-based mutagenesis, such as site- directed mutagenesis. X-ray crystallography results by Chen et al. (1999) show that only small changes induced by mutation can have significant effects on the energetics of Ab- Ag interactions. Chain shuffling involves the pairing of multiple light chains with heavy chains to give rise to functional antibodies with modulated affinity. Alternatively, extending the length of a CDR is useful for incorporating additional Ab-Ag contacts (Maynard and Georgiou, 2000). The immune system does not have an optimal way of producing small molecule binding pockets. The extension technique is useful for this, as shown by Lamminmaki et al. (1999), who added up to four amino acids to the CDR2 loop of an estradiol-specific 9 10 Ab, thereby increasing its affinity from a KD of 2.6 x 10" to 2.2 x 10" . Effects of beneficial mutations have been shown to be additive (Wells, 1990) and several of the above mentioned techniques can be combined to increase overall affinity of antibodies. 20 2.1.6. Antibody expression systems 2.1.6.1.Expression in E. coli Recombinant antibodies are expressed efficiently in E. coli. However, oxidation of cysteine thiols into disulfides is necessary for proper folding of proteins and can occur only in non-reducing environments. Complete protein folding in E. coli can be achieved by exporting the antibody fragments across the inner cell membrane into the periplasmic space, which is accomplished by attaching a bacterial leader peptide, such as pelB, phoA or ompA to the N-terminus of the antibody fragment. The fully folded antibodies are recovered from the periplasmic space by osmotic shock or from total cell lysates. Furthermore, prolonged high-level expression of antibodies at 37°C renders the outer membrane of E. coli permeable allowing the protein to be recovered from the culture media (Skerra and Pluckthun, 1988; Better et al, 1988). There are also mutant strains of E. coli with oxidizing conditions in the cytoplasm that give substantially higher yield of properly oxidised proteins than wildtype. However, these strains do not grow as rapidly as wildtype E. coli (Bessette et al, 1999). Very high concentrations of scFv, exceeding 1-2 g/L in a fermentor, have been achieved by periplasmic expression (Carter et al, 1992). However, in general, expression can vary greatly and often the antibodies fail to fold properly and may aggregate in the periplasm, thus becoming cytotoxic to the E. coli (Maynard and Georgiou, 2000). Co-expression with chaperone proteins that assist folding, such as GroESL chaperonins, can alleviate this problem. Minor amino acid substitutions can also have dramatic effects on protein solubility and expression levels, as was shown by Nieba et al. (1997), who only 21 substituted one solvent-exposed hydrophobic amino acid, yet increased the expression 25-fold. Furthermore, CDR grafting into the framework of well expressed antibodies has been used to improve expression of scFvs (Jung and Pluckthun, 1997). However, high expression often leads to aggregation of the antibodies into insoluble inclusion bodies. Nonetheless, in the rare case that an antibody is not negatively affected by denaruration and subsequent refolding, one can take advantage of these inclusion bodies as a purification step (Lilie et al, 1998). 2,1.6.2. Expression in yeast In general, carbohydrate moieties of glycoproteins play important roles in protein folding and assembly (De Wilde et al, 1999). Unlike the prokaryote E. coli, eukaryotic yeasts can provide advanced folding and secretion of proteins and, unlike mammalian expression systems, can be grown rapidly on simple growth media (Verma et al, 1998). Both whole chimeric antibodies and Fab fragments have been produced successfully in yeast (Horwitz et al, 1988). Proteins are also expressed in a more soluble state than in E. coli (Ridder et al, 1995) and post-translational degradation is reduced in yeasts (Verma et al, 1998). Yeasts are also capable of adding almost as many complex carbohydrates as hybridoma cell lines (Kukuruzinska et al, 1987). Furthermore, using either episomal vectors, which propagate extrachromosomally, or integrating vectors, which homologously recombine with the genome, concentrations of secreted antibody fragments have reached 100-250 mg/L (Eldin et al, 1997; Ridder et al, 1995). 22 Several different species of yeast have been used for heterologous protein expression (Cereghino and Cregg, 1999). The most common are Pichia pastoris (methanol utilising) and Saccharomyces cerevisiae (sugar/starch utilising) (Waterham et al, 1997; Shen et al, 1998; Sears et al, 1998; Lee et al, 1994), while lactose, xylose and alkane/fatty acid utilising yeasts, such as Kluyveromyces lactis, Pichia stipitis and Candida albicans, are used to a lesser extent (Muller et al, 1998; Den Haan and Van Zyl, 2001; Staib et al, 2006). Regardless of the species used, engineering expression of foreign proteins in yeast requires that the following steps are taken: DNA encoding the foreign DNA is cloned into a vector containing a yeast promoter, transcription termination sequences, a potential epitope tag and flanking genome-specific sequences for homologous recombination into the yeast genome; the vector is introduced into the yeast by chemical transformation or electroporation; the foreign protein expression is initiated in the host by supplying appropriate promoter-inducing substrates; and finally the protein of interest is purified using an introduced epitope tag or by specific affinity chromatography. Pichia pastoris has been a preferred protein expression system ever since it was first grown on a large scale by 'Phillips Petroleum' in the 1970s and protocols for the expression of proteins under the control of the alcohol oxidase gene were developed in conjunction with the Salk Institute (Macauley-Patrick et al, 2005; Cereghino and Cregg, 2000). Pichia pastoris is one of four methylotrophic yeasts capable of using methanol as a sole carbon source (Faber et al, 1995; Hollenberg and Gellissen, 1997). Two genes, AOX1 and AOX2, encode alcohol oxidase, the first enzyme in the methylotrophic pathway. The former gene encodes the majority of alcohol oxidase produced in P. 23 pastoris, is tightly regulated and induced by methanol, and thus has been altered for use in a variety of cloning vectors, such as pPICZa from Invitrogen. A notable feature of this vector is the secretion signal sequence from the Saccharomyces cerevisiae a factor peptide allowing for secretion into the relatively protein-scarce culture media ofPichia pastoris (Scorer et al, 1993). Glycosylation patterns of P. pastoris are favourable since it is capable of N-glycosylating at Asn-X-Ser/Thr sites along a protein, albeit at a lower frequency than found in S. cerevisiae (Grinna and Tschopp, 1989). O-glycosylation has been reported in P. pastoris with the addition of solely mannose residues, in contrast to the variety of sugars added in higher eukaryotes, including N-acetylgalactosamine, galactose and sialic acid (Cereghino and Cregg, 2000), but no studies showing negative effects on expression of proteins due O-glycosylation have been reported in the literature. What sets P. pastoris apart from other expression hosts is the quantity and diversity of proteins that it is capable of producing. Many complex proteins of various origin have been expressed at a high rate and concentration in P. pastoris. For example, E. coli and bovine proteins have been expressed at 6.4 and 350 g/1, respectively (Cereghino and Cregg, 2000; Chen et al., 2004; Peng et al., 2004). Thus, it is likely that P. pastoris will continue to be a widely used expression host in the future as the need for large quantities of proteins for medical, agricultural and industrial applications continues to grow. 24 2.1.6.3. Expression in cell culture There are two main cell culture systems in use for antibody expression, namely mammalian and insect-cell culture. The main advantage of mammalian cell culture is the proper synthesis, processing and secretion of mammalian proteins that are to be used as very specific human therapeutics. For example, many therapeutic proteins are required to be folded and glycosylated so as to avoid detection and clearance by the human immune system. The protein processing of murine cells results in addition of gala(l,3)gal epitopes, which are recognised by natural antibodies in humans, resulting in quick clearance of murine derived drugs from the patients body, thus limiting their usefulness. Yields of antibodies from mammalian systems are relatively low and typically four to five times less than from E. coli. They require a specific nutrient solution for growth and are very sensitive to any deviations from optimal conditions. Antibodies produced from mammalian cells can also carry human-compatible viral and bacterial diseases, thus posing a health risk to consumers. Therefore, only therapeutics that specifically require mammalian processing to function are usually produced in mammalian cell culture (Vermaefa/., 1998). Insect-cell culture can perform most of the post-translational alterations used by higher eukaryotes, but cannot process mature oligosaccharides to the extent found in mammals (Verma et al, 1998). In addition, some proteolytic cleavage sites are insufficiently or not at all recognised in insect-cell cultures (Kuroda et al, 1986). Using a transient baculovirus-mediated expression system, insect-cell cultures typically produce more protein than mammalian cell culture (1-500 mg/L) and functionally active antibodies 25 have been produced using this expression system (Verma et al, 1998; Hasemann and Capra, 1990; zu Putlitz et al, 1990). 2.1.6.4. Expression in plants Plants offer several advantages over the expression systems mentioned above. They are capable of eukaryotic modifications, are generally inexpensive to grow and do not require fermentors for growth (Fiedler and Conrad, 1995). Plantibodies undergo complex glycan modifications, albeit with differences compared to mammalian systems (Hein et al, 1991). However, the glycans added to plantibodies do not interfere with folding and do not seem to alter the solubility of the antibody (Hiatt et al, 1989; During et al, 1990; De Neve et al, 1993; Ma et al, 1995; van Engelen et al, 1994). Furthermore, a recent report by McLean et al. (2007) shows that a human anti-Pseudomonas aeruginosa serotype 06ad IgGl expressed in transgenic tobacco is indeed capable of recruiting immune system effector function in vitro. Downstream processing is a potential constraint, as impurities, such as secondary metabolites and pesticides, present in plant tissues can obstruct subsequent use (Sheedy and Hall, 2001). For a gene to be stably integrated into a plant genome, it is desirable to incorporate it into the nuclear DNA, although plastid expression may be suitable for some constructs. There are three main transformation methods used, namely Agrobacterium-mediated transformation, particle bombardment and transient viral transformation. Agrobacterium tumefaciens and rhizogenes, two soil-borne micro-organisms, naturally infect plants and cause crown gall and hairy root disease, respectively. A transformation technique, 26 derived from the understanding of how DNA is transferred to the plant host in this disease, is now most widely used to transform dicotyledonous plants (Hammond et al, 1999). Agrobacterium-mediated transformation allows insertions of one or a few copies of the transgene specifically into actively transcribed regions of the genome and insertions of up to 150 kb have been reported (Hamilton et al, 1996). The large tumour- inducing (Ti) or root-inducing (Ri) plasmids are the vectors for transformation and contain the transferred DNA (T-DNA) (Van Larebeke et al, 1974). T-DNA is transferred in a conjugation-like manner and covalently linked to the host DNA (Chilton et al., 1977). The original Ti plasmid has been modified extensively and it is evident that three main genetic components are necessary for cell transformation. First, two 25 bp long border sequences, called the left border and the right border, are required for Agrobacterium to recognise a sequence as T-DNA (Hammond et al, 1999). Second, a series of about 25 linked virulence genes, arranged into seven operons, are necessary for proper insertion of T-DNA (Stachel and Nester, 1986). Third, a series of virulence genes, which code for the synthesis of cellulose fibrils for attachment to the wounded plant, are necessary (Hammond et al, 1999). After infection is complete, callus formation is induced in cell culture to yield transgenic seedlings. A noteworthy example of Agrobacterium mediated antibody transformation is that of Yuan et al. (2000). They successfully transformed Arabidopsis with an anti-zearalenone scFv. This plantibody bound zearalenone with high affinity and is thus a good example of successful transformation of crops with mycotoxin-neutralising antibodies. However, gene silencing can account for instability in 27 plantibody expression. De Neve et al. (1999) have shown that antibody expression in homozygous transgenic Arabidopsis lines can be reduced by 100-fold compared to original expression levels. Monocotyledonous plants and conifers are classically difficult to transform with Agrobacterium and thus particle bombardment, also called biolistic transformation, is preferred for these plants. This direct DNA delivery system, where DNA coated projectiles are fired into cells, can stably transform plants and is especially advantageous when insertion of marker or vector genes is to be avoided. However, it is unfavourable in the sense that it tends to introduce multiple copies of DNA at one site. These multiple genes can rearrange themselves into complex arrays of fragmented genes, which are difficult to segregate by breeding (Hammond et al, 1999). Biolistic transformation is not limited to plants; transformations of bacteria, fungi and even animals have been reported (Smith et al, 1992; Toffaletti et al, 1993; Johnston, 1990). In particle bombardment, plasmid-coated gold or tungsten microparticles (0 = 0.5-5 um) are fired into the cells. The particles are small enough to not cause severe damage to the cells, but carry enough DNA for the purpose of transformation. The particles are washed and incubated with plasmid DNA, which, when precipitated with ethanol or CaCk, binds to the microparticles. These microparticles are adsorbed to a macro-carrier, such as a polymer sheet, and the whole assembly is fired through a screen toward the tissue. The macro-carrier is retained by the screen in front of the tissue, while the microparticles end up penetrating the tissue. The depth of penetration is controlled by modifying the 28 explosive burst, by changing the distance between the gun and the target, and by using different size particles (Hammond et ah, 1999). Once in the cell, the majority of the plasmids are degraded, some express independently and a few recombine with host plasmids or chromosomal DNA to make stable transformants. Several improvements to the particle gun have been made and today devices such as the particle inflow gun and the micro-targeting device have made it possible to directly accelerate the DNA-coated particles without the need for a macro-carrier (Finer et ah, 1992; Sautter et ah, 1991). Viral vectors are used to transiently transform cells, when stable transformation is either not desired or not possible. Tobacco mosaic virus (TMV) is a well-studied virus and was the first virus for which the virion structure and coat protein sequence was determined. It is a single-stranded virus with plus-sense RNA and can thus replicate directly after infection of the host. The genome consists of four open-reading frames (Goelet et ah, 1982), which code for two replicase proteins, one movement protein and one capsid protein. Together with its relatives in the tobamovirus group, TMV has several advantages for use as a transient transformation vector, including the malleability of its cDNA and the high rate of transmittance and plant infection (Hammond et ah, 1999). Recently, a novel TMV over-expression vector was constructed through gene deletions that eliminate the need for separation of protein from virion particles and reduces inadvertent plant-to-plant movement of the virion particles, all which simplify protein purification and improve biocontainment (Lindbo, 2007). Tobamoviruses can replicate for up to six months, but eventually all modified tobamoviruses return to their crippled wild-type state (Kearney et ah, 1993). This system can be beneficial for transient 29 production of proteins in a host, where stable transformation is not wanted. Transient systems are desirable if a certain therapeutic, such as an individual cancer drug, is required quickly and stable transformation is too costly and time-consuming. For short term production of therapeutics, viral expression systems yield more antibody than stable transformation systems, because the viruses take over the host protein manufacturing machinery (Plesha et al, 2007). The choice of protein expression system depends on the application intended for the produced protein. Applications can vary from in planta disease resistance, herbicide tolerance, modulation of metabolism, to ectopic protein production for ex planta uses. For example, Olea-Popelka (2005) showed that the highest expressors of transgenic anti- picloram scFv expressing tobacco could resist a constant exposure of 5 x 10"8 M of the pesticide picloram. Production of antibodies for ectopic uses may prove especially lucrative for the medical industry. 'Molecular pharming' focuses heavily on Nicotiana tabacum as a model expression system (Giddings et al, 2000). It is among the highest biomass and protein-producing crops and up to 5000 kg of protein per hectare can be produced per year (Turpen et al, 1995). Functional antibodies have been produced in tobacco in fractions up to 1.3% TSP (Hiatt et al, 1989; Schouten et al, 1996; Makvandi- Nejad et al, 2005). Fiedler and Conrad (1995) have produced scFv in seeds of tobacco and stored the seeds for one year at RT without loss of scFv protein or antigen-binding activity. In the future, commercial production of economically interesting biomolecules may occur in grain and oilseed crops, because of the ease of downstream processing. 30 2.2. Cellulose-protein scaffolds To develop antibodies as biotechnological tools it is useful to create a scaffold that allows antibodies to be displayed in various ways under different conditions. This section describes cellulose and cellulose binding domains, which together can form a low-cost and malleable scaffold for the presentation of antibodies to create bioactive filters for the capture of haptens, proteins and pathogens. 2.2.1. Structure of cellulose in plants Degradation of photosynthetically fixed carbon is mainly performed by bacterial and fungal glycosidic hydrolases (GHs), which cleave long and branched polysaccharides into small, soluble sugars. All GHs possess a non-catalytic carbohydrate binding module (CBM) in addition to their catalytic domain. GHs can degrade cellulose, hemicellulose, starch, maltose, xylan and chitin amongst others, but this review will focus only on the first two carbohydrates, because they constitute the major cell wall components of plants (Clarke, 1997). Cellulose is a linear homopolysaccharide composed of < 10,000 repeating 6-carbon 0-D- glucopyranose (glucose) units (Hilden and Johansson, 2004) forming chains by P-1,4- glucosidic bonding (see Figure 6) (Clarke, 1997). Adjacent chains are hydrogen bonded forming a straight, dense and insoluble cellulose microfibril (Hilden and Johansson, 2004). Crystalline regions refer to areas where chains are staggered to give optimal hydrogen bonding, whereas amorphous regions refer to less ordered areas (Clarke, 1997). 31 Figure 6. Molecular breakdown of a dense cellulose microfibril. Repeating 6-carbon glucose units linked by P-l,4-glucosidic bonds (circled) form a repeating chain with hydrogen bonds (dotted lines) to neighbouring chains. Adopted from Clarke, 1997. Other polysaccharides also exist in the plant cell wall. They often form hemicelluloses, consisting primarily xylanose, which in turn consists of repeating pentose xylan (P-l,4-D- xylopyranosyl). Xylanose branches frequently incorporates many different carbohydrates such as L-arabinose, D-galactose, D-galacturonic acid, D-glucuronic acid, D-glucose, D- mannose and D-ribose (see Figure 7) (Clarke, 1997). 32 Substituted xylanose backbone HO (n) HO HO—7-~^ 1 s, H0-V\0/rH S/ \ HOOC OH Glucuronic acid side chain Ribose side chain OH OH HO. Xylan Figure 7. An example of a cell wall hemicellulose. Xylanose, formed from repeating xylan units, is highly branched with a number of carbohydrate side chains. Adopted from Clarke, 1997. 2.2.2. Enzymatic digestion of plant cellulose Microbes convert cellulose and hemicellulose of plants using an array of enzymes: cellulases, aka endoglucanases (l,4-(3-D-glucan glucanohydrolases; EC 3.2.1.4), cellobiohydrolases, aka exoglucanases (1,4-p-D-glucan cellobiohydrolases; EC 3.2.1.91), xylanases (1,4-p-D-xylan xylanohydrolases; EC 3.2.1.8) and p-xylosidases (1,4-p-D- xylan xylohydrolases; EC 3.2.1.37) all hydrolyse P-l,4-glycosidic bonds between glucopyranose units (see Figure 8) and are thus termed glycosidic hydrolases (GHs) (Gilkes et al, 1991; Hilden and Johansson, 2004). 33 Beta-1,4-glycosidic bond Cellulose (n) Cellulase or cellobiohydrolasc + H20 Cellobiose Glucopyranose (glucose) Figure 8. Degradation of insoluble cellulose by hydrolysis of p-1,4-glycosidic bonds to single, soluble sugar units. Adopted from Clarke, 1997. 34 2.2.3. Cellulose binding modules 2.2.3.1. Background GHs possess a catalytic module and a cellulose binding module (CBM) connected by a flexible linker. The CBM functions to anchor and specifically concentrate the enzyme on the immobilised substrate (Hilden and Johansson, 2004), potentially accelerating polysaccharide hydrolysis (Bolam et al, 1998). Although the existence of CBMs was envisaged in 1949 (Reese et al, 1950), the first two were discovered in the late 1980s in the fungus Trichoderma reesei (van Tilbeurgh et al, 1986) and the bacterium Cellulomonas fimi (Gilkes et al, 1988). As of 2005 there are 43 defined families of CBMs, ranging in size from 4 to 20 kDa and grouped according to amino acid sequence similarities (CAZy, 2007). No universal style of nomenclature for CBMs has been adopted, although the style of Boraston (2001) is frequent in recent literature and describes the name of the GH and the family it belongs to; e.g. XynlOACBM2a refers to the xylanase 10A from Cellulomonas fimi belonging to the family 2a of CBMs. Several three-dimensional structures of CBMs from various families have been solved by nuclear magnetic resonance (NMR) and crystallography (Boraston et al, 2004). Generally, aromatic amino acids represent the primary drivers behind CBM specificity and affinity. For example, mutational studies have shown that the tryptophans of the CBM of xylanase A from Pseudomonas fluorescens play a primary role in hydrogen bond formation with hydroxyl groups of sugar residues (Poole et al, 1992). 35 2.2.3.2. Family 2a cellulose binding modules Family 2a CBMs are the best characterised family and they bind with high affinity to insoluble, highly crystalline cellulose, bacterial microcrystalline cellulose and with less affinity to partially crystalline cellulose, but display little or no binding to soluble carbohydrates (McLean et ah, 2002; Nagy et ah, 1998). This family is classified as surface-binding, because the planar binding site is complementary to the flat surfaces of crystalline cellulose (McLean et ah, 2000). The family 2a CBMs share several common features of their ligand binding site, such as linearly arranged and regularly spaced solvent-exposed aromatic residues (Reinikainen et ah, 1992; Poole et ah, 1992; Din et ah, 1994). The family 2a xylanase 10A CBM from Cellulomonas fimi (Xynl0ACBM2a; GenBank AAA56792) is a 108 amino acid, C-terminal CBM with an association constant (Ka) of ~106 M"1 for crystalline and amorphous cellulose (Ong et ah, 1993; Creagh et ah, 1996). It consists of a nine-stranded p-sheet polypeptide with p-barrel topology and possesses three solvent-exposed tryptophans, at positions 17, 54 and 72, which are involved in substrate binding (Xu et ah, 1995; McLean et ah, 2000). Xynl0ACBM2a adsorbs to crystalline cellulose according to equilibrium chemistry, but no desorption of CBM from the substrate is found during buffer exchange (Tomme et ah, 1998). The resulting high affinity is thought to be driven by the dehydration of the protein and the sorbent. This high affinity binding makes family 2a CBMs and Xynl0ACBM2a in particular popular tools for biotechnological applications (Creagh et ah, 1996). 