Chan, Ho Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 263-283). / 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 Abbreviations --- p.xvii / List of Figures --- p.xx / List of Tables --- p.xxv / Chapter Chapter I --- Introduction --- p.1 / Chapter 1.1 --- SUMO (Small Ubiquitin-like Modifier) and SUMOylation --- p.1 / Chapter 1.1.1 --- "Ubiquitin, Ubiquitin-like proteins and SUMO isoforms" --- p.2 / Chapter 1.1.2 --- SUMO cycle --- p.5 / Chapter 1.1.2.1 --- SUMO conjugation consensus sequence --- p.5 / Chapter 1.1.2.2 --- SUMO maturation --- p.6 / Chapter 1.1.2.3 --- SUMO conjugation cascade --- p.7 / Chapter 1.1.2.4 --- SUMO deconjugation --- p.9 / Chapter 1.1.3 --- Mode of SUMO action --- p.12 / Chapter 1.1.4 --- Biological functions of SUMO --- p.13 / Chapter 1.1.4.1 --- SUMO in cancer --- p.14 / Chapter 1.2 --- Human cervical cancer and human papillomavirus (HPV) --- p.17 / Chapter 1.2.1 --- Infectious cycle of HPV-16 --- p.18 / Chapter 1.2.1.1 --- Viral entry --- p.18 / Chapter 1.2.1.2 --- Maintenance --- p.18 / Chapter 1.2.1.3 --- Deregulation of cell cycle --- p.19 / Chapter 1.2.1.4 --- Amplification and virion release --- p.20 / Chapter 1.2.2 --- Viral cancer induction --- p.22 / Chapter 1.2.2.1 --- Integration into the host genome --- p.22 / Chapter 1.2.2.2 --- Viral oncoproteins E6 and E7 --- p.23 / Chapter 1.2.3 --- SUMOylation and HPV --- p.24 / Chapter 1.2.3.1 --- Known examples of virus-host SUMOylation system interaction --- p.24 / Chapter 1.2.3.2 --- Other possible mode of virus-SUMO interaction --- p.26 / Chapter 1.3 --- A novel labeling method: biarsenical-tetracysteine labeling in SUMO study --- p.28 / Chapter 1.3.1 --- Potential use of 2As-4Cys system in SUMO studies --- p.31 / Chapter 1.3.2 --- Potential use of 2As-4Cys system in SUMO proteomics --- p.31 / Chapter 1.4 --- Objectives of the present study --- p.34 / Chapter Chapter II --- Proteomics investigation of SUMOylation in human cervical carcinoma cell line HeLa --- p.35 / INTRODUCTION --- p.35 / Chapter 2.1 --- MATERIALS --- p.37 / Chapter 2.1.1 --- Vectors for expression of SUMO and SUMOylation enzymes in E. coli --- p.37 / Chapter 2.1.2 --- E.coli cell strains --- p.38 / Chapter 2.1.3 --- Mammalian cell lines --- p.39 / Chapter 2.1.4 --- E.coli growth mediums --- p.40 / Chapter 2.1.5 --- Mammalian cell growth medium --- p.41 / Chapter 2.1.6 --- Reagents and buffers --- p.41 / Chapter 2.1.6.1 --- Reagents and buffers for molecular cloning --- p.41 / Chapter 2.1.6.2 --- Reagents and buffers for E.coli protein expression --- p.43 / Chapter 2.1.6.3 --- Reagents and buffers for mammalian cell culture --- p.44 / Chapter 2.1.6.4 --- Reagents and buffers for Western blot study --- p.45 / Chapter 2.1.7 --- Reagents and solutions for two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) sample preparation --- p.46 / Chapter 2.1.7.1 --- Reagents and solutions for 2-DE --- p.46 / Chapter i. --- 2-DE sample preparation --- p.46 / Chapter ii. --- First dimensional gel electrophoresis -isoelectric focusing (IEF) --- p.46 / Chapter iii. --- Second dimensional gel electrophoresis -SDS-PAGE --- p.47 / Chapter iv. --- Silver staining --- p.47 / Chapter 2.1.7.2 --- Reagents and solutions for mass spectrometry sample preparation --- p.48 / Chapter i. --- Destaining of silver stained gel spots --- p.48 / Chapter ii. --- Trypsin