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Expression, purification and characterization of rat UDP-glucuronosyltransferase 1A8. / Expression, purification & characterization of rat UDP-glucuronosyltransferase 1A8

Lau San Shing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 113-120). / Abstracts in English and Chinese. / Title Page --- p.1 / List of Thesis Committee --- p.2 / Declaration Page --- p.3 / Acknowledgements --- p.4 / Table of Contents --- p.5 / Abstract --- p.10 / 論文撰要 --- p.12 / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Drug Metabolism --- p.14 / Chapter 1.2 --- Glucuronidation --- p.16 / Chapter 1.3 --- UDP-glucuronosyltransferase (UGTs) / Chapter 1.3.1 --- Nomenclature --- p.18 / Chapter 1.3.2 --- Tissue Distributions of UGTs --- p.20 / Chapter 1.3.3 --- Genetics --- p.26 / Chapter 1.3.4 --- Evolution of UGTs --- p.28 / Chapter 1.4 --- UDP-glucuronosyltransferase related Human Diseases --- p.33 / Chapter 1.4.1 --- Hyperbilirubinemia --- p.33 / Chapter 1.4.2 --- Cancer --- p.37 / Chapter 1.5 --- Rattus norvrgicus UDP-glucuronosyltransferase 1A8 --- p.38 / Chapter 1.6 --- Aims of the Project --- p.42 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials / Chapter 1. --- Rat liver mRNA Extraction --- p.43 / Chapter 2. --- RT-PCR of rat liver mRNA --- p.43 / Chapter 3. --- Amplification of UGT1A8 gene from the cDNA library --- p.43 / Chapter 4. --- Construction of bacterial expression vector --- p.43 / Chapter 5. --- Expression of recombinant protein in E.coli --- p.44 / Chapter 6. --- Purification of protein with Ni column --- p.44 / Chapter 7. --- Purification of protein with gel filtration column --- p.44 / Chapter 8. --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.44 / Chapter 9. --- Concentration and Desalting of protein --- p.45 / Chapter 10. --- Enzyme activity of glucuronidation --- p.45 / Chapter 11. --- Near UV and far UV circular dichroism (CD) spectroscopy --- p.45 / Chapter 12. --- Fluorescent properties studies --- p.45 / Chapter 13. --- Western Blotting --- p.46 / Chapter 14. --- 3D modeling of UGT1A8 and interactions with ligands --- p.46 / Chapter 2.2 --- Methods / Chapter 1. --- Rat liver mRNA extraction --- p.46 / Chapter 2. --- RT-PCR of rat liver mRNA --- p.47 / Chapter 3. --- Amplification of UGT1A8 gene from the cDNA library --- p.48 / Chapter 4. --- Cloning of UGT1A8 PCR product into expression vector pRSet B --- p.49 / Chapter 5. --- Confirmation of the presence of insert in the plasmid --- p.51 / Chapter 6. --- Sequence checking for UGT1A8 gene in the pRSet B vector --- p.52 / Chapter 7. --- Expression of recombinant protein in E.coli JM109(DE3) cell strain --- p.52 / Chapter 8. --- Purification of recombinant protein by Ni-column --- p.53 / Chapter 9. --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.53 / Chapter 10. --- Recombinant protein purification by gel filtration column --- p.54 / Chapter 11. --- Concentration or Desalting of Purified Protein --- p.54 / Chapter 12. --- Determination of Protein Concentration --- p.55 / Chapter 13. --- Far- UV Circular dichroism spectroscopy --- p.55 / Chapter 14. --- Intrinsic Fluorescence Studies of Proteins --- p.57 / Chapter 15. --- Chemical denaturation stability studies --- p.58 / Chapter 16. --- Glucuronidation protein activity assay --- p.59 / Chapter 17. --- Mutagenesis --- p.60 / Chapter 18. --- Western Blotting for the presence of protein --- p.61 / Chapter 19. --- Protein Modeling with Insight II / Chapter 