Chan, Kwok Ho. / Thesis submitted in: November 2007. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 124-129). / Abstracts in English and Chinese. / Statement --- p.I / Acknowledgements --- p.II / Abstract --- p.III / 摘要 --- p.IV / Table of Contents --- p.V / Abbreviations --- p.XIII / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- GTPase in general --- p.1 / Chapter 1.2 --- G proteins and GTP switch --- p.2 / Chapter 1.3 --- Structural similarities in GTPase --- p.3 / Chapter 1.4 --- G proteins in bacteria --- p.3 / Chapter 1.5 --- Background information of the protein family EngB --- p.4 / Chapter 1.6 --- Basic information of EngB in Thermotoga maritima --- p.5 / Chapter 1.7 --- Objectives of this work --- p.6 / Chapter Chapter 2 --- Materials and methods / Chapter 2.1 --- Materials / Chapter 2.1.1 --- Chemical reagents --- p.8 / Chapter 2.1.2 --- Buffers / Chapter 2.1.2.1 --- Preparation of buffers --- p.10 / Chapter 2.1.2.2 --- Buffers for common use --- p.11 / Chapter 2.1.3 --- Expression strains and plasmids --- p.14 / Chapter 2.1.4 --- Primer list --- p.14 / Chapter 2.2 --- Materials / Chapter 2.2.1 --- Preparation of competent cells --- p.15 / Chapter 2.2.2 --- Cloning / Chapter 2.2.2.1 --- Cloning of target genes by PCR --- p.15 / Chapter 2.2.2.2 --- Agrose gel electrophoresis --- p.17 / Chapter 2.2.2.3 --- Extraction and purification of DNA from agarose gel --- p.17 / Chapter 2.2.2.4 --- Restriction digestion of DNA --- p.18 / Chapter 2.2.2.5 --- Ligation of digested insert and expression vector --- p.18 / Chapter 2.2.2.6 --- Transformation and plating out transformants for miniprep --- p.19 / Chapter 2.2.2.7 --- Verification of insert by PCR --- p.20 / Chapter 2.2.2.8 --- Mini-preparation of plasmid DNA --- p.21 / Chapter 2.2.2.9 --- Confirmation of miniprep product by restriction enzyme digestion..… --- p.22 / Chapter 2.2.2.10 --- Sequencing of the plasmid DNA --- p.23 / Chapter 2.2.3 --- Expression of the recombinant MBP-TM EngB protein and SBP-CBP EC EngB / Chapter 2.2.3.1 --- Transformation for protein expression --- p.23 / Chapter 2.2.3.2 --- Preparation of starter culture --- p.24 / Chapter 2.2.3.3 --- Expression of recombinant protein --- p.24 / Chapter 2.2.3.4 --- Cell harvesting --- p.24 / Chapter 2.2.3.5 --- Releasing the cell content --- p.25 / Chapter 2.2.3.6 --- Check for protein expression by SDS-PAGE --- p.25 / Chapter 2.2.4 --- Purification of TM EngB / Chapter 2.2.4.1 --- SP ion-exchange chromatography --- p.27 / Chapter 2.2.4.2 --- Thrombin digestion to remove MBP tag --- p.28 / Chapter 2.2.4.3 --- Heparin affinity chromatography --- p.29 / Chapter 2.2.4.4 --- Gel filtration chromatography --- p.29 / Chapter 2.2.5 --- Purification of SBP-CBP EC EngB / Chapter 2.2.5.1 --- SP ion-exchange chromatography --- p.30 / Chapter 2.2.5.2 --- Gel filtration chromatography --- p.31 / Chapter 2.2.6 --- Protein concentration quantitation --- p.32 / Chapter 2.2.7 --- Crystallography of TM EngB / Chapter 2.2.7.1 --- Crystallization preparation --- p.32 / Chapter 2.2.7.2 --- Crystallization screening by sitting drop method --- p.32 / Chapter 2.2.7.3 --- Optimization of crystallization conditions --- p.33 / Chapter 2.2.7.4 --- X-ray diffraction --- p.33 / Chapter 2.2.8 --- Thermodynamics studies of proteins / Chapter 2.2.8.1 --- Preparation of protein sample --- p.34 / Chapter 2.2.8.2 --- Guanidine-induced denaturation experiment --- p.34 / Chapter 2.2.8.3 --- Thermal-induced denaturation experiment --- p.35 / Chapter 2.2.9 --- Binding assay to study affinity for ligands --- p.36 / Chapter 2.2.9.1 --- Using GDP analogue mant-GDP to detect formation of enzyme-ligand complex (TM EngB-mant-GDP) --- p.36 / Chapter 2.2.9.2 --- Basic information of Fluorescence spectroscopy --- p.36 / Chapter 2.2.9.3 --- Determination of λem and λex --- p.37 / Chapter 2.2.9.4 --- Studying ligand affinity by titration with ligand analogue --- p.37 / Chapter 2.2.10 --- Pull down experiment to study interacting partner of E. coli EngB --- p.38 / Chapter 2.2.10.1 --- Preparing protein extracts from E. coli --- p.38 / Chapter 2.2.10.2 --- Preparing streptavidin resin --- p.39 / Chapter 2.2.10.3 --- Binding of dual-tagged E. coli EngB to streptavidin resin --- p.39 / Chapter 2.2.10.4 --- Purifying protein using the prepared streptavidin resin --- p.40 / Chapter 2.2.10.5 --- Preparing calmodulin resin --- p.41 / Chapter 2.2.10.6 --- Binding of dual-tagged E.coli EngB to calmodulin resin --- p.41 / Chapter 2.2.10.7 --- Analysis of dual-tag affinity purified protein --- p.42 / Chapter 2.2.11 --- Silver staining of acrylamide gel / Chapter 2.2.11.1 --- Staining reagents --- p.42 / Chapter 2.2.11.2 --- Staining procedures --- p.43 / Chapter Chapter 3 --- Structure determination of T. maritima EngB by X-ray crystallography / Chapter 3.1 --- Introduction --- p.45 / Chapter 3.2 --- Generation of TM EngB expression construct --- p.45 / Chapter 3.3 --- Expression and purification of TM EngB --- p.46 / Chapter 3.4 --- TM EngB was crystallized with freshly purified TM EngB --- p.47 / Chapter 3.5 --- Data processing of diffraction data and structure refinement of TM EngB …… --- p.48 / Chapter 3.6 --- Apo-form TM EngB was obtained by unfolding and refolding --- p.49 / Chapter 3.7 --- Crystallization of apo-form TM EngB --- p.50 / Chapter 3.8 --- Data processing of diffraction data and structure refinement of apo-form TM EngB --- p.51 / Chapter 3.9 --- Producing EngB-GDP complex crystal from apo-from EngB --- p.52 / Chapter 3.10 --- TM EngB is a monomer in solution --- p.54 / Chapter 3.11 --- Summary of chapter three --- p.55 / Tables and figures of chapter three --- p.57 / Chapter Chapter 4 --- Structural details of TM EngB / Chapter 4.1 --- Introduction --- p.67 / Chapter 4.2 --- Overall fold of TM EngB --- p.67 / Chapter 4.3 --- Mode of nucleotide binding of TM EngB --- p.68 / Chapter 4.4 --- Structural differences in switch I region between chain A and chain B in crystal structure of TM EngB/GDP complex --- p.70 / Chapter 4.5 --- Structural difference between TM EngB/GDP complex and apo TM EngB --- p.73 / Chapter 4.6 --- Summary of chapter four --- p.73 / Tables and figures of chapter four --- p.76 / Chapter Chapter 5 --- Purified TM EngB is Active for binding guanine nucleotide but inactive for GTPase hydrolysis activity / Chapter 5.1 --- Introduction --- p.88 / Chapter 5.2 --- Studying ligand affinity by competitive binding experiment --- p.88 / Chapter 5.3 --- GDP binds to TMEngB with higher affinity than GTPyS --- p.91 / Chapter 5.4 --- TM EngB showed very low intrinsic GTPase activity --- p.92 / Chapter 5.5 --- Discussion --- p.93 / Tables and figures of chapter five --- p.95 / Chapter Chapter 6 --- Thermostability of EngB of T. maritima / Chapter 6.1 --- Introduction --- p.98 / Chapter 6.2 --- Guanidine hydrochloride - induced unfolding --- p.98 / Chapter 6.3 --- Thermal-induced unfolding --- p.99 / Chapter 6.4 --- Structural comparison of thermophilic and mesophilic EngB --- p.100 / Chapter 6.5 --- Discussion --- p.102 / Tables and figures of chapter six --- p.105 / Chapter Chapter 7 --- Construction of a dual-tag affinity pull-down system for finding interacting partner of EngB / Chapter 7.1 --- Introduction --- p.112 / Chapter 7.2 --- Preparation of dual-tagged E.coli EngB / Chapter 7.2.1 --- Cloning of SBP-CBP-EC EngB expression construct --- p.113 / Chapter 7.2.2 --- Expression and purification of SBP-CBP-EC EngB --- p.114 / Chapter 7.3 --- Pull down using dual tagged E.coli EngB as bait to isolate potential interacting partners of EngB --- p.114 / Chapter 7.4 --- Discussion --- p.115 / Tables and figures of chapter seven --- p.117 / Chapter Chapter 8 --- Conclusion --- p.122 / References --- p.124
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_326172 |
| Date | January 2008 |
| Contributors | Chan, Kwok-Ho., 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, xiii, 129 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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