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Protein engineering of active site residues of trichosanthin.

by Wong Kam Bo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 149-153). / Acknowledgments --- p.i / Abstract --- p.ii / Contents --- p.iii / Abbreviations --- p.vii / Short names for mutants --- p.viii / One letter symbol for amino acids --- p.x / Chapter Chapter 1 --- Introduction / Chapter 1.1. --- Chemical and Physical Properties of Trichosanthin --- p.1 / Chapter 1.2. --- Activities of Trichosanthin at the cellular level --- p.2 / Chapter 1.3. --- Activities of Trichosanthin at the molecular level --- p.3 / Chapter 1.4. --- Objective and Strategy of Protein engineering of Trichosanthin --- p.8 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1. --- General Techniques --- p.15 / Chapter 2.1.1. --- Ethanol Precipitation of DNA and RNA --- p.15 / Chapter 2.1.2. --- Spectrophotometric quantification of DNA and RNA --- p.15 / Chapter 2.1.3. --- Minipreparation of Plasmid DNA --- p.15 / Chapter 2.1.4. --- Preparation of Plasmid DNA using Qiagen-pack 100 Cartridge --- p.16 / Chapter 2.1.5. --- Preparation of Plasmid DNA using Magic´ёØ Minipreps DNA Purification kit from Promega --- p.17 / Chapter 2.1.6. --- Preparation and Transformation of Escherichia coli Competent Cell --- p.18 / Chapter 2.1.7. --- Agarose Gel Electrophoresis of DNA --- p.19 / Chapter 2.1.8. --- Purification of DNA from Agarose Gel using GeneClean® (BIO 101 Inc.) kit --- p.20 / Chapter 2.1.9. --- Polymerase Chain Reaction (PGR) --- p.21 / Chapter 2.1.10. --- Restriction Digestion of DNA --- p.23 / Chapter 2.1.11. --- Ligation of DNA fragments --- p.23 / Chapter 2.1.12. --- Autoradiography --- p.24 / Chapter 2.1.13. --- SDS-Polyacrylamide Gel Electrophoresis (SDS- PAGE) --- p.24 / Chapter 2.1.14. --- Staining of Protein in polyacrylamide gel --- p.25 / Chapter 2.1.15. --- Western Blot detection of TCS --- p.25 / Chapter 2.1.16. --- Liquid Scintillation Counting --- p.27 / Chapter 2.1.17. --- Minimization of Ribonuclease (RNAase) activity in experiments involving RNA --- p.27 / Chapter 2.2. --- Site-Directed Mutagenesis of Trichosanthin --- p.28 / Chapter 2.2.1. --- "Construction of E160D,El60A and SEAAR deletion mutants" --- p.28 / Chapter 2.2.2. --- Construction of E189A mutant and El60A E189A double mutant --- p.31 / Chapter 2.2.3. --- Construction of E189D mutant and El60A E189D double mutant --- p.36 / Chapter 2.2.4. --- Construction of Q156A mutant --- p.38 / Chapter 2.2.5. --- Construction of Q156A El60A mutant (Fig. 2.7) --- p.41 / Chapter 2.2.6. --- Construction of Q156A El89A mutant (Fig. 2.8) --- p.43 / Chapter 2.3. --- DNA sequencing --- p.45 / Chapter 2.3.1. --- DNA Sequencing Reaction --- p.45 / Chapter 2.3.2. --- DNA Sequencing Electrophoresis --- p.46 / Chapter 2.3.3. --- Resolving GC band compression --- p.48 / Chapter 2.4. --- Overexpression of mutated TCS in Escherichia coli --- p.48 / Chapter 2.5. --- Purification of mutated TCS --- p.49 / Chapter 2.6. --- Ribosome inactivating activity Assay using Rabbit Reticulocyte Lysate In Vitro Translation system --- p.50 / Chapter 2.7. --- N-glycosidase activity Assay --- p.51 / Chapter 2.7.1. --- Inactivation of ribosome in rabbit reticulocyte lysate --- p.51 / Chapter 2.7.2. --- RNA extraction --- p.51 / Chapter 2.7.3. --- Aniline Degradation --- p.52 / Chapter 2.7.4. --- Electrophoresis of RNA in Agarose Gel containing Formamide --- p.52 / Chapter 2.8. --- Reagents and buffers --- p.53 / Chapter 2.8.1. --- Nucleic Acid Electrophoresis Buffers --- p.53 / Chapter 2.8.2. --- Reagents for preparation of plasmid DNA --- p.54 / Chapter 2.8.3. --- Media for bacterial culture --- p.54 / Chapter 2.8.4. --- Reagents for SDS-PAGE --- p.55 / Chapter 2.8.5. --- Reagents for western blot --- p.56 / Chapter 2.8.6. --- Reagents for DNA sequencing --- p.57 / Chapter Chapter 3 --- Construction of TCS mutants / Chapter 3.1. --- Introduction --- p.59 / Chapter 3.2. --- Results --- p.59 / Chapter 3.2.1. --- "Construction of E160D,El60A and ASEAAR" --- p.59 / Chapter 3.2.2. --- Construction of E189A and E160AE189A mutants --- p.66 / Chapter 3.2.3. --- Construction of E189D and E160AE189D mutants --- p.80 / Chapter 3.2 --- A Construction of Q156A mutant --- p.82 / Chapter 3.2.5. --- Construction of Q156AE160A and Q156AE189A --- p.86 / Chapter 3.3. --- Discussion --- p.90 / Chapter Chapter 4 --- Expression and Purification of mutated TCS proteins / Chapter 4.1. --- Introduction --- p.94 / Chapter 4.2. --- Results --- p.95 / Chapter 4.2.1. --- Expression and purification of E160D and El60A mutants --- p.95 / Chapter 4.2.2. --- Expression and purifcation of E189D and E160AE189D mutants --- p.99 / Chapter 4.2.3. --- Expression and purifcation of E189A and E160AE189A mutants --- p.104 / Chapter 4.2.4. --- Expression and purifcation of Q156A and Q156AE160A mutants --- p.109 / Chapter 4.2.5. --- Expression and purifcation of Q156AE189A mutant --- p.114 / Chapter 4.2.6. --- Analysis of protein purity by SDS-PAGE and Western immunoblotting --- p.114 / Chapter 4.3. --- Discussion --- p.119 / Chapter Chapter 5 --- Biological Assay of mutated proteins / Chapter 5.1. --- Introduction --- p.125 / Chapter 5.2. --- Results --- p.125 / Chapter 5.2.1. --- Ribosome inactivating activity assay --- p.125 / Chapter 5.2.2. --- N-glycosidase activities of El60AE189A mutant --- p.131 / Chapter 5.3. --- Discussion --- p.133 / Chapter 5.3.1. --- Role of glutamate-160 --- p.133 / Chapter 5.3.2. --- A putative mechanism for N-glycosidase activity of TCS --- p.137 / Chapter 5.3.3. --- Role of glutamate-189 and glutamine-156 --- p.143 / Chapter 5.3.4. --- Prospective and future studies --- p.145 / Chapter 5.4. --- Concluding remarks --- p.147 / Appendix / Chapter A.l --- Size of molecule weight markers --- p.148 / Chapter A.2 --- Reference --- p.149

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_319125
Date January 1993
ContributorsWong, Kam Bo., Chinese University of Hong Kong Graduate School. Division of Biochemistry.
PublisherChinese University of Hong Kong
Source SetsThe Chinese University of Hong Kong
LanguageEnglish
Detected LanguageEnglish
TypeText, bibliography
Formatprint, x, 153 leaves : ill. (chiefly mounted col.) ; 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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