Identification of interacting partner(s) of SARS-CoV spike glycoprotein.

Chuck Chi-pang.Thesis (M.Phil.)--Chinese University of Hong Kong, 2006.Includes bibliographical references (leaves 138-160).Abstracts in English and Chinese.Thesis Committee --- p.iiAbstract --- p.iii摘要 --- p.vContents --- p.viiList of Figures --- p.xiList of Tables --- p.xiiiAbbreviations --- p.xivAcknowledgement --- p.xviiiIntroductionChapter 1. --- BackgroundChapter 1.1 --- SARSChapter 1.1.1 --- Outbreak and Influence --- p.1Chapter 1.1.2 --- Clinical Features --- p.4Chapter 1.2 --- SARS-CoVChapter 1.2.1 --- Genomic Organization --- p.5Chapter 1.2.2 --- Morphology --- p.7Chapter 1.2.3 --- Phylogenetic Analysis --- p.9Chapter 1.3 --- S GlycoproteinChapter 1.3.1 --- Functional Roles --- p.11Chapter 1.3.2 --- Structure and Functional Domains --- p.12Chapter 1.3.3 --- Interacting Partners --- p.15Chapter 1.3.4 --- Viral Entry Mechanism --- p.17Chapter 1.4 --- Aim of StudyChapter 1.4.1 --- Mismatch of SARS-CoV Tissue Tropism and Tissue Distribution of ACE2 --- p.20Chapter 1.4.2 --- Presence of Other Interacting Partner(s) --- p.22Chapter 1.4.3 --- Significance of the Study Materials and Methods --- p.22Chapter 2. --- Plasmid ConstructionChapter 2.1 --- Fragment DesignChapter 2.1.1 --- Functional Domain Analysis --- p.23Chapter 2.1.2 --- Secondary Structure and Burial Region Predictions --- p.24Chapter 2.2 --- Vector AmplificationChapter 2.2.1 --- E. coli Strain DH5a Competent Cell Preparation --- p.30Chapter 2.2.2 --- Transformation of E. coli --- p.30Chapter 2.2.3 --- Small-scale Vector Amplification --- p.31Chapter 2.3 --- Cloning of DNA Fragments into Various VectorsChapter 2.3.1 --- Primer Design --- p.32Chapter 2.3.2 --- DNA Amplification --- p.35Chapter 2.3.3 --- DNA Purification --- p.35Chapter 2.3.4 --- "Restriction Enzyme Digestion, Ligation and Transformation" --- p.36Chapter 2.3.5 --- Colony PCR --- p.37Chapter 2.4 --- DNA Sequence AnalysisChapter 2.4.1 --- Primer Design --- p.35Chapter 2.4.2 --- DNA Amplification and Purification for DNA Sequence Analysis --- p.39Chapter 2.4.3 --- Sequence Detection and Result Analysis --- p.40Chapter 3. --- "Protein Expression, Purification and Analysis"Chapter 3.1 --- Protein Expression in E. coliChapter 3.1.1 --- Molecular Weight and pI Predictions --- p.41Chapter 3.1.2 --- Glycerol Stock Preparation --- p.41Chapter 3.1.3 --- Protein Expression Induction --- p.41Chapter 3.1.4 --- Protein Extraction --- p.42Chapter 3.1.5 --- Affinity Chromatography --- p.42Chapter 3.1.6 --- Removal of GroEL --- p.43Chapter 3.1.7 --- Protein Solubilization and Refolding --- p.44Chapter 3.2 --- Protein Expression in P. pastorisChapter 3.2.1 --- Large-scale Plasmid Amplification --- p.46Chapter 3.2.2 --- Restriction Enzyme Digestion and Ethanol Precipitation --- p.47Chapter 3.2.3 --- Preparation of KM71H Competent Cells --- p.47Chapter 3.2.4 --- Electroporation --- p.48Chapter 3.2.5 --- Colony PCR --- p.48Chapter 3.2.6 --- Protein Expression Induction and Time Course Study --- p.49Chapter 3.2.7 --- Deglycosylation --- p.49Chapter 3.3 --- Protein