36 2.2.3.3. Applications ofCBMs Cellulose and related substrates exist in many forms, can be prepared to suit custom needs, yet are chemically inert and are relatively cheap. This makes CBMs versatile, safe for medical use and amenable to scale-up in industrial applications (Levy and Shoseyov, 2002). The high affinity and specificity of CBMs may account for their widespread use, of which the most prevalent is the fusion of a CBM to biologically active molecules, such as enzymes, antibodies, peptides, cytokines and even whole microorganisms. These applications are made possible because both fusion partners retain all or most of their native function (Jervis et al., 2005) and because of the ease of expression of such fusion products in both bacterial and yeast expression systems. These fusion products are primarily exploited for purification of target molecules by cellulose affinity chromatography (Linder et al, 1998; Levy et al, 2002). Many specific biotechnological and industrial applications of CBMs are only beginning to be exploited. One example is to immobilise whole cell catalysts on tailor made cellulose matrices as was demonstrated by Lehtio et al. (2001). They immobilised recombinant Staphylococcus on cotton fibres through cell surface anchoring and display of a CBM. Furthermore, Shpigel et al. (2000) demonstrated the use of Protein A-CBM for isolating IgG from serum and for detection of IgG with stained cellulose beads in Western blots. Berdichevsky et al. (1999) used a CBM that can bind cellulose in 6 M urea as a fusion partner for insoluble proteins, and was able to recover fully functional and soluble target proteins by first binding the fusion proteins to paper and slowly reduced the denaturing urea concentration to finally yield -60% properly folded and 37 functional protein. In another application, certain CBMs have shown to have dispersive effects on oral polysaccharides that comprise dental plaques (Fuglsang and Tsuchiya, 2001). Finally, Ofir et al. (2005) created protein-CBM fusion and showed improvement in folding and display of proteins and peptides on cellulose microarray chips compared to conventional protein arrays. The varied use of CBMs has attracted much interest from industry and several patents have been filed. For example, von der Osten et al. (2000) filed a patent for fusing different enzyme domains with CBMs to remove stains or bleach stains on cellulosic clothing like cotton, while Berry et al. (2001) filed a patent on binding fragrances to cellulosic clothing using CBMs. 38 2.3. Epidemiology of Pythium aphanidermatum To show that antibody-CBM fusions are feasible biotechnological tools for crop protection, it is important to develop a crop protection model that is simple to characterise, yet is applicable to solving the real problems of farmers and society at large. This section describes the epidemiology of the root rot causing pathogen Pythium aphanidermatum and the role of its necrosis inducing elicitor, whose neutralization by anti-PaNie antibodies could provide a model for crop protection using antibody-CBM fusions bound to paper. 2.3.1. Physiology of Pythium aphanidermatum Pythium aphanidermatum belongs to a class of parasitic microbes called the oomycetes. Its motile spores can move in water and moist soils to find their host by chemotaxis (Hardham, 1992). The oomycete class, which also includes the genus Phytophthora, is more closely related to the heterokont algae of the kingdom Chromista than to fungi. Therefore, the oomycetes have been placed into their own kingdom and are theoretically no longer classified as fungi (Van de Peer and De Wachter, 1997; Dick, 1997). The main body of P. aphanidermatum, the mycelium, colonises roots both internally and externally (Owen-Going, 2002). The mycelium is made up of filamentous branches, called hyphae, which are approx. 10 urn wide and off-white in appearance. During certain conditions, the hyphal branches swell and form terminal complexes called sporangia, approx. 20 urn wide (Plaats-Niterink, 1981). Sporangia are asexual reproductive structures, in which zoospores are formed. Sporangia of P. aphanidermatum have a 39 filamentous, inflated morphology. The female oospores are formed terminally on the hyphae in oogonia. Growth and reproduction of P. aphanidermatum are favoured by aquatic conditions (Plaats-Niterink, 1981). The zoospores of P. aphanidermatum are wall-less, small (0 = 5-20 um) and ovoid cells with a posterior whiplash ftagellum and an anterior tinsel flagellum (see Figure 9). Encysted zoospores are about 12 um in diameter (Hardham, 1992). Figure 9. Scanning electron micrograph of"a Phytophthora zoospore showing the ventral groove into which the anterior and posterior flagella are inserted. Source: The Biology Teaching Organisation, University of Edinburgh. The spherical oospores are 18-22 urn in diameter and have a cell wall of 1-2 um. The globose oogonium is 20-25 um in diameter and thus only contains one oospore. The oogonium ($) is sexually fertilised by antheridia (S) (Plaats-Niterink, 1981) (see Figure 10). 40 Figure 10. Fertilisation of oogonium by two antheridia in Pythium. Antheridia are indicated by arrows. Source: P.B. Hamm, Oregon State University 2.3.2. Associated disease The symptoms of disease caused by P. aphanidermatum include root browning followed by root stunting, root hair initiation, root swelling and tissue maceration (Owen-Going et ah, 2003). Root browning is likely caused by phenols and tannins produced by the plant as a response to infection (see Figure 11), although prolonged symptomless development of the disease below 18°C has been reported in hydroponic systems (Sutton et ah, 2006). Schnitzler and Seitz (1989) indicated that production of phenolic compounds by the host occurs through stimulation of the phenylpropanoid pathway, perhaps induced by elicitors released by the pathogen. The phenolic compounds produced act as signal molecules in the plant-microbe interaction and as fortification of cell walls against attack (Hahlbrock and Scheel, 1989). Symptoms that develop later in pathogenesis are likely caused by toxic substances such as pectinolytic and cellulolytic enzymes (Mellano et ah, 1970; Blok, 1973; Mojdehi et ah, 1990; Huet et ah, 1995). Microscopic investigations showed 41 that upon penetration of roots by a penetration peg, the protoplasm became granular and displayed a yellow and brown colour (Endo and Colt, 1974; Owen-Going et al, 2003) and after infection, haustorium-like structures are formed within the host cells (Kamoun et al, 1999). When root browning becomes severe, infection by P. aphanidermatum eventually leads to wilting and plant death in both soil and hydroponically grown crops (Paulitz, 1997). Figure 11. Extreme root rot of pepper caused by P. aphanidermatum compared to non-infected control. Source: Prof. J.C. Sutton, University of Guelph. 2.3.3. Occurrence and pathogenicity Species of the genus Pythium are found in soils and aquatic environments worldwide. P. aphanidermatum is a typical plant parasite of warm regions and its pathogenicity has been reported in many plants including conifers, corn, legumes, grasses, peppers, sugar beet, sugar cane, tobacco and tomato. It can cause diseases known as root rot and damping-off, stalk and rhizome rot, soft rot, fruit rot and cottony blight. Infection can occur at 18-30°C, but is optimal at 30-35°C (Plaats-Niterink, 1981; Sutton et al, 2006). 42 Release of zoospores often occurs after a temperature decrease of 2-5°C and only in wet conditions (Plaats-Niterink, 1981; Rahimian and Banihashemi, 1979) (see Figure 12). •a Figure 12. Release of zoospores from sporangium in P. undulatum. Source: P.B. Hamm, Oregon State University. Flooding of fields cause increases in gases such as ethylene and CO2 that play a role in plant growth (Glinski and Lipeic, 1990). It has been proposed that these conditions result in the initiation of Pythium root rot disease because these gases reduce nutrient uptake by roots, causing localised stress and making the roots more permeable; thus, releasing sugars and amino acids, which in turn may attract chemotactic zoospores of potential pathogens such as Pythium aphanidermatum (Jackson and Kowalewska, 1983; Jarvis, 1992). In hydroponic systems, the community of microbes is much less diverse, and often much sparser, than in field crops, leading to less natural suppression of pathogens by competition for nutrients and other mechanisms (Stanghellini and Rasmussen, 1994; Paulitz, 1997). Pathogenic species of Pythium, Phytophthora and Fusarium are estimated 43 to cause 80% of root disease epidemics in hydroponic systems (Schuerger, 1998). P. aphanidermatum is likely the most common Pythium species found in Ontario hydroponic systems (Zheng et al, 2000; Owen-Going et al, 2003) and has been reported as a hydroponic contaminant in Brittany, France (Rafin and Tirilly, 1995) and in Arizona, USA (Gold and Stanghellini, 1985). Contamination of advanced life-support testing facilities by Pythium species and root rot of associated plants have been reported (Jenkins et al, 2000). The higher incidence of root disease epidemics in hydroponic crops compared to field crops is due in part to the conditions of the root zone and facilitated dispersal of spores. A critical difference lies in the availability of root exudates as carbon sources. Roots can exude large fractions (~20%) of the carbon fixed during photosynthesis (Sutton et al, 2006), which is available to the pathogen with little competition from other microbes in hydroponics (Zheng et al, 2000). Investigations of several Pythium spp. in soil have shown that the disease develops mainly in seed and in seedlings (Larkin et al, 1995; Chun and Schneider, 1998), which do not normally survive the infection (Roncadori and McCarter, 1972). Roncadori and McCarter (1972) showed that the intensity of root rot generally decreased with plant age, although severe root rot has been observed in older plants. Studies on the encystment and germination of Pythium zoospores on glass showed that it is possible for Pythium spp. to survive and reproduce in the absence of plants when only an ionic nutrient solution is available (Donaldson and Deacon, 1993). 44 2.3.4. Mechanism of action Zoospores, the principal form of Pythium inoculum, are motile biflagellates, which locate roots or leaf stomata by chemotaxis. Their motility is sensitive to environmental conditions, such as turbulence (Sutton et ah, 2006), but under certain conditions, they are capable of sustaining motility for hours, covering distances of several centimetres, while using endogenous lipid stores (Hardham, 1992). Figure 13. Zoospore cysts of P. aphanidermatum germinating behind the root tip of wheat. Arrow indicates area of encystment. Source: Biology Teaching Organisation, University of Edinburgh. The zoospores readily attach to the surfaces of the host to begin the process of zoospore encystment (see Figure 13). The environmental trigger for encystment is still a highly debated topic among researchers. In Phytophthora, encystment may be triggered by ions, organic molecules, temperature extremes, low water potentials, water turbulence, frequent contact with soil particles and/or chemo-attractants (Hardham, 1992). Root mucilage polysaccharides and pectin have been reported to induce encystment in P. aphanidermatum (Deacon, 1996). Binding of a monoclonal antibody to the zoospore 45 surface also was found to induce encystment (Estrada-Garcia et al, 1990). Turbulence, which is common in the recharge tank and pipelines of hydroponic systems, can also trigger encystment (Holderness and Pegg, 1986). During encystment, the zoospore undergoes structural changes, including detachment of its flagella, build up of a cell wall and adhesive material, as well as large intracellular rearrangements resulting in disorganised organelle distribution (Hardham, 1992). Immunocytochemistry of P. aphanidermatum indicates that adhesive material is stored in large vesicles throughout the zoospore periphery (Estrada-Garcia et al, 1990). The release of the adhesive is triggered by spore hydration (Hardham, 1992). Spore germination in Phytophthora occurs immediately after encystment and can proceed without external supplies of nutrients (Hardham, 1992). A specialised hypha, called a penetration or infection peg, forms from the germinating cyst, the germ tube or the appressoria at the end of the germ tube. It penetrates either directly through the plant cell wall, through the wall at intercellular junctions or through the stomata. Upregulation of genes coding for wall-degrading enzymes aids the penetration process (Callow, 1983). The production of new oospores and zoospores completes the life cycle of P. aphanidermatum. Pythium oospores have been shown to be ingested and later excreted by shore flies and fungus gnats, which can thus transmit the disease between separate hydroponic troughs (Goldberg and Stanghellini, 1990; Jarvis et al, 1993). After initial and localised infection of the root, P. aphanidermatum may be dispersed to infect the entire primary root system, which often leads to death of the plant. P. 46 aphanidermatum is particularly aggressive and usually infects the cortex of the root (Endo and Colt, 1974). Its spore-forming ability permits high dispersal by flow of nutrient solution throughout troughs in hydroponic systems, dormancy over long periods of time and subsequent break-outs when conditions are right. These features allow Pythium aphanidermatum to appear as if it eludes even the strictest quarantine and allows the pathogen to abruptly cause destruction of entire crops (Wakeham et al, 1997). 2.3.4.1. Pythium aphanidermatum Necrosis inducing elicitor (PaNie) Koch et al. (1998) first discovered a 25 kDa protein in Pythium aphanidermatum exudates that caused cell death in carrot cell culture. Three years later, Veit et al. (2001) published the sequence of this Pythium aphanidermatum Necrosis inducing elicitor (PaNie), which consists of 234 amino acids, including a 21 amino acid long N-terminal secretory peptide that signals transport into the extracellular medium. When administered through the stomata of leaves, recombinant PaNie induces a hypersensitive-like response in dicotyledonous plants, such as tobacco, arabidopsis and tomato, but not in monocotyledonous plants such as maize, oat and bosse (Veit et al, 2001). Usually, when a potential pathogen triggers a hypersensitive response, the plant is successful in reducing the rate of infection in three ways; by sacrificing infected tissues through necrosis, blocking further invasion though cell wall depositions (i.e. localised quarantine), and combating the pathogen by production of phytoalexins. In nature, the hypersensitive-like response is unsuccessful in combating P. aphanidermatum infection although PaNie triggers host apoptosis and 4-hydroxybenzoic acid (4-HBA) production, 47 as demonstrated in cultured cells of carrot (Veit et al, 2001). 4-HBA is a precursor in the production of callose deposits in the cell wall and thus should decrease the colonization of roots by the pathogen. However, recent findings suggest that production of phenolic phytoalexins may aid in the proliferation of P. aphanidermatum, possibly by triggering sporangia formation (Owen-Going, 2005). PaNie shares 57-71% sequence similarity with elicitors from the genus Fusarium and Phytophthora (Table 1). The Fusarium oxysporum elicitor Nepl (24 kDa), which shares 57% of amino acid identity with PaNie, was the first of this growing family of elicitors to be described in detail and thus elicitors in this family are called Nepl-Like-Proteins (NLPs) (Gijzen and Nurnberger, 2006). There are presently 44 NLPs from 22 species with a wide taxonomic diversity. They can be identified by a central conserved hepta- peptide motif'GHRHDWE' called the NPP1 domain (Necrosis-inducing Phytophthora Protein 1 domain) (Qutob et al, 2006), and by the presence of two or four cysteine residues. However, amino acid point deletions and heat denaturation studies of the Phytophthora parasitica NLP NPP1 have shown that NLP activity cannot be reduced to this single motif and that the complete tertiary structure may be needed for function (Fellbrich et al, 2002; Gijzen and Nurnberger, 2006). All NLPs tested trigger a comprehensive defence response in Arabidopsis thaliana resulting in programmed cell death with reprogramming of the transcriptome similar to that evoked by bacterial flagellin (Qutob et al, 2006). Nepl induces ethylene production and necrosis in many species, including tomato, pepper, bean and crab apple (Bailey, 1995). Forty-eight hours after application of Nepl (200 nM) as foliar spray extensive necrosis of leaves of 48 Abutilon theoprasti, Papaver somniferum, Centaurea solstitialis, Centaurea maculosa and Sonchus oleraceus was observed. Delivery through the vascular system shows that this protein or an associated soluble downstream signal moves throughout the vascular tissue. Several weed species were infected and it was shown that the necrotic response is species-specific. Corn and wheat did not produce ethylene or become necrotic in response to Nepl, which indicates a resistance in monocotyledons and creates the possibility to use Nepl as a natural herbicide (Jennings et al, 2000). Other NLPs that have been characterised include NPP1 from Phytophthora infestans and Phytophthora parasitica, PsojNIP from Phytophthora sojae, BcNEPl and BcNEP2 from Botrytis cinerea and Nip from Erwinia carotovora and, in addition, putative NLPs have been found in Bacillus halodurans, Streptomyces coelicolor and Vibrio pommerensis sp. nov. CH-291 (Bailey, 1995; Fellbrich et al, 2002; Qutob et al., 2002; Veit et al, 2001; Schouten et al, 2008; Mattinen et al, 2004; Pemberton and Salmond, 2004; Jores et al, 2003). However, the exact mechanism of action of any NLP has not been determined. PsojNIP has been shown to only be expressed in later stages of infection, indicating that it may be involved in aiding the transition from biotrophy to necrotrophy. PsojNIP, lacking the secretory signal, was expressed in soybean and showed no necrosis induction, indicating that the protein has to be excreted for it to be active (Qutob et al, 2002). The fungus Colletotrichum coccodes showed a marked increase in virulence and a broader host range when transformed with Nepl. Strains of Erwinia carotovora lacking Nip cause far fewer lesions and less rot of tissues than strains expressing the protein (Gijzen and Nurnberger, 2006). 49 Table 2. Comparison of sequence similarity between PaNie and a selection of other NLPs. The homology is represented by the percentage positive alignments (Altschul et al., 1997; Henikoff and Henikoff, 1992). Elicitor Species of Similarity Source reference name NLP origin (% positives) Nepl Fusarium oxysporum 57 Bailey, 1995 NPP1 Phytophthora parasitica 69 Fellbrich et al, 2002 PsojNIP Phytophthora sojae 71 Qutob et al, 2002 No name Bacillus halodurans 61 NCBINP_241261gi|15612958 No name Streptomyces coelicolor 41 NCBIAL356932 gi|8246839 No name Vibrio pommerensis 36 NCBIAJ314791gi|14331088 Occurrence of NLPs in such a wide variety of primarily soil-borne organisms indicates that these organisms may share saprophytic or parasitic lifestyles and that this family of proteins aids the process of infection. Note that the percent positive alignments shown in Table 2 do not necessarily correlate with phylogenetic proximity. 2.3.5. Current detection and control strategies Currently prevalent detection strategies for P. aphanidermatum include baiting, culture plating and membrane filtration tests (Wakeham et al, 1997). Recently, an ELISA has been developed to quantify P. aphanidermatum in cucumber roots (Kyuchukova et al, 2006). Disease detection can also be accomplished by measuring growth responses of the host plant within 8 h of root inoculation (Ortiz-Uribe, 2007). 50 Chemical disease treatments are few and not generally effective at all life stages of P. aphanidermatum. There are two chemical fungicides currently registered for use on Pythium species in Canada, propamocarb and metalaxyl (PMRA, 2008). However, metalaxyl is not effective against all isolates of P. aphanidermatum (Brantner and Windels, 1998). The fungicide captafol was effective at reducing P. aphanidermatum in dieback disease of periwinkle (Kulkarni and Ravindra, 1997). Chitosan has been reported as a potential anti-microbial agent against P. aphanidermatum, by causing wall loosening, vacuolation and sometimes, protoplasm disintegration of hyphal cells (Elghaouth et ah, 1994). Chemical control of P. aphanidermatum with difluoromethylornithine can be effective when used in conjunction with CaCb or PEG 6000-7500 (Kumria et ah, 2000). A possible source of new chemical controls could be found from leaf extracts and essential oils that have proven to be fungitoxic to P. aphanidermatum under certain conditions (Pandey and Dubey, 1994). Zoospore lysis and prevention of vesicle formation in sporangia have been achieved by using non-ionic surfactants (Stanghellini and Tomlinson, 1987), soluble calcium-based chemical compounds (von Broembsen and Deacon, 1997) and silicon (Cherif and Belanger, 1992). Adding 3 cm2/l of silver coated cloth to the hydroponic solution three hours before zoospore inoculation created dissolved silver ions, promoted metallic silver compound formation on the surface of the roots and reduced infection by five-fold (Zhao et ah, 2000). Addition of silicon directly to the hydroponic solution is also effective at triggering heightened plant defence responses (Cherif et ah, 1994). 51 Methods such as UV sterilisation and/ or filtration (7 urn) are effective in small hydroponic systems (Stanghellini etal, 1983; Goldberg et al, 1992), but are not suitable for large-scale systems with nutrient solution flow rates in the order of magnitude of 1000 1/min. (Stanghellini and Rasmussen, 1994). Ozone treatment successfully kills the oomycete, but is expensive and creates reactive oxygen species, which in turn oxidise and reduce the life, expectancy of the equipment used (Zheng, 2002). Pythium infections are much less destructive in soil crops than in hydroponic crops partly because of the competition with other soil microorganisms (Paulitz, 1997). Thus, colonisation of the rhizosphere with non-detrimental spp., such as Pseudomonas chlororaphis or Bacillus cereus, results in niche competition and lowers the potential for infection (Martin and Loper, 1999; Liu et al, 2007) (see Figure 14). Figure 14. Effect of Pseudomonas chlororaphis biocontrol on root rot of pepper caused by P. aphanidermatum. From 11 r: Uninfected root, P. aphanidermatum infected root and root infected with both P. aphanidermatum and Ps. chlororaphis. Source: Prof. J Sutton, University of Guelph. 52 Studies of many rhizosphere inhabiting microbes show that Pseudomonads are amongst the best biocontrol agents of Pythium aphanidermatum caused disease (Gravel et al, 2005; Liu et al, 2007; Jayaraj et al, 2007). Ps. chlororaphis is an effective biocontrol agent in pepper plants (Khan et al, 2003) and induces plant-defence responses in cucumber and hot pepper (Ongena et al, 2000; Nakkeeran et al, 2006). Timing of applying Ps. chlororaphis is important (Chatterton et al, 2004). Pseudomonas spp. can exert both an indirect influence on P. aphanidermatum zoospore behaviour and infection via induced systemic resistance and a local influence via antibiosis or local induced resistance (Chen et al, 1998). Mycoparasitic species such as Pythiumperiplocum have also been shown to control P. aphanidermatum infection (Hockenhull et al, 1992). However, the search for novel biocontrol agents is not straight forward, because so far nobody has demonstrated a clear link between the ecophysiological traits of the biocontrol agent and its ability to successfully compete with a pathogen (Folman et al, 2003). All chemical control strategies that kill the P. aphanidermatum run the risk of creating pathogen resistance. Therefore, crop protection measures that reduce or neutralise the disease symptoms without killing are more sustainable in the long run. This thesis outlines the work to develop a neutralisation strategy for PaNie, with the aim to create a model biotechnology that can precisely disarm pathogens of their necrotrophic weaponry without creating a strong selection pressure. 53 2.4. Research objectives To create a biotechnological tool that is both precise and effective in neutralising PaNie and related NLPs, the following specific research objectives were chosen: 1. Design a conserved NLP peptide that is antigenic. 2. Clone and express full length PaNie. 3. Develop high-affinity VHHS against the conserved NLP peptide and full length PaNie. 4. Create and express functional VHH-CBM fusion proteins. 5. Demonstrate the use of VHH-CBMS in the construction of a bioactive paper filter for affinity-based capture of PaNie. 6. Demonstrate the use of VHH-CBMS for binding PaNie on the surface of plant roots. Objectives 1 and 2 are covered in chapter 3. Completion of objective 3 resulted in the major method development component of this thesis and was thus divided between chapters 3 and 4; chapter 3 covers the ribosome display panning of a naive VHH library against the NLP peptide, while chapter 4 covers the immune library construction and panning against PaNie by phage display. Objectives 4-6 are covered in chapter 5. 