Lee Chi-Fung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 154-167). / Abstracts in English and Chinese. / Thesis Committee --- p.I / Statement --- p.II / Acknowledgements --- p.II / Abstract --- p.IV / 摘要 --- p.VI / Content --- p.VIII / Abbreviations --- p.VIX / List of figures and tables --- p.XVII / Chapter Chapter 1 - --- Introduction --- p.1 / Chapter 1.1 --- How are the thermophilic proteins stabilized? --- p.2 / Chapter 1.1.1 --- Hydrophobic interactions --- p.2 / Chapter 1.1.2 --- Hydrogen bonds --- p.4 / Chapter 1.1.3 --- Electrostatic interactions --- p.6 / Chapter 1.1.4 --- Reduction in ΔCP --- p.9 / Chapter 1.2 --- Models of study: Thermococcus celer and yeast L30e --- p.12 / Chapter 1.2.1 --- Thermococcus celer ribosomal protein L30e --- p.12 / Chapter 1.2.2 --- Yeast ribosomal protein L30e --- p.13 / Chapter 1.2.3 --- Comparison between the two proteins --- p.13 / Chapter 1.3 --- Objective of this study --- p.20 / Chapter Chapter 2 - --- Materials and Methods --- p.21 / Chapter 2.1 --- General techniques --- p.21 / Chapter 2.1.1 --- Preparation and transformation of competent E. coli DH5α and BL21(DE3)pLysS --- p.21 / Chapter 2.1.2 --- Minipreparation of plasmid DNA (Invitrogen) --- p.22 / Chapter 2.1.3 --- Spectrophotometric quantitation of DNA --- p.24 / Chapter 2.1.4 --- Agarose gel electrophoresis --- p.24 / Chapter 2.1.5 --- Purification of DNA from agarose gel (Invitrogen) --- p.25 / Chapter 2.1.6 --- Restriction digestion of DNA fragments --- p.26 / Chapter 2.1.7 --- Ligation of DNA fragments into vector --- p.26 / Chapter 2.1.8 --- SDS-PAGE electrophoresis --- p.28 / Chapter 2.1.9 --- Native-PAGE electrophoresis --- p.32 / Chapter 2.2 --- Protein Engineering of Proteins --- p.35 / Chapter 2.2.1 --- Polymerase chain reaction (PCR) --- p.35 / Chapter 2.2.2 --- Site-directed mutagenesis of T. celer L30e --- p.37 / Chapter 2.2.3 --- Protein engineering of yeast L30e --- p.42 / Chapter 2.3 --- "Sub-cloning of mutation PCR fragment into expression vector, pET8c" --- p.45 / Chapter 2.4 --- Expression of recombinant proteins --- p.45 / Chapter 2.5 --- Purification of T. celer and its mutants --- p.46 / Chapter 2.5.1 --- Extraction of proteins by sonication --- p.46 / Chapter 2.5.2 --- Purification by ion-exchange chromatography --- p.47 / Chapter 2.5.3 --- Purification by affinity chromatography --- p.48 / Chapter 2.5.4 --- Purification by size exclusion chromatography --- p.49 / Chapter 2.6 --- Purification of yeast L30e and its mutants --- p.49 / Chapter 2.6.1 --- Extraction of proteins by sonication --- p.49 / Chapter 2.6.2 --- Purification yeast L30e variants by washing the inclusion bodies --- p.50 / Chapter 2.6.3 --- Purification by column chromatography --- p.51 / Chapter 2.7 --- Thermodynamic studies of proteins --- p.52 / Chapter 2.7.1 --- Guanidine-induced denaturation --- p.52 / Chapter 2.7.2 --- Thermal-induced denaturation --- p.53 / Chapter 2.7.3 --- Determination of protein stability curves by denaturant unfolding --- p.54 / Chapter 2.7.4 --- ΔCP and protein stability curve determination by thermal unfolding --- p.55 / Chapter 2.8 --- Media and buffer recipes --- p.56 / Chapter 2.8.1 --- Medium for bacterial culture --- p.56 / Chapter 2.8.2 --- Reagents for competent cell preparation --- p.58 / Chapter 2.8.3 --- Nucleic acid electrophoresis buffers --- p.58 / Chapter 2.8.4 --- Buffers for T. celer L30e variants purification --- p.59 / Chapter 2.8.5 --- Buffers for yeast L30e variants purification --- p.59 / Chapter 2.8.6 --- Reagents of SDS-PAGE --- p.60 / Chapter Chapter Three - --- Purification of T. celer and Yeast L30e --- p.63 / Chapter 3.1 --- Purification of T. celer L30e and its mutants --- p.63 / Chapter 3.2 --- Purification of yeast L30e and its mutants --- p.72 / Chapter Chapter Four - --- Thermodynamic Studies of T. celer and Yeast L30e --- p.77 / Chapter 4.1 --- Introduction --- p.77 / Chapter 4.2 --- Result --- p.79 / Chapter 4.3 --- Discussion --- p.85 / Chapter Chapter Five- --- Mutagenesis Study of a Charge Cluster in T. celer L30e --- p.92 / Chapter 5.1 --- Introduction --- p.92 / Chapter 5.2 --- Result --- p.92 / Chapter 5.3 --- Structure determination of T. celer L30e mutants --- p.99 / Chapter 5.4 --- Discussion --- p.105 / Chapter Chapter Six - --- Alanine Scanning Mutagenesis of Charge Residues of T. celer L30e --- p.114 / Chapter 6.1 --- Introduction --- p.114 / Chapter 6.2 --- Result --- p.114 / Chapter 6.3 --- Discussion --- p.121 / Chapter Chapter Seven - --- Protein Engineering of T. celer and Yeast L30e --- p.132 / Chapter 7.1 --- Introduction --- p.132 / Chapter 7.2 --- Result --- p.136 / Chapter 7.3 --- Discussion --- p.138 / Chapter Chapter Eight - --- Concluding Remarks --- p.141 / Appendix --- p.143 / Reference --- p.154
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_324828 |
| Date | January 2004 |
| Contributors | Lee, Chi-Fung., Chinese University of Hong Kong Graduate School. Division of Molecular Biotechnology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xx, 167 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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