Ng, Siu Fung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 206-211). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / 摘要 --- p.v / Table of contents --- p.vii / List of figures --- p.xv / List of tables --- p.xix / List of abbreviations --- p.xx / Chapter / Chapter 1. --- Introduction / Chapter 1.1 --- Growth hormone --- p.1 / Chapter 1.1.1 --- Historic discovery of growth hormone --- p.1 / Chapter 1.1.2 --- Structural and functional study of GH --- p.1 / Chapter 1.1.2.1 --- Molecular evolution of GH --- p.1 / Chapter 1.1.2.2 --- Two-dimensional and three dimensional structures --- p.5 / Chapter 1.1.2.3 --- Heterogeneity of GH --- p.8 / Chapter 1.1.2.4 --- Regulation and secretion pattern of GH --- p.9 / Chapter 1.1.2.5 --- Circulation of GH in blood --- p.11 / Chapter 1.1.2.6 --- Biological activity of GH in human --- p.12 / Chapter 1.2 --- GH receptor and signal transduction --- p.12 / Chapter 1.3 --- GH disorder --- p.15 / Chapter 1.4 --- Treatment for GH disorder --- p.16 / Chapter 1.5 --- GH assay --- p.17 / Chapter 1.6 --- Aims of study --- p.19 / Chapter 2. --- SUMO-hGH expression vector construction / Chapter 2.1 --- Introduction --- p.21 / Chapter 2.2 --- Fusion partner - SUMO --- p.23 / Chapter 2.3 --- Materials --- p.24 / Chapter 2.3.1 --- Reagents for bacterial culture --- p.24 / Chapter 2.3.2 --- Reagents for agarose gel electrophoresis --- p.26 / Chapter 2.3.3 --- 2'-deoxyribonucleoside 5'-triphosphate mix for polymerase chain reaction --- p.26 / Chapter 2.3.4 --- Sonication buffer --- p.26 / Chapter 2.3.5 --- Modified solubilization buffer --- p.27 / Chapter 2.3.6 --- Reagents for sodium dodecylsulphate polyacrylamide gel electrophoresis --- p.27 / Chapter 2.4 --- Methods --- p.29 / Chapter 2.4.1 --- General techniques in molecular cloning of hGH gene --- p.29 / Chapter 2.4.2 --- Expression of SUMO-hGH fusion protein - small scale --- p.42 / Chapter 2.4.3 --- General protein analysis --- p.43 / Chapter 2.5 --- Results --- p.45 / Chapter 2.5.1 --- Molecular cloning of hGH gene into expression vector --- p.45 / Chapter 2.5.2 --- Expression of SUMO-hGH --- p.46 / Chapter 2.5.3 --- Modification of the expression conditions --- p.46 / Chapter 2.6 --- Discussion --- p.50 / Chapter 2.6.1 --- Expression vector --- p.53 / Chapter 2.6.2 --- Protein expression --- p.53 / Chapter 2.7 --- Conclusion --- p.54 / Chapter 3. --- SUMO-hGH purification and downstream processing / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Immobilized-metal affinity chromatography --- p.55 / Chapter 3.3 --- SUMO protease --- p.57 / Chapter 3.4 --- Materials --- p.59 / Chapter 3.4.1 --- Reagents for IMAC purification of SUMO-hGH fusion protein --- p.59 / Chapter 3.4.2 --- Reagents for IMAC purification of mature rhGH --- p.60 / Chapter 3.4.3 --- Reagents for Western blotting --- p.60 / Chapter 3.4.4 --- Gel filtration running buffer --- p.62 / Chapter 3.5 --- Methods --- p.62 / Chapter 3.5.1 --- Purification of SUMO-hGH fusion protein by Ni2+-NTA affinity chromatography --- p.62 / Chapter 3.5.2 --- Cleavage of His-SUMO fusion partner to generate mature rhGH --- p.63 / Chapter 3.5.3 --- Purification of mature rhGH by 2nd round of Ni2+-NTA affinity chromatography --- p.64 / Chapter 3.5.4 --- Purification of rhGH by size exclusion chromatography - gel filtration chromatography --- p.64 / Chapter 3.5.5 --- General protein analysis --- p.65 / Chapter 3.6 --- Results --- p.67 / Chapter 3.6.1 --- Purification of SUMO-hGH fusion protein by Ni2+-NTA affinity chromatography --- p.67 / Chapter 3.6.2 --- Cleavage of His-SUMO fusion partner to generate mature rhGH --- p.69 / Chapter 3.6.3 --- Digestion efficiency of different constructs of SENP1C --- p.73 / Chapter 3.6.4 --- Purification of mature rhGH by 2nd round of Ni2+-NTA affinity chromatography --- p.77 / Chapter 3.6.5 --- Purification of rhGH by size exclusion chromatography -gel filtration chromatography --- p.78 / Chapter 3.7 --- Discussion --- p.81 / Chapter 3.7.1 --- Purification of SUMO-hGH fusion protein by Ni2+-NTA affinity chromatography --- p.81 / Chapter 3.7.2 --- Cleavage of His-SUMO fusion partner to generate mature rhGH --- p.82 / Chapter 3.7.3 --- Purification of mature rhGH by 2nd round of Ni2+-NTA affinity chromatography --- p.82 / Chapter 3.7.4 --- Purification of rhGH by size exclusion chromatography -gel filtration chromatography --- p.85 / Chapter 3.8 --- Conclusion --- p.85 / Chapter 4. --- Fermentation expression of SUMO-hGH and scale-up of downstream process / Chapter 4.1 --- Introduction --- p.86 / Chapter 4.2 --- Bioreactor system for E.coli host cultivation --- p.87 / Chapter 4.3 --- Mechanical cell disruption for cell --- p.88 / Chapter 4.4 --- rhGH binding assay --- p.88 / Chapter 4.5 --- Materials --- p.89 / Chapter 4.5.1 --- Reagents for bacterial culture by fermenter --- p.89 / Chapter 4.5.2 --- Reagents for HEK293 Hi cultivation --- p.91 / Chapter 4.5.3 --- Reagents for Dual-Luciferase® Reporter Assay System --- p.92 / Chapter 4.5.4 --- Reagents for silver stain of SDS-PAGE mini-gel --- p.93 / Chapter 4.6 --- Methods --- p.94 / Chapter 4.6.1 --- Bioreactor system and fixed volume fed-batch fermentation --- p.94 / Chapter 4.6.2 --- Large scale mechanically disruption of cell membrane --- p.97 / Chapter 4.6.3 --- Downstream processing of SUMO-hGH --- p.97 / Chapter 4.6.4 --- Culture of HEK293 Hi cells --- p.97 / Chapter 4.6.5 --- Dual-Luciferase® Reporter Assay System --- p.98 / Chapter 4.6.6 --- Silver staining of SDS-PAGE mini-gels --- p.101 / Chapter 4.7 --- Results --- p.101 / Chapter 4.7.1 --- Fed-batch fermentation of E. coli BL21 --- p.101 / Chapter 4.7.2 --- Comparison on disruption methods and the purification of SUMO-hGH from cell lysate --- p.106 / Chapter 4.7.3 --- Optimization of His-MBP-SENP1C digestion condition --- p.108 / Chapter 4.7.4 --- Optimization of rhGH purification in 2nd round of IMAC --- p.110 / Chapter 4.7.5 --- Characterization of mature rhGH --- p.112 / Chapter 4.8 --- Discussion --- p.116 / Chapter 4.8.1 --- Fed-batch fermentation of E. coli BL21 --- p.118 / Chapter 4.8.2 --- Downstream processing of fermentation culture and characterization of rhGH --- p.120 / Chapter 4.8.3 --- M9 based defined medium fermentation study --- p.122 / Chapter 4.8.4 --- rhGH production yield estimation --- p.128 / Chapter 4.8.5 --- Comparison of our fermentation expression system to the published data --- p.130 / Chapter 4.9 --- Conclusion --- p.132 / Chapter 5. --- His-MBP-SENPIC expression and purification / Chapter 5.1 --- Introduction --- p.133 / Chapter 5.2 --- Materials --- p.134 / Chapter 5.2.1 --- Reagents for bacterial culture --- p.134 / Chapter 5.2.2 --- Reagents for immobilized metal affinity chromatography purification of His-MBP-SENP1C --- p.135 / Chapter 5.3 --- Methods --- p.136 / Chapter 5.3.1 --- Expression of His-MBP-SENP1C --- p.136 / Chapter 5.3.2 --- Semi-purification of His-MBP-SENP1C by Ni2+-NTA affinity chromatography --- p.138 / Chapter 5.4 --- Results --- p.139 / Chapter 5.4.1 --- Expression of His-MBP-SENP1C --- p.139 / Chapter 5.4.2 --- Digestion activity of His-MBP-SENP1C expressed --- p.139 / Chapter 5.5 --- Discussion --- p.141 / Chapter 5.5.1 --- Expression and purification of His-MBP-SENP1C --- p.141 / Chapter 5.5.2 --- His-MBP-SENP1C production yield estimation --- p.143 / Chapter 6. --- Production and characterization of monoclonal antibodies against rhGH / Chapter 6.1 --- Introduction --- p.145 / Chapter 6.2 --- Materials --- p.146 / Chapter 6.2.1 --- Reagents for Sp2/0-Ag14 cultivation --- p.146 / Chapter 6.2.2 --- Reagents for PEG fusion --- p.147 / Chapter 6.2.3 --- Reagents for enzyme linked immunosorbent assay --- p.149 / Chapter 6.2.4 --- Reagents for mAbs purification by HiTrap´ёØ Protein G HP Column --- p.150 / Chapter 6.3 --- Methods --- p.151 / Chapter 6.3.1 --- ELISA --- p.151 / Chapter 6.3.2 --- Immunization --- p.152 / Chapter 6.3.3 --- Culturing of myeloma fusion partner cells --- p.153 / Chapter 6.3.4 --- Isolation of splenocyte --- p.153 / Chapter 6.3.5 --- PEG fusion --- p.154 / Chapter 6.3.6 --- Limiting dilution --- p.155 / Chapter 6.3.7 --- Cryopreservation of hybridoma cell lines --- p.156 / Chapter 6.3.8 --- Mass production of monoclonal antibodies --- p.157 / Chapter 6.3.9 --- Purification of IgG mAbs from ascites --- p.157 / Chapter 6.3.10 --- MAbs isotyping --- p.159 / Chapter 6.3.11 --- Determination of kinetic parameters of mAbs --- p.159 / Chapter 6.4 --- Results --- p.162 / Chapter 6.4.1 --- Production of murine anti-rhGH monoclonal antibodies --- p.162 / Chapter 6.4.2 --- Characterization of anti-rhGH mAbs --- p.170 / Chapter 6.5 --- Discussion --- p.178 / Chapter 6.5.1 --- Mass production of mAbs --- p.179 / Chapter 6.5.2 --- Future works on mAbs --- p.179 / Chapter 6.6 --- Conclusion --- p.181 / Chapter 7. --- Development of sandwich ELISA for rhGH / Chapter 7.1 --- Introduction --- p.182 / Chapter 7.2 --- Materials --- p.184 / Chapter 7.2.1 --- Reagents for sandwich ELISA --- p.184 / Chapter 7.3 --- Methods --- p.184 / Chapter 7.3.1 --- Production of rabbit polyclonal antiserum against rhGH --- p.184 / Chapter 7.3.2 --- Sandwich ELISA --- p.185 / Chapter 7.4 --- Results --- p.186 / Chapter 7.4.1 --- Production of rabbit antiserum against rhGH --- p.186 / Chapter 7.4.2 --- Sandwich ELISA --- p.188 / Chapter 7.4.3 --- Optimization of sandwich ELISA --- p.190 / Chapter 7.4.4 --- Specificity of sandwich ELISA --- p.194 / Chapter 7.4.5 --- Cross reactivity of sandwich ELISA to E.coli cell lysate --- p.196 / Chapter 7.4.6 --- Measurement of SUMO-hGH with sandwich ELISA --- p.198 / Chapter 7.5 --- Discussion --- p.201 / Chapter 7.5.1 --- Application of sandwich ELISA --- p.203 / Chapter 7.5.2 --- Future works on sandwich ELISA --- p.205 / Chapter 7.6 --- Conclusion --- p.205 / References --- p.206 / Appendix - pJ2:G01458 nucleotide sequence --- p.213
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_326553 |
| Date | January 2008 |
| Contributors | Ng, Siu Fung., 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, 213 leaves : ill. ; 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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