54 3. SCREENING OF A NAIVE LLAMA RIBOSOME DISPLAY LIBRARY AGAINST AN NLP PEPTIDE 3.1. Introduction The detection and neutralisation of virulence factors using antibodies has major potential as novel means to manage plant diseases. The aim of this chapter is to design a conserved NLP peptide and to develop VHH antibodies against this peptide that may be used to bind PaNie and other NLPs. Such VHHS could then be used by plant pathologists and farmers for disease detection and treatment in greenhouse hydroponic systems. NLPs were once considered to be associated with avirulence, but increasingly it has been seen that they can also act as virulence factors: Instead of initiating cell death to limit infection, the necrosis aids the spread of the pathogen in the host (Greenberg and Yao, 2004). This idea that avirulence proteins could in some instances, such as during the switch of biotrophy to necrotrophy, act as virulence factors has been controversial in the past, but mounting evidence supports the relatively common occurrence of this idea (Espinosa and Alfano, 2004). VHH antibodies against NLPs would be useful both for the detection of NLPs at different stages of infection and for crop protection through capture of NLPs. To ensure that the developed antibodies have a wide applicability, the PaNie sequence was compared to other NLPs to find peptide sequences of high homology. Developing a VHH antibody against such a 'conserved' peptide would be useful not only for fighting Pythium aphanidermatum in Ontario, but also for plant pathologists and farmers dealing with NLP-induced disease the world over. 55 Ribosome display was chosen as a method for developing anti-NLP antibodies because a naive, ribosome-displayed, VHH library with the required diversity was immediately available (Yau et al, 2003). In case that this naive library would not yield very high affinity binders, the ribosome display format would allow the in vitro affinity maturation of different binders without any need for subcloning. In addition, the nai've VHH ribosome display library is suitable for development of antibodies for agricultural use, because VHH antibodies are stable under adverse conditions such as extreme pH and temperature fluctuations (see section 2.1.2.2). The results of this chapter show that panning of the nai've VHH library against a conserved, 20 amino acid-long NLP peptide yielded several antibodies with essentially the same amino acid sequence and identical CDR3s. This VHH not only bound the constructed peptide, but also to the full length PaNie (KD =15.4 uM) showing that the NLP peptide is exposed on the surface of PaNie. These antibodies are the first anti-NLP recombinant antibodies developed. They may be developed to diagnose NLP presence during plant infections and thus help elucidate the action of NLPs in the development of NLP-induced plant disease. 3.2. Materials and methods 3.2.1. Peptide design & preparation To find regions of conservation in the NLP family, the PaNie gene was compared to other known NLP sequences using the default BLAST search available through the online resources of the US National Center for Biotechnology Information (Altschul et al, 56 1997). To fulfil the aim of targeting NLPs that are relevant in plant disease, BLAST search results were cross-referenced with the APSnet plant disease database (APS, 2002). The design was based on seven NLP sequences including PaNie (see Table 2) and the secondary structure of each NLP. The design was carried out using proteomics tools from the online Expert Protein Analysis System (ExPASy) (ExPASy, 2002). Since the peptide represents a conserved domain within all seven NLPs and because it was designed with an 'SGSG' C-terminal linker for biotinylation, it was named NLPsg2 and had the sequence GHRHDWENVVVWLDNSGSGK-biotin. NLPsg2 was synthesized by Invitrogen (Burlington, ON) and shipped in lyophilised form. NLPsg2 was dissolved in DMSO (10 mg/ml), diluted to 1 mg/ml (= final 10% v/v DMSO) with heparin block buffer (HB; 200 mg/ml heparin in 50 raM Tris-acetate, 150 mM NaCl, 50 mM Mg- acetate, 0.1% (v/v) Tween 20, pH 7.5) and stored at -80°C. 3.2.2. Selection of antibodies by ribosome display This section describes the panning of an already constructed naive library using ribosome display. Since the library construction has already been described by Yau et al. (2003), the construction is only described in brief: RNA was extracted from nai've llama leukocytes and reverse transcribed by RT-PCR to make a cDNA library which was added into the library framework by splice overlap extension PCR. As shown in Figure 5 and described in section 2.1.4.3, the ribosome display selection cycle begins with amplified library DNA, which is transcribed and translated to form 57 ARM complexes that provide the phenotype-genotype linkage that is exploited during panning. The particular library framework of Yau et al. (2003) is outlined in Figure 15. T7B T6Te DNA 5' w—^MMfwa^ 3' 5 RNA 3' ^O—^iRii. ^f^^f^^f *%&&£} ' RNA & Protein 3' TTFOWmieWfy'^SpSf Figure 15. Basic structure and function of the ribosome display library and its components. T7 = T7 polymerase binding site; RBS = ribosome binding site; F = flag tag; forward and reverse primers shown in red; stem loops after the T7 binding site and stem loops in the spacer are indicated by bulges in the RNA; the t-RNA containing ribosome forms part of the Antibody-Ribosome-mRNA (ARM) complex by translating the sequence and remaining attached to the mRNA that lacks a stop codon. All primers used during ribosome display panning are shown in Table 3. The library was amplified by PCR using T7B and T6te (Ta = 62°C) or alternatively T7B2 and T6te2 (Ta = 64°C) with 10 ul of library DNA template in a 100 ul volume. Reaction conditions for the first round were: Melting of double helix at 94°C, 4 min.; Main cycle amplification for 15 cycles at (94°C, 30 sec; 62 or 64°C, 1 min.; 72°C, 1 min.); Final extension at 72°C, 10 58 min.; 4°C, oo. In subsequent rounds the main cycle was performed 35 instead of 15 times to allow sufficient amplification. Table 3. Primers used in the ribosome display cycle. Palindromes exist in T6te, T6te2 and T7B2, but these did not seem to interfere with the ribosome display cycle. Name Oligonucleotide sequence 5' -> 3' T6te CCGCACACCAGTAAGGTGTGCGGTATCACCAGTAGCACCATTA T6te2 CCGCACACCAGTAAGGTGTGCGGTATCACCAGTAGCACCATTA CCATTAGCAAGGCCGG T7B ATACGAAATTAATACGACTCACTATAGGGAGACCACAACGG T7B2 ATACGAAATTAATACGACTCACTATAGGGAGACCACAACGGT TTCCCTCTAGA SDA2 GAAGGAGATATATCCATGGACTAC G3Notl CACCACCCGCGGCCGCCGAGGC SDApagl GAAGGAGATATATTCATGAGTGACTAC A 1.5% agarose gel (1/3 low melting agarose) of the PCR products (5 ul) and a mass marker was run every round to assure no non-specific amplification occurred and that DNA concentrations were at least 10-20 ng/ul. These amplicons were sometimes stored at -20 °C. Glass test tubes were preincubated with 4% w/v milk in phosphate buffered saline (PBS, pH 7.4) for at least 2 h. Milk (10 ml; 10% w/v) was autoclaved and de- biotinylated overnight (o/n, 12-16 h) using streptavidin beads (Dynabeads M-280,200 ul; Dynal - Invitrogen, Burlington, ON, Canada). 59 The amplicons were used in 10-ul aliquots as templates for in vitro transcription using the T7 RiboMax Large Scale RNA Production System (Promega, Madison, WI). The mRNA produced was purified using Probe Quant G-50 Micro-columns (GE Healthcare, Piscataway, NJ) and quantified at A26o- Concentrations of 1.00 ug/ul were either immediately translated or stored at -80°C for up to one week. Blocked glass tubes were washed with 3 x 1 ml of HB, followed by 3 ml of HB for 15 minutes and then placed on ice. The mRNA was diluted to 1 ug/ul in nuclease free water and added to the following mastermix in nuclease free water: 220 raM K-glutamate; 7.6 mM Mg-acetate; 10 ug protein disulphide isomerase; 4 uM anti-ssrA oligo; a premixture of 8.8 mM adenosine triphosphate, 2.2 mM guanidine triphosphate, 4.4 mM cyclic adenosine monophosphate, 132 mM acetyl-phosphate (final concentrations) and 40 ul E. coli S30 extract. Protein disulphide isomerase encourages disulphide bond formation and the anti-ssrA oligo blocks ssrA RNA, which normally releases nascent polypeptides from the ribosome. The sequence of the anti-ssrA oligonucleotide is 5'- 3' TTAAGCTGCTAAAGCGTAGTTTTCGTCGTTTGCGACTA. In vitro translation was carried out using cell contents, hereafter known as S30 extract, from the ribonuclease I deficient E. coli strain MRE 600 (Wade and Robinson, 1966). The harvesting of S30 extract and the in vitro translation were performed according to methods described by Hanes et al. (2000) and Yau et al. (2003). The translation mixture was incubated in a 37°C waterbath for 7 minutes. To stop the reaction the translation mixture (110 ul) was added to chilled HB (460 ul) contained in a chilled microfuge tube. 60 All subsequent procedures after translation were carried out in the cold room (4°C) and all ingredients and tools pre-chilled to safeguard the stability of the ARM complex. The chilled microfuge tubes containing the terminated translation mixture (570 ul) were spun down at 10,000 g for five minutes. The supernatant (530 ul) was added to the blocked and washed glass test tubes containing debiotinylated milk (50 ul) and biotinylated NLPsg2 (1.5 ul yielding a final concentration of 1 uM) and incubated at 4°C for 2 h and 45 min. A solution of magnetic streptavidin beads (100 ul/6.7 x 107 Dynabeads M-280; Dynal - Invitrogen, Burlington, ON) was prepared by washing with 3x 3 ml HB using a magnetic seperator. After 30 min of incubation in HB, the magnetic beads were resuspended in approx. 100 ul HB and transferred to the glass tubes. The antigen-ARM complexes from the translation-antigen mixture were captured on the beads by incubating the magnetic beads in the glass test tubes for 10-15 min. Using a magnetic separator, the beads covered in antigen-ARM complexes were washed five times with HB (3 ml ea). Competitive washes and elutions were incorporated during some rounds (10-118X competition). The final solution was transferred to a fresh microfuge tube. EDTA chelates magnesium ions, destabilising the ribosome complex and releasing the RNA. This was achieved by incubating the ARM complexes bound to the magnetic beads for 10 min with elution buffer containing EDTA (50 mM Tris-acetate, 150 mM NaCl, 30 mM EDTA, 50 ug/ml yeast RNA, pH 7.5). The eluate containing the RNA was aspirated while the beads were retained by the magnet. The RNA was purified using the High Pure RNA isolation kit (Roche, Laval, QC) and stored at -80°C for up to one week. 61 Purified RNA (12 ul) was mixed with dNTP (0.5 jo.1; 25 mM), T6te(2) and T7B(2) primers (0.5 ul; 100 uM) and denatured at 85°C for 5 min, placed on ice for at least 1 min, spun for 1 min and finally added to a Superscriptase III mix (7 ul) according to the manufacturers instructions (Invitrogen, Burlington, ON). The reverse transcription reaction was performed at 55°C for 1 h and terminated at 70°C for 15 min. The amplification PCR for the following round was done as described above but with 35 cycles instead of 15 and in presence of 5% DMSO. Samples were taken following the 15l and 25th cycle to estimate enrichment between rounds of panning. 3.2.3. Cloning and expression of VHH sequences PCR products from the 5th round of panning were amplified using SDA2 and G3Notl primers and cloned using the TOPO TA cloning system (vector pCR2.1) according to manufacturers instructions (Invitrogen, Burlington, ON). Successful insertion of the antibody genes was affirmed by colony PCR using primers T6te and T7B. Plasmid preparation of pCR2.1-VnH was performed using a Qiagen plasmid mini/midi kit (Qiagen, Mississauga, ON). Subcloning was achieved by PCR amplification with SDApagl and G3Notl (for sequences see Table 3), subsequent cutting with Pagl and Notl restriction enzymes (37°C, 1 h; Fermentas Canada Inc. Burlington, ON, Canada) and ligation (16°C, o/n) into pET22b+ and pET28b+ (Novagen, Madison, WI). Transformation into E.coli was achieved in a two-step process; first DH5a and secondly BL21 strains of E.coli were made to be electrocompetent and used according to Current Protocols in Molecular Biology (Ausubel, 2003). Glycerol stocks were prepared with 10% glycerol and stored at -80°C. 62 Protein expression from BL21 in Luria-Bertani (LB) media (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, pH 7.5) containing carbenicillin (CARB, 75 jxg/ml final concentration in 50 - 1000 ml) was induced in the log phase with ImM IPTG and grown at 37°C for an additional 4-6 hours. Cells were harvested by centrifugation, frozen in - 20°C and later sonicated in 10 mM HEPES, 500 mM NaCl, pH 7.5. VHH was visualised, both before and after sonication, by SDS-PAGE (12% Tris-Glycine). VHH concentrations were determined by A280 using calculated molar absorption coefficients based on the amino acid sequence for each VHH clone (ExPASy, 2002). 3.2.4. Cloning and expression of PaNie Dried pepper roots containing Pythium aphanidermatum (Edson) Fitzp. isolates were used to inoculate selective corn meal agar medium containing 250 mg/1 ampicillin (AMP) and 10 mg/1 rifampicin and grown for 48 h beneath fluorescent lamps at 22°C. The identity of P. aphanidermatum was confirmed by microscopy. Discs of the colonized medium were transferred to V-8 juice agar medium (12 g agar, 200 ml V8 juice, 3 g CaC03, 800 ml deionised water), incubated for 48 h as before, discs bearing mycelium transferred to sterile malt-dextrose broth (2 discs per 100 ml broth) and cultured while shaking (150 rpm) for a week at 28°C. Mycelial tissue was harvested from the broth, washed and ground in liquid nitrogen using a mortar and pestle. Genomic DNA was prepared from dried mycelium (120-200 mg) using the DNeasy plant mini kit (Qiagen, Mississauga, ON). 63 Xhol and Ncol restriction sites were added to the PaNie gene by PCR with primers PaNie forward and reverse (5'-CCAACGCCATGGCCGTGATCAACCATGATGC-3'; 5'-GACAGCCTCGAGCTGGAAAAACGCCTTCACGA-3') as follows: 94°C for 5 min; 30 cycles of 94°C for 30 sec, 60°C for 30 sec, 72°C for 15 sec; 72°C for 5 min; 4°C. The amplicons were digested and cloned into the pET22b+ vector (Novagen, Madison, WI). The pET22b+PaNie construct was transformed into DH5a and BL21 E. coli by electroporation (25 uF, 200 Q. and 1200 V) and streaked onto Petri plates containing LB- CARB (75 ug/ml CARB). Transformed colonies growing on LB-CARB plates were screened by colony PCR and the PaNie gene verified by DNA sequencing. Optimal PaNie protein expression conditions were determined by varying temperature, induction time and concentration of IPTG in 250-ml cultures (see Table 5 in section 0). In preparation for large scale expression, the BL21 pET22b+PaNie E. coli were grown o/n at 37°C, 200 rpm in 10 ml of LB-CARB (75 ug/ml CARB) with additional 34 ug/ml chloramphenicol. The starter culture was added to LB-CARB (1 L), grown at 37°C, 200 rpm to an OD600 of 0.6-0.7, induced with IPTG (1 mM) and harvested after a further 4 h of growth. Frozen pellets were disrupted by ultrasonication (Model 550 Sonic Dismembrator, Fisher Scientific Ltd., Nepean, ON) in 10 mM HEPES buffer containing 500 mM NaCl and 8M urea (pH 7). The lysate was loaded onto a 5 ml Ni-NTA HiTrap column and purified using an AKTA FPLC system (GE Healthcare Bio-sciences AB, Uppsala, Sweden). The column was washed and eluted using a stepwise gradient of imidazole (50, 75, 100, 125, 150, 250, 350 and 500 mM) in HEPES buffer (10 mM HEPES, 500 mM NaCl, 8 M urea, 50-500 mM imidazole, pH 7). Elution occurred most markedly at 150 mM imidazole. Samples of eluted fractions were analyzed by SDS- 64 PAGE. The eluate fractions containing PaNie were desalted into PBS using Sephadex G25 or by dialysis. Concentrations were determined by absorbance at 280 nm and confirmed by the Bradford method. 3.2.5. Anti-peptide ELISA Microtitre wells were coated with anti-NLPsg2 VHH cl05 (i.e. clone #05) at a concentration of 1.4 and 14 uM in PBS (100 ul) o/n at 8°C followed by 3.5 h at RT. After decanting, the wells were blocked with 2% w/v casein in PBS (200 ul) for 4.5 h at RT followed by o/n at 8°C. The wells were washed four times with PBST (0.05% Tween) and decanted. NLPsg2-biotin (50 ug/100 ul 1% casein-PBS; 187 uM) or PaNie (2.5 uM) was added to the wells and left to incubate at RT for 1 h. After three washes with PBST, streptavidin-HRP (2.5 or 25 ug/ml in 100 ul) was added and left to incubate for 1 h at RT. After three final washes with PBST, TMB (100 ul; Pierce, Rockford, IL) was added and left to develop for 7 min. The level of binding was determined spectrophotometrically at 450 nm after neutralising with 1.5 M H2SO4 (100 ul). 3.2.6. Anti-PaNie inhibition ELISA Microtitre wells were coated with VHH cl05 (1.4 uM; 100 ul PBS) o/n at 8°C followed by 1.5 h at RT. After decanting, the wells were blocked with 2% w/v casein in PBS (200 ul) for 4 h at RT. The wells were washed three times with PBST and decanted. A 0.1 uM PaNie-HRP solution, with and without free PaNie, was diluted in 1% w/v casein-PBS. The unconjugated, i.e. free PaNie, ranged in concentration from 1.25 - 20 uM. This primary step, including inhibition, was performed at RT for one hour, followed by five 65 washes with PBST. After decanting, TMB (150 ul; Pierce, Rockford, IL) was added and left to develop for one hour. The level of binding was determined spectrophotometrically at 450 nm after neutralising with an equal volume of 1.5 M H2SO4. All dilutions were done in triplicate. 3.3. Results 3.3.1. NLPsg2 peptide characteristics BLAST searches of the PaNie (GenBank accession AAD53944) revealed sequence homology to several protein elicitors and uncharacterised proteins from related and unrelated species. As illustrated in Table 2 in chapter 1 the % positives are high but greatly varied amongst different species. Figure 16 shows the alignment of selected NLPs (PsojNIP, AAM48171; NPP1, AAK19753; Bacillus halodurans, NP_241261; Nepl, AAC9738; Streptomyces coelicolor, AL356932; Vibrio pommerensis, AJ314791) used to determine a suitable peptide sequence that was common among all NLPs. It is important to mention that the design of the peptide preceded any of the recent literature on the NPP1 domain that is common to all NLPs. In other words, too few NLP sequences were available at the time of peptide design (September 2002) to draw any definite conclusions about the relative importance of common domains. 66 10 20 60 70 T T T K Bbaladumisi'f-ize MM - FF ',.' lOFUt ' I A|| V|I wdF 8^ pB 't B ^ii HPPV1-2V • • • UN vi 7L I AAA LAJ •-VVPF • |PTjMtSElKgPM H PCKf H t i hi PstyWp/i-23? • - • MNLFFALL ABL F IKPQJHIS - N| mp 1/1-253 «H < POT I ' ' 100% COAttfttUS MM- + + V+-FLL- - LAA+GSLAPVNA AVINHDAVVPFEQPTPTTA+QKAAVBF +PQL+ I S-+6C + ON Bh3loduniti&>1-23& S - ~ O C V P f PAViA D MPP1/1-237 S- N*CI»IPAUIAN £>saf/ilip/1-23? S - '•0c|pflpAvfltlN Vep *"?-253 A - " ©C : PpA VIT N "ajMfe/^-M* V- '-frC-P|PAVHN2 lpofl«»eremws^-26f D N : CIPIA£ I S K'Y ScoelicQlQt/1-258 0 T : GCBPBP A I {A 0 100% Conservation 4 - S 3 56:9 535 5684 44 9 81876 31450- -078573 434 2 5 5 4 6 ' 5 i 6 5 5 4 8 100% Consensus S-+GC+PYPAVDANGNTSGGLNPTGS-++GC+DSSYL-e+QVY+RAA+ - - - -YNGVYAIMYSWYFPKD+P+TO- -LLGHRHD Figure 16 continues on the next page. 150 160 170 180 190 200 210 Bhatodtirans/1-238 - • I VVWVMF AN P1PQ|_%B* NPP1/1-237 - 1 tfVVWlflr I K L D«P S -HlJI' N I vlpPESNT ll|fYSfB|Dls Psq/Wp/1-237 -1 N KY|PPS SIYF SBN sp||[>ls Nep 1/1-253 vjj NVWF 'BpQSP&TllllfcAj DIKKTKNPQRI---BNHJMIEIF PsMe/1-234 - j NVVVVKLIAA- -BANJIIISI T S K HVAiuflkWV I KKSSPIDKIYLBI SH | I Scoelico>o//1-258 L|I HVWW^REl TW|Q | KgN - TKP^TQHAR^DHiBvlHKD 100% Conservation 100% Consensus! LLGHRHDWEHVVVWVDN+A- PSPT IVA+ SASAHGGYNK++PP++S++DGN+AKI+YSSSWV++ NHALRFTT+A-E- - ENG Bhalodtiransf 1-238 ON NPPV1-237 00 PsqNip/1-237 Nep 1/1-253 PaMe/1-234 Vpommetensis/1-261 T A E NlYgYWV| ScaelicQloi/1-258 HI E N H KBAWQ 100% Conservation .. .4 .... 2 100% Consensus f I..,ML ii!:-jjiMi-jij . - -ENG-G-WETQPL I+WDQ+ - T+AAR-AL E+TDFG+ANVPFKD+NFQT+L+ KA+ P- YAP Figure 16. Jalview sequence alignment showing conservation and consensus of selected NLPs. Conservation #'s (0-9,10 = *) provide a numerical scale for amount of conservation. Consensus letters refer to amino acid homology present in more than 2 sequences for that position. A '+' shows that there are 2 or more conflicting homologies for that position (Clamp et al., 2004). The numbering of amino acids in the Jalview alignment will hereafter be referred to as 'NLP positions". In general, the putative secretion signals of different species span between NLP positions 1 - 35 in the Jalview alignment (Veit et al, 2001), which may explain the lack of any completely conserved amino acids in this section. The sequence after NLP position 35 was analysed for the longest stretches of conserved amino acids that could be used as an antigen for the development of a VHH that may detect all NLPs. To limit the analysis, sequences greater or equal to 6 amino acids and having at least five amino acids conserved amongst the oomycetes and at least two positions with sequence similarity amongst non-oomycetes were considered as possible antigens. Based on the fact that NLPs from V. pommerensis and S. coelicolor are the most different from NLPs of other species in terms of extra lengths of amino acid sequences and because the NLPs from these two species are not known to cause plant disease, their homology was given lower priority (APS, 2002). The six peptides chosen for further study are shown in Figure 17. The longest, almost entirely conserved sequence is GHRHDWE and it is followed by eight relatively conserved amino acids (NVVVWLDN). PYPAVD__GNTSGGL may be important for NLP function and would form a very long (13 amino acids) stretch of relatively conserved amino acids were it not for two intervening amino acids with little or no conservation. AIMYSWY, which is followed by highly conserved amino acids amongst the NLPs other than PaNie, could also prove important for NLP structure or function. The latter two peptides become more attractive if less importance is given to V. pommerensis and S. coelicolor, which are largely responsible for the low conservation of AIMYSWY and to a lesser extent PYPAVD GNTSGGL. 69 10 20 30 40 50 60 IWRFVSALLLAAAGVLASTNAAVINHDAVPVWPQPEPADATQALAVRFKPQLDVVNGCQ| 36 76 7 0 80 9£ 100 110 120 YPAVDPQGNTSGGLKPSGSQAAACRDMSKAQVYSRSGTYNGYYAIMYSWYMPKDSPSTGI 8i 127 130 14() 150 160 170 180 GHRHDWENVVVWLDNAASANIVALSASAHSGYKKSFPADKSYLDGITAKISYKSTWPLDH 1.47 175 190 200 210 22£ 230 ELGFTTSAGKQQPLIQWEQMTQAARDALESTDFGNANVPFKSNFQDKLVKAFFQ 26£ Figure 17. Selected conserved peptides displayed within the PaNie amino acid sequence. N-terminal secretion signal and peptides highlighted in dark and light grey, respectively. Numbers above the sequence refer to the specific PaNie positions, while the numbers below the sequence refer to the NLP positions as seen in Figure 16. In order to have another means of discriminating amongst the six peptides, the hydrophobicity of the PaNie sequence was modelled according to the method of Kyte & Doolittle (ExPASy, 2002). Figure 18 shows the output for the PaNie sequence. The lowest hydrophobicity (= highest hydrophilicity) occurs near PaNie position 125. Therefore, it was determined that, since GHRHDWE spans PaNie positions 121-127, this sequence is most likely exposed on the surface of PaNie and is the prime candidate for development of an anti-NLP VHH. 70 ProtSoale output for user sequence Hpi'i. J t & %-. Boo i i. %•'% 1 e ---—— £.5 1.5 1 h 0.5 h 'At i * -0.5 . ! I .' • ,'lt •" ' f ?"t<"V' ijl tj u M -1.5 '•V \ -£.5 50 150 800 Figure 18. Hydrophobicity plot of PaNie according to Kyte & Doolittle (ExPASy, 2002). To supplement or confirm the choice of peptide several secondary structure prediction tools were used. The SOPM, SOPMA, GOR1 and the 'Hierarchical Neural Network from Pole Bio-Informatique Lyonnais' algorithms indicate a possible turn at PaNie positions 66 and 67 between PYPAVD and GNTSGGL. The putative loop that would result from such a turn could be a feature shared by all NLPs, especially in light of the conservation of two cysteines (NLP positions 74 and 100; PaNie positions 58 and 84) that may form a disulphide bridge. In contrast to multi-domain antibodies, VHH antibodies are generally more suitable for binding epitopes that are pockets rather than loops. GHRHDWE and the following amino acids are mostly predicted to be either extended strand or random coil. 71 Thus, GHRHDWE was confirmed as the prime candidate, because extended strands and random coils may be more likely to form pockets that could bind a VHH (ExPASy, 2002). It is worth noting that these seven amino acids constitute the NPP1 domain, which, although unknown at the time of peptide design, has recently been found to be present in all NLPs (Gijzen and Nurnberger, 2006). Table 4 shows the Kyte and Doolittle hydrophobicity scores for the six peptides including the tail of the chosen NLPsg2 candidate GHRHDWE-NVVVWLDN. It must be noted that although GHRHDWE (PaNie positions 121-127) is the most hydrophilic peptide (most negative hydrophobicity score), the NLPsg2 tail (PaNie positions 128-135) is somewhat hydrophobic (positive score). Nevertheless, the tail was included in peptide synthesis to ensure that the prime antigen GHRHDWE would not be disturbed by subsequent biotinylation and binding to streptavidin beads (i.e. the C-terminal end of the tail NWVWLDN was biotinylated). In addition, the high homology of the tail was thought to be indicative of an important structural or functional role, which may be required for VHH recognition of PaNie and other NLPs. 72 Table 4. Kyte & Doolittle hydrophobicity scores for six peptides of PaNie. The final peptide GHRHDWENVVVWLDN is also included. Neg. scores = hydrophilicity. PaNie Amino Average PaNie Amino Average Score Score Position acid score Position acid score 60 P -0.378 121 G -1.356 61 Y -0.378 122 H -1.367 62 P -0.511 123 R -1.678 -0.720 63 A -1.178 124 H -2.022 -1.535 64 V -0.833 125 D -2.056 65 D -1.044 126 W -1.544 127 E -0.722 68 G -1.133 69 N -1.644 147 S 0.356 70 T -0.833 148 A 0.011 71 S -1.089 -1.011 149 H -0.844 -0.704 72 G -0.878 150 S -1.189 73 G -0.922 151 G -1.478 74 L -0.578 152 Y -1.078 104 A -0.044 216 A -0.100 105 I 0.244 217 N -0.144 106 M 0.144 218 V -0.544 -0.441 107 Y 0.500 0.013 219 P -0.889 108 S 0.467 220 F -0.189 109 w -0.167 221 K -0.778 110 Y -1.056 Amino Average Position Score acid score 121 G -1.356 122 II -1.367 123 R -1.678 124 II -2.022 -1.535 125 1) -2.056 • 126 \V -1.544 i 127 E -0.722 128 N -0.322 -0.438 129 V 0.456 130 V 0.456 131 V 0.167 0.521 132 w 0.756 133 L 1.344 134 D 0.789 \ 135 N 0.522 i 73 3.3.2. Ribosome display selection and sequence analysis Conventional antibody selection from ribosome display libraries as described by Yau et al. (2003) resulted in non-specific background binding. Therefore, a subtractive and competitive selection protocol was developed. The first three rounds of selection against NLPsg2 were conducted as described by Yau et al. (2003) and progress of library selection was monitored by DNA agarose electrophoresis of RT-PCR products after each round of selection. In the third round of selection, the RT-PCR product was visible after 25 as opposed to 35 cycles of PCR amplification as was the case in round 1 and 2, indicating enrichment of antibody sequences in the selected pool (see Figure 19). Figure 19. Third round ribosome display results. Amplicon enrichment is evidenced by the visualisation of a 730 b band in lane 1 after 25 cycles, not seen until after 35 cycles in round 1 and 2. Lane 1 = NLPsg2 peptide selection; Lane 2 = no antigen neg. cnt.; Lane 3 = mock reverse transcription; Lane 4 = original library as positive control; Lane 5 = mastermix PCR without template. Arrows indicate lanes of interest. 