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Characterization of spike glycoprotein fusion core and 3C-like protease substrate specificity of the severe acute respiratory syndrome (SARS) coronavirus: perspective for anti-SARS drug development.

Chu Ling Hon Matthew. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 201-223). / Abstracts in English and Chinese. / Declaration --- p.i / Thesis/Assessment Committee --- p.ii / Abstract --- p.iii / 摘要 --- p.vi / Acknowledgements --- p.viii / General abbreviations --- p.xi / Abbreviations of chemicals --- p.xv / Table of Contents --- p.xvi / List of Figures --- p.xxiii / List of tables --- p.xxviii / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Severe Acute Respiratory Syndrome (SARS) - Three Years in Review --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.1 / Chapter 1.1.2 --- Clinical presentation --- p.3 / Chapter 1.1.3 --- Diagnostic tests --- p.5 / Chapter 1.2 --- Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) --- p.7 / Chapter 1.2.1 --- SARS - Identification of the etiological agent --- p.7 / Chapter 1.2.2 --- The coronaviruses --- p.9 / Chapter 1.2.3 --- The genome organization of SARS-CoV --- p.11 / Chapter 1.2.4 --- The life cycle of SARS-CoV --- p.13 / Chapter 1.3 --- Spike Glycoprotein (S protein) of SARS-CoV --- p.15 / Chapter 1.3.1 --- SARS-CoV S protein --- p.15 / Chapter 1.3.2 --- S protein-driven infection --- p.17 / Chapter 1.4 --- SARS-CoV S Protein Fusion Core --- p.22 / Chapter 1.4.1 --- Heptad repeat and coiled coil --- p.22 / Chapter 1.4.2 --- The six-helix coiled coil bundle structure --- p.25 / Chapter 1.5 --- 3C-like Protease (3CLpro) of SARS-CoV --- p.28 / Chapter 1.5.1 --- Extensive proteolytic processing of replicase polyproteins --- p.28 / Chapter 1.5.2 --- SARS-CoV 3CLpro --- p.30 / Chapter 1.5.3 --- Substrate Specificity of SARS-CoV 3CLpro --- p.31 / Chapter 1.6 --- SARS Drug Development --- p.32 / Chapter 1.6.1 --- Drug targets of SARS-CoV --- p.32 / Chapter 1.6.2 --- Current anti-SARS drugs --- p.36 / Chapter 1.7 --- Project Objectives --- p.39 / Chapter 1.7.1 --- Characterization of SARS-CoV S protein fusion core --- p.39 / Chapter 1.7.2 --- Characterization of SARS-CoV 3CLpr0 substrate specificity --- p.40 / Chapter 2 --- Materials and Methods --- p.42 / Chapter 2.1 --- Characterization of SARS-CoV S Protein Fusion Core --- p.42 / Chapter 2.1.1 --- Bioinformatics analyses of heptad repeat regions of SARS- CoV S protein --- p.42 / Chapter 2.1.2 --- Recombinant protein approach --- p.43 / Chapter 2.1.2.1 --- Plasmids construction --- p.43 / Chapter 2.1.2.2 --- Protein expression and purification --- p.52 / Chapter 2.1.2.3 --- Amino acid analysis --- p.57 / Chapter 2.1.2.4 --- GST-pulldown experiment --- p.58 / Chapter 2.1.2.5 --- Laser light scattering --- p.61 / Chapter 2.1.2.6 --- Size-exclusion chromatography --- p.62 / Chapter 2.1.2.7 --- Circular dichroism spectroscopy --- p.62 / Chapter 2.1.3 --- Synthetic peptide approach --- p.64 / Chapter 2.1.3.1 --- Peptide synthesis --- p.64 / Chapter 2.1.3.2 --- Native polyacrylamide gel electrophoresis --- p.65 / Chapter 2.1.3.3 --- Size-exclusion high-performance liquid chromato-graphy --- p.66 / Chapter 2.1.3.4 --- Laser light scattering --- p.66 / Chapter 2.1.3.5 --- Circular dichroism spectroscopy --- p.67 / Chapter 2.2 --- Identification of SARS-CoV Entry Inhibitors --- p.70 / Chapter 2.2.1 --- HIV-luc/SARS pseudotyped virus entry inhibition assay --- p.70 / Chapter 2.2.2 --- Recombinant protein- and synthetic peptide-based biophysical assays --- p.74 / Chapter 2.2.3 --- Molecular modeling --- p.75 / Chapter 2.3 --- Characterization of SARS-CoV 3CLpro Substrate Specificity --- p.79 / Chapter 2.3.1 --- Protein expression and purification --- p.79 / Chapter 2.3.2 --- """Cartridge replacement"" solid-phase peptide synthesis" --- p.80 / Chapter 2.3.3 --- Peptide cleavage assay and mass spectrometric analysis --- p.83 / Chapter 3 --- Results --- p.84 / Chapter 3.1 --- Characterization of SARS-CoV S Protein Fusion Core --- p.84 / Chapter 3.1.1 --- Bioinformatics analyses of heptad repeat regions of SARS- CoV S protein --- p.84 / Chapter 3.1.2 --- Recombinant protein approach --- p.87 / Chapter 3.1.2.1 --- "Plasmids construction of pET-28a-His6-HRl, pGEX-6P-l-HR2 and pGEX-6P-l-2-Helix" --- p.87 / Chapter 3.1.2.2 --- Protein expression and purification --- p.92 / Chapter 3.1.2.3 --- GST-pulldown experiment --- p.101 / Chapter 3.1.2.4 --- Laser light scattering --- p.103 / Chapter 3.1.2.5 --- Size-exclusion chromatography --- p.105 / Chapter 3.1.2.6 --- Circular dichroism spectroscopy --- p.107 / Chapter 3.1.3 --- Synthetic peptide approach --- p.112 / Chapter 3.1.3.1 --- Peptide synthesis --- p.112 / Chapter 3.1.3.2 --- Native polyacrylamide gel electrophoresis --- p.116 / Chapter 3.1.3.3 --- Size-exclusion high-performance liquid chromatography --- p.117 / Chapter 3.1.3.4 --- Laser light scattering --- p.122 / Chapter 3.1.3.5 --- Circular dichroism spectroscopy --- p.124 / Chapter 3.2 --- Identification of SARS-CoV Entry Inhibitors --- p.129 / Chapter 3.2.1 --- HIV-luc/SARS pseudotyped virus entry inhibition assay --- p.129 / Chapter 3.2.2 --- Recombinant protein- and synthetic peptide-based biophysical assays --- p.131 / Chapter 3.2.3 --- Molecular modeling --- p.135 / Chapter 3.3 --- Characterization of SARS-CoV 3CLpro Substrate Specificity --- p.141 / Chapter 3.3.1 --- Protein expression and purification --- p.141 / Chapter 3.3.2 --- Substrate specificity preference of SARS-CoV 3CLpr0 --- p.142 / Chapter 3.3.3 --- "Primary and secondary screening using the ""cartridge replacement strategy""" --- p.142 / Chapter 4 --- Discussion --- p.149 / Chapter 4.1 --- Characterization of SARS-CoV S Protein Fusion Core --- p.149 / Chapter 4.1.1 --- Design of recombinant proteins and synthetic peptides of HR regions --- p.149 / Chapter 4.1.2 --- Recombinant protein approach --- p.151 / Chapter 4.1.3 --- Synthetic peptide approach --- p.153 / Chapter 4.1.4 --- Summary of the present and previous studies in the SARS-CoV S protein fusion core --- p.157 / Chapter 4.2 --- Identification of SARS-CoV Entry Inhibitors --- p.167 / Chapter 4.2.1 --- HIV-luc/SARS pseudotyped virus entry inhibition assay --- p.167 / Chapter 4.2.2 --- Identification of peptide inhibitors --- p.168 / Chapter 4.2.3 --- Identification of small molecule inhibitors --- p.172 / Chapter 4.3 --- Characterization of SARS-CoV 3CLpro Substrate Specificity --- p.183 / Chapter 4.3.1 --- A comprehensive overview of the substrate specificity of SARS-CoV 3CLpro --- p.184 / Chapter 4.3.2 --- The development of the rapid and high-throughput screening strategy for protease substrate specificity --- p.188 / Appendix --- p.191 / Chapter I. --- Nucleotide Sequence of S protein of SARS-CoV --- p.191 / Chapter II. --- Protein Sequence of S protein of SARS-CoV --- p.194 / Chapter III. --- Protein Sequence of 3CLpro of SARS-CoV --- p.195 / Chapter IV. --- Vector maps --- p.196 / Chapter 1. --- Vector map and MCS of pET-28a --- p.196 / Chapter 2. --- Vector map and MCS of pGEX-6P-l --- p.197 / Chapter V. --- Electrophoresis markers --- p.198 / Chapter 1. --- GeneRuler´ёØ 1 kb DNA Ladder --- p.198 / Chapter 2. --- GeneRuler´ёØ 100bp DNA Ladder --- p.198 / Chapter 3. --- High-range Rainbow Molecular Weight Markers --- p.199 / Chapter 4. --- Low-range Rainbow Molecular Weight Markers --- p.199 / Chapter VI. --- SDS-PAGE gel preparation protocol --- p.200 / References --- p.201

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_325704
Date January 2006
ContributorsChu, Ling Hon Matthew., Chinese University of Hong Kong Graduate School. Division of Molecular Biotechnology.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xxviii, 223 leaves : ill. (some col.) ; 30 cm.
RightsUse 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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