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
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_325785 |
| Date | January 2006 |
| Contributors | Lau, San Shing., 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, 120 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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