74 Enrichment indicated that enough copies of individual antibody sequences and their associated ARM complexes were present for effective competition to occur. Therefore, following round three, two more rounds of panning were conducted in which stringent washing and competitive selection were performed to remove as many of the non-specific binders as possible and to determine if specific binders could be eluted by competition with free antigen (see Figure 20 and Figure 21). After panning, the streptavidin beads with bound ARM-complexes were washed three times with HB, followed by two incubations with 10X and 50X competitition with free antigen (10 and 50 uM unbiotinylated NLPsg2) and a final elution with EDTA. The competition with free antigen served to elute entire ARM-complexes from the biotinylated antigen, while EDTA breaks the ARM-complexes to release the RNA coding for bound VHHS. PCR Cycle 15 I PCR Cycle 25 I PCR Cycle 35 12 3 4 5 6 7 8 9 lolbi 12 3 4 5 6 7 8 9 lol 12 3 4 5 6 7 8 9 10 750 b 500 b 250 b Figure 20. Fourth round ribosome display results, when competition as well as EDTA elution was used. A 730 b band appears faintly in lane 6 after 25 cycles, while lane 4 and 5 only show faint bands after 35 cycles. Lane 1 = HB Wash 1; Lane 2 = HB Wash 2; Lane 3 = HB Wash 3; Lane 4 = lOx competition; Lane 5 = 50x competition; Lane 6 = EDTA elution; Lane 7 = no antigen negaive control; Lane 8 = mock reverse transcription; Lane 9 = original library as positive control; Lane 10 = mastermix PCR without template. Arrows indicate lanes of interest. 75 In the 4 round a band from the EDTA elution arises after 25 cycles of PCR indicating the presence of binders that withstand 5 Ox competition with free antigen. After 35 cycles bands are also faintly visible in the 50x and lOx peptide competition lanes, indicating that some VHHs are eluted by competition. All amplicons represented in lanes 4, 5 and 6 after 35 cycles in Figure 20 were pooled and amplified to use as template for round 5. In round 5 the stringency of washing was increased to a total of two hours of washing and competition: lx 30 min of washing with HB, followed by 3x 30 min of 118x competition with free, unbiotinylated NLPsg2 (118 uM) and a final elution with EDTA. Figure 21. Fifth round ribosome display selection results. Lane 1 = 30 min HB wash; Lane 2-4 = successive 30 min washes with 118 uM NLPsg2; Lane 5 = EDTA elution; Lane 6 = unrelated peptide selection followed by 5x 25 min HB washes & EDTA elution; Lane 7 = mock reverse transcription as negative control; Lane 8 = original library as positive control; Lane 9 = mastermix PCR without template as negative control. Arrows indicate lanes of interest. 76 The only lane yielding a band was the EDTA elution lane, indicating the presence of only binders that have endured 4 wash cycles amounting to 2 h of stringent washing and competition. A negative control that was washed and eluted in the same way as the selection with NLPsg2, but using an unrelated, biotinylated peptide, yielded no bands (lane 6 in Figure 21), supporting the presence of specific binders rather than non-specific background binders. The 5th round EDTA elution was amplified and inserts recovered for subcloning by TA cloning and subsequent cloning into the pET22b+ expression vector. Of 16 clones picked, 7 had an insert with the correct length (-440 bp) and were sequenced. Two sequences named cl03 and ell4 were truncated and/or contained repeats and were discarded. The remaining sequences have high percentage identity and are most similar to the VHH subfamily 3 (see Figure 22) (Harmsen et al, 2000). I FRl | CDRl | FR2 I CDR2 10 20 30 40 50 60 cl02 DVQLQASGGGLVQPGGSLRLSCAASGFTLAIYAIGWFRQAPGKEREGVSCMHSFDSSTYYTA cl04 ...V V N G C105 ...V A N G cllO ...A V I G C115 ...V V N G | FR3 | CDR3 | PR4 | 70 80 9(3 100 110 118. Cl02 SVKGRFSISRDNAMGTVYLQMNNLEGSDTAVYTCAADPDGEGYGFWGQGTQVTVSS cl04 N cl05 D cllO N cll5 N Figure 22. Amino acid alignment of the five full length anti-NLPsg2 VHH clones. The dots in the sequence represent identities with the other clones. Sequential numbering system was used and the CDR regions were identified according to the alignment described in Saerens et al. (2005). 77 There are differences among the five clones at four amino acid positions in the framework regions (2 in FR1; 1 in FR3; 1 in FR4) and one amino acid position in each of CDR1 and CDR2. C103 and cllO have an isoleucine substitution at position 31 in CDR1 (consensus (CS) = asparagine) and cl03 has a serine substitution at position 56 in CDR2 (CS = glycine). CDR3 was conserved amongst all clones and because CDR3 is regarded to be involved in specific antigen recognition, it is likely that all clones display the same paratope. The substitutions in CDR1 and CDR2 as well as at positions 2,12 and 84 in the framework may be neutral substitutions that do not affect antigen-antibody interaction. The fact that ell5 is identical to cl04 in protein sequence meant that it was not further characterized. However, it must be noted that since the DNA sequence of ell 5 is not identical to cl04, the selection of DNA sequence was indeed convergent to a particular protein sequence, which indicates that selection of NLPsg2 binders through ribosome display was likely successful. 3.3.3. VHH protein expression The four clones (cl02, cl04, cl05, cllO) were successfully expressed (see Figure 23). Since the CDR comparisons indicate that all clones likely display the same paratope and because there was no significant difference in binding of the different clones to PaNie in conventional ELISAs, further studies focused on cl05, the highest expressor. 78 kDa iaA4 ^^^K «HK 4tfHhr ^^HK *•••**. flHHE Figure 23. SDS-PAGE (left) and Western (right) analysis of VHH clones from the fifth round ribosome display panning against NLPsg2. Lane 1 = No insert vector control pre-induction; Lane 2 = No insert vector control 4 h induction; Lane 3 = VHH cl02 pre-induction; Lane 4 = VHH cl02 4 h induction; Lane 5 = VHH cl04 pre- induction; Lane 6 = VRH CI04 4 h induction; Lane 7 = VRH C105 pre-induction; Lane 8 = VHH cl05 4 h induction; Lane 9 = VHH cllO pre-induction; Lane 10 = VHH cllO 4 h induction. 3.3.4. Cloning and expression of PaNie The cloning of PaNie from Pythium aphanidermatum genomic DNA using the Plant DNeasy genomic DNA preparation kit was successful in isolating genomic DNA from homogenised mycelium of Pythium aphanidermatum. Primers used in cloning were synthesized based on the PaNie sequence published by Veit et al. (2001). Although the amount of DNA yield from mycelium was lower than from plants, it was of sufficient quality to consistently amplify the correct 683 bp band corresponding to the PaNie gene and primer sequences (see Figure 24). 79 Figure 24. Genomic DNA preparation (A) and PaNie gene amplification (B) from Pythium aphanidermatum. Lanes Al, A2 and A3 show total genomic DNA from two mycelial samples and a tobacco leaf control sample, respectively. Lanes Bl, B2, B3 and B4 show the amplified 683 bp PaNie gene from four different genomic DNA gel purifications, while lanes B5 and B6 represent negative (no template) and positive (known template and primers) controls, respectively. After successful cloning of the 683 bp PaNie gene into the pET22b+ vector and £". coli, protein expression at different temperature and induction regimes was monitored (see Figure 25 and Table 5). Lane 10, which served as a negative control without induction, showed no leaky expression of PaNie and no expression of anti-penta HIS detectable proteins in the absence of IPTG. The highest level of expression was seen in lanes 3 and 5 as would be expected from an o/n induction, but with a concomitant loss in purity compared to lanes 6-9. The o/n inductions seemed to result in some form of PaNie aggregate of approx. 60 kDa. This could be a dimer of PaNie, even though the theoretical size of a dimer should be 50 kDa, as dimers or multimers often do not run according to their size. It is important to note that such a dimer or aggregate is formed even under 80 reducing conditions. Alternatively, it could be a native HIS-containing protein, but it is not seen in the non-induced control (lane 10). The purest expressions are shown in lane 2 and 4 indicating that a lower temperature (25 °C) and shorter harvest times after induction are the best conditions for producing PaNie with few contaminants, at some loss of total protein expression. Furthermore, at 37°C it is better to harvest 4 instead of 6 h after induction to avoid PaNie aggregation, i.e. the 60 kDa band. 22 -%^\ 16 -W:_ kDa Figure 25. PaNie expression under various temperature and induction regimes. The top panel shows a coomassie stain and the bottom panel a Western blot using anti- HIS IgG-AP. Lane 1 = Protein standard; Lane 2-9 = PaNie expressions (see Table 5 for temperature and induction conditions); Lane 10 = no induction negative control. Note that lanes 6 and 8 as well as 7 and 9 are replicates. 81 Table 5. Growth and induction conditions for PaNie expressions (see Figure 25 for Coomassie and Western blot analysis of each lane). ______ Condition 2 3 4 5 6&8 7&9 10 Starter culture temp 25°C 25°C 25°C 25°C 37°C 37°C 37°C Scale-up temp 32°C 32°C 32°C 32°C 37°C 37°C 37°C Induction temp 32°C 32°C 32°C 32°C 37°C 37°C 37°C IPTG(mM) 1 1 0.5 0.5 1 10 ______^^ ______ The presence of a faint band below PaNie (24.5 kDa) in the SDS-PAGE may be PaNie that has been processed for expression into the periplasm of E.coli, i.e. PaNie without the pelB leader sequence of 22 amino acids (~2.2 kDa). Periplasmic extraction of native PaNie was unsuccessful, due to an inability to purify the PaNie using Ni-NTA columns under native conditions (data not shown). The majority of the PaNie remains unprocessed as inclusion bodies (aggregates) in the cytoplasm of E.coli. Sonication and subsequent Ni-NTA purification in the presence of 6 M urea was successful in recovering all PaNie from the cytoplasm and periplasm. Once renatured the PaNie was functional when injected into leaves of tobacco (see Chapter 5). The co-cloning of the chaperone GroESL did not improve the purification of native PaNie under non-denaturing conditions (data not shown). Veit et al. (2001) also had to use denaturing conditions to purify PaNie. This suggests that perhaps the C-terminal HIS-tag is not fully accessible in native PaNie and that this is the reason for poor purification from Ni-NTA under native conditions. 82 3.3.5. Binding of VHH cl05 to NLPsg2 C105 was expressed, purified and run over a Sephadex 75 gel filtration column in preparation for surface plasmon resonance. However, no results were obtained using surface plasmon resonance, because the DMSO, required to dissolve NLPsg2, interfered with the readings by creating a high surface density that masked all measurements (data not shown). Thus, the antibody-antigen interactions were studied solely by ELISA. Initial attempts at coating NLPsg2 on microtitre plates and performing a conventional ELISA as well as avidity-enhancing ELISAs using magnetic histidine coated beads were not successful. Therefore, a modified ELISA was developed, where the cl05 VHH was used to coat polystyrene microtitre plates and probed with NLPsg2-biotin followed by streptavidin-HRP. The results shown in Figure 26 indicate specific binding of cl05 to NLPsg2, albeit at high antigen and antibody concentrations. 83 1.6 • 25 (xg/ml strep-HRP 1.4 • 2.5 ug/ml strep-HRP 1.2 O M1.0 |0.8 §0.6 0.4 0.2 0.0 H^ 14 uM 1.4 uM 14 uM cl05 cl05 cl05 & 187 uM & 187 uM & 2.5 uM NLPsg2 NLPsg2 PaNie Figure 26. Binding of NLPsg2 to VHH cl05. Concentrations of 14 and 1.4 uM cl05 were coated on a 96-well microtitre plate as displayed on the x-axis. Binding of biotinylated NLPsg2 (187 uM) or unbiotinylated PaNie (2.5 uM; control) was probed with 25 or 2.5 ug/ml streptavidin-HRP. Background subtraction includes non-specific binding of NLPsg2-biotin to uncoated, blocked wells. 3.3.6. Determination of VHH cl05-PaNie binding kinetics by CI-ELISA An inhibition ELISA was not conducted to determine the antibody binding kinetics of cl05 to NLPsg2 because too little unbiotinylated NLPsg2 was available to inhibit the large concentrations of NLPsg2-biotin (187 uM). However, once PaNie was cloned, expressed and successfully purified, it was used to determine an IC50 value for the interaction of cl05 with full length PaNie in an inhibition ELISA (see Figure 27). Since cl05 binds NLPsg2, and this peptide represents a putatively exposed region of PaNie, it is 84 likely that cl05 will bind to the NLPsg2 epitope on the full length PaNie. Thus, the kinetics of cl05 binding to PaNie should be indicative of the binding kinetics to the NLPsg2 peptide alone or when it is present on the surface of PaNie and other NLPs. 70% -, ymax = 0.2238Ln(x) - 0.0512 60% - I1 yave = 0.2238Ln(x) - 0.1113 50% - c ymin = 0.2238Ln(x) - 0.1731 o 40% - 30% - hibit i c 20% - 10% - 0% - 100 l.7<15.4<20.2 Concentration of free PaNie (|JM) Figure 27. Competitive inhibition ELISA (CI-ELISA) of VHH cl05 vs. PaNie. The equation for the trendlines are shown and the IC50 is calculated from these equations in accordance with the logit method. The IC50 refers to the inhibitory concentration of free PaNie necessary to reduce the absorbance at 0 jiM PaNie (Ao) by 50%. The logit method was used to transform the free PaNie inhibition rates and the output is displayed graphically in Figure 27 (Hubert, 1992). The IC50 was calculated using the equations of the average trendline. The equations for the maximum and minimum trendlines were calculated based on one standard deviation from the mean. Thus, the mean IC50 is 15.4 JJM and the range 11.7 to 20.2 uM. 85 3.4. Discussion The screening of a nai've llama library by ribosome display was successful in obtaining a binder to the NLPsg2 peptide. The VHH antibody (cl05) had an affinity for PaNie of 15.4 uM and may be developed as a diagnostic for NLPs by plant pathologists or greenhouse hydroponic growers. However, to prevent disease through capture of PaNie or other NLPs, the affinity of cl05 must be improved or it must be combined with other VHHS recognizing non-overlapping epitopes to capture NLPs using avidity. Ribosome display is a powerful method for the selection of antibodies from large repertoires. However, due to the absence of a reliable functional assay, the antibodies could only be assessed for function by subcloning, soluble expression and subsequent ELISA. This makes ribosome display a less attractive method than phage display, where polyclonal phage ELISA and phage titrations between rounds of panning can be used to track the enrichment via the number of specific clones and via the improved affinity of clones. Access to a radioimmunoassay may have made it possible to monitor ARM- complex binding, thus allowing changes in the panning protocol to be made according to results obtained. In this project for example, no binders could be found by conventional panning without background subtraction and stringent washing, including competition with free antigen. To elucidate what changes are ultimately responsible for the improvement in panning, a functional and practical working assay is required after each round of panning. For example, a radioimmunoassay could be developed using 125I- labelledNLPsg2. 86 Elution of weak VHH binders with free NLPsg2 helped increase VHH specificity. However, the concentration of competitor that was used was too low to eliminate low micromolar binders as indicated by the cl05 antibody binding kinetics. Therefore, competition with higher concentrations (i.e. 1000 fold or more) would have been useful to displace low affinity binders like cl05, but this could not be done because too little peptide was available. The minor differences in the amino acid sequence among the sequenced clones can be explained in three ways; the display of NLPsg2 only allows for a single epitope-paratope interaction, the relatively small library did not contain any other binders, or there were other binders but their sequences were not amplified. The first and second scenarios may be investigated by panning another library with NLPsg2, for example, an immune library raised against PaNie or another full length NLP. The third scenario could be investigated by subcloning end-of-round amplicons into a phage display vector and continue panning this way. In fact, the strengths of ribosome display and phage display could be combined by using this strategy. For example, a very large naive library could be panned one or two rounds using ribosome display and then subcloned into a phage library vector where the enriched (and reduced) library could be panned and monitored using phage ELISA, thus eliminating PCR amplification as a bias during selection. With access to other NLPs, the panning strategy could be further refined so that, for example, the first two rounds are panned with a conserved peptide such as NLPsg2 and subsequent rounds panned with different, specific NLPs, in sequence or in parallel, to elucidate the exposure and function of the conserved peptide. 87 To verify the IC50 range of 11.7 - 20.2 uM, surface plasmon resonance could be performed on the binding of PaNie with cl05 or the binding of other NLPs with cl05. The failure of the surface plasmon resonance analysis with NLPsg2 can be attributed to the large surface bulk change that occurs due to the solvent DMSO. DMSO was required to dissolve the hydrophobic tail of NLPsg2, whose sequence (NVVVWL) within PaNie is predicted to be a beta sheet using the Jpred2 algorithm (ExPASy, 2002). This may mean that cl05 does not have access to its complete epitope, when binding to full-length PaNie, because the beta sheet may not be exposed. Thus, it may be useful to create a smaller version of NLPsg2, which contains only hydrophilic amino acids: i.e. GHRHDWENSGSGK-biotin (essentially the NPP1 domain with a short linker and biotin). Injections of the NLPsg2 peptide into leaves of tobacco did not cause any necrosis (data not shown) and Fellbrich et al. (2002) show that the complete tertiary structure of NPP1 is necessary for necrosis to occur. Thus, the selection of antibodies against NLPsg2 may have resulted in a good NLP binder but will not necessarily lead to the development of an antibody-based NLP antidote because an epitope essential for phytotoxicity is not blocked. Other studies, such as site-directed mutagenesis of conserved domains within the full length NLP will likely answer more of the physiological questions of disease development due to NLPs. For example, it may be possible to construct a NLP-phage display or ribosome display library and use site directed mutagenesis and random mutagenesis to add variety to the NLP sequences. If several anti-peptide antibodies were developed, successive panning of the NLP library with different antibodies used as 88 antigens, could allow a 'functional consensus sequence' of NLPs to be determined. However, this idea assumes that conserved portions of NLPs are actually involved with binding to a putative necrosis eliciting target. At least, such a strategy could determine what portions of NLPs are purely of structural importance and which are of functional importance, which is akin to determining framework and CDR regions of antibodies. Alternatively, if a crystal structure of one or several NLPs was to become known, binding of different antibodies to any portion of the NLP could be modelled and amino acid sequences important for binding could be determined. These important amino acids could then be used as targets for site directed mutagenesis to determine their importance in necrosis elicitation. An alternate, but more expensive way to determine which parts of the NLP protein may be involved in necrosis elicitation would be to synthesise several of the conserved peptides, especially the longer ones such as PYPAVDPGGNTSGGL, and raise antisera against them. NLPs could be injected alone and in conjunction with anti-peptide sera into plant leaves to determine which peptides are more important for necrosis elicitation. However, this assumes that the binding of an antibody blocks a specific site from interacting with its target, rather than blocking the entire NLP from reaching its target by steric hindrance. If sensitive enzyme immunoassays are to be used to either detect NLP presence under various conditions or to elucidate mechanism of action of NLPs, a high affinity binder is required. Typically, a binder in the nanomolar range is sufficient for sensitive ELISA applications. Mechanism of action studies require an antibody that has a binding affinity equal to or lower (better) than the physiological concentration of NLP found under 89 natural conditions. As shown in section 5.3.6, PaNie can induce necrosis in tobacco at concentrations of 50-5000 nM. Thus, it is important to develop an antibody with nanomolar affinity, especially for applications intended to neutralise the action of PaNie. The VHH clones derived from the naive ribosome display library could be matured by in vitro molecular evolution using ribosome display coupled with error prone PCR or by DNA shuffling of early round amplicons (2nd, 3rd or 4th round), where the gene pool is still varied. Alternatively, an immune library could be constructed by injecting NLPsg2 conjugated to bovine serum albumin (BSA) or by injecting full length PaNie into mice or other mammals and isolating the antibody genes from the spleen or peripheral lymphocytes. To optimise use of an immune library it would serve to inject the full length PaNie to generate many binders against various epitopes, so that the library can be panned with a variety of synthesized peptides in the future. 90 4. CONSTRUCTION AND SCREENING OF A HYPERIMMUNE LLAMA PHAGE DISPLAY LIBRARY AGAINST PANIE 4.1. Introduction Historically, antibodies have been used mainly as diagnostics and for a few specific medical therapeutics. Increasingly, research is geared towards developing immunomodulators of host metabolism in animals and plants. In contrast to some agricultural applications of antibodies such as immunoassays for detection of agrochemicals (Chin et al, 2002) or affinity chromatography for the discovery of lead chemistries for new pesticides (Webb et al, 2000; Sheedy and Hall, 2001), plant immunomodulation or disease protection will require antibodies with high affinities that can withstand host or environmental degradation. The previous chapter showed that it was possible to develop a VHH that binds an NLP peptide and PaNie, albeit at low affinity. In addition, last chapter's panning yielded essentially only one amino acid sequence, thus, limiting options for affinity maturation. Thus, it was decided to immunise a llama and create a hyperimmunized library from which several anti-PaNie and perhaps anti-NLP VHHS with high affinity could be isolated. It was decided to create the library from llama rather than mice or rabbits, because of the potential downstream gain of having a single domain antibody that could be easily expressed and that could withstand adverse conditions such as pH and temperature fluctuations. Recently, it has been shown that VHHS will rapidly refold into functional states after complete denaturation and some can even be purified using a heat denaturation and renaturation step (Dumoulin et al, 2002; Olichon et al, 2007). This 91 makes VHHS very attractive for the development of bioactive filters or surfaces, which can be exposed to denaturating conditions and even operate under high temperature and pressure conditions such as are used for sterilization in industry. The phage display format was chosen for library construction because the functional monitoring of antibodies after each round of panning, both by titration of phage particles and by phage ELISA, provides insight for adapting the panning protocol for best results. Furthermore, the library size limitation inherent with phage display is not of concern with highly enriched immune libraries. To develop strong and diverse VHH binders against PaNie it is vital that there is not only a conventional IgG (ConvIgG) response but also a HcIgG response from the llama before the library is constructed. It is as yet unknown if the type of antigen can influence the relation of HcIgG to ConvIgG responses in camelids. This chapter shows that even a relatively small HcIgG immune response is enough to create a large and diverse immune VHH library against an NLP. Different panning strategies yielded three different binders from VHH subfamilies 1 and 2, although no correlation between specific strategies and final antibody properties could be drawn without further investigation. The results of this chapter show that PaNie is immunogenic and that single domain antibodies can be developed from llama HcIgG variable domain sequences. The results show that the constructed phage display library is diverse and that panning can be performed in various ways to obtain different antibodies. One antibody, P10, has an 92 affinity of around ten times that of the others, approaching the nanomolar range, which may be sufficient for testing in downstream applications for the elucidation and neutralisation of the effects of PaNie and other NLPs in hydroponic systems. 