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
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_319125 |
| Date | January 1993 |
| Contributors | Wong, Kam Bo., Chinese University of Hong Kong Graduate School. Division of Biochemistry. |
| Publisher | Chinese University of Hong Kong |
| Source Sets | The Chinese University of Hong Kong |
| Language | English |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, x, 153 leaves : ill. (chiefly mounted 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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