Study of PE15 and PPE20 of Mycobacterium tuberculosis.

• Other Authors: Hon, Ching Yi.
• Format: Others
• Language: English; Chinese
• Published: 2010
• Subjects: Mycobacterium tuberculosis > Genetic aspects; Proteins > Analysis; Mycobacterium Tuberculosis > genetics; Proteins > analysis
• Online Access: http://library.cuhk.edu.hk/record=b5896642; http://repository.lib.cuhk.edu.hk/en/item/cuhk-327120

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