Chong, King Wai Isaac. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 99-104). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 論文摘要 --- p.iv / Table of Contents --- p.vi / Lists of Figures --- p.ix / List of Tables --- p.xi / List of Abbreviations --- p.xii / Chapter Chapter One: --- Introduction --- p.1 / Chapter 1.1 --- Overview of Glyceraldehyde 3-phosphate Dehydrogenases --- p.1 / Chapter 1.2 --- Properties And Molecular Structures of GAPDH --- p.3 / Chapter 1.3 --- Action Mechanism of GAPDH --- p.6 / Chapter 1.4 --- Novel Functions of GAPDH Unrelated to Carbohydrate Metabolism --- p.8 / Chapter 1.5 --- Effects of Heavy Metal on Enzyme Activity And Gene Expression of GAPDH --- p.10 / Chapter 1.6 --- Metal Binding Properties And Metal Binding Sites of GAPDH --- p.12 / Chapter 1.7 --- Isolation And Purification of GAPDH from Different Organisms --- p.13 / Chapter 1.8 --- Development of New Purification Method of GAPDH Using Immobilized Metal Affinity Chromatography --- p.15 / Chapter 1.9 --- Study of GAPDH from Chironomidae Larvae --- p.16 / Chapter 1.10 --- Aims of Study --- p.18 / Chapter Chapter Two: --- Methods And Materials --- p.19 / Chapter 2.1 --- Isolation of Native Chironomidae GAPDH --- p.19 / Chapter 2.1.1 --- Chemicals And Reagents --- p.19 / Chapter 2.1.2 --- Reagents --- p.19 / Chapter 2.1.3 --- Preparation of Crude Protein Extract from Chironomidae Larvae --- p.24 / Chapter 2.1.4 --- Immobilized Metal Affinity Chromatography --- p.24 / Chapter 2.1.5 --- Large Scale Preparation of Crude Protein Extract --- p.25 / Chapter 2.1.6 --- Ammonium Sulfate Fractionation --- p.25 / Chapter 2.1.7 --- Copper Affinity Column Chromatography --- p.26 / Chapter 2.1.8 --- Dye Affinity Column Chromatography --- p.26 / Chapter 2.2 --- Identification of Chironomidae GAPDH --- p.27 / Chapter 2.2.1 --- Chemicals And Reagents --- p.27 / Chapter 2.2.2 --- Reagents --- p.28 / Chapter 2.2.3 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis --- p.32 / Chapter 2.2.4 --- Non-Denaturing Polyacrylamide Gel Electrophoresis --- p.33 / Chapter 2.2.5 --- Protein Extraction from Coosmassie Blue Stained Polyacrylamide Gel --- p.33 / Chapter 2.2.6 --- N-terminal Amino Acid Analysis --- p.33 / Chapter 2.2.7 --- Sequence Analysis --- p.34 / Chapter 2.3 --- Kinetic Analysis of Chironomidae GAPDH --- p.34 / Chapter 2.3.1 --- Chemcials And Reagents --- p.34 / Chapter 2.3.2 --- Reagents --- p.34 / Chapter 2.3.3 --- Determination of Enzyme Concentration And GAPDH Activity --- p.35 / Chapter 2.4 --- Molecular Cloning of Chironomidae GAPDH --- p.36 / Chapter 2.4.1 --- Chemicals And Reagents --- p.36 / Chapter 2.4.2 --- Reagents --- p.37 / Chapter 2.4.3 --- RNA Extraction from Chironomidae Larvae --- p.41 / Chapter 2.4.4 --- DNase I Removal of Genomic DNA Contamination --- p.42 / Chapter 2.4.5 --- RNA Concentration Determination And RNA Agarose Electrophoresis --- p.42 / Chapter 2.4.6 --- First Strand cDNA Synthesis --- p.43 / Chapter 2.4.7 --- pRSet A B C Vectors --- p.43 / Chapter 2.4.8 --- Cloning Primer Design --- p.45 / Chapter 2.4.9 --- Polymerase Chain Reaction --- p.45 / Chapter 2.4.10 --- DNA Agarose Electrophoresis --- p.46 / Chapter 2.4.11 --- Restriction Enzyme Digestion of Insert And Plasmid --- p.46 / Chapter 2.4.12 --- Ligation of Plasmid And Insert DNA --- p.46 / Chapter 2.4.13 --- Preparation of Chemically Competent E. coli --- p.47 / Chapter 2.4.14 --- Transformation of Plasmid by Heat Shock --- p.47 / Chapter 2.4.15 --- Colony PCR --- p.48 / Chapter 2.5 --- Recombinant Protein Expression And Purification --- p.48 / Chapter 2.5.1 --- Chemicals And Reagents --- p.48 / Chapter 2.5.2 --- Reagents --- p.49 / Chapter 2.5.3 --- Protein expression by IPTG --- p.51 / Chapter 2.5.4 --- Protein purification by Nickel Affinity Column Chromatography --- p.52 / Chapter 2.5.5 --- EnterokinaseMax ´ёØ Removal of Polyhistidine Fusion Tag --- p.52 / Chapter 2.5.6 --- Western Blotting of Protein --- p.53 / Chapter Chapter Three: --- Results --- p.54 / Chapter 3.1 --- Two Affinity Chromatography Methods for GAPDH Purification --- p.54 / Chapter 3.2 --- Isolation And Purification of Native Chironomidae GAPDH --- p.54 / Chapter 3.3 --- Identification of Chironomidae GAPDH --- p.62 / Chapter 3.3.1 --- N-terminal amino acid analysis --- p.62 / Chapter 3.3.2 --- Sequence Analysis --- p.62 / Chapter 3.4 --- Molecular Cloning of Chironomidae GAPDH --- p.63 / Chapter 3.5 --- Isolation And Purification of recombinant Chironomidae GAPDH --- p.70 / Chapter 3.6 --- Protein Gel Electrophoresis Analysis of GAPDHs --- p.74 / Chapter 3.7 --- "Effects of Heavy Metals, pH And Temperature on GAPDHs" --- p.76 / Chapter 3.7.1 --- Heavy Metal Effect --- p.76 / Chapter 3.7.2 --- pH Effect --- p.76 / Chapter 3.7.3 --- Temperature --- p.77 / Chapter 3.8 --- Kinetic Analysis of GAPDHs --- p.84 / Chapter Chapter Four: --- Discussion --- p.89 / Chapter 4.1 --- New Method for The Isolation and Purification of Chironomidae GAPDH --- p.89 / Chapter 4.2 --- "Effects of Heavy Metals, pH And Temperature on GAPDHs" --- p.91 / Chapter 4.3 --- Kinetic Analysis of GAPDHs --- p.91 / Chapter 4.4 --- Zinc Activation of Chironomidae GAPDH --- p.92 / Chapter 4.5 --- Future Study --- p.93 / Chapter 4.5.1 --- Sequence Analysis Using Prediction Programmes --- p.94 / Chapter 4.5.2 --- Protein Crystallization --- p.95 / Chapter 4.5.3 --- Site-Directed Mutagenesis --- p.95 / Chapter 4.5.4 --- Biacore Surface Plasmon Resonance --- p.95 / Chapter Chapter Five: --- Conclusion --- p.98 / Chapter Chapter Six: --- References --- p.99