4.2. Materials and methods 4.2.1. Immunisation and serum preparation Four CD-I outbred mice (female, <25 g, 7-8 weeks old) and three NZW outbred rabbits (female, 2.5-3.0 kg, 11-13 weeks old) were injected subcutaneously with 25 ug PaNie in a 1:1 PBS:Titermax solution (Sigma-Aldrich, Oakville, ON, Canada). Mice and rabbits were injected three times (100 ul ea) and four times (250 ul ea), respectively, at a different injection site every two weeks. The animals were bled in the weeks between injections. The final two injections into rabbits were performed without Titermax adjuvant. Terminal heart bleeds (and splenectomy for the mice) were performed two weeks following the last injection, resulting in a total of 7 and 9 weeks of immunisation and bleeding for mice and rabbits, respectively. Serum was prepared by centrifugation at 2700 g for 10 minutes at 4°C following clotting at RT for 1-4 h. A two-year-old, male llama (150 kg), was injected subcutaneously in the lower rear back four times; the first three times with a 100 p,g PaNie-Titermax emulsion (1:1 v/v) and the last time with 80 ug PaNie in PBS without adjuvant. The first injection was made on Day 1, the second on Day 14, the third on Day 42 and the fourth Day 56. Approximately 150 ml of pre-immune blood (Day -56) and, on alternate weeks between injections, 150 ml of blood for specific antibody titrations were collected from the jugular vein. 93 Llama serum was prepared by centrifugation at 2700 g for 10 minutes at 4°C after clotting and stored at -20°C. Part of the serum was fractionated twice by protein G chromatography and twice by protein A chromatography (Hitrap, GE Healthcare, Uppsala, Sweden) using a gradient pH elution (Hamers-Casterman et al, 1993), dialysed against PBS, filtered through 0.22 um and stored at 4°C. The protein concentration was determined using the BCA-protein assay (Pierce, Rockford, IL). 4.2.2. Serum titre ELISAs Microtitre wells were coated with PaNie (1 jxg/100 ul PBS) o/n at 8°C. After decanting, the wells were blocked with 3% w/v milk in PBS (200 pi) for 4 h at RT. The wells were washed ten times with PBST. Diluted sera (in 100 ul PBS) were added to each well for 1 h at RT. Following ten washes with PBST, secondary antibody (100 ul) was added to each well for 1 h at RT. The secondary antibody differed depending on which species the serum was derived from: For mouse- and rabbit-based ELISAs, goat anti-mouse (Pierce, Rockford, IL, USA) and goat anti-rabbit IgG (Jackson Labs, West Grove, PA) conjugated to HRP was added at a 1/2500 and 1/6000 dilution, respectively. The llama-based ELISA was performed using goat anti-llama IgG (Bethyl Labs, Montgomery, TX) as the secondary antibody and swine anti-goat - HRP conjugate (Cedarlane Labs, Burlington, ON, Canada) as the tertiary antibody, at 1/1000 and 1/3000 dilutions, respectively. Prior to adding the secondary and tertiary antibodies, the wells were washed 5 to 10 times with PBST (100 ul). After decanting and 5-10 washes with PBST, TMB (100 ul; KPL, Gaithersburg, MD) was added and left to develop for one hour. The antibody binding was 94 determined spectrophotometrically at 450 nm after neutralising with 1.5 M H2SO4 (100 ul). 4.2.3. Library construction RNA for library construction was prepared from the second (Day 21) and fourth (Day 63) llama bleeds: Approximately 100 ml of fresh blood was mixed with EDTA (final 6 mM) to prevent clotting. Total RNA was prepared from 1.5-ml aliquots of blood according to the QIAamp RNA Blood Mini Kit (QIAGEN Inc., Mississauga, Canada), with the exception that lysed leukocytes were stored in the provided buffer (Qiagen buffer RLT) at -80°C until the first day of library construction, when the lysed leukocytes were thawed and incubated at 37°C for 10 minutes. Total RNA was prepared according to the manufacturer's protocol and eluted twice with distilled water (30 ul each), but the second elution was left to soak in the spin tube's membrane for 10 min before centrifugation. First strand cDNA was prepared using the First-Strand cDNA Synthesis Kit (GE Healthcare Biosciences, Uppsala, Sweden) using random hexamers as primers for the transcription of total RNA (3 ^g/20 JLXI) to cDNA. The cDNA (1-5 ul) was used as template for six 50-ul amplifications of the variable heavy (VHH) domains of the heavy chain immunoglobulin genes by polymerase chain reaction using various primers (10 pmol each); in three reactions the forward primers MJ1, MJ2 and MJ3 were used together with the CH2 reverse primer, while in the other three reactions the same forward primers were used in conjunction with the Cn2b3 reverse primer (for primer sequences see Table 6). DNA Taq Polymerase was added only after the first three minutes of the following cycles: 94°C 3 min; 30 cycles of: 94°C for 45 sec, 55°C for 45 sec, 72°C for 1.5 min; 72°C 95 for 7 min; 4°C a>. The 650 and 750 bp amplicons from the CH2 reactions and the 650 bp amplicons from the Cn2b3 reactions were purified using the Qiagen QIAquick Gel Extraction kit and QIAquick PCR purification kit, respectively (QIAGEN Inc., Mississauga, ON, Canada). Approximately 1.1 p.g/31 ul of amplified heavy chain immunoglobulin genes were obtained; 50% from the CH2 amplification and 50% from the CH2b3 amplification. Table 6. Primer oligos used in library construction. S = C/G; M = A/C; K = G/T. Source: Dr. Mehdi Arbabi-Ghahroudi. Name Oligonucleotide sequence 5' -•> 3' MJ1 GCCCAGCCGGCCATGGCCSMKGTGCAGCTGGTGGAKTCTGGGGGA™ MJ2 GCCCAGCCGGCCATGGCCCAGGTAAAGCTGGAGGAGTCTGGGGGA MJ3 GCCCAGCCGGCCATGGCCCAGGCTCAGGTACAGCTGGTGGAGTCT CH2 CGCCATCAAGGTACCAGTTGA CH2b3 GGGGTACCTGTCATCCACGGACCAGCTGA MJ7 CATGTGTAGACTCGCGGCCCAGCCGGCCATGGCC MJ8 CATGTGTAGATTCCTGGCCGGCCTGGCCTGAGGAGACGGTGACCTGG PN2 CCCTCATAGTTAGCGTAACGATCT Ml 3 CAGGAAACAGCTATGACC All of the amplified immunoglobulin genes were used as templates for 31 PCR reactions (1 ul or 35.5 ng per reaction). The PCR amplifications were performed in 50 ul using the Expand High Fidelity PCR System (Hoffmann-La Roche Ltd., Mississauga, ON, Canada) and 10 pmol each of the MJ7 forward and MJ8 reverse primer (see Table 6). The following thermal cycles were employed: 94°C for 3 min; 35 cycles of: 94°C for 30 sec, 96 55°C for 30 sec, 72°C for 1 min; 72°C for 7 min; 4°C. Amplicons were purified using the QIAquick PCR purification kit to yield 22.8 jug/370 ul DNA, of which 20 ug was restriction digested using Sfil. The digestion was done 2 ug at a time in 50 ul (32.5 ul DNA, 2.5 ul H20, 5 ul lOx NEB buffer 2, 5 ul lOx BSA, 5 ul/100 units Sfil; New England Biolabs, Ipswich, MA) overlayed with 100 ul mineral oil and incubated for 5 h at 50°C. Digested DNA (20 ug) was purified using 4 spin columns from the QIAquick PCR purification kit to yield 18.5 ug/80 ul DNA after concentration by speed vacuum. The vector pMEDl (see Figure 28) was double digested using Sfil at 50°C o/n. To reduce the likelihood that any remnants of the excised multiple cloning site (MCS) would re-ligate with the vector and decrease the library insert ratio, the MCS was double digested with Pstl and Xhol at 37°C for 5 h prior to vector purification using the QIAquick PCR purification kit. Two reactions of 5.92 ug of Sfil digested VHH each were ligated into 34.28 jag of Sfil-digested pMEDl each at an approximate molar ratio of 1.6:1 insert:vector, in a total volume of 250 ul with 15ul (45 U) of ligase and 125 ul 2x buffer from the LigaFast Rapid DNA Ligation System (Promega, Madison, WI). After 3 h at RT the reaction products were purified using 12 spin columns from the QIAquick PCR purification kit, elutions pooled and concentrated by speed vacuum to yield approximately 71 ug/360 ul. 97 Plac Ml3 origin PelB leader of replication l K. / NcoIPstI amp pMEDl 4574 bp HArTag fd gene III E. coli origin of replication EcoRI TTTCAAGGAGACAGTCATAATGAAATACCTATTGCCTACGGCAGCCGCTGGA RBS PelB leader signal peptide TTGTTATTACTCGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGCAGTCTA | Sfil | |NcoI | | PstI | Xbal GACTCGAGGGCCAGGCCGGCCAGCACCATCACCATCACCATGTCTCCAGCG | Xhol | Sfil | ( His-Tag ) |NotI GCCGCTACCCGTACGACGTTCCGGACTACGGTTCCGGCCGAGCATMG-fdgUI |( HA-Tag ) Amber Figure 28. pMEDl vector map. The multiple cloning site (MCS) is shown below the map, displaying gene segments, PelB signal peptide (underlined), restriction sites (grey) and purification/detection tags. Source: Dr. Mehdi Arbabi-Ghahroudi. 98 Subsequently, 50 reactions each with 1 ul (197 ng) DNA and 50 ul electrocompetent TGI E.coli cells (Stratagene, La Jolla, CA) were prepared and electroporated using Gene Pulser cuvettes with 0.1-cm gaps (Bio-Rad Laboratories, Mississauga, ON, Canada) at 25 uF, 200 Q. and 1200 V. The transformed cells were immediately transferred to 1 ml of pre-warmed SOC medium and 10 reactions pooled into a 50-ml sterile tube and glucose added to a final concentration of 2% w/v. The five tubes were incubated at 37°C for 2 h at 250 rpm, then centrifuged at 1000 g for 25 min at 4°C. The pellets were resuspended in a total of 30 ml 2xYT containing 100 [ig/ul AMP; a 10-ul aliquot was taken for dilution and the rest was mixed with glycerol (30% final concentration) and frozen as 5-ml aliquots at -80°C. E. coli (100 ul of 10"4, 10'6, 10"8,10"10, 10"12) were plated on LB-AMP plates and incubated at 32°C o/n to determine the library size. Colony PCR was performed on 16 colonies from the dilution plates using the PN2 forward primer and Ml3 reverse primer (see Table 6) and the following cycles: 94°C for 5 min; 30 cycles of: 94°C for 30 sec, 55°C for 30 sec, 72°C for 1 min; 72°C for 7 min; 4°C. The insert ratio was determined to be 50% by agarose gel electrophoresis. DNA fingerprinting using Haelll (Invitrogen Inc., Burlington, ON, Canada) and DNA sequencing were performed on the colony PCR amplicons (10 colonies; 8 ul per clone). 4.2.4. Selection by phage display Library stock (5 ml), representing 2.5 x 109 cells, was thawed, used to inoculate 300 ml 2xYT containing 100 ug/ul AMP and 1% glucose, and grown at 37°C with shaking at u 250 rpm until OD60o was 0.340. M13K07 helper phage (9 x 10 plaque forming units 99 (pfu); New England Biolabs, Ipswich, MA) was used to infect the cells at 37°C without shaking for 30 min followed by shaking for 1 h at 250 rpm. The culture was centrifuged at 3300 g for 10 min and resuspended in 300 ml 2xYT-AMP-KAN (100 ug/ul AMP, 50 ug/ul kanamycin) and grown o/n at 37°C with shaking at 250 rpm. The o/n culture was pelleted at 4400 g for 15 min at 4°C and the supernatant containing phage was filtered through a 0.22 urn filter (Millipore, Billerica, MA). An aliquot (500 ul) was kept for titration of the original phage library. The remaining phage were aliquoted into six tubes (25 ml ea) and precipitated by adding a mixture of polyethylene glycol and NaCl (20% w/v PEG 6000, 2.5 M NaCl; 5 ml per tube), mixed by inversion and incubating on ice for 1 h. After centrifugation at 13800 g for 15 min at 4°C, the 6 white pellets were aspirated and redissolved in a total volume of 2 ml sterile PBS and spun at 20000 g for 3 min at 4°C to remove any remaining bacterial debris. A 10 (j.1- aliquot of the supernatant was used for titration, 300 ul used for panning, and the remaining solution was kept at 8°C after mixing with NaN3 (0.02% v/v). Titrations were performed by adding 10 ul of a dilution (from 10"6 to 10"12) to 90 ul of exponentially growing TGI E. coli cells, placed at 37°C for 15 min and plated on LB-AMP (100 ug/ul) and LB-KAN (50 ug/ul) to determine titre and helper phage packaging ratio, respectively. Two wells of a 96-well microtitre plate (Nunc A/S, Roskilde, Denmark) were coated with recombinant PaNie (100 ul) in sterile PBS and one well coated with sterile PBS (100 ul) o/n at 4°C. See Table 8 for details of the amount of PaNie coated, type of blocking agent 100 used and stringency of washing in each round. Wells were blocked with respective blocking agent (250 ul) in sterile PBS for > 2 h at 37°C, after which precipitated phage (100 ul; 0.17-14 x 1012 pfu) were added to each well and incubated for 2 h at 37°C. The wells were washed with PBST according to the regime shown in Table 8. The phage were eluted from each well using 100 mM triethylamine (100 ul) for a duration of 10 min, with lOx pipetting up and down after 8 min., after which the eluted phage were transferred to a microspin tube containing 1M Tris-HCl pH 7.5 (200 ul) to neutralise the pH. During certain rounds of panning E coli were infected with phage remaining in the well to recover any potentially irreversible binders: The wells were washed with PBST to remove elution buffer, filled with exponentially growing TGI E.coli (100 ul) and incubated at RT for 15 min. These TGI were removed from each well, a 2 ul-sample taken for titration and the remaining solution was kept on ice until being recombined with the 'eluted phage/is. coW mix described below. All eluted phage (600 ul) were added to exponentially growing TGI E. coli cells (2 ml) and placed at RT for 15 min. After this incubation, 2 ul of infected cells were added to 2xYT (200 ul), of which 10 and 100 ul were used for titration on LB-AMP plates. The remaining mixture (2.598 ml) was added to pre-warmed 2xYT to make a final volume of 8 ml; AMP was added to a final concentration of 50 ug/ul and the culture grown for 30 min at 37°C with shaking at 250 rpm. In all cases except for the last round of panning, the remaining E. coli infected by non-eluted phage in the well were combined with the E. coli infected by eluted phage. More AMP was added to a final concentration of 100 ug/ul and the culture grown for an additional 30 min at 37°C with shaking at 250 rpm. 101 M13K07 helper phage (900 ^1; 9x 1010 pfu) was added to infect the culture at 37°C for 5 min without shaking. The 8 ml of infected culture were made up to 100 ml with 2xYT- AMP (100 ug/ul) and grown for approx. 1.25 h at 37°C with shaking at 250 rpm. KAN was added to a final concentration of 50 ug/ul and the culture was grown o/n at 37°C with shaking at 250 rpm to amplify phage for the next round of panning. 4.2.5. Monoclonal phage ELISA Colony PCR was performed on 20 colonies as described in section 4.2.3 and all were shown to have inserts. A further 50 colonies (i.e. a total of 70) were grown in 2 ml of 2x YT-AMP (100 ug/ul) With 0.1% glucose until OD6oo ~ 0.5, after which M13K07 helper phage (10 ul; 1 x 109 pfu) were added and left to infect at 37°C for 15 min without shaking, followed by 30 min with shaking at 250 rpm. KAN was added to a final concentration of 50 ug/ul and the culture grown o/n at 37°C with shaking at 250 rpm. The cultures were centrifuged at -1500 g for 20 min at 4°C and the supernatant used directly in a monoclonal phage ELISA. Microtitre plates (96-well; Nunc A/S, Roskilde, Denmark) were coated o/n with 0.5 ug recombinant PaNie (100 ul) in sterile PBS and negative control wells coated with sterile PBS (100 ul) at 4°C followed by blocking with either 2% fish gelatin (round 4a and 4b) or 1% casein (round 4c) in PBS (250 ul) for at least 2 h at 37°C (see Table 8). Supernatant of monoclonal phage amplifications or 1/1000 dilution of polyclonal phage amplifications were added to 3x PBST washed wells and incubated at 37°C for 2 h. 102 Negative controls consisted of no phage and positive controls consisted of 1:10000 rabbit serum. The wells were washed 5x with PBST (250 ul) and anti-M13 IgG conjugated to horseradish peroxidase (HRP; 100 ul; GE Healthcare, Baie d'Urfe, QC, Canada) was added at 1:5000 dilution in PBS (round 4a and 4b) or in 1% casein-PBS (round 4c) and incubated for 1 h at RT. The wells were again washed 5x with PBST (250 ul) and TMB substrate (100 ul; KPL Inc., Gaithersburg, USA) was added and left to react between 3-7 minutes. The reaction was terminated by addition of 1M phosphoric acid (100 ul) and the optical density was measured at 450 nm. 4.2.6. Soluble expression of selected clones Single colonies of selected clones were picked from LB-CARB plates and used to inoculate 5 ml of LB-G-CARB (0.1% glucose, 75 ug/ml CARB). After growing o/n at 30°C with shaking at 150 rpm, a sample (600 ul) was used to inoculate LB-G-CARB (60 ml). These cultures were induced with IPTG (final cone, of 1 mM) at OD60o = 0.8 and grown o/n at 30°C with shaking at 150 rpm. Cultures were centrifuged at 3000 g for 10 min and expression in both sonicated pellets and the supernatant were analyzed by SDS- PAGE and Western blotting. Good expressors were purified using the BD Talon benchtop purification system (Becton Dickinson, Oakville, ON, Canada) according to the manufacturer's instructions. Large scale cultures were started in 2x 10 ml LB-G-CARB as described above and all 20 ml used to inoculate 1 litre of LB-G-carb. At OD600 = 0.8 the culture was centrifuged at 1500 g for 10 min, fresh LB-CARB without glucose added, and IPTG added after 10 min of growth. Expression, harvesting, analysis and purification were done as described above. 103 4.2.7. Soluble ELISA and surface plasmon resonance Conventional competitive ELISA was not possible for clones P16 and P31, so a novel protocol was developed. A 10 uM PaNie solution was covalently linked to HRP using EZ link Plus Activated Peroxidase (Pierce, Rockford, IL) at a molar stoichiometry of 1:4. The competitive inhibition ELISAs (CI-ELISAs) were performed by coating microtitre wells (Nunc A/S, Roskilde, Denmark) with VHH (100 ul; 5 uM) o/n at 4°C followed by 1 h at RT. After decanting, all wells were blocked with PBS-casein (2% w/v casein-PBS) for 4 h at RT. Following three washes with PBST, 0.1 uM PaNie-HRP in 1% w/v casein- PBS (100 ul) was added, with or without PaNie, and left to incubate for 1 h at RT. PaNie varied from 1.25 - 20 uM, which is equal to a 12.5-200x competition compared to PaNie- HRP. Following five washes with PBST, TMB substrate (150 ul; KPL Inc., Gaithersburg, MD) was added and left to react for 1 h at RT. The reaction was terminated with 1.5 M H2SO4 (150 Jul) and the optical density was measured at 450 nm. The three VHHS PI0, PI6 and P31 were immobilized on three different flow cell surfaces of a CM5 chip using standard amine coupling with 5 ug/ml antibody in 10 mM acetate- buffer, pH 5.0, to a final surface density of 260 resonance units (RU). The reference flow cell FC1 was activated with NHS/EDC and deactivated with ethanolamine. PaNie, at ten concentrations ranging from 24 nM to 2.4 uM, was dissolved in running buffer (150 ul; PBS, 150 mM NaCl, 1 mg/ml CM-Dextran, 0.05% Tween 20) and injected at a flow rate of 30 ul/min onto the chip surface. The surface was regenerated for 3x 20 sec with glycine (10 mM; pH 2.2). All procedures were carried out at RT and all solutions were 104 filtered and degassed. Kinetic constants were determined using a 1:1 Langmuir binding model using the Biaevaluation software 4.0.1 (GE Healthcare Bio-sciences AB, Uppsala, Sweden). After reference subtraction of the control surface, an additional double referencing using a blank buffer injection was applied on the data to subtract matrix effects. 4.3. Results 4.3.1. Anti-PaNie polyclonal serum titres Mice, rabbits and a llama were immunized with PaNie as described in section 4.2.1 and their sera used in ELISAs to determine antibody titres. All animals developed anti-PaNie titres (see Figure 29) with the llama serum being the most sensitive at detecting PaNie. At a dilution of 106, the llama serum from day 63 could detect 200 nM PaNie with a signal to noise ratio greater than four. A similar signal to noise ratio was obtained at a maximum serum dilution of 1:12,800 and 1:102,400 for mice and rabbits, respectively. The rabbits responded more quickly to PaNie than the mice, even though they are larger animals and were injected with the same dose of PaNie. There was no increase in titre after day 35 (3 injections) and day 21 (2 injections) in the rabbits and the llama, respectively. 105 o Q O 100 400 1600 6400 25600 50 200 800 3200 12800 51200 o IO Q O 3200 6400 12800 25600 51200 102400 2.5 KCMaratUW^Jdmarw^LlW*..^^ minimum nil n i O Q O 256 4096 65536 1048576 16777216 1024 16384 262144 4194304 Serum Dilution Figure 29. Titres of anti-PaNie antibodies in serum from immunized mice, rabbits and llama. Lines represent preimmune (Pre) serum and sera collected 7, 21,35,49 and 63 days after the first immunization. 106 To determine the suitability of creating a VHH library, the sera were fractionated into HcIgGs and ConvIgGs by Protein G and Protein A chromatography using a gradient pH elution (Hamers-Casterman et al, 1993). The fractions were visualised for purity by SDS-PAGE and Western blotting (see Figure 30 panels A and B). Purity of the HcIgG fraction is crucial since contamination with ConvIgG can lead one to conclude that there is a HcIgG response when the signal is really due to contaminating ConvIgG. In Figure 30, the individual IgG chains are observed to migrate as distinctly separate molecular weight bands. Due to the high concentration of protein loaded and the concomitant overlapping bands one cannot be certain that the ConvIgG fraction is free of HcIgG, but the HcIgG fraction is likely very pure, since no trace of conventional light chains are seen in either the SDS-PAGE or the Western blot. Panels C and D of Figure 30 show the ELISA results of the heavy chain and conventional IgG serum fractions, when analyzed separately for binding against 200 nM PaNie. The HcIgG fraction is ca. 10X less concentrated than the ConvIgG fraction and a discernable difference from preimmune titres can be observed only above 25 ^g/ml of HcIgG compared to 1 ug/ml of ConvIgG. 107 Serum fractions - Coomassie B Serum fractions - Western Day: -56 -7 21 63 -56 -7 21 63 Day: -56 -7 21 63 -56 -7 21 63 +~* Conv H ConvH .A. _A_ •N» te ^~ , J Conv L V^ J ConvL _A_ HcIgG r ^ HcIgG r ^ %* HcIgG ELISA vs. PaNie D ConvIgG ELISA vs. PaNie 100 1 0.01 0.0001 100 1 0.01 0.0001 jig/ml HcIgG (ig/ml ConvIgG Figure 30. Purity and activity of HcIgG and ConvIgG fractions from the PaNie immunized llama. Panels A & B, respectively, show the Coomassie-stained SDS- PAGE and Western blot probed with goat anti-llama IgG and swine anti-goat IgG- HRP. HcIgGs can be seen at 46 kDa, while conventional heavy (Conv H) and light chains (Conv L) are seen at 50 and 25 kDa, respectively. Panels C & D, respectively, show the binding of HcIgG and ConvIgG to 200 nM PaNie as assessed by ELISA. Sera from day 63 are green, Day 21 are red, while preimmune sera are black. 108 4.3.2. Library construction Although the titre of the llama HcIgGs was much lower than that of ConvIgGs (see Figure 30), a library of 1.5 x 1010 clones (5 x 108 clones/ml) with 50% insert ratio and high sequence diversity was constructed as described in section 4.2.3. Agarose gel electrophoresis of the DNA fingerprinting showed that 8 of 10 sequences were unique, while sequencing showed 100% diversity among the same 10 clones (see Table 7). The least diversity was seen in CDR1, both in terms of identities and length, while both CDR 2 and 3 were highly diverse in both respects. Thus, even though HcIgG titre was low, probably because of the lack of combinatorial diversity in HcIgGs and because it is likely that fewer pocket-forming paratopes are possible without light chains, a large and diverse library was constructed. An immune scFv library from the four mouse spleens was made as well. However, the construction of the scFv library was limited by inefficient linker PCR and low transformation efficiency, resulting in a very small library of 10 clones. Screening of this scFv library indicated that no clones with affinity for PaNie could be found (data not shown). 109 Table 7. DNA sequences of ten random pre-panning clones from the anti-PaNie VRH library. The complementarity determining regions (CDRs) are shown to illustrate sequence diversity between the clones sampled. Clone # CDR1 CDR2 CDR3 1 GGTFSSLGMG VSWSGGRTAYA GERGGSNWYLRGPD 2 GFTFSNYGMS FINSGSTFKNYG TTSGYYDS 3 EFNSETDGIG DDRAGISCIKLSDGTTYYGDP TAEVIPASPLLPLWYDK 4 GSIISIHVMG RVTSHDTTNYA NFFLYAGYGPSSE 5 GFTSDDYAIG GCISKSEDSTYYR DDTTAWNCQDYGHGMD 6 GRTFSDYVMG SITRFDTATIYAAPVR NVRTEYGSKVYD 7 GDTICISAMA RSFKDGRTTSADSVK DPGRRTAMTARCDAV 8 APGSIFSLDD RITLGGTPTYA RGYDLELLGRTYD 9 GSFFSIDTMA TTTPLGKSNYA FIRAEPESGSRA 10 GFTFSGYDMS GIDRGGGVTNYA RGFWSDYSA 4.3.3. Panning titre results Panning was performed using a standard protocol and a modified protocol using well infection as described in section 4.2.4. Polyclonal phage ELISA performed on eluted phage after each round of panning showed a saturated signal after the 2nd round of panning (data not shown). Thus, the progress of panning was monitored solely by phage titres in the elutions from each round of panning (see Table 8). 