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.ii / Abstract --- p.iii / 摘要 --- p.v / Contents --- p.vii / List of Figures --- p.xi / List of Tables --- p.xiii / Abbreviations --- p.xiv / Acknowledgement --- p.xviii / Introduction / Chapter 1. --- Background / Chapter 1.1 --- SARS / Chapter 1.1.1 --- Outbreak and Influence --- p.1 / Chapter 1.1.2 --- Clinical Features --- p.4 / Chapter 1.2 --- SARS-CoV / Chapter 1.2.1 --- Genomic Organization --- p.5 / Chapter 1.2.2 --- Morphology --- p.7 / Chapter 1.2.3 --- Phylogenetic Analysis --- p.9 / Chapter 1.3 --- S Glycoprotein / Chapter 1.3.1 --- Functional Roles --- p.11 / Chapter 1.3.2 --- Structure and Functional Domains --- p.12 / Chapter 1.3.3 --- Interacting Partners --- p.15 / Chapter 1.3.4 --- Viral Entry Mechanism --- p.17 / Chapter 1.4 --- Aim of Study / Chapter 1.4.1 --- Mismatch of SARS-CoV Tissue Tropism and Tissue Distribution of ACE2 --- p.20 / Chapter 1.4.2 --- Presence of Other Interacting Partner(s) --- p.22 / Chapter 1.4.3 --- Significance of the Study Materials and Methods --- p.22 / Chapter 2. --- Plasmid Construction / Chapter 2.1 --- Fragment Design / Chapter 2.1.1 --- Functional Domain Analysis --- p.23 / Chapter 2.1.2 --- Secondary Structure and Burial Region Predictions --- p.24 / Chapter 2.2 --- Vector Amplification / Chapter 2.2.1 --- E. coli Strain DH5a Competent Cell Preparation --- p.30 / Chapter 2.2.2 --- Transformation of E. coli --- p.30 / Chapter 2.2.3 --- Small-scale Vector Amplification --- p.31 / Chapter 2.3 --- Cloning of DNA Fragments into Various Vectors / Chapter 2.3.1 --- Primer Design --- p.32 / Chapter 2.3.2 --- DNA Amplification --- p.35 / Chapter 2.3.3 --- DNA Purification --- p.35 / Chapter 2.3.4 --- "Restriction Enzyme Digestion, Ligation and Transformation" --- p.36 / Chapter 2.3.5 --- Colony PCR --- p.37 / Chapter 2.4 --- DNA Sequence Analysis / Chapter 2.4.1 --- Primer Design --- p.35 / Chapter 2.4.2 --- DNA Amplification and Purification for DNA Sequence Analysis --- p.39 / Chapter 2.4.3 --- Sequence Detection and Result Analysis --- p.40 / Chapter 3. --- "Protein Expression, Purification and Analysis" / Chapter 3.1 --- Protein Expression in E. coli / Chapter 3.1.1 --- Molecular Weight and pI Predictions --- p.41 / Chapter 3.1.2 --- Glycerol Stock Preparation --- p.41 / Chapter 3.1.3 --- Protein Expression Induction --- p.41 / Chapter 3.1.4 --- Protein Extraction --- p.42 / Chapter 3.1.5 --- Affinity Chromatography --- p.42 / Chapter 3.1.6 --- Removal of GroEL --- p.43 / Chapter 3.1.7 --- Protein Solubilization and Refolding --- p.44 / Chapter 3.2 --- Protein Expression in P. pastoris / Chapter 3.2.1 --- Large-scale Plasmid Amplification --- p.46 / Chapter 3.2.2 --- Restriction Enzyme Digestion and Ethanol Precipitation --- p.47 / Chapter 3.2.3 --- Preparation of KM71H Competent Cells --- p.47 / Chapter 3.2.4 --- Electroporation --- p.48 / Chapter 3.2.5 --- Colony PCR --- p.48 / Chapter 3.2.6 --- Protein Expression Induction and Time Course Study --- p.49 / Chapter 3.2.7 --- Deglycosylation --- p.49 / Chapter 3.3 --- Protein Analysis / Chapter 3.3.1 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis --- p.50 / Chapter 3.3.2 --- Western Blotting --- p.50 / Chapter 3.3.3 --- Mass Spectrometry --- p.51 / Chapter 3.3.4 --- N-terminal Sequencing --- p.52 / Chapter 3.3.5 --- Size Exclusion Chromatography --- p.52 / Chapter 4. --- Identification of Interacting Partner(s) / Chapter 4.1 --- VeroE6 Preparation / Chapter 4.1.1 --- Cell Culture --- p.53 / Chapter 4.1.2 --- Protein Extraction and Western Blotting --- p.53 / Chapter 4.2 --- Pull-down Assay --- p.54 / Chapter 4.3 --- Two-dimensional Gel Electrophores --- p.is / Chapter 4.3.1 --- Isoelectric Focusing --- p.56 / Chapter 4.3.2 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis --- p.56 / Chapter 4.3.3 --- Silver Staining --- p.57 / Chapter 4.4 --- Mass Spectrometry / Chapter 4.4.1 --- Destaining --- p.58 / Chapter 4.4.2 --- In-gel Digestion --- p.58 / Chapter 4.4.3 --- Desalting by Zip-tip --- p.59 / Chapter 4.4.4 --- Loading Sample --- p.59 / Chapter 4.4.5 --- Peptide Mass Detection and Data Analysis --- p.59 / Results / Chapter 5. --- S Protein Expression / Chapter 5.1 --- Plasmid Construction --- p.61 / Chapter 5.2 --- Molecular Weight and pi Predictions --- p.63 / Chapter 5.3 --- Protein Expression and Optimization in E. coli / Chapter 5.3.1 --- "Comparison of Expression Levels, Solubility and Purities of S Protein Fragments" --- p.64 / Chapter 5.3.2 --- "Alteration of the Solubility in Various Cell Strains, Expression Conditions and Lysis Buffers" --- p.68 / Chapter 5.3.3 --- Identification and Remove of the non-target proteins --- p.72 / Chapter 5.3.4 --- Unfolding and Refolding --- p.79 / Chapter 5.4 --- Protein Expression and Optimization in P. pastoris / Chapter 5.4.1 --- "Expression Levels, Solubility and Purities of Various S Protein Fragments" --- p.85 / Chapter 5.4.2 --- Characterization of De-N-glycosylated Recombinant Proteins --- p.89 / Chapter 6. --- Identification of Interacting partners / Chapter 6.1 --- Practicability of Pull-down Assay / Chapter 6.1.1 --- ACE2 Extraction --- p.95 / Chapter 6.1.2 --- Pull-down of ACE2 by the P. pastoris-expressed recombinant RBD --- p.96 / Chapter 6.2 --- Pull-down Assay and Two-dimensional Gel Electrophoresis --- p.97 / Chapter 6.3 --- Identification of Putative Interacting Partners by MALDI-TOF-TOF --- p.107 / Chapter 7. --- Discussion / Chapter 7.1 --- S Protein Expression in E. coli / Chapter 7.1.1 --- Improving Recombinant Protein Expression Level and Solubility --- p.114 / Chapter 7.1.2 --- S Recombinant Protein Bound by GroEL --- p.117 / Chapter 7.2 --- S Protein Expression in P. pastoris / Chapter 7.2.1 --- Advantages of Using P. pastoris --- p.119 / Chapter 7.2.2 --- Variation of S Fragment Expression Levels --- p.120 / Chapter 7.2.3 --- Sizes of S Protein Fragments --- p.123 / Chapter 7.3 --- Identification of Interacting Partners / Chapter 7.3.1 --- Relationship between S Protein and Putative Interacting Partners --- p.124 / Chapter 7.3.2 --- Failure of Finding ACE2 --- p.125 / Chapter 7.3.2 --- Difficulty in the Identification of Protein Spots --- p.126 / Chapter 7.4 --- Conclusion --- p.131 / Chapter 7.5 --- Future Perspective --- p.132 / Chapter 8. --- Appendix --- p.133 / Chapter 9. --- References --- p.138
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_325712 |
| Date | January 2006 |
| Contributors | Chuck, Chi-pang., Chinese University of Hong Kong Graduate School. Division of Biochemistry. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xviii, 160 leaves : ill. (some col.) ; 30 cm. |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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