digestion --- p.48 / Chapter iii. --- Peptide extraction --- p.48 / Chapter iv. --- Desalting and concentration of peptide mixture --- p.49 / Chapter 2.2 --- METHODS --- p.50 / Chapter 2.2.1 --- Molecular cloning of SUMO-1 into pET-28m and pHM6 vectors --- p.50 / Chapter 2.2.1.1 --- Design of primers for the cloning of SUMO-1 --- p.50 / Chapter 2.2.1.2 --- DNA amplification by polymerase chain reaction (PCR) --- p.51 / Chapter 2.2.1.3 --- DNA extraction from agarose gels --- p.52 / Chapter 2.2.1.4 --- Restriction digestion of vectors and purified PCR products --- p.54 / Chapter 2.2.1.5 --- Ligation of SUMO cDNA into expression vector pET-28m and pHM6 --- p.55 / Chapter 2.2.1.6 --- Preparation of competent cells --- p.56 / Chapter 2.2.1.7 --- Transformation of ligated mixture into competent DH5a --- p.56 / Chapter 2.2.1.8 --- Preparation of plasmid DNA --- p.57 / Chapter 2.2.1.8.1 --- Mini-preparation of plasmid DNA --- p.57 / Chapter 2.2.1.8.2 --- Midi-preparation of plasmid DNA --- p.58 / Chapter 2.2.1.8.3 --- DNA quantification and quality measurement --- p.60 / Chapter 2.2.2 --- "Expression of His6-tagged SUMO, ubc9, TDG, GST-tagged El and MBP-tagged Prdx 1 with E.coli" --- p.60 / Chapter 2.2.3 --- "Purification of His6-tagged SUMO, ubc9, TDG, GST-tagged El and MBP-tagged Prdx 1" --- p.62 / Chapter 2.2.3.1 --- Affinity chromatography --- p.65 / Chapter 2.2.3.1.1 --- Ni-NTA affinity chromatography --- p.65 / Chapter 2.2.3.1.2 --- Heparin affinity chromatography --- p.66 / Chapter 2.2.3.1.3 --- Glutathione affinity chromatography --- p.66 / Chapter 2.2.3.1.4 --- Amylose affinity chromatography --- p.67 / Chapter 2.2.3.2 --- Ion exchange chromatography --- p.68 / Chapter 2.2.3.2.1 --- Anion exchange chromatography --- p.68 / Chapter 2.2.3.2.2 --- Cation exchange chromatography --- p.68 / Chapter 2.2.3.3 --- Size exclusion chromatography --- p.69 / Chapter 2.2.3.4 --- Purification strategies --- p.70 / Chapter 2.2.3.4.1 --- Purification of His6-tagged SUMO --- p.70 / Chapter 2.2.3.4.2 --- Purification of His6-tagged TDG --- p.71 / Chapter 2.2.3.4.3 --- Purification of His6-tagged ubc9 --- p.72 / Chapter 2.2.3.4.4 --- Purification of GST-tagged El --- p.73 / Chapter 2.2.3.4.5 --- Purification of MBP-tagged Prdx 1 --- p.74 / Chapter 2.2.4 --- HeLa and C-33A cell culturing and protein extraction --- p.75 / Chapter 2.2.4.1 --- HeLa and C-33A cell culturing --- p.75 / Chapter 2.2.4.2 --- Protein extraction for in vitro SUMOylation assay --- p.76 / Chapter 2.2.5 --- Protein quantification with Bradford assay --- p.76 / Chapter 2.2.6 --- In vitro SUMO conjugation assay --- p.77 / Chapter 2.2.6.1 --- In vitro SUMO conjugation system optimization --- p.77 / Chapter 2.2.6.2 --- In vitro SUMO conjugation of HeLa cell extract --- p.78 / Chapter 2.2.7 --- Transient transfection of pHM6-SUMO-l into HeLa cells and protein extraction from HeLa cells --- p.79 / Chapter 2.2.7.1 --- Transfection with lipofection method --- p.79 / Chapter 2.2.7.2 --- Determination of transfection efficiency --- p.80 / Chapter 2.2.7.3 --- Whole cell protein extraction of transfected cells --- p.81 / Chapter 2.2.8 --- Protein quantification with BCA assay --- p.81 / Chapter 2.2.9 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.83 / Chapter 2.2.10 --- Western blot analysis --- p.84 / Chapter 2.2.10.1 --- Electro-transfer blotting --- p.84 / Chapter 2.2.10.2 --- Immunoblotting with antibodies --- p.84 / Chapter 2.2.10.3 --- ECL detection --- p.85 / Chapter 2.2.10.4 --- Mild stripping for re-probing --- p.86 / Chapter 2.2.11 --- Two-dimensional gel electrophoresis (2-DE) --- p.86 / Chapter 2.2.11.1 --- Sample preparation --- p.86 / Chapter 2.2.11.2 --- First dimension gel electrophoresis -isoelectric focusing (IEF) --- p.87 / Chapter 2.2.11.3 --- Second dimension gel electrophoresis -SDS-PAGE --- p.88 / Chapter 2.2.11.3.1 --- Strip equilibration --- p.88 / Chapter 2.2.11.3.2 --- 16 x 18cm SDS-PAGE --- p.88 / Chapter 2.2.11.4 --- Visualization of proteins on SDS-polyacrylamide gel --- p.90 / Chapter 2.2.11.4.1 --- Silver staining --- p.90 / Chapter 2.2.11.4.2 --- Coomassie Blue® R250 staining --- p.91 / Chapter 2.2.12 --- Sample preparation for mass spectrometry analysis --- p.92 / Chapter 2.2.12.1 --- Destaining and trypsin digestion --- p.92 / Chapter 2.2.12.2 --- Extraction of peptide mixture --- p.93 / Chapter 2.2.12.3 --- Desalting and concentration of peptide mixture --- p.93 / Chapter 2.3 --- RESULTS --- p.95 / Chapter 2.3.1 --- Construction of recombinant pET-28m-SUMO-l and pHM6-SUMO-l --- p.95 / Chapter 2.3.2 --- "Purification of His6-tagged SUMO, ubc9, TDG and GST-tagged El" --- p.98 / Chapter 2.3.2.1 --- Purification of His6-SUMO --- p.98 / Chapter 2.3.2.2 --- Purification of His6-TDG --- p.101 / Chapter 2.3.2.3 --- Purification of His6-ubc9 --- p.104 / Chapter 2.3.2.4 --- Purification of GST-El --- p.106 / Chapter 2.3.3 --- In vitro SUMO conjugation assay --- p.108 / Chapter 2.3.3.1 --- Optimization of in vitro SUMO conjugation system --- p.108 / Chapter 2.3.3.2 --- In vitro SUMO conjugation of HeLa cell protein extract --- p.111 / Chapter 2.3.3.2.1 --- Protein extraction for in vitro sumoylation assay --- p.111 / Chapter 2.3.3.2.2 --- In vitro SUMOylation of HeLa cell lysate --- p.114 / Chapter 2.3.4 --- Differential proteomes of control and in vitro SUMOylated HeLa total cellular extract --- p.116 / Chapter 2.3.4.1 --- Mass spectrometric identification of differential protein candidates --- p.123 / Chapter 2.3.5 --- Overexpression of SUMO-1 in HeLa cells by transient transfection --- p.127 / Chapter 2.3.6 --- Differential proteomes of total cellular protein extract from control and SUMO-1 transfected HeLa cells --- p.128 / Chapter 2.3.6.1 --- Mass spectrometric identification of differential protein candidates --- p.132 / Chapter 2.4 --- Proteins identified in proteomic study with in vitro SUMOylation -Analysis of protein candidate --- p.133 / Chapter 2.4.1 --- Proteins identified from the in vitro investigation --- p.133 / Chapter 2.4.2 --- Verification of putative SUMO substrate Prdx 1 --- p.139 / Chapter 2.4.2.1 --- Purification of Prdx 1 --- p.139 / Chapter 2.4.2.2 --- In vitro SUMOylation of Prdx 1 --- p.142 / Chapter 2.4.3 --- Highlights of the proteins identified --- p.145 / Chapter 2.4.3.1 --- DJ-1 protein --- p.145 / Chapter 2.4.3.2 --- nm23A --- p.145 / Chapter 2.4.3.3 --- v-crk protein of CT10 --- p.146 / Chapter 2.4.3.4 --- Annexin I --- p.146 / Chapter 2.4.3.5 --- "Enolase 1, aldolase A, triosephosphate isomerase (TIM) and phosphoglycerate mutase 1" --- p.147 / Chapter 2.4.3.6 --- CyclophilinA(CypA) --- p.148 / Chapter 2.4.3.7 --- Stress induced phosphoprotein 1 (Stip 1) --- p.148 / Chapter 2.4.3.8 --- TSA and peroxiredoxin 1 (Prdx 1) --- p.149 / Chapter 2.5 --- Proteins identified in proteomic study with overexpression of SUMO-1 in HeLa cells -Analysis of protein candidate --- p.150 / Chapter 2.5.1 --- Proteins identified from the in vivo investigation --- p.150 / Chapter 2.5.2 --- Verification of upregulation of keratin 17 --- p.157 / Chapter 2.5.2.1 --- Immunoblotting against keratin 17 --- p.157 / Chapter 2.5.3 --- Highlights of the proteins identified --- p.159 / Chapter 2.5.3.1 --- "Heat shock proteins (Hsp 60, 70 and 27)" --- p.159 / Chapter 2.5.3.2 --- 14-3-3σ protein (SFN protein) --- p.161 / Chapter 2.5.3.3 --- PDZ-RGS3 --- p.162 / Chapter 2.5.3.4 --- "Keratins 8, 17" --- p.163 / Chapter 2.5.3.5 --- XIAP-1 --- p.164 / Chapter 2.5.3.6 --- ISG15 --- p.164 / Chapter 2.6 --- DISCUSSION --- p.166 / Chapter Chapter III --- Characterization of a novel fluorescent labeling method: Biarsencial-tetracysteine labeling in SUMO study --- p.182 / INTRODUCTION --- p.182 / Chapter 3.1 --- MATERIALS --- p.184 / Chapter 3.1.1 --- "Molecular cloning, protein expression and purification of pET-28m-4Cys 1 -SUMO-1 and pET-28m-4Cys2-SUMO-1" --- p.184 / Chapter 3.1.2 --- Mammalian cell culture and transient transfection of pHM6-4Cysl-SUMO-1 and pHM6-4Cys2-SUMO-l into HeLa cells --- p.184 / Chapter 3.1.3 --- Reagents and buffers --- p.184 / Chapter 3.1.3.1 --- Reagents and buffers for Lumio´ёØ in-gel labeling --- p.184 / Chapter 3.1.3.2 --- Reagents and buffers for Lumio´ёØ in cell labeling --- p.185 / Chapter 3.1.3.3 --- Reagents and buffers for immunostaining --- p.186 / Chapter 3.2 --- METHODS --- p.187 / Chapter 3.2.1 --- Molecular cloning of tetracysteine-tagged SUMO (4Cys-SUMO) into pET-28m and pHM6 vectors --- p.187 / Chapter 3.2.1.1 --- Design of primers and oligonucleotides encoding tetracysteine tag --- p.187 / Chapter 3.2.1.1.1 --- For 4Cysl-SUMO-1 --- p.187 / Chapter 3.2.1.1.2 --- For 4Cys2-SUMO-l --- p.188 / Chapter 3.2.1.2 --- DNA amplification of 4Cysl-SUMO-1 by Polymerase chain reaction (PCR) --- p.189 / Chapter 3.2.1.3 --- Restriction digestion of vectors and purified PCR products of 4Cysl-SUMO-1 --- p.191 / Chapter 3.2.1.4 --- Ligation of 4Cysl-SUMO into expression vector pET-28m and pHM6 --- p.191 / Chapter 3.2.1.5 --- Restriction digestion of pET-28m-SUMO and pHM6-SUMO for ligation with 4Cys2 oligos --- p.192 / Chapter 3.2.1.6 --- Ligation of 4Cys2 oligos to the digested pET-28m-SUMO and pHM6-SUMO plasmids --- p.193 / Chapter 3.2.1.6.1 --- Self-annealing of the 4Cys oligonucleotides --- p.193 / Chapter 3.2.1.6.2 --- Phosphorylation of ds 4Cys2 oligos and ligation to the plasmids --- p.193 / Chapter 3.2.2 --- Expression and purification of pET-28m-4Cys 1 -SUMO-1 and pET-28m-4Cys2-SUMO-1 in E.coli expression system --- p.195 / Chapter 3.2.3 --- Immunohistochemistry (IHC) staining of endogenous SUMO in HeLa cells --- p.196 / Chapter 3.2.4 --- In-cell labeling of 4Cysl/2-SUMO with Lumio´ёØ Reagent --- p.197 / Chapter 3.2.4.1 --- Preparation --- p.197 / Chapter 3.2.4.2 --- In-cell Lumio´ёØ labeling --- p.198 / Chapter 3.2.4.3 --- Detection and imaging of the labeled cells --- p.199 / Chapter 3.2.5 --- In-gel labeling of 4Cysl/2-SUMO with Lumio´ёØ Reagent --- p.199 / Chapter 3.2.5.1 --- Lumio´ёØ in-gel labeling --- p.199 / Chapter 3.2.5.2 --- Visualization and imaging of the labeled gel --- p.200 / Chapter a. --- UV illumination at 302 nm --- p.200 / Chapter b. --- Typhoon Trio TMLaser-scanning at 532 nm --- p.201 / Chapter 3.2.5.3 --- Detection limit of fluorescent 4Cys2-SUMO-l in SDS-PAGE --- p.201 / Chapter 3.2.5.4 --- In-gel labelling in two-dimensional electrophoresis (2-DE) --- p.202 / Chapter 3.2.5.4.1 --- Modification of equilibration buffer before SDS-PAGE --- p.202 / Chapter 3.3 --- RESULTS --- p.203 / Chapter 3.3.1 --- Adoption of old version of 4Cys-tag (4Cys 1) in SUMO study --- p.203 / Chapter 3.3.1.1 --- Construction of recombinant pET-28m-4Cys 1 -SUMO-1 and pHM6-4Cysl-SUMO-1 --- p.203 / Chapter 3.3.1.2 --- In vivo HA-4Cysl-SUMO-1 Lumio´ёØ labelling --- p.205 / Chapter 3.3.1.3 --- Immunohistochemistry (IHC) staining of endogenous SUMO in HeLa cells --- p.207 / Chapter 3.3.1.4 --- Expression and purification of His6-4Cysl-SUMO-1 --- p.208 / Chapter 3.3.1.5 --- Validation of 4Cys1-SUMO-1 conjugate by Lumio´ёØ in-gel labeling --- p.211 / Chapter 3.3.2 --- Adoption of a modified version of 4Cys-tag (4Cys2) in SUMO study --- p.213 / Chapter 3.3.2.1 --- Construction of recombinant pET-28m-4Cys2-SUMO-l and pHM6-4Cys2-SUMO-l --- p.213 / Chapter 3.3.2.2 --- In vivo HA-4Cys2-SUMO-l Lumio´ёØ labelling --- p.216 / Chapter 3.3.2.3 --- Expression and purification of His6-4Cys2-SUMO-1 --- p.219 / Chapter 3.3.2.4 --- Validation of 4Cys2-SUMO-l conjugate Lumio´ёØ in-gel labeling --- p.221 / Chapter 3.3.3 --- 2As-4Cys labeling in two-dimensional electrophoresis (2-DE) --- p.223 / Chapter 3.3.3.1 --- Detection limit of 4Cys2-SUMO-l in SDS-PAGE --- p.224 / Chapter 3.3.3.2 --- Lumio´ёØ labeling in 2-DE --- p.226 / Chapter 3.4 --- DISCUSSION --- p.232 / Chapter Chapter IV --- Conclusion and Future Perspectives --- p.242 / Chapter 4.1 --- Conclusion on proteomic study of SUMOylation --- p.242 / Chapter 4.2 --- Future perspectives of proteomic study of SUMOylation --- p.245 / Chapter 4.2.1 --- In vitro study --- p.245 / Chapter 4.2.2 --- In vivo study --- p.246 / Chapter 4.3 --- Conclusion of the investigation of biarsencial-tetracysteine (2As-4Cys) system application on SUMO study --- p.247 / Chapter 4.4 --- Future perspectives of the application of 2As-4Cys system application on SUMO study --- p.249 / Chapter 4.4.1 --- In cell study --- p.249 / Chapter 4.4.2 --- In gel study --- p.250 / Appendices --- p.251 / Chapter 1. --- Genotype of E.coli strains --- p.251 / Chapter 2. --- Vector maps --- p.252 / Chapter a. --- Vector map and MCS of pET-28a --- p.252 / Chapter b. --- Vector map and MCS of pHM6 --- p.253 / Chapter c. --- Vector information of pTwo-E --- p.254 / Chapter 3. --- Primers used in this study --- p.255 / Chapter 4. --- Nikon TE2000 filter sets spectrums --- p.257 / Chapter a. --- FITC/GFP filter set --- p.257 / Chapter b. --- RFP filter set --- p.257 / Chapter c. --- UV/DAPI/Hoechst filter set --- p.258 / Chapter 5. --- Akt signalling pathway diagram --- p.259 / Chapter 6. --- DNA sequence of SUMOs and 4Cys2 oligonucleotide --- p.260 / Chapter 7. --- Electrophoresis markers --- p.261 / References --- p.263
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_326043 |
| Date | January 2007 |
| Contributors | Chan, Ho Yin., 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, xxvi, 283 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/) |
Page generated in 0.0032 seconds