19.1 --- Construction of substrate 1-napthol structure --- p.62 / Chapter 19.2 --- Obtaining UDP-glucuronic acid in PDB file --- p.63 / Chapter 19.3 --- Obtaining rat UGT1A8 model structure in PDB file --- p.63 / Chapter 19.4 --- Optimization of rat UGT1A8 structure --- p.63 / Chapter 19.5 --- Docking studies of interaction between ligands and protein / Chapter 19.5.1 --- Setting up a Grid --- p.66 / Chapter 19.5.2 --- Docking of 1-napthol to UGT1A8 --- p.67 / Chapter 19.5.3 --- Docking of UDP-glucuronic acid in the complex of UGT1A8 and1- napthol --- p.68 / Chapter 19.5.4 --- Definition of Subsets --- p.68 / Chapter Chapter 3 --- Results --- p.70 / Figure 3.1 The extracted RNA from rat liver tissue --- p.76 / Figure 3.2 DNA gel of PCR amplified gene product --- p.77 / Figure 3.3 Colony PCR of UGT1 A8-pRSetB transformed DH5 a bacteria --- p.78 / Figure 3.4 The alignment of amplified gene sequence with the rat UGT1A8 sequence on NCBI database --- p.79 / Figure 3.5 SDS-PAGE of cell lysates with different expression temperature and time duration --- p.82 / Figure 3.6 SDS-PAGE of bacterial cell lysates --- p.83 / Figure 3.7 SDS-PAGE of Ni-column eluted protein --- p.84 / Figure 3.8 Elution Profile of Gel Filtration Chromatography --- p.85 / Figure 3.9 SDS-PAGE analysis of UGT1A8 fractions from Ni-column and gel filtration column --- p.86 / Figure 3.10 Sequence Alignment of UGTs in the rat UGT1A family and 2D structure prediction of UGT1A8 --- p.88 / Figure 3.11 Circular Dichroism (CD) measurements on rat UGT1A8 --- p.89 / Figure 3.12 Western Blotting of UGT1A8 wild-type and mutant proteins --- p.91 / Table 3.1 The specific activity of wild-type and mutated proteins --- p.92 / Figure 3.13 Fluorescence spectrum of wild type and two charged-residue mutants ofUGTlA --- p.93 / Figure 3.14 Fluorescence spectrum of wild type and Trp mutants of UGT1A8 --- p.94 / Figure 3.15 Chemical denaturation of wild type and Trp-mutated UGT1A8 proteins --- p.95 / Figure 3.16 Resolved Stern-Volmer plot of UGT1A8 on acrylamide quenching --- p.96 / Figure 3.17 The 3D modeling structure of rat UGT1A8 --- p.97 / Figure 3.18 Modeling simulated the interaction between UDP-glucuronic acid and UGT1A8 --- p.98 / "Figure 3.19 Modeling simulated the interaction between UDP-glucuronic acid, 1-napthol and UGT1A8" --- p.99 / Chapter Chapter 4 --- Discussion / Chapter 1. --- Successful Expression of Rat UGT1A8 --- p.100 / Chapter 2. --- The recombinant rat UGT1A8 protein was properly folded and enzymatic functioning --- p.102 / Chapter 3. --- Purified recombinant rat UGT1A8 protein contained well-ordered structure --- p.103 / Chapter 4. --- "Relative positions of Trp38, Trp64, Trp98 and Trp208 in the protein" --- p.105 / Chapter 5. --- Contribution of Trp residues in the folding and stability of the protein --- p.106 / Chapter 6. --- Probing of substrate coupling region by mutagenesis --- p.108 / Chapter 7. --- Interaction studies of substrates and UDP-glucuronic acid with UGT1A8 by computer modeling and docking simulation --- p.109 / Chapter Chapter 5 --- Conclusion --- p.111 / Chapter Chapter 6 --- References --- p.113

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_325785
Date January 2006
ContributorsLau, San Shing., Chinese University of Hong Kong Graduate School. Division of Biochemistry.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, 120 leaves : ill. (some 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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