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I Construction of a Synthetic Human VL Phage Display Library And Construction of a Synthetic Human VL Phage Display Library and Isolation of Potential Neuropilin-1-specific VL Therapeutics from the Library Artine Keklikian Thesis Submitted to the Faculty of Graduate and Postdoctoral Studies In partial fulfillment of the requirements For the M.Sc. degree in Microbiology & Immunology Department of Biochemistry, Microbiology & Immunology Faculty of Medicine University of Ottawa ©Artine Keklikian, Ottawa, Canada, 2011 i 2 ABSTRACT Antibody phage display technology mimics the natural immune system, and has been widely used for rapid isolation of single-domain antibodies (sdAbs) with various binding specificities and affinities in the micromolar to low nanomolar range. SdAbs are the variable regions of immunoglobulins (e.g., VH, VL, VHH) and serve as potential probes with therapeutic value. The small size, high solubility, high expression and stability, and high specificity and affinity for the cognate antigen, make sdAbs ideal in improving drug delivery and the overall therapeutic value of antibodies. The main objective of this thesis was to construct a large VL phage display library (~1010 diversity); analyze it via sequence analysis, and to subtractively pan the library for isolation of Neuropilin-1 (NRP1)-specific VLs. Neuropilin-1 (NRP1), a cell-surface receptor for both vascular endothelial growth factor (VEGF) and class 3 Semaphorins (Sema3A), contributes to neuron cell death through its interaction with Sema3A in stroke patients. Disruption of this NRP1-Sema3A interaction would allow for axonal outgrowth and neuron regeneration in the area of the brain affected by stroke. Construction of the synthetic phage antibody library utilized a single VL framework with selected positions in the complementarity-determining regions (CDRs) targeted for randomization in vitro using synthetic oligonucleotides that introduced sequence degeneracy. Specific VLs were then selected from the repertoire through subtractive panning against a cell line endogenously expressing NRP1 (PC12) as well as a negative cell line that does not express NRP1 (HEK293) with competitive elution carried out using a synthetic Sema3A-derived peptide. Fifteen VL clones were isolated, cloned in E. coli, expressed and purified, and of these, nine were determined to be non-aggregating by size exclusion chromatography. Further studies will determine the potential therapeutic use of these VL sdAbs as agents in recovery from stroke and neuron degeneration. ii ACKNOWLEDGEMENTS I would like to thank my supervisor, Dr. Jamshid Tanha, for having given me the opportunity to work on a project at the NRC and for the patient guidance, constructive comments and advice he has provided throughout my time as his student. I am also deeply grateful to the entire Antibody Engineering group for their friendship and assistance in introducing me to the various laboratory procedures, and for helping to make my stay at NRC a memorable experience. Many thanks to Amy Aylsworth, Susan Jiang, and Sheng T. Hou, for preparing and providing the cell lines that were used as part of this project. Also, thank you to Rebecca To for cloning of the “VL-24” template into the Fd-tet vector, Henk van Faassen for assistance with flow cytometry, Dr. Dae Young Kim for protocols on TG1 electrocompetent cell preparation, protein expression and protein purification, and Sonia Leclerc for DNA sequencing. I owe my loving gratitude to my mom and dad for their unending encouragement and support, and also to my three brothers who will always be my best of friends. iii TABLE OF CONTENTS ABSTRACT……………………………...………………………………………………………ii ACKNOWLEDGEMENTS…………………………………………………………………….iii I. LIST OF ABBREVIATIONS……………..………………………………………..…….…5 II. LIST OF TABLES.....………………………..……………….…………………...……..…..8 III. LIST OF FIGURES……...…..…….……….….……………………………………………9 IV. INTRODUCTION………………...………………………………….…..…………..........11 1. Immunoglobulin structure and function……………………………………………….....11 a. Types of antibody fragments/domains……………………………………………….15 b. Fab fragment………………………………………………………………………....15 c. scFv fragment….….………………….……………………………………………...16 d. Single domain antibodies………………………………………………………….....19 2. Antibody phage display......................................................................................................20 a. Filamentous bacteriophage………………………………………………….…….....21 b. Phage vectors………………………………………………………………………...21 c. Coat proteins of filamentous phage………………….………………………………25 3. Development and types of antibody libraries….…..….….....…….….….……………….25 a. Naïve libraries………………………………………………………………..………25 b. Immune libraries…………………………………………………………..…………26 c. Synthetic libraries…………………………….……………………………………...27 d. Library size…………………………………………………………………………..28 e. Kunkel’s method of mutagenesis…….