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Expression of mature human growth hormone using a novel fusion vector and characterization of MAb against it.

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

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_326553
Date January 2008
ContributorsNg, Siu Fung., Chinese University of Hong Kong Graduate School. Division of Biochemistry.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xxiii, 213 leaves : ill. ; 30 cm.
RightsUse 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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