Tse, Yuk Tin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 141-149). / Abstracts in English and Chinese. / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- The influenza virus --- p.1 / Chapter 1.1.1 --- Influenza NA and its inhibitors --- p.3 / Chapter 1.1.2 --- Follow-up on the use of NAIs --- p.9 / Chapter 1.2 --- Sources of NA for experimental studies --- p.11 / Chapter 1.2.1 --- Viral sources --- p.11 / Chapter 1.2.2 --- NA isolation --- p.12 / Chapter 1.2.3 --- Recombinant NA expressed in cell lines --- p.12 / Chapter 1.2.4 --- Glycosylation on NA functionality --- p.13 / Chapter 1.2.5 --- Recombinant NA expressed in yeast --- p.15 / Chapter 1.3 --- Research objectives --- p.16 / Chapter 2 --- Cloning of Influenza Neuraminidase and Expression in P. pastoris --- p.17 / Chapter 2.1 --- Background --- p.17 / Chapter 2.1.1 --- Full-length cloning of the A/HongKong/483/97(H5N 1) NA --- p.17 / Chapter 2.1.2 --- Identification of the H274 equivalent --- p.19 / Chapter 2.1.3 --- The experiment --- p.22 / Chapter 2.2 --- Materials and methods --- p.23 / Chapter 2.2.1 --- Preparation of chemically competent Escherichia coli --- p.23 / Chapter 2.2.1.1 --- Reagents --- p.23 / Chapter 2.2.1.2 --- Reagent setup --- p.23 / Chapter 2.2.1.3 --- Equipment --- p.23 / Chapter 2.2.1.4 --- Procedure --- p.23 / Chapter 2.2.2 --- Amplification of N1 NA and EGFP genes --- p.24 / Chapter 2.2.2.1 --- Reagents --- p.24 / Chapter 2.2.2.2 --- Reagent setup --- p.25 / Chapter 2.2.2.3 --- Equipment --- p.25 / Chapter 2.2.2.4 --- Procedure --- p.26 / Chapter 2.2.2.4.1 --- Amplification of the full-length N1 NA gene from cDNA --- p.26 / Chapter 2.2.2.4.2 --- Amplification of the EGFP gene --- p.26 / Chapter 2.2.3 --- TA cloning of PCR products --- p.27 / Chapter 2.2.3.1 --- Reagents --- p.27 / Chapter 2.2.3.2 --- Reagent setup --- p.27 / Chapter 2.2.3.3 --- Equipment --- p.28 / Chapter 2.2.3.4 --- Procedure --- p.28 / Chapter 2.2.3.4.1 --- TA cloning of PCR products --- p.28 / Chapter 2.2.3.4.2 --- Site-directed mutagenesis by overlapping PCR --- p.30 / Chapter 2.2.4 --- Construction of P. pastoris expression vectors --- p.31 / Chapter 2.2.4.1 --- Reagents --- p.31 / Chapter 2.2.4.2 --- Reagent setup --- p.31 / Chapter 2.2.4.3 --- Procedure --- p.32 / Chapter 2.2.4.3.1 --- Generation of N1 NA expression vectors --- p.32 / Chapter 2.2.4.3.2 --- Generation of EGFP expression vectors --- p.34 / Chapter 2.2.5 --- Transformation of P. pastoris --- p.37 / Chapter 2.2.5.1 --- Reagents --- p.37 / Chapter 2.2.5.2 --- Reagent setup --- p.37 / Chapter 2.2.5.3 --- Equipment --- p.38 / Chapter 2.2.5.4 --- Procedure --- p.38 / Chapter 2.2.5.4.1 --- Preparation and transformation of electrocompetent P. pastoris --- p.38 / Chapter 2.2.5.4.2 --- PCR analysis of P. pastoris transformants (colony PCR) --- p.39 / Chapter 2.2.6 --- Expression of N1 NA and EGFP in P. pastoris --- p.40 / Chapter 2.2.6.1 --- Reagents --- p.40 / Chapter 2.2.6.2 --- Reagent setup --- p.40 / Chapter 2.2.6.3 --- Procedure --- p.41 / Chapter 2.2.6.3.1 --- Small-scale