Ma Hoi-Wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 113-120). / Abstracts in English and Chinese. / Acknowledgement --- p.I / Abstract --- p.II / 摘要 --- p.III / Content --- p.IV / Abbreviations --- p.X / List of Figures --- p.XII / List of Tables --- p.XIV / Chapter Chapter One --- Introduction --- p.1 / Chapter 1.1 --- Interactions that stabilize proteins --- p.1 / Chapter 1.2 --- Some common strategies of protein engineering to improve thermostability --- p.6 / Chapter 1.3 --- Ribosomal protein T. celer L30e as a study model for thermostability --- p.7 / Chapter 1.4 --- Extra proline residue is one of the insights by comparing the two proteins --- p.10 / Chapter Chapter Two --- Materials and Methods --- p.13 / Chapter 2.1 --- General Techniques --- p.13 / Chapter 2.1.1 --- Preparation of Escherichia coli competent cells --- p.13 / Chapter 2.1.2 --- Transformation of Escherichia coli competent cells --- p.14 / Chapter 2.1.3 --- Spectrophotometric quantitation of DNA --- p.14 / Chapter 2.1.4 --- Agarose gel electrophoresis --- p.14 / Chapter 2.1.5 --- DNA extraction from agarose gel electrophoresis using Viogene Gene Clean kit --- p.15 / Chapter 2.1.6 --- Plasmid DNA minipreperation by Wizard® Plus SV Minipreps DNA Purification System from Promega --- p.16 / Chapter 2.1.7 --- Polymerase Chain Reaction (PCR) --- p.17 / Chapter 2.1.8 --- Ligation of DNA fragments --- p.18 / Chapter 2.1.9 --- Sonication of pellet resuspension --- p.18 / Chapter 2.1.10 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.19 / Chapter 2.1.11 --- Native polyacrylamide gel electrophoresis --- p.20 / Chapter 2.1.12 --- Staining of protein in polyacrylamide gel by Coommassie Brillant Blue R250 --- p.22 / Chapter 2.1.13 --- Protein Concentration determination --- p.22 / Chapter 2.2 --- Cloning the Mutant Genes --- p.22 / Chapter 2.2.1 --- Site-directed mutagenesis --- p.22 / Chapter 2.2.1.1 --- Generation of full length mutant gene by megaprimer --- p.23 / Chapter 2.2.1.2 --- Generation of mutant gene by QuikChange® Site-Directed Mutagenesis Kit from Stratagene --- p.26 / Chapter 2.2.2 --- Restriction Digestion of DNA --- p.27 / Chapter 2.2.3 --- Ligation of DNA fragments --- p.27 / Chapter 2.2.4 --- Screening for successful inserted plasmid clones from ligation reactions --- p.28 / Chapter 2.2.4.1 --- By PCR --- p.28 / Chapter 2.2.4.2 --- By restriction digestion --- p.28 / Chapter 2.2.5 --- DNA sequencing --- p.29 / Chapter 2.3 --- Expression and Purification of Protein --- p.29 / Chapter 2.3.1 --- "General bacterial culture, harvesting and lysis" --- p.29 / Chapter 2.3.2 --- Purification of recombinant wild type TRP and mutants --- p.30 / Chapter 2.3.3 --- Purification of recombinant wild type YRP and mutants --- p.32 / Chapter 2.4 --- Thermodynamic Studies by Circular Dichroism (CD) Spectrometry --- p.34 / Chapter 2.4.1 --- Thermodynamic studies by guanidine-induced denaturations --- p.34 / Chapter 2.4.2 --- Themodynamic studies by thermal denaturations --- p.36 / Chapter 2.4.3 --- ACp measurement of the TRP mutants --- p.37 / Chapter 2.4.3.1 --- By Gibbs-Helmholtz analysis --- p.37 / Chapter 2.4.3.2 --- By van't Hoff analysis --- p.37 / Chapter 2.5 --- Crystal Screen for the Mutant T. celer L30e --- p.38 / Chapter 2.5.1 --- T. celer L30e Pro→Ala and Pro→Gly mutants --- p.38 / Chapter 2.5.2 --- Yeast L30e K65P mutant --- p.38 / Chapter 2.6 --- Sequences of Primers --- p.39 / Chapter 2.6.1 --- Primers for TRP and its mutants --- p.39 / Chapter 2.6.2 --- Primers for YRP and its