110 Table 8. Polyclonal phage titres for two trials of panning against PaNie by phage display. In the standard trial triethylamine was used to elute phage from uncoated (BG) and PaNie-coated (PC) wells, while in the well-infection trial triethylamine elution was followed by infection of the well with E. coli. Shaded rounds were screened for VHH clones. Standard trial Round 1 Round 2 Round 3 Round 4a Round 4b BG elution 2.8 x 105 7.8 x 104 7.8 x 104 3.6 xlO6 1.3 xlO4 PC elution 8.8 x 105 3.9 xlO6 8.7 xlO7 9.8 x 106 1.3 x 107 ug PaNie coated 28 /well 20 /well 10/well 5 /well 10 /well Blocking agent 1% casein 1% OVA 2% fish gelatin 2% fish gelatin 1% casein # washes 6 7 8 13 13 Well-infection trial Round 1 Round 2 Round 3 Round 4c BG elution 1.1x10* 6.6 xlO4 7.2 xlO4 no elution BG well infection 8.3 x 104 1.3 x 104 7.1 xlO4 2.0 x 102 PC elution 1.1 xlO6 8.8 xlO4 2.1 xlO5 no elution PC well infection 3.1 x 105 1.2 xlO5 1.6 xlO5 2.6 xlO4 ug PaNie coated 60 /well 30 /well 15 /well 10 /well Blocking agent 1% fish gelatin 1% fish gelatin 1% fish gelatin 1% casein # washes 6 7 8 12 (2 h 40 min)* O - 1st and 2nd wash 30 min each, 3rd and 4th wash 15 min each, followed by 8 washes of 5 min each. Table 8 shows that during both trials panning stringency was increased, both by decreasing the antigen coated and by increasing the number and duration of washes (i.e. the time the wash solution remained in each well). Generally, this increase in stringency had the desired effect of reducing the phage titre of the background controls, while not inhibiting the enrichment of PaNie binders. An exception to this was found in round 4a, where the background increased 50X compared to the previous round, presumably due to not switching the blocking agent. Furthermore, the PaNie-specific titre decreased in round 4a, when only 5 u.g of PaNie was used to coat the microtitre wells and the washing stringency was increased to 13, thus resulting in a signal to noise ratio of only 3:1. In contrast, round 4b showed a signal to noise ratio of 1000:1. The titration signal to noise ratio provides a measure of the number of colony screenings needed to find specific binders amongst non-specific binders in the subsequent monoclonal phage ELISA. Well infection trials are not frequently used in the published literature but the results of Table 8 suggest that this panning strategy warrants further investigation. Although a significant reduction in background titres was not observed until the blocking agent was changed in round 4c, the background titres were consistently lower than those from the PaNie-coated wells. The low output titres of round 4c can be largely attributed to the very long duration of washing, i.e. 2 h and 40 min. Moreover, these lower titres favourably shifted the final signal to noise ratio to approximately 100:1, which made screening of individual clones by monoclonal phage ELISA less difficult. 112 4.3.4. Monoclonal phage ELISA 4^ Round 4a Round 4b KULIIKI 4C 25.0 44 0 20.0 •-c « u 1 15.Q ftl 56 64 C8 60 C St & 10.0 3032 35 38 5.0 4- - 5 4.0 4 —"io"-y-i8, 3.0 Minium It .njii aiUIUUL Ul P10 in green P31inred P16 in blue Clones screened Figure 31. Monoclonal phage ELISA from the three different panning rounds compared by signal to noise ratio. The noise is defined as the average background signal of an uncoated well. Seventy clones are shown, with the 22 sequenced clones highlighted (label = clone number). Sequence comparisons showed three unique amino acid sequences: P10 (green bars), P16 (blue bars) and P31 (red bars). Grey, unlabelled bars were not selected for further study. Monoclonal phage ELISA is a quick way to assess the binding affinity of an individual antibody clone, but it is important to note that the overall binding is relative to the titre of the Ml3 phage displaying the antibody. In the ELISA shown in Figure 31 individual 113 phage amplifications were not titred to save labour and time; this explains how the same clone can yield different signals. To minimise this bias, the monoclonal phage ELISA results are represented as signal to noise ratio, because this takes into account the proportionally higher background titre due to the non-specific binding of phage particles to the microtitre plate. From Figure 31 it is clear that the round 4c ELISA had markedly improved signal to noise ratios compared to round 4a and 4b ELISAs. Since the P31 clone was found in all three selections, some comparisons can be made. It is unlikely that the increase in signal to noise ratio is solely due to higher titre because the same phage amplification method was used and non-specific background binding did not increase, as would be expected if phage titre increased. Instead, the improved signal to noise ratio was likely due to improved blocking with casein instead of fish gelatin, thus reducing non-specific binding (i.e. noise). By round 4, panning selected for three unique anti-PaNie VHH antibodies: P10, P16 and P31, which, respectively, accounted for 2/22 (9%), 5/22 (23%) and 15/22 (68%) of the sequenced clones. 4.3.5. Sequence analysis of selected VHH antibodies Comparison of sequences of P10, P16 and P31 VHHS with the consensus and variable VHH subfamily sequences in Harmsen et al. (2000) revealed that P10 belongs to subfamily VHHI, while P16 and P31 belong to subfamily VHH2. The cl05 VHH described in chapter 2 belongs to subfamily VHH3. Thus, the four anti-PaNie antibodies shown in Figure 32 represent three of four VHH subfamilies. The sequence diversity of P10, PI 6 114 and P31 reflects the excellent quality of the library and the success of the different panning procedures. Even though it is theoretically possible that different paratopes can bind the same epitope, the diversity of the four antibodies likely reflects the existence of multiple antigenic epitopes on the 25 kDa PaNie molecule. I FRl | CDRl I FR2 | CDR2 10 20 3£ 40 50^ 60 P10 (1) QVQLVESGGGLVQAGGSLRLSCAGSGRTFSNHNMGWFRQAPGKEREFVAAINWSGSHTYYAD P16 (2) K...E P TP SV...NFNI W E W H...TSAD-S...M...R... P31 (2) K...E S P T INVGRFAL Y Q...DL E...ARG-GS...N cl05 (3) D QA A...P A F...LA...YAI G...SCMHSFDGS TA I FR3 | CDR3 | FR4 | 70 80 9(3 10C) 110 12£ P10 (1) SVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAEDTISGVSNAEGYRYWGQGTQVTVSS P16 (2) M ASA...YPL...G...NYVKF P31 (2) I H NARV...T TD cl05{3) S MG DN...EGS T A...P DG GF Figure 32. Amino acid alignment of the three anti-PaNie VHH clones and the anti- NLPsg2 VHH C105. VHH subfamily number is shown in brackets (Harmsen et al., 2000). The dots in the sequence represent 100% homology with P10. The Sequential numbering system was used and the CDR regions were identified according to the alignment in Saerens et al. (2005). There are eight conserved amino acids in the CDRs of the four antibodies, none of which are in CDR3. The conserved amino acids are G26 and G35 at the opposite ends of CDRl, as well as T58 and Y60 at the center and S63, V64, K65, G66 at the C-terminal end of CDR2. Except for T58 and Y60, these amino acids may simply function as extensions of the framework regions. There is little sequence similarity in the CDR3 between any clones, except for four amino acids, including a consecutive stretch of three, in the CDR3 of P10 115 and cl05 (D99; E108-G109-Y110). It is worth noting that both P31 and cl05 have shorter CDR3s: 7 and 9 amino acids, respectively, vs. 14 amino acids in P10 and PI6. Protein characteristics for the antibodies were calculated using proteomics tools from the online Expert Protein Analysis System (ExPASy) and are shown in Table 9 (ExPASy, 2002). All antibodies are hydrophilic as indicated by the negative scores of GRAVY (GRand AVerage hYdropathicity). P10 and PI6 exhibit very similar protein characteristics, with neutral pi and a near balance of charges. P31 has a pi of 9.30 due to the presence of more positively charged amino acids, while cl05 has a pi of 5.90 due to its excess of negatively charged amino acids. Panning was carried out at pH ~ 7.5, close to the pi of P10 and P16, but at this pH P31 has an overall positive charge and cl05 has an overall negative charge. Table 9. Protein parameters for the selected anti-PaNie VRHS. VHH clone P10 P16 P31 c!05 Molecular Weight (Da) 15051.5 14906.6 13838.3 14042.3 Molar extinction coefficient 26992.5 31002.5 18512.5 21492.5 Theoretical pi 7.87 7.86 9.30 5.90 Negatively charged amino acids 10 10 9 12 Positively charged amino acids 11 11 13 8 GRAVY* -0.596 -0.495 -0.529 -0.449 * GRAVY = GRand AVerage hYdropathicity; negative values indicate hydrophilicity 116 4.3.6. Soluble expression of selected anti-PaNie VHHS All VHHS expressed well in culture volumes of 60 - 1000 ml, and culture supernatants could be visualised on SDS-PAGE and by Western blot. VHHS readily leaked into the growth medium from the periplasm and were harvested from the supernatant by centrifugation. A significant concentration of VHH remained in the cytoplasm or periplasm but was not purified due to the potential of aggregation in inclusion bodies. Larger cultures (1 L) yielded 1.8-2.8 mg/L of pure protein from the culture supernatant after purification by nickel affinity chromatography. kDa 37 26 19 15 6- P10 P16 P31 P10 P16 P31 Figure 33. Reducing SDS-PAGE of VHH P10, P16 and P31 purified from E coli. Left panel shows Coomassie stained gel and right panel shows a Western blot probed with anti-penta-HIS IgG. Figure 33 shows an SDS-PAGE and Western blot of purified VHHs: apart from the correct size band, all solubly expressed VHHS also show a weak band between 19-26 kDa, which is most evident for P31. The Western blot shows that this band does react with the anti-penta HIS secondary antibody, indicating that it may be an aggregate of P31. The band's location just beneath the 26 kDa marker indicates that it may be a dimer, 117 which should have a theoretical weight of 21.1 kDa. It should be noted that this putative aggregate has withstood boiling and the reducing conditions of the SDS-PAGE. 4.3.7. Kinetic studies of selected anti-PaNie VHHS VHHS P10, P16 and P31 were analysed by competitive inhibition ELISA as shown in Figure 34. The affinities determined from the ELISA were verified by surface plasmon resonance (see Figure 35). Preceding the kinetic studies, checkerboard ELISAs were performed to determine working concentrations for the VHHS. Minimum coating concentrations of PaNie lie between 0.25 and 1 uM, with consistently high signals at 1 uM. Minimum concentrations of VHH to accurately detect a 1 uM coating of PaNie were determined to be 0.4 uM. Maximum coating and VHH working concentrations could not be determined as the ELISA signal could not be saturated at the concentrations tested (8.3 uM PaNie coated and 13.8 uM VHH applied). CI-ELISAs of all three VHHs vs. PaNie showed the relative affinities of the clones to be P10 > P31 > PI 6 (> cl05) as detailed in Table 11. The logit method (Hubert, 1992) was used to transform and analyze the free PaNie inhibition of VHHs P10, PI6 and P31 (Figure 34). The IC50 values for each antibody were calculated using the equations of the average trendline. The equations for the maximum and minimum trendlines were calculated based on one standard deviation from the mean. Figure 34 shows that for P10 the mean ICsois 1.1 uM and the range 0.8 to 1.6 uM; for P16 the mean IC50 is 13.3 uM and the range 9.9 to 17.7 uM; and for P31 the mean IC50 is 10.3 uM and the range 7.6 to 13.8 uM. 118 < 90% • j CI-ELISA of VHH P10 vs. PaNie 3 .-li^t ' 70% ,.£^^^'' T- ...•••^S%*-"''' ymax = 0.1369Ln(x) +0.5327 § 50% ----- .-.-.-^gggf^ yave = 0.1369Ln(x) + 0.4836 it i ..•••<^i^ll i 5 5 30% - E^^ ' ymin = 0.1369Ln(x) +0.4345 .c ill -10% - IC -»0.8<1.K1.6MM 0^ 50 0.1 1 1015 Concentration of free PaNie (\iM) B "o < 70%- CI-ELISA of VHH P16 vs. PaNie ••••••'T ^ 50% -L X* ,^ ?90%i CI-ELISA of V H P31 vs. PaNie < H | 70%- ,1 50%- ymax = 0.3486Ln(x) - 0.2090 S.*' -"'[s^ .,-•••*' = 30%- yave = 0.3486Ln(x) - 0.3124 0 --"'^' -••" 'i 10%- T , <%A : ymin = 0.3486Ln(x) - 0.4158 : J 1-10% "X^ NP Qno/ _ IC50^7.6<10.3<13.8|JM •-"" : 1 1 o>-OU /o T •• 1 1() 15 20 Concentration of free PaNie (uM) Figure 34. Competitive inhibition ELISA (CI-ELISA) of VHHs P10 (A), P16 (B) and P31 (C) vs. PaNie. The equations for the trendlines are shown and the IC5o calculated from these equations in accordance with the logit method. The IC50 refers to the concentration of free PaNie required to reduce the absorbance of 0 uM PaNie (A0) by 50%. 119 To compare and verify the affinities obtained from CI-ELIS A, the VHHS were analysed by surface plasmon resonance as described in section 4.2.7. Figure 35 shows the response of PaNie binding to the three VHHs over time. Due to the very low signals, saturation of the surfaces (Rmax) could not be reached. Since the Rmax value is required for an accurate determination of the kinetic constants this value was estimated based on the ligand density. Comparison of the molecular masses of PaNie and the VHHS results in a theoretical maximum binding signal of 440 RU for PaNie based on 260 RU of immobilised VHH. Since steric restrictions for the antigen/antibody binding on the surface cannot be excluded, a stoichiometry for the active ligand density on the chip surface was estimated to be about 25% yielding a Rmax value of 110 RU. This Rmax value was set constant for all three antibodies for calculating kass and KD. For PaNie binding to P10 a strongly biphasic dissociation can be seen, where the surface plasmon resonance response drops off rapidly at 300 sec and the dissociation slows around 350 sec. Thus a second kdiSS2 can be calculated based on the slow dissociation phase. For the determination of the KD, the fast dissociation kdjSS was used. PaNie has the highest affinity to P10 with a KD of about 2.8 uM, whereas the binding to PI 6 and P31 show KDS of about 11.1 and 6.6 uM, respectively. The lower KD of P10 is due to it having a faster association rate than the other two clones (see Table 10 and Table 11). Note that if kdiss2 was used to calculate the KD of P10 it would be ca. 240 nM. 120 PaNie binding to P10 ^ 40- •Z4MM D •1.4yM 863nM •518nM • c . 3 20- •311nM m * • 187nM tn •112nM | 10- in 67nM i i i i II 100 200 300 400 500 600 Time {sec} 15n PaNie binding to P31 24MM 3 10- 1.4MM oi ***< 863nM it s 5- 518nM c 3 311nM «i 0- 187nM 01 c 112nM o -ft- 67nM Ma. 40nM 0C -10- 24nM -15- 100 100 200 300 400 500 600 Time {sec} Figure 35. Surface plasmon resonance of immobilised VRH P10, P16 and P31 vs nM - 2.4 uM PaNie. 121 Table 10. Association (kass) and dissociation (kdiss) rate constants for the interaction of PaNie and VHHS during surface plasmon resonance. The kdiss2 for the latter portion of the biphasic dissociation of PaNie from P10 is shown in brackets. Constant "^~~~VHHP10~~~~~~THB7ir~~~~~THHP3r 3 kass (10 M"'s"') 5.4 ±1.5 0.85 ±0.27 0.62 ±0.14 1^^ (10'V) 1.5 ±0.1 0.94 ±0.23 0.41 ±0.09 (kdiaa; lOV) (0.13 ±0.02) The affinity of the VHH antibodies, as calculated by KD = kdiss / kass from the surface plasmon resonance data, is shown in Table 11 in comparison to the KD ~ IC50 obtained from CI-ELISA. According to all measurements, the three VHHS derived from the hyperimmune llama library have stronger affinity than cl05 (11.7<15.4<20.2 uM) derived from the nai've llama library described in chapter 2. Table 11. Comparison of affinity constants (KDS) for the anti-PaNie VHHS P10, P16 and P31. The data were obtained by CI-ELISA and surface plasmon resonance (SPR). It is assumed for the comparison that the IC50 obtained by CI-ELISA is an approximation for the KD. Data are shown as mean (bold) and range. KD(uM) VHH P10 VHH PI 6 VHH P31 CI-ELISA 0.8<1.1<1.6 9.9 < 13.3 < 17.7 7.6 < 10.3 < 13.8 SPR 1.9 < 2.8 < 4.1 6.3 < 11.1 < 20.2 4.2 < 6.6 < 10.4 122 4.4. Discussion The construction and screening of the hyperimmune phage display library was successful in yielding three unique anti-PaNie VHHS representing two VHH subfamilies. All three VHHS expressed solubly and their affinities were determined to be in the low micromolar range by CI-ELISA and surface plasmon resonance. It was concluded that clone P10, which had the fastest association rate and lowest KD (low uM), and which also binds to the NLPsg2 peptide (see section 5.3.4), was most suitable for development of assays for the capture of PaNie and other NLPs. Understanding of the immune response is of primary importance for recombinant antibody development, because a strong immune response in the immunized animal provides the basis for construction of a hyperimmune library with diverse, high affinity binders. All immunisations of mice, rabbits and a llama with PaNie resulted in sera with affinity for PaNie. The titres of both the rabbit and llama sera indicate that three injections of PaNie sufficed to saturate the response as seen by ELISA. The fractionation of the llama serum into conventional and heavy chain IgGs afforded a limited view of the different immunogenicities of PaNie in relation to IgG subclasses. The HcIgG response was lower than the ConvIgG response, which was expected due to the nature of the HcIgG molecule. Antibody-antigen interactions are often referred to as being analogous to 'lock and key' mechanisms. The HcIgGs lack light chains and compensate by having, on average, longer CDR loops than ConvIgGs. Thus, the HcIgG may be more likely to present the proverbial 'key' and PaNie acts as the 'lock', while ConvIgGs may also present a 'lock' using the VH and VL as a cleft with PaNie being the 'key'. If PaNie 123 presents several attractive protrusions and less attractive clefts, then ConvIgG will have more attractive epitopes to bind to than HcIgGs. One such protrusion may exist because all NLPs have two conserved cysteines (NLP positions 74 and 100; PaNie positions 58 and 84) that may form a loop by disulphide bridging. It is noteworthy that the preimmune ConvIgGs had higher background binding than the preimmune HcIgGs, perhaps due to the presence of more malleable and thus non-specific binding clefts in ConvIgGs. Solving the crystal structure for PaNie and modeling the antibody-PaNie interaction may provide sufficient information to determine why HcIgGs have lower affinities than ConvIgGs. After all, it may be that PaNie does not present epitopes that elicit high- affinity antibody-based responses and that the llama immune system, as well as that of mice and rabbits, relies on the combined weak affinities (avidity) of multiple IgGs. The HcIgG response of the llama was sufficient to construct a diverse VHH library as shown by random clone sequences and by the diversity of anti-PaNie VHHS selected. However, how a library is panned is also very important; i.e. the blocking agents used, the type and concentration of antigen used, washing stringency and the elution strategy used. It is interesting to note that P10 with the highest kass came from round 4a, in which the lowest concentration of PaNie was used to coat wells. Even though this round resulted in worse phage titres overall, it did yield the best binder; i.e. P10. As described in section 4.3.3, the background titre increased and the PaNie-specific titre decreased in round 4a, possibly because the 5 ug coating of PaNie was too low to retain low affinity binders during 13 stringent washes. The background titre may have increased because the blocking agent was not switched from the previous round. 124 It is possible that large amplifications of phage create a bias for easily copied and well packaged sequences. Round 4b may be an example of this. Although clone P10 was present in the amplified pool of phages used as input for round 4b, clone P31 was preferentially selected, even though it has a lower affinity. This may be explained by the high occurrence of P31 relative to P10, rather than by an absence of P10 in round 4b eluate. In conclusion, it is important to switch the blocking agent to lower background noise, and to reduce the amount of PaNie coated to the wells, so that specific and diverse antibodies can be found. Modifying the standard phage display trial, by infecting E. coli with phage remaining in the well after triethylamine elution, provides a method for selecting antibodies by phage display. Using this method, a third anti-PaNie VHH was found; namely PI6. This shows that enrichment of specific binders is possible using this method of panning. By using this method, it may also be possible to find potentially irreversible binders, which remain bound to the coated PaNie after very stringent washing and triethylamine elution. VHHS selected in this chapter and in chapter 2 represent three out of four VHH subfamilies and have varied CDR sequences, which indicates that there may be several different immunogenic epitopes on PaNie. However, note that cl05 and P10 have four amino acids (3 consecutive) in common in CDR3, which could indicate that they bind to the same or overlapping epitope(s). The three amino acids 'EGY' in the CDR3 of P10 and cl05 could be mutated to determine if this sequence is crucial for the binding to NLPsg2 and the part of PaNie corresponding to NLPsg2. Comparing P31 and cl05, it 125 may not be a coincidence that both have much shorter CDR3s and have lower affinities for PaNie than P10 and PI6. Thus, extending the CDR3s could improve the binding affinity of P31 and cl05, which would be especially interesting in the case of cl05 as it may bind the same epitope as P10. These kinds of investigations could prove very important in determining the protein structure-function relationships, particularly when combined with growing bioinformatics data on the emerging NLP and VHH families. The two methods of CI-ELISA and surface plasmon resonance used to measure the affinities of each of the three anti-PaNie VHHS were in general agreement. However, regardless of the method used, uncertainty in these affinity values was exacerbated by two potentially linked phenomena: the increase rather than decrease of absorbance at low concentrations of competition during CI-ELISA and the biphasic nature of the dissociation of VHH and PaNie. The increase in absorbance at low concentrations of competition during CI-ELISA can be almost entirely accounted for by error in the measurements as seen from Figure 34. Alternatively, specific aggregation/polymerization between free PaNie and PaNie-HRP may create an avidity effect at low concentrations of the competitor, i.e. PaNie. This polymerization may create an avidity effect, which in turn may contribute to the resistance of PaNie-HRP to being washed away during the ELISA. The biphasic nature of the dissociation of PaNie from the VHH, seen most clearly in the surface plasmon resonance results using clone P10, may also be due to polymerization of PaNie. These putative PaNie-PaNie interactions may compete with PaNie-VHH interactions. Therefore, at high concentrations of PaNie, there would seem to be a faster off rate, because more PaNie molecules are there to compete, through polymerization 126 with surrounding PaNie molecules, with the binding of PaNie molecules to the VRH. This hypothesis is supported by the fact that the biphasic nature of the dissociation is most pronounced at higher concentrations of injected PaNie. In addition, any PaNie that may have polymerized while bound to P10, may create a polymer between different P10- PaNie pairs. This 'bridging' or avidity effect would result in PaNie remaining bound longer to P10 on the chip, thus contributing to the biphasic nature of the dissociation. The affinity constant (KD) calculated for each phase was 2.8 uM and 240 nM and the average was 1.5 uM, which falls within the range of the KD calculated by CI-ELISA. It is thus concluded that CI-ELISA is an accurate method for determining the KD, while surface plasmon resonance can give additional insight into the nature of the Ab-Ag interaction. Of the three VHHS selected from the hyperimmune llama library by phage display, clone P10 had the highest affinity and was also shown to bind to the NLPsg2 peptide. Since the NLPsg2 peptide contains the NPP1 domain shared by all NLPs described in the literature, VHH P10 represents a very good candidate for the development of anti-NLP diagnostics and therapeutics. This antibody not only provides a building block for crop protection measures, but may also help elucidate the mechanism of action of PaNie and related NLPs. 127 5. CONSTRUCTION OF VHH-CBM FUSIONS FOR CAPTURE OF PANIE ON CELLULOSE 5.1. Introduction To utilise the VHH antibodies developed in this thesis for crop protection in hydroponic systems, a simple and cheap assay is needed. The objectives of this chapter are to demonstrate that VHH-CBM protein fusions, when bound to cellulose surfaces, provide a reliable mechanism for capture of PaNie. The research aims to build a strategy to neutralize PaNie and other NLPs before they reach the plant, thus providing hydroponic greenhouse growers with a single technology that may be used to protect any crop species against PaNie and perhaps other NLPs. This chapter describes the construction of VHH-CBMS, their expression and purification, and their function in solution and while bound to cellulose. In addition, the characterization of necrosis induced by PaNie in the model plant tobacco, Nicotiana tabacum, is described. Tobacco was chosen as a model system, because it is fast growing, well studied, amenable to hydroponic cultivation and known to be susceptible to PaNie- induced necrosis (Veit et ah, 2001). The results show that all VHH-CBMS express well in the yeast P. pastoris, that functional VHH can be recovered by thrombin cleavage and that the binding kinetics to PaNie are unchanged from the VHHS produced in E. coli. P10- CBM can be bound to cellulose filter paper and can be used to detect PaNie in the filtrate. The filtration assay was performed in less than half an hour and was shown to be semi quantitative. Anti-PaNie polyclonal sera neutralized necrosis elicited by both exogenous and native PaNie in leaves, promising that necrosis prevention using antibody-CBM 128 fusions should be possible in principle. The results of confocal laser scanning microscopy show that P10-CBM bound to the surface of tobacco roots is capable of binding PaNie. An in-line bioactive paper filter in a hydroponic system may be able to protect plants from NLP-induced disease or at least act as an early warning system for greenhouse growers. Binding exogenously produced VHH-CBM directly to the surface of plant roots could mimic expression of VHH-CBM from the roots of a transgenic plant, but without having to carry out lengthy, complicated transformation steps and without the vegetable crop product being transgenic. In short, this chapter concludes that applications of VHH- CBMs for crop protection are technically feasible and may provide a versatile alternative or addition to current pest management practices. 