by Chan Wai Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 170-185). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / 摘要 --- p.v / Table of Content --- p.vii / Abbreviations --- p.xii / for Nucleotides --- p.xii / for Amino acids --- p.xii / for Standard genetic codes --- p.xiii / for Units --- p.xiii / for Prefixes of units --- p.xiv / for Terms commonly used in the report --- p.xiv / List of Figures --- p.xvii / List of Tables --- p.xxiii / Chapter Chapter I --- Introduction --- p.1 / Chapter 1.1 --- Epidemiology of the Severe Acute Respiratory Syndrome --- p.1 / Chapter 1.2 --- The SARS Coronavirus --- p.3 / Chapter 1.3 --- Cell Biology of Coronavirus Infection and Replication and the Role of Nucleocapsid Protein --- p.9 / Chapter 1.4 --- Recent Advances in the SARS-CoV Nucleocapsid Protein --- p.16 / Chapter 1.5 --- The Sumoylation System --- p.24 / Chapter 1.6 --- Objectives of the Present Study --- p.28 / Chapter Chapter II --- SARS-CoV N protein and Fragment Purification --- p.29 / Chapter 2.1 --- INTRODUCTION --- p.29 / Chapter 2.2 --- METHODOLOGY --- p.31 / Materials --- p.31 / Methods --- p.39 / Chapter 2.2.1 --- Construction of the pMAL-c2P vector --- p.39 / Chapter 2.2.2 --- Sub-cloning of the N protein into expression vectors --- p.42 / Chapter 2.2.2.1 --- Design of primers for the cloning of N protein --- p.43 / Chapter 2.2.2.2 --- DNA amplification using Polymerase Chain Reaction (PCR) --- p.44 / Chapter 2.2.2.3 --- DNA extraction from agarose gel --- p.45 / Chapter 2.2.2.4 --- Restriction digestion of purified PCR product and vectors --- p.46 / Chapter 2.2.2.5 --- Ligation of N protein into expression vectors --- p.47 / Chapter 2.2.2.6 --- Preparation of competent cells --- p.48 / Chapter 2.2.2.7 --- Transformation of plasmids into competent Escherichia coli --- p.49 / Chapter 2.2.2.8 --- Preparation of plasmid DNA --- p.49 / Chapter 2.2.2.8.1 --- Mini-preparation of plasmid DNA --- p.49 / Chapter 2.2.2.8.2 --- Midi-preparation of plasmid DNA --- p.51 / Chapter 2.2.3 --- Expression of tagged and untagged N protein --- p.53 / Chapter 2.2.3.1 --- Preparation of E. coli competent cells for protein expression --- p.53 / Chapter 2.2.3.2 --- Expression of N protein --- p.53 / Chapter 2.2.3.3 --- Solubility tests on the fusion proteins expressed --- p.54 / Chapter 2.2.4 --- Purification of N protein Chromatographic methods --- p.55 / Chapter 2.2.4.1 --- Affinity chromatography --- p.55 / Chapter 2.2.4.1.1 --- Ni-NTA affinity chromatography --- p.55 / Chapter 2.2.4.1.2 --- Glutathione affinity chromatography --- p.56 / Chapter 2.2.4.1.3 --- Amylose affinity chromatography --- p.56 / Chapter 2.2.4.2 --- Ion exchange chromatography --- p.57 / Chapter 2.2.4.2.1 --- Cation exchange chromatography --- p.57 / Chapter 2.2.4.2.2 --- Anion exchange chromatography --- p.58 / Chapter 2.2.4.3 --- Heparin affinity chromatography --- p.58 / Chapter 2.2.4.4 --- Size exclusion chromatography Purification strategies --- p.60 / Chapter 2.2.4.5 --- Purification of His6-tagged N proteins --- p.60 / Chapter 2.2.4.6 --- Purification of MBP-tagged N proteins --- p.60 / Chapter 2.2.4.7 --- Purification of GST-tagged N proteins --- p.61 / Chapter 2.2.4.8 --- Purification of untagged N proteins --- p.61 / Chapter 2.2.5 --- Trypsin digestion assay for the design of stable fragment --- p.64 / Chapter 2.2.6 --- Partial purification of the N protein amino acid residue 214-422 fragment --- p.65 / Chapter 2.2.7 --- Sumoylation of the SARS-CoV N protein --- p.67 / Chapter 2.2.7.1 --- In vitro sumoylation assay --- p.67 / Chapter 2.2.7.2 --- Sample preparation for mass spectrometric analysis --- p.68 / Chapter 2.3 --- RESULTS --- p.70 / Chapter 2.3.1 --- Construction of the vector pMAL-c2P --- p.70 / Chapter 2.3.2 --- "Construction of recombinant N protein-pAC28m, N-protein- pGEX-6P-l,N protein-pMAL-c2E and N protein-pMAL-c2P plasmids" --- p.72 / Chapter 2.3.3 --- Optimization of expression conditions --- p.79 / Chapter 2.3.4 --- Screening of purification strategies --- p.82 / Chapter 2.3.4.1 --- Purification of