Hon, Ching Yi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 144-157). / Abstracts in English and Chinese. / Examination Committee List --- p.i / Acknowledgements --- p.ii / Abstract --- p.iv / 摘要 --- p.vi / Table of Contents --- p.viii / List of Tables --- p.xiv / List of Figures --- p.xv / List of Abbreviations --- p.xvii / List of Chemicals --- p.xx / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Mycobacterium tuberculosis --- p.1 / Chapter 1.2 --- Tuberculosis --- p.2 / Chapter 1.3 --- Pathogenesis of TB --- p.3 / Chapter 1.3.1 --- Mycobacterial entry to the host macrophage --- p.3 / Chapter 1.3.2 --- Modulation of the host endocytic pathway --- p.4 / Chapter 1.3.2.1 --- The fusion between lysosome and mycobacterial phagosomeis blocked --- p.4 / Chapter 1.3.2.2 --- The endosomal pH of mycobacterial phagosome is preserved --- p.5 / Chapter 1.3.2.3 --- Mtb successfully mediates host cell apoptosis inhibition --- p.6 / Chapter 1.3.3 --- Cell migration and granuloma formation --- p.7 / Chapter 1.4 --- Insights from the complete genome sequence of Mtb H37Rv --- p.9 / Chapter 1.4.1 --- PE protein family --- p.9 / Chapter 1.4.2 --- PPE protein family --- p.10 / Chapter 1.5 --- Interaction between PE and PPE proteins --- p.11 / Chapter 1.5.1 --- Integrated bioinformatics prediction --- p.11 / Chapter 1.5.2 --- In vitro interaction studies of PE25/PPE41 protein complex --- p.12 / Chapter 1.5.3 --- Structural characterization of PE/PPE complex as revealed by PE25/PPE41 crystal structure --- p.14 / Chapter 1.6 --- Biological roles of PE and PPE proteins --- p.15 / Chapter 1.6.1 --- Immunological roles of PE family proteins --- p.15 / Chapter 1.6.2 --- Immunological roles of PPE family proteins --- p.16 / Chapter 1.6.3 --- Immunological roles of PE/PPE protein complex --- p.16 / Chapter 1.7 --- Sub-cellular localization of PE and PPE proteins --- p.17 / Chapter 1.8 --- PE and PPE as exported proteins --- p.18 / Chapter 1.8.1 --- Association of Mtb secreted proteins to pathogenesis of tuberculosis --- p.18 / Chapter 1.8.2 --- Exported PE and PPE proteins --- p.19 / Chapter 1.9 --- Differential expression of PE and PPE genes --- p.20 / Chapter 1.10 --- Objective of project --- p.21 / Chapter CHAPTER 2 --- "CLONING, EXPRESSION, PURIFICATION AND VERIFICATION OF PE15 AND PPE20 AS COMPLEX" --- p.24 / Chapter 2.1 --- Materials --- p.24 / Chapter 2.1.1 --- Bacterial expression plasmids for expression of PE or PPE proteins --- p.24 / Chapter 2.1.2 --- E. coli strains --- p.27 / Chapter 2.1.3 --- Reagents and buffers for molecular cloning --- p.27 / Chapter 2.1.4 --- Reagents and buffers for E. coli protein expression --- p.29 / Chapter 2.1.5 --- Buffers for protein purification --- p.30 / Chapter 2.2 --- Methods --- p.31 / Chapter 2.2.1 --- Molecular cloning --- p.31 / Chapter 2.2.1.1 --- Primers --- p.31 / Chapter 2.2.1.2 --- Gene Amplification by Polymerase Chain Reaction (PCR) --- p.33 / Chapter 2.2.1.3 --- Agarose gel electrophoresis --- p.34 / Chapter 2.2.1.4 --- Extraction and purification of DNA from agarose gel by QIAquick Gel Extraction Kit --- p.34 / Chapter 2.2.1.5 --- Restriction digestion of DNA --- p.35 / Chapter 2.2.1.6 --- Purification of DNA by QIAquick PCR Purification Kit --- p.36 / Chapter 2.2.1.7 --- Ligation of DNA and expression vector to produce recombinant plasmid --- p.37 / Chapter 2.2.1.8 --- Transformation of plasmid