5.2. Materials And Methods 5.2.1. VHH-CBM construction Antibody sequences (P10, P16, P31) described in section 4.3.5 were modified by PCR to include terminal Xhol and Sail restriction sites and a C-terminal thrombin site. PCR was performed using primers GH16 + GH18 for VHH P10 and GH17 + GH18 for both VHH P16 and P31 (see Table 12). The PCR conditions were as follows: 95°C for 5 min; 26 cycles of: 95°C for 30 sec, 54°C for 30 sec, 72°C for 45 sec; 72°C for 10 min; 4°C oo. The amplified VHH DNA was purified using the Qiagen PCR purification kit (Qiagen, Mississauga, ON, Canada), digested With. Xhol and Sail, and gel-purified with the Qiagen gel-purification kit (Qiagen, Mississauga, ON, Canada). The VHH fragments were ligated into a similarly digested vector, pPICZaA (Invitrogen Inc., Burlington, ON, Canada), 129 which contains the S, cerevisiae a-factor secretion signal, AOX1 inducible promoter, zeocin antibiotic resistance gene and a previously inserted cellulose binding domain XynlOACBM2a (Hussack, 2008). After ligation at 16°C o/n, the constructs were transformed into TOP 10 F' E, coli and plated on YPD agar + zeocin (25 ug/ml) for selection. Clones were screened by colony PCR, grown o/n in low-salt LB and plasmids purified for DNA sequencing. The purified constructs were linearized by Sacl, transformed into chemically competent P. pastoris strain X-33 (Invitrogen Inc., Burlington, ON, Canada), and selected on YPD agar + zeocin (200 ug/ml) plates. The transformants were incubated at 30°C for 72 h. Table 12. Primers used in VHH-CBM cloning Primer Oligonucleotide sequence 5' -> 3' GH16 TGTGATTCTCTCGAGAAAAGAGAGGCTGAAGCTATGGCCCA GGCTCAGGTACAGCTG GH17 TGTGATTCTCTCGAG AA AAGAGAGGCTG AAGCTATGGCCC A G TAAAGCTGGAGGAG GH 18 TTTTGTGTCGACCTATC AAGAACCTCTTGGAACCAAGGAACC GTAGTCCGGAACGTCGTACGG To identify positive clones, three colonies from each of the three VHH constructs were used to inoculate BMGY media (10 ml; 1% w/v yeast extract, 2% w/v peptone, 2% w/v glucose, 0.1 M sodium phosphate buffer, 1.0% glycerol, 0.04 mg/ml biotin; pH 6.0) and grown at 30°C for 18 h with shaking at 250 rpm. This culture was used to inoculate BMMY media (100 ml; 1% w/v yeast extract, 2% w/v peptone, 0.1 M sodium phosphate buffer, 1.0% (v/v) methanol, and 0.04 mg/ml biotin; pH 6.0) and grown at 30°C for 5 days with shaking at 250 rpm, with additions of methanol every 24 h (0.5 % v/v). After 5 130 days of induction, Pichia-VHH-CBM supernatant was analyzed by SDS-PAGE and Western immunoblotting to confirm expression. 5' AOX1 a - signal sequence Anti-PaNie VHH 6x His tag HA tag Thrombin site E. coli ori XynlOACBM2a 6x His tag c-myc tag zeocin Figure 36. Construction of an anti-PaNie VHH-CBM expression plasmid based on pPICZaA. The highest expressing clone of each of the VHH and VHH-CBM constructs was chosen for large-scale expression (300 - 1000 ml) and for making glycerol stocks (30% glycerol, frozen at -80°C). For large scale expression, the clones selected from small-scale (100 ml) cultures were started in BMGY as described above, centrifuged (1500 g, 10 min) after 18 h and resuspended in fresh BMGY (100 ml) and grown for an additional 12-16 h at 30°C with shaking at 250 rpm. This BMGY culture was centrifuged (1500 g, 10 min) and resuspended in BMMY (300-1000 ml, OD600 ~ 1.0) and grown in bevelled flasks at 30°C for 5-7 days with shaking at 250 rpm; methanol was added every 24 h (0.5 % v/v). The large-scale culture was terminated by centrifugation at 8000 g for 20 min. The supernatant was sterile filtered through a 0.22 um filter (Corning Inc. Corning, NY) and the VHHS and VHH-CBMs purified from the filtrate using nickel affinity chromatography 131 (5-ml Ni-NTA HisTrap column; FPLC Akta; GE Healthcare Bio-sciences AB, Uppsala, Sweden). VHHs and VHH-CBM fusions were eluted using a step gradient of imidazole. The imidazole contained in the eluted fractions was removed on a Sephadex G-25 desalting column and presence of VHH or VHH-CBM confirmed by SDS-PAGE and Western immunoblotting. 5.2.2. VHH-CBM binding to cellulose and thrombin cleavage Purified P10 and P10-CBM (175 jig) were added to CP-102 cellulose beads (100 mg; Asahi Kasei Chemicals Corp., Tokyo, Japan) in 1.5 ml of PBS and left to incubate at RT o/n on a rocker. Samples of the supernatant (80 ul) were taken before and after this incubation. In addition, samples (25 ul) of CP-102 beads were taken after the incubation and the beads washed 3X with PBST. The samples of beads were resuspended in IX loading dye buffer (150 ul; 62.5 mM Tris-HCl, 2% w/v SDS, 10% glycerol and 0.01% w/v bromophenol blue, 13 mM DTT, pH 6.8) and all samples boiled for 5 min prior to loading the SDS-PAGE gel. Antibody was also immobilized onto CP-102 cellulose beads directly during culture. CP- 102 beads (2X 1 g) were autoclaved in distilled water (300 ml each in two 1-L bevelled flasks). The sterile water was aspirated and a 'day 3' large-scale P. pastoris culture added to the sterile flask containing the CP-102 beads and left to incubate with the culture for 3 days at 30°C with shaking at 250 rpm. Harvesting of beads and bound antibodies was performed as follows: Supernatant was decanted and CP-102 beads washed with PBST 132 (50 ml) until the supernatant was clear (at least five times). The washed beads were resuspended in PBST (2 ml) and samples (40 ul) taken for SDS-PAGE. CP-102 cellulose beads (~1 g/2 ml PBST) with bound antibody were used in a 24-h thrombin cleavage test. CP-102 beads (1.9 ml) were added to a thrombin cleavage solution (4 ml; 400 ul 10X cleavage buffer, 20 ul thrombin, 180 ul dilution buffer, 3400 ul distilled water; Novagen, Madison, WI) in a glass test tube (10 ml) and incubated at RT for 24 h. Samples of the supernatant (120 ul) were taken every 4 h, mixed with 5X loading dye (30 ul; 312.5 mM Tris-HCl, 10% w/v SDS, 50% glycerol and 0.05% w/v bromophenol blue, 65 mM DTT, pH 6.8) and immediately boiled. Samples of the beads (100 ul) were taken before and after the thrombin cleavage and resuspended in PBS and loading dye (160 ul PBS, 30 ul 5X loading dye). The SDS-PAGE gel (Tris-Glycine; 12% acrylamide) was loaded with 30 ul of sample per lane. 5.2.3. Antibody functionality by ELISA Microtitre plates (96-well) were coated with PaNie (0.2-4 uM; 100 ul PBS) o/n at 8°C followed by 1.5 h at RT. After decanting, the wells were blocked with 2% w/v casein in PBS (200 ul) for at least 2 h at RT. The wells were washed three times with PBST (200 ul). VHH or VHH-CBM was made up in 1% w/v casein-PBS (100 ul) and added to the wells for 1 h at RT. Following five washes with PBST (200 ul), rabbit anti-HA IgG conjugated to HRP (100 ul; 0.4 ug/ml in 1% casein-PBS; Bethyl Labs, Montgomery, TX) was added for 1 h at RT. Following another five washes with PBST (200 ul), TMB (150 ul; Pierce, Rockford, IL) was added and left to develop for 15 min to 1 h at RT in the 133 dark. The level of binding was determined spectrophotometrically at 450 nm after neutralising with 1.5 M H2S04 (150 ul). 5.2.4. Surface plasmon resonance PaNie (10 ul; 50 ng/ul) was immobilized on a CM5 chip using standard amine coupling in 10 mM acetate-buffer, pH 4.5, to a final surface density of 440 resonance units (RU). A reference flow cell FC1 was coated with ovalbumin. All cells were blocked by deactivation with ethanolamine (1 M, pH 8.5). Pure VHH P10 obtained from P. pastoris culture was further purified by size exclusion chromatography (Superdex 75) to exclude any potential multimers. VHH P10 (40 ul; 0.2 - 8.8 uM) dissolved in running buffer (lOmM HEPES, 150mM NaCl, 3mM EDTA, 0.005% surfactant P20, pH7.4) was injected at a flow rate of 40 ul/min onto the chip surface. All procedures were carried out at RT and all solutions were filtered and degassed. Analysis was conducted using the Biaevaluation software 4.0.1 (GE Healthcare Bio-sciences AB, Uppsala, Sweden). 5.2.5. Bioactive paper assay development Paper discs were cut using a core-borer of 5-mm diameter and inserted as filters for individual wells in a vacuum manifold (Bio-Dot apparatus, Bio-Rad Laboratories, Mississauga, ON, Canada). Alternatively, strips of paper acting as filters for several wells at a time were cut and sandwiched in the Bio-Dot apparatus. Unused wells were covered with tape to create a seal. All steps were carried out at RT. The filter papers were coated with P10-CBM or P10 alone as control by applying the desired concentration in PBS to the wells of the Bio-Dot apparatus for 2 min to 1 h. Vacuum was applied to pull the liquid through the filters. Filters were blocked with 2% w/v casein-PBS or 3% w/v milk- 134 PBS (200 |il) for 10 min to 2 h. Filters were washed with PBST (300 ul) five times, applying vacuum between each wash. PaNie-HRP (50 nM; 100 ul) was added for 10 min and pulled through by vacuum. Filters were washed with PBST (300 ul) ten times and then removed from the Bio-Dot apparatus to a microtitre well. TMB (150 ul; KPL, Gaithersburg, MD) was added and left to develop for 5 min to 1.5 h, during which the developing solution was mixed by pipetting 10 times. The liquid (120 ul) was removed from the well containing the filter to a new microtitre well and the level of binding was determined spectrophotometrically at 450 nm after neutralising with 1.5 M H2SO4 (120 ul). Competitive bioactive paper assays were performed as described above with the exception that free, unconjugated PaNie (2.5 uM) was applied to the paper for 1 h before the addition of PaNie-HRP. 5.2.6. PaNie leaf injections and analysis of necrosis Tobacco plants, 81V9 wildtype Nicotiana tabacum, were grown from seed and injected with PaNie at 5-7 weeks of age. Injections were performed using a 10 ul Hamilton syringe with its needle piercing and protruding by less than 0.5 mm from a rubber stopper. Injections were made to the top of the leaf using a foil covered rubber stopper as a backing for the bottom of the leaf as shown in Figure 37. Plants were injected during the day to ensure light-dependent necrosis could occur. When different solutions were injected together (e.g. PaNie and anti-PaNie serum), they were mixed immediately before injection. 135 Figure 37. Leaf injection system mounted on a micromanipulator. Early onset of necrosis was monitored using a fluorimager (Model FISY5006; Technologia Ltd., Essex, UK). Injected plants were placed in the dark 30 min after injection and left there for 30 min before measuring chlorophyll fluorescence according to the method of Maxwell and Johnson (2000). 5.2.7. PaNie root assays and root preparations Tobacco plants, 81V9 wildtype N. tabacum, were seeded on sterile soil and transferred at 2 weeks of age to hydroponic nutrient solution (0.487 g/L Ca(NC>3)2, 0.73 g/L Plant-Prod 7:11:27 - N:P:K, pH 5.8). The hydroponic assembly is shown in Figure 38. 136 Figure 38. Hydroponic plant growth unit with a 6-week-old tobacco plant. The roots and two drinking straws were positioned in a 3-ml container and secured with foam. The straws provided support and functioned as conduits for application of nutrient solution and treatments. Roots for immunofluorescence experiments were prepared by washing roots from 2- week-old tobacco plants with PBS and then removing a 5 mm section of secondary root with a scalpel. 5.2.8. Production of Pythium aphanidermatum exudates P. aphanidermatum was grown in sterile malt-dextrose broth (100 ml) with shaking at 150 rpm for one week at 28°C as described in section 3.2.4. Mycelium was removed by filtration through cheese cloth and the filtrate centrifuged at 3000 g for 10 min. The presence of PaNie in the supernatant was verified by SDS-PAGE and Western blot analysis using mouse anti-PaNie serum (1/5000) and goat anti-mouse IgG conjugated to alkaline phosphatase (AP) (1/2500; Pierce, Rockford, IL). 137 5.2.9. Confocal laser scanning microscopy Freshly cut tobacco-root sections (5 mm; secondary roots) were incubated with P10- CBM (6 uM) for 3.5 h at RT, washed in PBST (3 x 10 min; 1 ml each) and then incubated with anti-HA IgG-FITC (4.5 |xg/ml; Sigma-Aldrich, Oakville, ON, Canada) for 1 h at RT in the dark. After washing the sections in PBST (3X 10 min; 1 ml ea), they were transferred to a microscope slide and visualised using a confocal laser scanning microscope (CLSM) (Leica TCS-SP2, Leica Microsystems GmbH, Wetzlar, Germany). PaNie conjugation to Alexa Fluor 594 (Invitrogen, Burlington, ON, Canada) was performed according to manufacturers instructions with a final degree of labelling of 2.21 Alexa Fluor molecules to one PaNie molecule. Briefly, PaNie (15.5 ug) was reacted with 8 ul of Alexa Fluor dye and filtered three times over the same resin, with wash steps of PBS between each filtration to clear unbound dye from the resin. The degree of labelling was determined by measuring the absorbance at 590 nm. 5.3. Results 5.3.1. Expression and purification of VHH-CBM constructs Figure 39 shows the expression of full-length P10-, P16- and P31-CBM. The truncated and smeared bands run on the SDS-PAGE between 15-20 kDa, which may indicate that they are cleaved or prematurely truncated around the introduced thrombin site, which would create both VRH and CBM monomers of slightly different sizes. Full-length VHH- CBMs appear slightly larger than they should in theory (36-38 kDa instead of 31.5-32.7 138 kDa). This shift maybe due to glycosylation of any uncleaved a-signal sequence, which contains four known glycosylation sites. * 1 2 3 4 5 6 82 *•• 36 **• 25 19 15 Figure 39. SDS-PAGE showing crude purifications of VHH-CBM protein and BSA standards. Lanes 1-3 show P10-, P16- and P31-CBM, respectively, while lanes 4-6 show 2,1, 0.5 jig BSA, respectively. Prestained benchmark molecular weight marker bands (Invitrogen, Burlington, ON, Canada) are labelled on the left in kDa. Attempts were made to optimize the yeast expression with regard to full-length VHH- CBM vs. truncated proteins. No difference was found between using non-bevelled vs. bevelled culture flasks, 150 rpm instead of 250 rpm of shaking, or pH 5, 6 or 7 in terms of the amount of truncated compared to full-length proteins, but the overall protein expression was maximised by using bevelled flasks, 250 rpm shaking and a pH of 6. Under these conditions protein expression was consistently above 10 mg/L for all VHHS and VHH-CBMS. Further work concentrated on the highest affinity binder P10 and hence 139 P10-CBM. Purification of P10 and P10-CBM by nickel affinity chromatography was successful at eliminating non-antibody proteins (see Figure 40). However, less full-length P10-CBM could be purified than P10 alone, due to the truncation of P10-CBM into constituent protein fragments. Unfused P10 is purified as several different bands around the correct size of 17 kDa, indicating that perhaps some contain uncleaved a-signal sequences, which may be glycosylated. 36 P10-CBM 25 19 #•*$§« P10 15 Figure 40. Coomassie-stained SDS-PAGE of VHH P10 and P10-CBM after purification from a large-scale P. pastoris culture. Pre-stained benchmark molecular weight marker bands (Invitrogen, Burlington, ON, Canada) are labelled on the left in kDa. 140 5.3.2. Binding of VHH-CBM to cellulose and cleavage with thrombin Purified P10-CBM bound to CP-102 cellulose beads as described in section 5.2.2. As shown in Figure 41, P10-CBM binds to the CP-102 cellulose beads (protein in lane 3b » 2b), while the PI0 (not fused to CBM) only weakly associates with the CP-102 beads (lane 3 a compared to lane 2a). kDa la lb 2a 2b 3a 3b la lb 2a 2b 3a 3b 82 64 48 —* P10-CBM 36 25 19 14 P10 Figure 41. Western blot (left) and Coomassie stained gel (right) of P10 (a) and P10- CBM (b) binding to CP-102 cellulose beads. Lane la/b = before binding to beads; lane 2a/b = supernatant after binding to beads; lane 3a/b = elution from beads by boiling after washing. Note the Western blot and the Coomassie were loaded with 3 and 6 fig of protein per lane, respectively. The Western blot was probed with rabbit anti-HA IgG (0.2 ug/ml; Jackson Labs, West Grove, PA) and goat anti-rabbit IgG- AP (0.1 ug/ml; Bethyl Labs, Montgomery, TX). Since purified P10-CBM can bind to the CP-102 cellulose beads, an experiment was done to purify P10-CBM directly from yeast culture and cleave functional P10 from the beads using thrombin. The thrombin cleavage of P10 from the P10-CBM construct immobilised on cellulose beads during culture is illustrated in Figure 42. By analyzing the two blots 141 using different anti-tag antibodies, namely anti-HA and anti-HIS, it was determined that there was a large quantity of truncated protein, consisting of the CBM portion ofP10- CBM only, bound to the cellulose beads before and after thrombin cleavage. The anti-HA blot shows only proteins containing the P10-HA-HIS part (17.3 kDa) of the construct, while the anti-HIS blot shows both P10-HA-HIS (17.3 kDa) and the CBM-HIS (15.4 kDa) as well as the full construct (PlO-HA-HIS-thrombin-CBM-HIS; 32.7 kDa) containing two HIS tags. Anti-HA blot Anti-HIS blot kDa l 2 3 4 5 6 7 12 3 4 5 6 7 64 48 3g *• •**- P10-CBM 25 19 H P10&CBM 14 Figure 42. Western blots showing the thrombin cleavage of P10 from P10-CBM directly purified from culture using cellulose beads. The left blot was probed using an anti-HA IgG-AP (Bethyl Labs, Montgomery, TX) and the right blot using mouse anti-HIS IgG (Qiagen, Mississauga, ON, Canada) together with goat anti-mouse IgG-AP (Pierce, Rockford, IL). Lane 1 = P10-CBM eluted from beads by boiling before thrombin cleavage; lane 2-6 = thrombin cleavage supernatant after 2, 4, 8,12 and 24 h, respectively; lane 7 = uncleaved P10-CBM eluted from beads by boiling after 24 h thrombin cleavage and washing. The theoretical MWs of P10, CBM and P10-CBM are 17.3,15.4,32.7 kDa, respectively. 142 5.3.3. Verification of VHH and VHH-CBM functionality To ensure that the fusion of the VHH to the CBM and expression in P. pastoris does not alter the antibody function, comparative ELISAs and surface plasmon resonance were performed with P10 constructs. The ELIS A in Figure 43 shows that PI 0 and P10-CBM, at concentrations of 0.5, 1.0 and 2.0 uM, display the same amount of binding to 0.2 to 4 \\M PaNie. Furthermore, there is no statistical difference between P10-CBM (0.5 [iM) and PlOcieaved (0.5 u,M) binding to 4 uM PaNie coated in microtitre wells. 0.5 uM VHH 1 uM VHH 2 uM VHH 3 F> 3.0 • P10 • P10CBM m 2.5 • © 2.0 1.5 Q O 1.0 0.5 0.0 HU 0.2 2 4 uM PaNie coated to microtitre well Figure 43. Comparison of P10 and P10-CBM binding to PaNie as determined by ELISA. Three different VHH and PaNie concentrations were used in the ELISA. To further test if the introduction of the thrombin site and any possible glycosylation differences introduced by P. pastoris to P10 affected the binding kinetics of the antibody, surface plasmon resonance was also performed. In this experiment, PaNie rather than the VHH was immobilised to avoid any potential polymerization of PaNie as discussed in chapter 4. By doing this, the on and off rates should not be influenced by any 143 polymerization of PaNie during the injection into the flow cell. Figure 44 shows the response over time of P10 (0.2 - 8.8 uM) from P. pastoris binding to PaNie (0.5 jag). 0.2-8.8uM P10 binding to PaNie 0 10 20 30 40 50 Time (s) Figure 44. Surface Plasmon resonance of VHH P10 expressed in P. pastoris. Unlike the binding of PaNie to immobilised VHH as shown in section 4.3.7, Figure 44 does not display a biphasic dissociation. Furthermore, there seems to be little or no dip in the response units at the initiation of P10 injection into the flow cell. However, both on and off rates were too fast to calculate using the Langmuir binding model, so instead the KD was calculated by drawing a Scatchard plot using the binding response at steady state (Req). Table 13 and Figure 45 show the Req values and the Scatchard plot, respectively. Table 13. Response units of P10 binding to PaNie at steady state (Req) P10 (pJM) ~j^"fl^J)"" H2~ ' ' 7.9? 0.5 15.8 1 23.5 2 34.7 3 42.4 5 51.7 7 58.5 8.8 61.1 144 a o '•5 35 i. y = -0.4617X + 34.695 S3 O) 30 C o u 251 a? 20 S rj '-3 5 15 .5 lo 10 <« >> "O vel CO 10 20 30 40 50 60 70 Steady state binding (RU) Figure 45. Scatchard plot of VHH P10 from P. pastoris binding to PaNie. The KD (1/slope) and Rmax (x-intercept) were calculated from the equation of the trendline. P10 showed binding to immobilized PaNie with a KD of 2.17 uM. The theoretical Rmax (440 x MW of P10/MW of PaNie) was 313 RUs while the empirical value was 75.1 RUs. This means that 24% of the immobilized PaNie surface was used for binding P10. In summary, the ELISA and surface plasmon resonance results indicate that there is no difference in function of VHH P10 when presented as soluble VHH, VHH-CBM, or as VHH following cleavage from the CBM, nor is there a difference between P10 expressed in E.coli or in P. pastoris. 145 5.3.4. Binding of VHH-CBM to NLPsg2 Since P10-CBM and P16-CBM could be purified from P. pastoris, an anti-NLPsg2 ELISA was performed with minor modifications from that described in section 3.2.5 and 3.3.5. Specifically, 1.25 uM VHH-CBM was coated to wells of 96-well microtitre plates, 25 |^g/ml streptavidin-HRP was used for detection, and substrate was developed for 3 min. The results shown in Figure 46 indicate that both P10-CBM and P16-CBM bound NLPsg2-biotin with P10-CBM showing a significantly higher absorbance than PI 6- CBM. 1.8 1.6 1.4 O 1.2 tt ' 1.0 o ^ 0.8 j Q O 0.6 0.4 0.2 0.0 P10-CBM P16-CBM PBS control Figure 46. Binding of NLPsg2-biotin to P10-CBM and P16-CBM. VHH-CBMs (1.25 uM) were coated in wells of a microtire plate and NLPsg2-biotin (187 uJM) binding was measured using streptavidin-HRP (25 ug/ml). The background subtraction represents binding of streptavidin-HRP to coated, blocked wells, while the PBS control shows binding of NLPsg2-biotin to uncoated, blocked wells. 146 5.3.5. Functionality of P10-CBM while bound to cellulose paper Since P10-CBM can be immobilized on cellulose beads and can bind both PaNie and NLPsg2, it may be used to create a bioactive cellulose filter paper for the capture of PaNie and other NLPs in hydroponic systems. P10-CBM bound well to Whatman cellulose paper (#3), while there was little or no non-specific binding of PI 0. In the first experiment, capture of PaNie by the immobilized P10-CBM was detected using an anti- PaNie rabbit serum (1:3000) and a goat-anti-rabbit IgG-HRP (0.1 ug/ml; Bethyl Labs, Montgomery, TX), but non-specific binding of the probes was high. Thus, the bioactive filter assay was modified using PaNie-HRP for direct detection of binding to immobilized P10-CBM. This assay was performed in only 13 minutes (including 5 min development time) and resulted in a signal to noise ratio that allows clear distinction between specific binding of PaNie to immobilized PI 0-CBM vs. non-specific binding to entrapped P10 or the paper itself (see Figure 47). This assay also revealed that the PI 0-CBM, once bound to the paper retains its function even after dehydration for several hours. PI 0-CBM P10 Figure 47. Binding of PaNie to a bioactive paper filter. Whatman paper (#3) was loaded with 4 fig P10-CBM (top) and 4 fig P10 (bottom) and probed with PaNie- HRP. Background noise was subtracted by adjusting the contrast of the picture. 147 A bioactive filter paper for binding PaNie and other NLPs would be very useful if the NLP could be quantified by e.g. spectrophotometry to correlate NLP presence with disease symptoms and help elucidate progression of disease. To measure the binding spectrophotometrically, the TMB substrate was transferred from the developing paper filter to a well of a clear polypropylene microtitre plate for subsequent neutralisation with H2SO4 and reading at 450 nm. However, it was not possible to reliably extract TMB from Whatman paper (#3) even though soluble TMB substrate was used. This retention of substrate resulted in poor signal to noise ratios for Whatman paper (#3). Therefore, three new papers, two cotton cellulose papers (Ahlstrom Grade 601, 55 ml/min filter rate and Ahlstrom Grade 631, 200 ml/min) and a wood cellulose paper (Ahlstrom Grade 950, 14 ml/min), were tested. Figure 48 shows that using P10-CBM bound to the cotton paper 'Ahlstrom 601' provides a clear qualitative assay for PaNie detection and the results in Table 14 show that 'Ahlstrom 601', with its signal almost three times above background (S/N ratio = 2.85), is the best candidate for the development of a quantitative assay for PaNie. 148 P10-CBM W No VHH Figure 48. Anti-PaNie bioactive filter paper. Binding of PaNie-HRP to P10-CBM (4 ug) immobilized on Ahlstrom 601 paper is visualised by placing the filters in soluble TMB substrate (left; 4X magnification). TMB substrate is extracted from the developing filters (centre) and transferred to new wells for neutralization (right) and spectrophotometric quantification (see Table 14). Table 14. Comparison of four papers for use as bioactive filters. The TMB representing the binding of PaNie-HRP to immobilized P10-CBM was quantified spectrophotometrically (450 nm) after extraction and neutralisation of TMB from the filters as shown in Figure 48. The signal to noise (S/N) ratio represents the relative strength of the signal due to PaNie-HRP binding to P10-CBM vs. the non specific binding of PaNie-HRP to the paper. Filter paper used Ahlstrom 601 Ahlstrom 631 Ahlstrom 950 Whatman 3 P10-CBM 0.680 0.556 0.302 1.132 NoVHH 0.239 0.677 0.199 1.084 S/N ratio 2.85 0.83 1.52 1.04 149 There are two ways in which this bioactive paper assay could be expanded to quantify native PaNie produced by P. aphanidermatum. One way is to develop a sandwich assay using a second antibody recognising a different epitope on PaNie. Another is to develop a competitive assay using PaNie-HRP. Since the use of secondary antibodies adds to background noise by non-specific binding to the paper, it was decided to develop a competitive assay using PaNie-HRP as a probe and unconjugated PaNie as competitor. The binding of PaNie-HRP (0.05 uM) to P10-CBM on Ahlstrom 601 paper (OD450-BG = 1.095) could be inhibited by 43% (OD450-BG = 0.624) when a 50X competition of free PaNie (2.5 uM final) was applied. A rough estimation of this competition puts the IC50 of this paper assay at 2.75 uM, which is in line with the KDS obtained for P10 by CI- ELISA and surface plasmon resonance (see section 4.3.7). The background signal in the PaNie-HRP based assay was consistently high, with OD450 values typically greater than 0.500. Attempts to reduce this non-specific binding using various blocking agents did not result in significant improvements. The blocking agents resulting in no difference were, in combination and alone: 1% w/v casein, 1M MgCb, 0.1% Tween and 0.1% polyvinylpyrrolidone in PBS and 0.1% Si(OEt)3-PEG(8K) as a preatreatment of the paper. Blocking agents that resulted in higher background than 1% casein-PBS included: protein-free block and superblock (Pierce, Rockford, IL), poly-L- lysine (1-10 % w/v) and polyvinyl amine (0.0001-0.1% w/v). 