Protein engineering of active site residues of trichosanthin.

• Other Authors: Wong, Kam Bo.
• Format: Others
• Language: English
• Published: Chinese University of Hong Kong 1993
• Subjects: Trichosanthin; Protein Engineering
• Online Access: http://library.cuhk.edu.hk/record=b5895773; http://repository.lib.cuhk.edu.hk/en/item/cuhk-319125

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