His6-N protein --- p.82 / Chapter 2.3.4.2 --- Purification of MBP-N protein --- p.84 / Chapter 2.3.4.3 --- Purification of GST-N protein --- p.85 / Chapter 2.3.4.4 --- Purification of untagged N protein --- p.87 / Chapter 2.3.5 --- Limited trypsinolysis for the determination of discrete structural unit --- p.91 / Chapter 2.3.6 --- Partial purification of the N protein 214-422 fragment --- p.94 / Chapter 2.3.7 --- Sumoylation of N protein --- p.97 / Chapter 2.2.7.1 --- Sumoylation site prediction --- p.97 / Chapter 2.2.7.2 --- In vitro sumoylation assay --- p.99 / Chapter 2.2.7.3 --- Mass spectrometric identification of sumoylated SARS-CoV N protein --- p.103 / Chapter 2.4 --- DISCUSSION --- p.109 / Chapter Chapter III --- Characterization of the Nucleic Acid Binding Ability of N protein --- p.119 / Chapter 3.1 --- INTRODUCTION --- p.119 / Chapter 3.2 --- METHODOLOGY --- p.120 / Materials --- p.120 / Methods --- p.124 / Chapter 3.2.1 --- Spectrophotometric Measurement of ratio OD260/ OD280 --- p.124 / Chapter 3.2.2 --- Native gel electrophoresis --- p.124 / Chapter 3.2.3 --- Quantitative determination of nucleic acids content --- p.125 / Chapter 3.2.3.1 --- Dische assay - quantitative determination of DNA content --- p.125 / Chapter 3.2.3.2 --- Orcinol assay - quantitative determination of RNA content --- p.126 / Chapter 3.2.4 --- RNase digestion of the N protein-bound RNA --- p.128 / Chapter 3.2.5 --- Isolation of RNA from purified GST-N proteins --- p.128 / Chapter 3.2.6 --- In vitro transcription of SARS-CoV genomic RNA fragment --- p.129 / Chapter 3.2.7 --- Vero E6 cell line maintenance and total RNA extraction --- p.131 / Chapter 3.2.8 --- Electrophoretic mobility shift assay (EMSA) --- p.131 / Chapter 3.3 --- RESULTS --- p.133 / Chapter 3.3.1 --- Detection of nucleic acids in the purified N proteins byspectrophotometric Measurement of ratio OD260/ OD280 --- p.133 / Chapter 3.3.2 --- Native gel electrophoresis --- p.135 / Chapter 3.3.3 --- Quantitative determination of nucleic acids content in purified GST-N proteins --- p.136 / Chapter 3.3.3.1 --- Dische assay for the determination of DNA --- p.136 / Chapter 3.3.3.2 --- Orcinol assay for the determination of RNA --- p.138 / Chapter 3.3.4 --- RNase digestion treatment --- p.139 / Chapter 3.3.5 --- Extraction of RNA from GST-N proteins --- p.140 / Chapter 3.3.6 --- In vitro transcription of SARS-CoV genomic RNA fragment --- p.142 / Chapter 3.3.7 --- Electrophoretic mobility shift assay (EMSA) --- p.144 / Chapter 3.4 --- DISCUSSION --- p.147 / Chapter Chapter IV --- Discussion --- p.154 / Chapter 4.1 --- "Purity, Aggregation and RNA Binding Property of the SARS-CoV Nucleocapsid Protein" --- p.154 / Chapter 4.2 --- Future perspectives --- p.156 / Chapter 4.2.1 --- Structural study of the SARS-CoV N protein through x-ray crystallography --- p.156 / Chapter 4.2.2 --- Mapping the RNA binding domain in the SARS-CoV N protein --- p.156 / Chapter 4.2.3 --- Determination of aggregation state by lateral turbidimetry analysis --- p.156 / Chapter 4.2.4 --- Exploring protein interacting partners that enhance RNA binding specificity --- p.157 / Appendix --- p.159 / Chapter I. --- Sequence of the SARS-CoV N protein --- p.159 / Chapter II. --- Sequence of the SARS-CoV genome fragment used for RNA binding assay in section 3.37.1 --- p.161 / Chapter III. --- Vector maps --- p.161 / Chapter a) --- Vector map of pACYC177 --- p.161 / Chapter b) --- Vector map and MCS of pET28a --- p.163 / Chapter c) --- Vector map and MCS of pAC28 --- p.164 / Chapter d) --- Vector map and MCS of pGEX-6P-1 / Chapter e) --- Vector map of pMAL-c2X and MCS of pMAL-c2E / Chapter IV. --- Electrophoresis markers --- p.166 / Chapter V. --- SDS-PAGE gel parathion protocol --- p.169 / References --- p.170
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_325201 |
| Date | January 2005 |
| Contributors | Chan, Wai Ling., 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, xxiii, 185 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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