……………….……………….…………….29 4. Selection of binders from antibody libraries: “panning”……...…….….…………….…32 1 5. Protein expression in E. coli.….…..….….………..…….….….…………………………36 a. Protein expression in the periplasm of E. coli.………………….……………….......37 b. Factors influencing protein expression……..……….….……………………………38 c. Protein purification by Immobilized Metal Affinity Chromatography (IMAC)…....38 6. Semaphorins as promoters of neurodegeneration.…..…….…….….…..………….……39 a. Neuropilins: structure and function.…..……………..…..……………………..……40 b. The role of NRP1/Sema3A interaction in neurodegenerative diseases……………...43 c. Synthetic Sema3A “Ig” inhibitory peptide…….……….………..……………….…43 7. Objectives……..….…..…….….….……….…………………………………………......47 V. MATERIALS AND METHODS…………………………………….……………………48 1. Reagents and Solutions….….…....….…….……………………………………………..48 2. Media, bacterial strains, and mammalian cell lines……….…………..………….….…...49 3. Preparation of E. coli TG1 electrocompetent cells….……....…….….…….………....…50 4. Construction of the CDR3/3a/3b-randomized synthetic VL phage display library….………..…….………………………………………………………...………..52 a. ssDNA production using E. coli CJ236…………………………………...…………52 b. In vitro mutagenesis (CDR3/3a/3b randomization)………………………………….53 c. Transformation of phage heteroduplex DNA into E. coli TG1……………………...57 5. Construction of the CDR1/2/3/3a/3b-randomized synthetic VL phage display library..….….…...…….…….……………………..………………………………..……58 a. Isolation of ssDNA from constructed CDR3/3a/3b-randomized synthetic VL phage display library…..…...…….….…………….……………………………..…58 b. In vitro mutagenesis (CDR1/2 randomization)…..….…..…..….…..………………59 2 c. Transformation of phage heteroduplex DNA into E. coli TG1….….…….………...60 6. Identification and sequencing of VL library clones..……..………………...………….....61 7. Cloning of VL inserts………….…………………………………………………….…...63 8. Expression of His6-tagged VL proteins……………….…….……………………………66 9. Extraction of VL proteins………….……..………………………………………….…..67 a. Total Cell Lysis…….……….………………………………………………............67 b. Periplasmic Extraction…………….……………………….……………………….68 10. Western blot………….….…………….………………………………………………...68 11. Purification by Immobilized Metal Affinity Chromatography…..……….……………..69 12. Size Exclusion Chromatography…….…….….….…….….……….……………………70 13. Subtractive panning of the constructed VL synthetic phage display library….….….…..71 14. Flow Cytometry……….…….….…...………….……………………………………….76 VI. RESULTS……………………………………………………………………………….77 1. Efficiency of transformation for freshly prepared electrocompetent E. coli TG1 cells…………………………………………………………………………………77 2. Synthetic VL phage display library……………………………………………………....79 a. Construction of the CDR3/3a/3b-randomized synthetic VL phage display library..…………………………………………………………………………….…82 b. Construction of the CDR1/2/3/3a/3b-randomized synthetic VL phage display library..………………………………….……………………………………………88 3. Sequencing and statistical analysis of the constructed synthetic VL phage display library................................................................................................................................90 4. Expression and protein analysis of randomly selected VL clones from the constructed synthetic library……..…….……………...……………………..……...…122 5. Identification and expression of subtractive panning isolates from the constructed synthetic VL phage display library………....…..….……….……..…..…..136 3 6. Size exclusion chromatography analysis of VL subtractive panning isolates………….148 7. Flow cytometric analysis of VL subtractive panning isolates………………………….151 VII. DISCUSSION………………………………………………………………………….157 1. Efficiency of transformation for electrocompetent cells…………...........………..……159 2. Design, construction and characterization of the synthetic VL library.…....….….….…160 3. VL expression analysis…………....….…...……….………….…....……………..……169 4. Subtractive panning for NRP1-specific VL isolates…..…..…....…..…..…….…………171 VIII. CONCLUSIONS AND FUTURE DIRECTIONS……………………………..……176 IX. REFERENCES………………………………………………………………………..180 X. SUPPLEMENTAL………………………………………………………..…………..210 XI. CURRICULUM VITAE…………………………………………………..………….225 4 I. LIST OF ABBREVIATIONS Ab Antibody Ag Antigen Amp Ampicillin AP Alkaline Phosphatase BBB Blood-Brain Barrier bp Base Pair(s) BSA Bovine Serum Albumin CH Constant Domain of the Heavy Chain CL Constant Domain of the Light Chain CDR Complementarity-Determining Region CFU Colony-Forming Unit Chlor Chloramphenicol CT C-terminal Da Dalton D-MEM Dulbecco’s Modified Eagle Medium DNA Deoxyribonucleic Acid dNTP Deoxynucleoside Triphosphate DRG Dorsal Root Ganglion dsDNA Double-Stranded DNA DTT Dithiothreitol ELISA Enzyme-Linked Immunosorbent Assay Fab Antigen-Binding Fragment 5 FBS Fetal Bovine Serum Ff Filamentous Bacteriophage FPLC
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