Wong Kwong Fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 97-104). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / List of Figures --- p.x / List of Tables --- p.xi / List of Abbreviations --- p.xii / Chapter Chapter One --- Introduction --- p.1 / General background and objective of this study --- p.1 / Chapter Part One --- Early studies on ferrochelatase from chironomidae larvae / Chapter 1.1 --- Purification of ferrochelatases from different organisms --- p.5 / Chapter 1.2 --- Purification of ferrochelatase from chironomidae larvae --- p.5 / Chapter 1.3 --- Molecular cloning of ferrochelatase from chironomidae larvae --- p.6 / Chapter 1.4 --- Expression of recombinant chironomidae ferrochelatase --- p.8 / Chapter Part Two --- Study on the iron-sulfur cluster of chironomidae ferrochelatase / Chapter 2.1 --- Presence of iron-sulfur cluster at chironomidae ferrochelatase --- p.13 / Chapter 2.2 --- Possible roles of iron-sulfur cluster at ferrochelatase --- p.13 / Chapter 2.3 --- Mutagenesis study of iron-sulfur cluster at chironomidae ferrochelatase --- p.14 / Chapter Part Three --- Study on the catalytic mechanism of ferrochelatase / Chapter 3.1 --- Binding and distortion of porphyrin --- p.17 / Chapter 3.2 --- Binding of ferrous iron to ferrochelatase --- p.18 / Chapter Part Four --- Study on the effect of metal ions on ferrochelatase / Chapter 4.1 --- Previous studies on the effects of metal ions on different ferrochelatases --- p.24 / Chapter 4.2 --- Effects of metal ions on chironomidae ferrochelatase --- p.24 / Chapter Chapter Two --- Methods and Materials --- p.28 / General procedure --- p.28 / Chapter Part One --- Cloning of chironomidae ferrochelatase coding fragment / Chapter 1.1 --- Amplification of chironomidae ferrochelatase coding cDNA --- p.29 / Chapter 1.2 --- Construction of the expression vector (pETBlue-FECH) --- p.30 / Chapter Part Two --- Mutagenesis / Chapter 2.1 --- Choices of residue replacements --- p.31 / Chapter 2.2 --- Constructions of mutant plasmids --- p.31 / Chapter 2.3 --- Screenings of mutant plasmids --- p.32 / Chapter Part Three --- Construction of chironomidae-human hybrid plasmid / Chapter 3.1 --- Human RNA isolation --- p.33 / Chapter 3.2 --- Human cDNA synthesis --- p.33 / Chapter 3.3 --- Construction of human ferrochelatase expression plasmid (pETBlue-HFECH) --- p.34 / Chapter 3.4 --- Construction of chironomidae-human hybrid plasmid (pETBlue-HYB-FECH) --- p.34 / Chapter 3.4.1 --- First round PCR --- p.34 / Chapter 3.4.2 --- Second round PCR --- p.34 / Chapter 3.4.3 --- Overlapping PCR --- p.35 / Chapter 3.4.4 --- Cloning of HYB-FECH --- p.35 / Chapter Part Four --- Protein expression and purification / Chapter 4.1 --- Protein expression in bacterial expression system --- p.36 / Chapter 4.2 --- Cell harvest and ammonium sulfate fractionation --- p.36 / Chapter 4.3 --- Protein purifications by Blue Sepharose CL-6B chromatography --- p.37 / Chapter 4.4 --- Precipitation of protein samples for SDS-PAGE analysis --- p.37 / Chapter 4.5 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.38 / Chapter 4.6 --- Fast protein liquid chromatography (FPLC) --- p.38 / Chapter Part Five --- Functional study of expressed ferrochelatase / Chapter 5.1 --- Kinetic measurements --- p.40 / Chapter 5.2 --- Metal titration and fluorescence spectroscopy --- p.40 / Chapter Part Six --- Homology modeling of chironomidae ferrochelatase --- p.42 / Chapter Part Seven --- Other routine laboratory methods / Chapter 7.1 --- Determination of nucleic acid --- p.44 / Chapter 7.2 --- Determination of protein --- p.44 / Chapter 7.3 --- Agarose gel electrophoresis of DNA --- p.44 / Chapter 7.4 --- Agarose gel electrophoresis of RNA --- p.45 / Chapter 7.5 --- Nucleic acid purification --- p.45 / Chapter 7.6 --- Preparation of chemically competent bacterial cells --- p.46 / Chapter 7.7 --- Heat shock transformation of competent bacterial cells --- p.46 / Chapter 7.8 --- Colony PCR --- p.47 / Chapter 7.9 --- Plasmid rescue by alkaline lysis --- p.48 / Chapter 7.10 --- Washing of Blue Sepharose CL-6B column --- p.48 / Chapter Chapter Three --- Results --- p.49 / Chapter Part One --- Protein expression and purification --- p.49 / Chapter Part Two --- Kinetic study of chironomidae ferrochelatase --- p.53 / Chapter Part Three --- Mutagenesis study of iron-sulfur cluster --- p.56 / Chapter Part Four --- Mutagenesis study of metalation by ferrous iron / Chapter 4.1 --- Mutagenesis study at His234 --- p.58 / Chapter 4.2 --- "Mutagenesis study at Glu310, Glu313 and Glu317" --- p.58 / Chapter Part Five --- Copper activation of chironomidae ferrochelatase / Chapter 5.1 --- Kinetic analysis of copper activation --- p.62 / Chapter 5.2 --- Copper activation on chironomidae-human hybrid (HYB-FECH) --- p.63 / Chapter 5.3 --- Mutagenesis study on copper activation --- p.63 / Chapter Part Six --- Homology modeling of chironomidae ferrochelatase --- p.70 / Chapter Chapter Four --- Discussion --- p.72 / Chapter Part One --- Protein expression and purification --- p.72 / Chapter Part Two --- Mutant constructions by QuikChange´ёØ approach --- p.73 / Chapter Part Three --- Kinetic study of expressed chironomidae ferrochelatase --- p.74 / Chapter Part Four --- Mutagenesis study of iron sulfur cluster / Chapter 4.1 --- Location of cysteine residues forming the iron-sulfur cluster --- p.77 / Chapter Part Five --- Mutagenesis study of metalation by ferrous iron / Chapter 5.1 --- Role of His234 --- p.79 / Chapter 5.2 --- Catalytic roles of Glu310，Glu313 and Glu317 --- p.80 / Chapter 5.3 --- "Substrate binding on Glu310, Glu313 and Glu317" --- p.83 / Chapter 5.4 --- Partial picture of metalation and future work on active site residues --- p.84 / Chapter 5.5 --- Future works on the active site residues --- p.84 / Chapter Part Six --- Copper activation of chironomidae ferrochelatase / Chapter 6.1 --- Chironomidae ferrochelatase interaction with copper (II) ion --- p.88 / Chapter 6.2 --- Putative copper-binding sites on chironomidae ferrochelatase --- p.89 / Chapter 6.3 --- The hypothesized model of copper activation --- p.90 / Chapter 6.4 --- Direct copper involvement in catalysis --- p.91 / Chapter Chapter Five --- Summary --- p.94 / Chapter Chapter Six --- References --- p.97
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_324931 |
| Date | January 2004 |
| Contributors | Wong, Kwong Fai., Chinese University of Hong Kong Graduate School. Division of Biochemistry. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xii, 104 leaves : ill. (some 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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