into E. coli competent cell --- p.38 / Chapter 2.2.1.9 --- PCR Screening of recombinant clones --- p.39 / Chapter 2.2.1.10 --- Plasmid DNA purification using the QIAprep Spin Miniprep Kit --- p.40 / Chapter 2.2.1.11 --- DNA sequencing --- p.41 / Chapter 2.2.2 --- Expression of PE15 and PPE20 proteins --- p.41 / Chapter 2.2.2.1 --- Small scale protein expression of PE 15 and PPE20 proteins --- p.41 / Chapter 2.2.2.2 --- Small scale co-expression of PE 15 and PPE20 proteins --- p.42 / Chapter 2.2.2.3 --- Protein solubility analysis --- p.43 / Chapter 2.2.2.4 --- Large scale expression of PE and PPE proteins --- p.43 / Chapter 2.2.3 --- SDS-PAGE --- p.44 / Chapter 2.2.4 --- Bradford assay --- p.45 / Chapter 2.2.5 --- Protein purification of PE15 --- p.46 / Chapter 2.2.5.1 --- Sonication and extraction of proteins --- p.46 / Chapter 2.2.5.2 --- Protein purification of PE15 by gutathione-sepharose affinity chromatography --- p.46 / Chapter 2.2.5.3 --- Protein purification of PE15 by re-binding to glutathione-sepharose --- p.47 / Chapter 2.2.5.4 --- Protein purification of PE15 by size exclusion chromatography --- p.47 / Chapter 2.2.5.5 --- Protein purification of GST-PE15 --- p.48 / Chapter 2.2.6 --- Protein purification of PPE20(PPE) --- p.49 / Chapter 2.2.6.1 --- Sonication and extraction of proteins --- p.49 / Chapter 2.2.6.2 --- Protein purification of PPE20(PPE) by glutathione-sepharose affinity chromatography --- p.49 / Chapter 2.2.6.3 --- Protein purification of PPE20(PPE) by re-binding to glutathione-sepharose --- p.49 / Chapter 2.2.6.4 --- Protein purification of PPE20(PPE) by size exclusion chromatography --- p.50 / Chapter 2.2.6.5 --- Protein purification of GST-PPE20(PPE) --- p.50 / Chapter 2.2.7 --- Verification of PEPPE protein complex --- p.50 / Chapter 2.2.7.1 --- Size exclusion chromatography --- p.50 / Chapter 2.2.7.2 --- Cross-linking --- p.50 / Chapter 2.2.7.3 --- Pull-down assay --- p.51 / Chapter 2.3 --- Results --- p.52 / Chapter 2.3.1 --- Construction of bacterial expression plasmids for PE15 and PPE20 genes --- p.52 / Chapter 2.3.2 --- Expression of PE15 and PPE20 proteins --- p.54 / Chapter 2.3.2.1 --- Small scale protein expression of PE15 --- p.55 / Chapter 2.3.2.2 --- Small scale protein expression of PPE20 --- p.56 / Chapter 2.3.2.3 --- Small scale co-expression of PE15 and PPE20 proteins --- p.60 / Chapter 2.3.3 --- Large scale purification of PE15 and PPE20(PPE) proteins --- p.66 / Chapter 2.3.3.1 --- Large scale purification of PE15 --- p.66 / Chapter 2.3.3.2 --- Large scale purification of GST-PE15 --- p.68 / Chapter 2.3.3.3 --- Large scale purification of PPE20(PPE) --- p.69 / Chapter 2.3.3.4 --- Large scale purification of GST-PPE20(PPE) --- p.70 / Chapter 2.3.4 --- Verification of PE15/PPE20 complex --- p.71 / Chapter 2.3.4.1 --- Size exclusion chromatography --- p.71 / Chapter 2.3.4.2 --- Cross-linking study --- p.74 / Chapter 2.3.4.3 --- Pull-down assay --- p.77 / Chapter 2.4 --- Discussion --- p.79 / Chapter 2.4.1 --- Expression of PE15 and PPE20 proteins --- p.79 / Chapter 2.4.2 --- Co-expression of PE15 and PPE20 proteins --- p.81 / Chapter 2.4.3 --- Prediction of PE/PPE interaction --- p.82 / Chapter 2.4.4 --- Study ofPE15 and PPE20(PPE) interaction --- p.83 / Chapter CHAPTER 3 --- PRELIMINARY X-RAY ANALYSIS OF PPE20(PPE) PROTEIN CRYSTAL --- p.86 / Chapter 3.1 --- Materials --- p.86 / Chapter 3.1.1 --- Crystallization screening kits --- p.86 / Chapter 3.1.2 --- Crystallization chemicals --- p.86 / Chapter 3.2 --- Methods --- p.87 / Chapter 3.2.1 --- Crystallization screening using Phoenix´ёØ RE --- p.87 / Chapter 3.2.2 --- Optimization of PPE20(PPE) crystals by grid screening --- p.88 / Chapter 3.2.3 --- Optimization using Additive screen for PPE20(PPE) --- p.88 / Chapter 3.2.4 --- X-ray diffraction and data collection --- p.88 / Chapter 3.3 --- Results --- p.89 / Chapter 3.3.1 --- Crystallization screening --- p.89 / Chapter 3.3.2 --- Crystallization optimization --- p.90 / Chapter 3.3.3 --- Preliminary X-ray diffraction analysis --- p.92 / Chapter 3.3.4 --- Attempts to solve the phase by molecular replacement --- p.96 / Chapter 3.4 --- Discussion --- p.97 / Chapter CHAPTER 4 --- ISOLATION OF INTERACTING PARTNERS OF PE15 AND PPE20(PPE) FROM HUMAN MACROPHAGE U-937 --- p.99 / Chapter 4.1 --- Materials --- p.99 / Chapter 4.1.1 --- Mammalian cell line --- p.99 / Chapter 4.1.2 --- Mammalian cell growth medium --- p.99 / Chapter 4.1.3 --- Reagents and buffers for mammalian cell culture --- p.100 / Chapter 4.1.4 --- Reagents and buffers for mass spectrometry sample preparation --- p.100 / Chapter 4.2 --- Methods --- p.101 / Chapter 4.2.1 --- U-937 cell culturing --- p.101 / Chapter 4.2.1.1 --- Thawing U-937 cells --- p.101 / Chapter 4.2.1.2 --- Monitoring cell growth --- p.102 / Chapter 4.2.1.3 --- Cell differentiation --- p.102 / Chapter 4.2.1.4 --- Cell Harvesting --- p.103 / Chapter 4.2.2 --- In-vitro pull-down to identify interacting partners of PE15 or PPE20(PPE) --- p.103 / Chapter 4.2.2.1 --- Preparation of cellular proteins from U-937 cells --- p.103 / Chapter 4.2.2.2 --- Pre-clearing of U-937 supernatant --- p.104 / Chapter 4.2.2.3 --- Pull-down of U-937 cellular proteins with immobilized GST-PE15 --- p.104 / Chapter 4.2.2.4 --- Pull-down of U-937 cellular proteins with immobilized GST-PPE20(PPE) --- p.106 / Chapter 4.2.2.5 --- SDS-PAGE analysis --- p.106 / Chapter 4.2.2.6 --- Silver staining --- p.106 / Chapter 4.2.3 --- Mass- Spectrometry --- p.107 / Chapter 4.2.3.1 --- De-staining of silver stained gel spots --- p.107 / Chapter 4.2.3.2 --- Trypsin digestion --- p.108 / Chapter 4.2.3.3 --- Peptide Extraction --- p.108 / Chapter 4.2.3.4 --- Desalting and concentration of peptide mixture --- p.109 / Chapter 4.3 --- Results --- p.110 / Chapter 4.3.1 --- U-937 differentiation --- p.110 / Chapter 4.3.2 --- In-vitro pull-down --- p.113 / Chapter 4.3.2.1 --- Pull-down with immobilized GST-PE15 --- p.113 / Chapter 4.3.2.2 --- Pull-down with immobilized GST-PPE20(PPE) --- p.116 / Chapter 4.3.2.3 --- Mass spectrometry identification of protein --- p.120 / Chapter 4.4 --- Discussion --- p.122 / Chapter 4.4.1 --- Differentiation of U-937 --- p.122 / Chapter 4.4.2 --- Isolation of PE15 and PPE20(PPE) interacting partners from U-937 --- p.125 / Chapter CHAPTER 5 --- CONCLUSION AND FUTURE PERSPECTIVES --- p.133 / Chapter 5.1 --- Conclusion --- p.133 / Chapter 5.2 --- Future perspectives --- p.137
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_327120 |
| Date | January 2010 |
| Contributors | Hon, Ching Yi., Chinese University of Hong Kong Graduate School. Division of Life Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xxi, 157 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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