mutantsReagents and buffers --- p.40 / Chapter 2.7 --- Reagents and Buffers --- p.40 / Chapter 2.7.1 --- Reagents for competent cell preparation --- p.40 / Chapter 2.7.2 --- Nucleic acid eletrophoresis buffers --- p.41 / Chapter 2.7.3 --- Media for bacterial culture --- p.41 / Chapter 2.7.4 --- Reagents for SDS-PAGE --- p.42 / Chapter 2.7.5 --- Buffers for TRP purification --- p.44 / Chapter 2.7.6 --- Buffers for YRP purification --- p.45 / Chapter 2.7.7 --- Buffer for Circular Dichroism (CD) Spectrometry --- p.46 / Chapter Chapter Three --- Results --- p.48 / Chapter 3.1 --- "Cloning, expression and purification of the mutant proteins" --- p.48 / Chapter 3.1.1 --- "Mutagenesis, cloning and purification of the thermophilic proteins - T. celer L30e protein and its mutants" --- p.48 / Chapter 3.1.2 --- "Mutagenesis, cloning and purification of the mesophilic proteins - yeast L30e protein and its mutants" --- p.52 / Chapter 3.2 --- Stability of Pro→Ala/Gly mutants of T. celer L30e at 298K --- p.55 / Chapter 3.2.1 --- Design of alanine and glycine mutants from thermophilic homologue --- p.55 / Chapter 3.2.2 --- "Among alanine mutants, only P59A was destabilized" --- p.55 / Chapter 3.2.3 --- Ala→Gly mutations destabilized the protein --- p.59 / Chapter 3.3 --- Stability of Xaa→Pro mutants of yeast L30e at 298K --- p.61 / Chapter 3.3.1 --- Design of proline mutants from mesophilic homologue --- p.61 / Chapter 3.3.2 --- "K65P, corresponding to P59 in T. celer L30e, stabilized yeast L30e" --- p.62 / Chapter 3.3.3 --- Yeast L30e mutated with thermophilic consensus sequence did not give a more stable protein --- p.65 / Chapter 3.4 --- Temperature dependency of the stability of the mutants of T. celer L30e --- p.67 / Chapter 3.4.1 --- The trend of ΔGU was consistence through 25 to 75°C --- p.67 / Chapter 3.4.2 --- Melting temperatures of T. celer mutants determined by thermal denaturations --- p.68 / Chapter 3.5 --- pH dependency of melting temperatures --- p.75 / Chapter 3.5.1 --- ΔCP values of the P59A/G mutants determined by van't HofF's analyses increased significantly --- p.77 / Chapter 3.6 --- No structural change was observed in the crystal structure of P59A --- p.80 / Chapter Chapter Four --- Discussion --- p.84 / Chapter 4.1 --- The trend of stability from guanidine-induced denaturation agreed with that from thermal denaturations --- p.86 / Chapter 4.2 --- The magnitude of destabilization of P59A and Ala→Gly mutation was consistent with the expected destabilization due to entropy --- p.87 / Chapter 4.3 --- Entropic effect had little effect for residues in flexible region --- p.93 / Chapter 4.4 --- Stabilization forces that compensate the entropic effect --- p.96 / Chapter 4.5 --- Compensatory stabilization due to the release of amide group --- p.99 / Chapter 4.5.1 --- Intra-molecular H-bond in P88A --- p.99 / Chapter 4.5.2 --- Solvent-protein H-bond in P43A --- p.103 / Chapter 4.6 --- Consensus concept was not applicable in our model --- p.110 / Chapter 4.7 --- "Pro→Ala mutation destabilized the protein increase the protein's ACP value, however enthalpy and entropy change were difficult to be decomposed" --- p.111 / Chapter 4.8 --- Concluding Remarks --- p.112 / References --- p.113
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_325313 |
| Date | January 2005 |
| Contributors | Ma, Hoi-Wah., 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, xiv, 120 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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