AnalysisChapter 3.3.1 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis --- p.50Chapter 3.3.2 --- Western Blotting --- p.50Chapter 3.3.3 --- Mass Spectrometry --- p.51Chapter 3.3.4 --- N-terminal Sequencing --- p.52Chapter 3.3.5 --- Size Exclusion Chromatography --- p.52Chapter 4. --- Identification of Interacting Partner(s)Chapter 4.1 --- VeroE6 PreparationChapter 4.1.1 --- Cell Culture --- p.53Chapter 4.1.2 --- Protein Extraction and Western Blotting --- p.53Chapter 4.2 --- Pull-down Assay --- p.54Chapter 4.3 --- Two-dimensional Gel Electrophores --- p.isChapter 4.3.1 --- Isoelectric Focusing --- p.56Chapter 4.3.2 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis --- p.56Chapter 4.3.3 --- Silver Staining --- p.57Chapter 4.4 --- Mass SpectrometryChapter 4.4.1 --- Destaining --- p.58Chapter 4.4.2 --- In-gel Digestion --- p.58Chapter 4.4.3 --- Desalting by Zip-tip --- p.59Chapter 4.4.4 --- Loading Sample --- p.59Chapter 4.4.5 --- Peptide Mass Detection and Data Analysis --- p.59ResultsChapter 5. --- S Protein ExpressionChapter 5.1 --- Plasmid Construction --- p.61Chapter 5.2 --- Molecular Weight and pi Predictions --- p.63Chapter 5.3 --- Protein Expression and Optimization in E. coliChapter 5.3.1 --- "Comparison of Expression Levels, Solubility and Purities of S Protein Fragments" --- p.64Chapter 5.3.2 --- "Alteration of the Solubility in Various Cell Strains, Expression Conditions and Lysis Buffers" --- p.68Chapter 5.3.3 --- Identification and Remove of the non-target proteins --- p.72Chapter 5.3.4 --- Unfolding and Refolding --- p.79Chapter 5.4 --- Protein Expression and Optimization in P. pastorisChapter 5.4.1 --- "Expression Levels, Solubility and Purities of Various S Protein Fragments" --- p.85Chapter 5.4.2 --- Characterization of De-N-glycosylated Recombinant Proteins --- p.89Chapter 6. --- Identification of Interacting partnersChapter 6.1 --- Practicability of Pull-down AssayChapter 6.1.1 --- ACE2 Extraction --- p.95Chapter 6.1.2 --- Pull-down of ACE2 by the P. pastoris-expressed recombinant RBD --- p.96Chapter 6.2 --- Pull-down Assay and Two-dimensional Gel Electrophoresis --- p.97Chapter 6.3 --- Identification of Putative Interacting Partners by MALDI-TOF-TOF --- p.107Chapter 7. --- DiscussionChapter 7.1 --- S Protein Expression in E. coliChapter 7.1.1 --- Improving Recombinant Protein Expression Level and Solubility --- p.114Chapter 7.1.2 --- S Recombinant Protein Bound by GroEL --- p.117Chapter 7.2 --- S Protein Expression in P. pastorisChapter 7.2.1 --- Advantages of Using P. pastoris --- p.119Chapter 7.2.2 --- Variation of S Fragment Expression Levels --- p.120Chapter 7.2.3 --- Sizes of S Protein Fragments --- p.123Chapter 7.3 --- Identification of Interacting PartnersChapter 7.3.1 --- Relationship between S Protein and Putative Interacting Partners --- p.124Chapter 7.3.2 --- Failure of Finding ACE2 --- p.125Chapter 7.3.2 --- Difficulty in the Identification of Protein Spots --- p.126Chapter 7.4 --- Conclusion --- p.131Chapter 7.5 --- Future Perspective --- p.132Chapter 8. --- Appendix --- p.133Chapter 9. --- References --- p.13