protein expression in P. pastoris --- p.41 / Chapter 2.2.6.3.2 --- Sequence alignment --- p.42 / Chapter 2.2.6.3.3 --- Data processing --- p.42 / Chapter 2.3 --- Results and Discussion --- p.43 / Chapter 2.3.1 --- Cloning of NA and EGFP into the pPICZB expression vector --- p.43 / Chapter 2.3.2 --- Growth of P. pastoris transformants --- p.51 / Chapter 3 --- Physical Characterization of Influenza Neuraminidase Expressed in P. pastoris --- p.53 / Chapter 3.1 --- Background --- p.53 / Chapter 3.1.1 --- Structural significance of disulphide bonds in NA --- p.53 / Chapter 3.1.2 --- Localization of recombinant N1 NA in P. pastoris --- p.55 / Chapter 3.1.3 --- The experiment --- p.56 / Chapter 3.2 --- Materials and methods --- p.57 / Chapter 3.2.1 --- Differential centrifugation --- p.57 / Chapter 3.2.1.1 --- Reagents --- p.57 / Chapter 3.2.1.2 --- Reagent setup --- p.57 / Chapter 3.2.1.3 --- Equipment --- p.57 / Chapter 3.2.1.4 --- Procedures --- p.58 / Chapter 3.2.1.4.1 --- Cell harvesting and lysis --- p.58 / Chapter 3.2.1.4.2 --- Preparation of crude membrane --- p.58 / Chapter 3.2.1.4.3 --- Preparation of plasma membrane --- p.58 / Chapter 3.2.2 --- Sodium dodecyl sulphate polyaciylamide gel electrophoresis (SDS-PAGE)… --- p.59 / Chapter 3.2.2.1 --- Reagents --- p.59 / Chapter 3.2.2.2 --- Reagent setup --- p.60 / Chapter 3.2.2.3 --- Equipment --- p.61 / Chapter 3.2.2.4 --- Procedure --- p.61 / Chapter 3.2.3 --- Immunoblotting --- p.61 / Chapter 3.2.3.1 --- Reagents --- p.61 / Chapter 3.2.3.2 --- Reagent setup --- p.62 / Chapter 3.2.3.3 --- Equipment --- p.62 / Chapter 3.2.3.4 --- Procedure --- p.62 / Chapter 3.2.3.4.1 --- Electroblotting --- p.62 / Chapter 3.2.3.4.2 --- Blocking and probing --- p.63 / Chapter 3.2.3.4.3 --- Immunodetection --- p.63 / Chapter 3.2.3.4.4 --- Molecular weight determination --- p.63 / Chapter 3.2.4 --- Confocal microscopy --- p.64 / Chapter 3.2.4.1 --- Equipment --- p.64 / Chapter 3.2.4.2 --- Procedure --- p.64 / Chapter 3.2.4.2.1 --- Image acquisition --- p.64 / Chapter 3.2.4.2.2 --- Image processing --- p.65 / Chapter 3.3 --- Results --- p.66 / Chapter 3.3.1 --- Localization of recombinant N1 NA in P. pastoris sub-cellular fractions --- p.66 / Chapter 3.3.2 --- Molecular weight determination for the N1 NA expressed in P. pastoris --- p.69 / Chapter 3.3.3 --- Cellular localization of recombinant N1 NA in P. pastoris --- p.71 / Chapter 3.4 --- Discussion --- p.77 / Chapter 3.4.1 --- Molecular weight determination for N1 NA expressed in P. pastoris --- p.77 / Chapter 3.4.2 --- Disulphide bond formation in N1 NA expressed in P. pastoris --- p.78 / Chapter 3.4.3 --- Cell-surface association of recombinant N1 NA in P. pastoris --- p.79 / Chapter 3.5 --- Conclusion --- p.81 / Chapter 4 --- Functional Characterization of Influenza Neuraminidase Expressed in P. pastor --- p.is / Chapter 4.1 --- Background --- p.82 / Chapter 4.1.1 --- Fluorometric NA activity assay --- p.82 / Chapter 4.1.2 --- Colorimetric assay of NA activity --- p.84 / Chapter 4.1.3 --- The experiment --- p.85 / Chapter 4.2 --- Materials and methods --- p.86 / Chapter 4.2.1 --- Fluorometric assay of N1 NA expressed in P. pastoris --- p.86 / Chapter 4.2.1.1 --- Reagents --- p.86 / Chapter 4.2.1.2 --- Reagent setup --- p.86 / Chapter 4.2.1.3 --- Equipment --- p.86 / Chapter 4.2.1.4 --- Procedure --- p.87 / Chapter 4.2.1.4.1 --- Calibrating cell density with viable cell counts --- p.87 / Chapter 4.2.1.4.2 --- End-point measurement of NA activity --- p.87 / Chapter 4.2.1.4.2.1 --- Determination of expression yield --- p.89 / Chapter 4.2.1.4.2.2 --- End-point assay of NAI sensitivity --- p.89 / Chapter 4.2.1.4.3 --- Kinetic measurement of NA activity --- p.90 / Chapter 4.2.1.4.3.1 --- Derivation ofV0 --- p.92 / Chapter 4.2.1.4.3.2 --- Graphical determination of KM --- p.93 / Chapter 4.2.1.4.3.3 --- Graphical determination of KI --- p.94 / Chapter 4.2.2 --- Colorimetric assay of N1 NA expressed in P. pastoris --- p.96 / Chapter 4.2.2.1 --- Reagents --- p.96 / Chapter 4.2.2.2 --- Reagent setup --- p.96 / Chapter 4.2.2.3 --- Equipment --- p.96 / Chapter 4.2.2.4 --- Procedure --- p.96 / Chapter 4.3 --- Results --- p.98 / Chapter 4.3.1 --- CFU determination --- p.98 / Chapter 4.3.2 --- Fluorescent NA activity assay for N1 NA expressed in P. pastoris --- p.98 / Chapter 4.3.2.1 --- End-point measurement of NA activity --- p.98 / Chapter 4.3.2.1.1 --- Course of N1 NA expression in P. pastoris --- p.102 / Chapter 4.3.2.1.1.1 --- NA activity per unit cell mass --- p.102 / Chapter 4.3.2.1.1.2 --- Yield of NA --- p.102 / Chapter 4.3.2.1.2 --- End-point assay for NAI sensitivity --- p.105 / Chapter 4.3.2.2 --- Kinetic measurement of NA activity and NAI sensitivity --- p.107 / Chapter 4.3.2.2.1 --- Graphical determination of KM --- p.107 / Chapter 4.3.2.2.2 --- Graphical determination of KI --- p.107 / Chapter 4.3.2.3 --- Colorimetric NA activity assay --- p.111 / Chapter 4.4 --- Discussion --- p.114 / Chapter 4.4.1 --- Fluorescent NA activity assay of N1 NA expressed in P. pastoris --- p.115 / Chapter 4.4.1.1 --- End-point measurement of NA activity --- p.115 / Chapter 4.4.1.1.1 --- Time course of expression --- p.115 / Chapter 4.4.1.1.2 --- Effect of H275Y mutation on NA activity and NAI sensitivity --- p.117 / Chapter 4.4.1.1.3 --- Effect of C-terminal tags on NA activity and NAI sensitivity --- p.117 / Chapter 4.4.1.2 --- Kinetic measurement of NA activity --- p.118 / Chapter 4.4.1.2.1 --- Graphical determination of KM --- p.119 / Chapter 4.4.1.2.2 --- Graphical determination of KI --- p.120 / Chapter 4.4.1.3 --- Comparison of fluorometric NA activity assays for use with whole P pastoris cells --- p.122 / Chapter 4.4.2 --- Colorimetric NA activity assay --- p.124 / Chapter 4.5 --- Conclusion --- p.126 / Chapter 5 --- Conclusions and Discussions --- p.127 / Chapter 5.1 --- General conclusions --- p.127 / Chapter 5.2 --- Follow-up --- p.127 / Chapter 5.2.1 --- Studies of influenza NA with enhanced activity --- p.128 / Chapter 5.2.2 --- NAI screening using yeast-expressed NA --- p.132 / Appendix --- p.134 / References --- p.141