150 Using P10-CBM and P16-CBM simultaneously may create avidity, which would enable the capture of lower concentrations of PaNie-HRP, which in turn would decrease non specific background binding. However, several attempts to create this avidity failed, either due to the sparse spacing of VHH-CBMS on the internal surface of the paper or because P10-CBM and P16-CBM share the same or overlapping epitopes. 5.3.6. PaNie necrosis elicitation in tobacco 5.3.6.1. Necrotic effect of PaNie in leaves To determine the level of PaNie to be detected or immobilised it is necessary to establish a dose-response assay for PaNie necrosis elicitation in plants. Tobacco leaves (Nicotiana tabacum) have previously been infiltrated with PaNie (Veit et al, 2001). This was repeated and it was found that a 10 ul solution of 100 nM PaNie was sufficient to cause repeatable and visible necrosis after 48 hours (see Figure 49). Even injections of 50 nM PaNie caused visible symptoms in some leaves, but symptom intensity was variable. The onset of symptoms was proportional to the dose of PaNie injected, as can be seen by the paper-thin, almost transparent necrotic area of the 10 uM treatment vs. the green-brown necrotic area visible with 50 nM treatments (see Figure 49). Symptoms were visible 4-6 h post injection of 10 uM PaNie, thus supporting the results of Veit et al. (2001). A 10 uM BSA solution was also injected in PBS as a control and no necrosis was observed (data not shown). 151 10 uM 100 nM 50 nM 10 nM Figure 49. Necrosis induced by PaNie injection into the leaf of tobacco. Pictures were taken 48 hours after injections (10 ul) of 10,50,100 nM and 10 uM PaNie. An attempt to monitor the onset of necrosis in a more quantitative manner included measuring chlorophyll fluorescence, a measure of photosynthesis detected by a fluorimager. PaNie (100 nM) infiltration did not significantly perturb any parameters of photosynthesis, but a 27 uM injection of PaNie significantly decreased the non- photochemical quenching (NPQ) value from 1.53 to 1.09 after only 1 h. This technique could be used to quantify early onset of necrosis due to high concentrations of PaNie or other NLPs, but since low concentrations of PaNie (100 nM) had no effect on NPQ, this technique was not subsequently used to quantify early onset of necrosis in leaves. 5.3.6.2.Necrotic effect of PaNie in roots To establish a dose response of PaNie infiltrated through intact roots, small-scale hydroponic systems were built. Figure 50 depicts the effects on 7-week-old plants (from sowing) of a 5 uM PaNie root treatment for one week. 152 Figure 50. Necrotic effect due to a week-long 5 uM PaNie application to the roots of hydroponically growing tobacco. Chlorosis and stunting of growth was visible in the PaNie treated plants compared to untreated controls. The chlorosis of leaves caused by the PaNie treatment (5 uM) of the roots was monitored daily using a chlorometer, yielding significant differences in chlorometer readings at day 7. Root symptoms were limited to stunting and no root browning was observed. Thus, leaves and roots were harvested at day 7 for further analysis. Growth and chlorophyll parameters compared to control are shown in Figure 51. Leaf wet weight, dry weight, area index and root dry weight were significantly reduced by 50% or more in the PaNie treated plants. The concentration of chlorophyll in the leaves was also significantly reduced, in line with the visible chlorosis. 153 Leaf and Root parameters Chlorophyll parameters 6OO1 n = 3 — 9 \~JT \ 2 8 n = 3. ei g Leaf wet Leaf dry Leaf area Root dry 0 * 7 s- t 6 weight weight index weight 20 £ 5 *S 4 n = 3 ua> H > J 3 V bf • Control • 5 uM PaNie 2 g/m i Rel a 1 3. 0 Figure 51. Growth and chlorophyll parameters of tobacco after a week-long 5 uM treatment with PaNie. 5.3.6.3.Inhibition of PaNie-induced necrosis in leaves Necrosis of leaves induced by 100 nM PaNie could be inhibited by murine anti-PaNie serum raised according to the schedule described in section 4.2.1. Furthermore, P. aphanidermatum exudates from a malt broth culture containing ca. 200 nM PaNie were also inhibited by the serum (see Figure 52). The malt broth culture was grown as described in chapter 3 and the concentration of PaNie determined by comparison to recombinant PaNie by Western blot analysis. 154 100 nM PaNie + P. aph. exudate + 100 nM PaNie + P. aph. exudate + pre-immune serum pre-immune serum anti-PaNie serum anti-PaNie serum Figure 52. Polyclonal serum neutralisation of PaNie necrosis in leaves of tobacco. Pre-immune serum was used as a control. 'P. aph. Exudate' refers to the supernatant of a one-week Pythium aphanidermatum culture in malt broth. 5.3.7. Confocal laser scanning microscopy of VHH-CBM and PaNie bound to roots Since a comparatively large amount of PaNie is required for necrosis induction in roots (5 uM compared to 100 nM in leaves) a similarly large amount of VHH-CBM bound to a filter is expected to be required to neutralise root necrosis due to PaNie. As construction of such a filter was not permissible due to the limited amount of antibody available, it was decided to test if P10-CBM could bind directly to the surface of tobacco roots and capture PaNie before it reaches its putative target on the roots. Such a demonstration would make it possible to release PI0-CBM from a bacterial or yeast source in the rhizosphere, without transforming the crop of interest and without having to produce VHH-CBM exogenously. To determine both binding and functionality of PI 0-CBM on tobacco roots, a two-part experiment using CLSM was undertaken. First, PI 0-CBM was bound to the roots and detected using an anti-HA IgG conjugated to the fluorophore FITC. Figure 53 shows 155 more P10-CBM than P10 was bound to roots, suggesting that the CBM provides specific binding to the surface of roots. P10-CBM binding seemed to be saturated at 0.5 |iM, while, within these concentration limits, the putatively non-specific binding of the control P10 seemed to be directly proportional to the concentration applied. There was no background binding due to anti-HA IgG-FITC on its own (data not shown). Figure 53. Confocal laser scanning microscopy depicting the binding of P10-CBM and P10 (ctr) VHHS to the surface of tobacco roots. The VHHs were probed with anti-HA IgG-FITC (Sigma-Aldrich, Oakville, ON, Canada). The grey panels on the right of each pair show the roots in the visible spectrum. 156 Following the success of P10-CBM binding to the surface of the roots, attempts were made to detect PaNie binding to the P10-CBM displayed on the root surface. First, PaNie was incubated with PlO-CBM-coated roots and detected using rabbit anti-PaNie immune serum, followed by goat anti-rabbit IgG conjugated to the fluorophore TRITC. However, due to the high background binding of the immune serum and goat anti-rabbit IgG- TRITC, this treatment did not yield any signal differences compared to controls. In another experiment Alexa Fluor 594 was conjugated to PaNie, which resulted in significant reductions in non-specific background signal. Figure 54 depicts the binding of PaNie-Alexa 594 to three roots that have been coated with P10-CBM vs. three roots that have not been coated. 157 P10-CBM + PaNie-Alexa 594 PaNie-Alexa 594 only Figure 54. Confocal laser scanning microscopy depicting the binding of Alexa Fluor 594-labelled PaNie to VHH-CBM displayed on the surface of tobacco roots. The grey panels on the right of each pair show the roots in the visible spectrum. 158 5.4. Discussion The results demonstrate that genetic engineering of VHHs to CBMs resulted in full-length fusion proteins being expressed that bind to cellulose beads, paper and tobacco roots. Furthermore, the affinity of the VHH for PaNie was unaffected by the fusion to the CBM or by expression of the fusion protein in P. pastoris. PaNie can be captured by the VHH- CBM, when immobilized on paper or roots, thus enabling the development of bioactive cellulose surfaces for the detection and amelioration of NLP-induced plant disease. Expression in P. pastoris provided a means of producing large amounts of VHH-CBM, which were harvested using cellulose beads and cleaved with thrombin to yield pure and functional VHH. When compared to metal affinity chromatography, the cellulose bead harvesting system may provide a cheaper means for purification of recombinant proteins such as VHHS. Even though the full-length VHH-CBM was expressed intact as a single pure band, there were also breakdown products that could represent the VHH or CBM portion of the parent molecule. KEX 2 endopeptidase, which cleaves the a-signal sequence at Lysine-Arginine or Arginine-Arginine, also has some affinity for cleavage at Proline-Arginine (Brenner and Fuller, 1992), and thus could cleave the thrombin site which consists of'Leucine-Valine-Pro/me-/irg/m'ne-Glycine-Serine'. This could explain why there are breakdown products and that some of them appear to be glycosylated due to the remaining a-signal sequence, which contains glycosylation sites. Full-length VHH- CBM seems not to be glycosylated, because, unlike the breakdown products, the a-signal sequence has likely been removed before secretion into the media. However, because CBM-HIS bound to the CP-102 cellulose beads during P. pastoris culture indicates that 159 some form of fusion protein cleavage must occur during or after the protein is secreted from the yeast, or else the CBM-HIS portion would remain in the cytoplasm and not be secreted into the medium that is harvested. It would be worthwhile to make constructs with different cleavage sites to elucidate if this mechanism occurs generally or is specific to the thrombin site, which is especially important, since a more stable cleavage site may improve the yield of full length VHH-CBM. TO investigate if sugars are present on the VHH-CBM and/or its breakdown products, concanavalin A-HRP could be used as a probe for the sugars or the sugars could be removed using PNGaseF (Stork et ah, 2008). Surface plasmon resonance showed that VHH P10 purified from P. pastoris had the same affinity (KD) as that from E.coli. Putative PaNie polymerization was eliminated by immobilising PaNie rather than the VHH on the chip and it was shown that the fast off rate is an inherent feature of the PaNie-VHH interaction and not a result of PaNie polymerization 'competing' with PaNie binding to the VHH. In addition,using this method, the biphasic dissociation was no longer observed, which supports the hypothesis that PaNie may polymerize. Thus, the antibodies may help to elucidate the structure and function of PaNie. P10-CBM bound to various filter papers and was able to withstand dehydration, indicating that bioactive paper filters may be stored and shipped dry until used. A quantitative assay was developed for the capture of PaNie, but the error in quantitative measurements due to non-specific PaNie adsorption to the paper limits the assay to being semi-quantitative unless a reduction of background noise is possible. One gram of filter 160 paper contains a complex matrix of different size cellulose fibres that collectively present several square metres of surface area (van de Ven, 2007). The putative polymerization of PaNie may exacerbate the non-specific adsorption of PaNie to internal paper surfaces. For trapping of PaNie, the non-specific binding may be beneficial, but if the bioactive paper assay is used as a measure of disease progression, the quantitation needs to be accurate. However, the IC50 obtained by the inhibition of PaNie binding to the bioactive filter paper agreed with the affinity (KD) determination of P10 by CI-ELISA and surface plasmon resonance. This serves as a proof of concept for the further development of a reliable quantitative bioactive filter assay for NLPs and shows that the sensitivity and accuracy of the paper filter assay will be directly related to the affinity of the antibodies used. Heteropentamerisation of the antibodies may increase the avidity so that PaNie could be captured from hydroponic nutrient solutions at very low concentrations. Thus, by passing many litres of hydroponic solution through a bioactive filter, low concentrations of PaNie and other NLPs may be captured and subsequently detected at a very early stage of disease development when PaNie concentrations are very small. Concentrations as low as 50-100 nM caused necrosis in leafs after 48 h, while roots had to be exposed to concentrations of 5 uM for one week to cause visible necrosis. Higher concentrations of PaNie (10 uM) injected into leaves caused necrosis after 6 h, supporting previous work by Veit et al. (2001). The symptoms of the root treatment with PaNie indicate that the plants stop growing after exposure to PaNie and that they gradually lose chlorophyll from the leaves, ending with less than half the chlorophyll per unit leaf wet weight after one week of treatment. The chlorosis and halting of growth may 161 be due to a disruption of nutrient uptake by the roots through local action of PaNie, or it could be due to systemic effects of translocated PaNie. Translocation could be investigated using radiolabelled PaNie. Disruption of nutrient uptake could be due to PaNie creating porosity in root membranes and this could also be investigated using radiolabelled molecules of certain sizes that normally cannot traverse the root membrane. Alternatively, metabolite and ion loss could be investigated using liquid chromatography and selective ion electrodes, respectively. P. aphanidermatum secretes approximately 200 nM PaNie when cultured in malt broth, but the expression may be increased in the presence of e.g. host phytoalexins. However, these results indicate that a bioactive paper filter must capture PaNie at concentrations ranging from 50-5000 nM to reduce disease symptoms. Since roots were sensitive to 5 uM but not to 1 uM PaNie, a five fold concentration reduction of 5 uM PaNie will be sufficient to reduce its necrotic effects. However, if P. aphanidermatum creates a high local concentration of PaNie at a site of infection, e.g. in response to host phytoalexins, or if it provides access for PaNie via mechanical penetration, the capture of PaNie directly on the surface of roots may provide a more sensitive strategy for reducing the effects of PaNie. Indeed, CLSM showed that root-immobilized P10-CBM could bind PaNie. Although this root display of P10-CBM may be more effective at preventing necrosis, it may also concentrate PaNie on the root surface, which may accelerate necrosis. A more sensitive root assay is currently under development in our laboratory (Hall, 2008), which promises to elucidate the protective function of the anti-PaNie antibodies. 162 6. OVERALL CONCLUSIONS AND FUTURE DIRECTIONS The research objectives of this thesis were to develop high-affinity VHHS against NLPsg2 and full length PaNie and apply these antibodies for the construction of biotechnological tools for the capture of PaNie and other NLPs in hydroponic systems. Four VHHS with affinities to PaNie ranging from 800 nM to 20.2 uM were selected by panning a naive ribosome display library and by constructing and panning a hyperimmune phage display library. The highest affinity VHH, which also binds to NLPsg2, was fused to a CBM and captured PaNie when bound to cellulosic filter paper and tobacco roots. Beyond fulfilling these main objectives, this thesis has provided insights into the structure and function of PaNie and provided a proof of concept for various antibody-CBM fusion-based applications in the future. Immunization of the llama and construction of the phage display library was more successful at developing diverse VHHS against PaNie than the panning of the naive llama library by ribosome display (Chapters 3 and 4). Although a hyperimmune library requires much work to construct, it provides a far greater chance of developing diverse, high affinity antibodies. It is also easier to find VHHS that bind to PaNie using phage display, mainly because polyclonal phage ELISA and phage titration can be used as immediate measures of panning success. The four VHH antibodies produced represent the first recombinant antibodies produced against any NLP. They represent three of four VHH subfamilies and have varied amino acid sequences (see section 4.3.5). The diversity of their CDR3s would indicate that there 163 are many different epitopes on PaNie, yet at least three of the four anti-PaNie VHHs also bind the NLPsg2 peptide, which includes the NLP-conserved NPP1 domain. This result indicates that the NPP1 domain is especially antigenic. If the NPP1 domain is involved in binding to a putative receptor or transmembrane protein in the plant host, then the CDR3s of the anti-PaNie VHHS could mimic portions of such transmembrane proteins. On the basis of this information, a BLAST search (Altschul et al, 1997) of the Arapidopsis thaliana protein database was performed and yielded sequence similarity to some transporter proteins. However, this sequence is similar to many other types of proteins, including KH domain-containing proteins that bind nucleic acids; thus, based on amino acid sequence, it is unlikely that the CDR3 is a true mimic of PaNie's target site. The kinetic studies of the antibodies by CI-ELISA and surface plasmon resonance indicate that PaNie may polymerize (see section 4.4), which is supported by previous reports that PaNie may form homotetramers (Koch et al, 1998) and that the related NLP from V. pommerensis is a putative polymerizing hemolysin (Jores et al, 2003). In addition, this raises the possibility that PaNie is a pore-forming toxin (PFT), which has recently been independently suggested by Schouten et al. (2008) on the basis that NLPs are associated with cell and nuclear membranes. Koch et al. (1998) show that the effect of PaNie can be blocked by the calcium channel blocker nifedipine, which raises the possibility that PaNie is either dependent on Ca2+ for function or that it may bind to calcium channel proteins, locking the channel into a permanently 'open' position. If NLPs bind to several different kinds of transmembrane transporters, this may explain why leaves are more sensitive to PaNie than roots (see section 5.3.6); i.e. there may be 164 more target sites in leaves as opposed to roots. Furthermore, if PaNie binds to transmembrane transporter proteins, it may be that necrosis is not strictly light-dependent, as Veit et al. (2001) suggest, but that PaNie sensitivity is much higher during the day due to the activity of e.g. light adsorption-driven transporters. Thus, if PaNie binds to targets in the host cell membrane, it is very likely that sufficient anti-PaNie VHH-CBMS, displayed on the host's roots as shown in section 5.3.7, would prevent binding of PaNie to its target and in so doing prevent possible pore formation and necrosis. The fusion of the VHHS to CBMs offers many additional opportunities for capture of PaNie on various cellulose surfaces. The fusion did not alter the function of the antibody and a large concentration of P10-CBM (>10 mg/L) was expressed in Pichia pastoris and purified using cellulose beads (see section 5.3). Through the construction of a bioactive filter paper displaying P10-CBM on its surface, PaNie could be captured and quantified with an affinity similar to that determined by CI-ELISA and surface plasmon resonance (see section 5.3.5). This affinity may suffice to capture a large portion of the 5 uM PaNie needed to cause necrosis in root treatments, but will need to be improved if lower concentrations of PaNie are to be neutralised (see section 5.3.6). Lower concentrations, such as the 200 nM that are secreted by P. aphanidermatum during malt broth culture, may prove to cause damage in roots if the P. aphanidermatum penetrates the host's cell wall for example. The thesis' results suggest that there are several viable strategies, based on the use of VHH-CBM fusions, to combat NLP-induced plant disease. Plants may be protected from 165 NLPs by using root display of antibodies, bioactive filters or even bioactive cellulose beads. To determine which of these strategies are most suitable for reducing the effects of PaNie, it may be important to understand the function of NLPs and their role in plant disease progression. For example, to discover whether pores are formed in the process of PaNie binding to a target, one could determine if fluorescently labelled proteins enter plant cells during the cells' exposure to PaNie. Furthermore, an immunoaffinity column or cellulose beads covered with P10-CBM could capture any PaNie-protein complexes from infected membranes that have been lysed. Any complexes found could be micro- sequenced at their N-terminus and the sequence compared to the existing Arabidopsis thaliana protein database. These comparisons may reveal membrane proteins that are PaNie targets. The techniques used in this thesis could prove useful in the determination of functionally important parts of NLPs. Surface plasmon resonance of PaNie to PaNie binding in combination with mutations in the PaNie molecule itself may elucidate which amino acids are responsible for polymerization. It would also be interesting to see if two or more different NLPs could polymerise to each other, uncovering possible synergisms between NLP-producing pathogens. Through collaboration all or most of the known genes of NLPs could be pooled, a ribosome display library displaying the various NLPs could be created and artificially diversified using error-prone PCR. The NPP1 domain, which due to its conserved sequence amongst all NLPs must play an important role in efficacy, could be preserved by using e.g. P10 as an 'antigen' during selection of diversified NLP sequences. After several rounds of diversification and selection, the ribosome display 166 library could be cloned into the pMEDl vector, which would allow both phage display and soluble expression in E. coli. Infection with NLPs displayed on phage may cause necrosis and could act as a functional assay between rounds of panning. If phage- displayed NLPs do not cause necrosis, due to an inability to form pores while attached to the phage particle, the diversified NLP clones could be solubly expressed and injected into leaves. Those clones that cause necrosis can be sequenced and a synthetic NLP database created. Such a database could identify crucial domains or specific amino acids involved in PaNie polymerization or target binding. It may also be used to discover new lead chemistries for the control of dicotyledonous weeds in monocotyledonous crops. The development of antibody-based bioactive cellulose surfaces is not limited to crop protection and serves as a proof of concept for many other applications. Since antibodies with very high specificity and affinity can be produced against even small molecular haptens (Yau et ah, 2003), bioactive paper filters could be produced to quantitate almost any contaminant in any water system. This could be useful for e.g. environmental scientists and ecologists who want to measure very low concentrations of chemicals in rivers, where bioactive filters could be installed to sample thousands of litres of water, something that conventional ELISAs cannot do. For all antibody-based biotechnological applications, the cost of antibody production itself is the limiting factor. Molecular farming of plantibodies promises to reduce this cost significantly (Sheedy and Hall, 2001; Almquist et ah, 2006), but still requires purification of antibodies from the host expression system. When compared to purification by metal 167 affinity chromatography, the CBM-cellulose bead purification system offers a new and perhaps less costly purification method (see section 5.3.2). In addition, since P10-CBM binds to the surface of tobacco roots and can bind PaNie, it may be possible to express and secrete an anti-PaNie VHH-CBM directly from a non-pathogenic microbe naturally present in the rhizosphere of plants. This would further reduce the cost of crop protection, by requiring only an inoculum of transgenic biocontrol agent, rather than a steady supply of purified antibodies for the production of bioactive paper filters. Microalgae, which are naturally found growing in hydroponic systems, represent largely unexplored hosts for recombinant protein production. Microalgal metabolism is very malleable and protein expression levels can be maintained in the range of 20-70% of biomass. Unicellular algae like Chlorella sorokiniana and Spirulina platensis are used for food production and waste remediation in closed environment life support systems (CELSS) for space travel and have been successfully transformed with recombinant proteins (Gitelson, 1999; Dawson et al, 1997; Kawata et al, 2004). Thus it may be possible to generate transgenic algae, with much higher antibody expression levels than would be possible for transgenic plants, yeast or bacteria. These antibody producing algae could act as detoxifiers of terrestrial and space-based hydroponic systems. CELSS test systems in orbit and on the ground have been reported to be contaminated with common greenhouse pathogens, including Pythium species (Fjallman and Hall, 2005). 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