by Chung Wilson. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 126-149). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract (English) --- p.ii / Abstract (Chinese) --- p.v / Content --- p.vii / Abbreviations --- p.xii / List of Figures --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Alzheimer's disease --- p.1 / Chapter 1.1.1 --- History of Alzheimer's disease --- p.1 / Chapter 1.1.2 --- Definition of Alzheimer's disease --- p.2 / Chapter 1.1.3 --- Symptoms of Alzheimer's disease --- p.6 / Chapter 1.1.3.1 --- Memory deficit --- p.6 / Chapter 1.1.3.2 --- Difficulty in learning --- p.6 / Chapter 1.1.3.3 --- Language difficulties --- p.7 / Chapter 1.1.3.4 --- Decline in ability to perform routine tasks --- p.7 / Chapter 1.1.4 --- Prevalence of Alzheimer's disease --- p.8 / Chapter 1.2 --- Present treatment of Alzheimer's disease --- p.9 / Chapter 1.2.1 --- Acetylcholine and dementia --- p.9 / Chapter 1.2.2 --- Tacrine as first drug approved by US Food and Drug Administration --- p.9 / Chapter 1.3 --- Proposed theory of Alzheimer's disease formation --- p.10 / Chapter 1.3.1 --- The amyloid cascade hypothesis --- p.10 / Chapter 1.3.1.1 --- The amyloid precursor protein --- p.10 / Chapter 1.3.1.2 --- The processing of amyloid precursor protein --- p.12 / Chapter 1.3.1.3 --- Neurotoxic effect of amyloid plaque --- p.15 / Chapter 1.3.1.4 --- Genetic factors --- p.15 / Chapter 1.3.1.4.1 --- The amyloid precursor protein --- p.15 / Chapter 1.3.1.4.2 --- Apolipoprotein E (ApoE) --- p.16 / Chapter 1.3.1.4.3 --- Presenilin genes --- p.17 / Chapter 1.3.2 --- Tau and tangle hypothesis --- p.19 / Chapter 1.3.2.1 --- Tau protein --- p.19 / Chapter 1.3.2.2 --- Paired helical filaments (PHF) --- p.20 / Chapter 1.3.2.3 --- Tau protein kinase --- p.20 / Chapter 1.3.2.3.1 --- Glycogen synthase kinase-3 (GSK-3) --- p.21 / Chapter 1.3.2.3.2 --- Cyclin-dependent kinase 5 (CDK5) --- p.21 / Chapter 1.3.2.4 --- Tangle leads to dementia --- p.22 / Chapter 1.4 --- Cross-talk between the two hypotheses --- p.24 / Chapter 1.5 --- β -secretase (BACE) --- p.24 / Chapter 1.5.1 --- Discovery of β-secretase (BACE) --- p.24 / Chapter 1.5.2 --- Detailed structure of BACE --- p.25 / Chapter 1.5.3 --- Comparsion of human and mouse BACE --- p.27 / Chapter 1.5.4 --- Comparsion of BACE-1 with BACE-2 --- p.27 / Chapter 1.5.5 --- Properties of BACE-1 --- p.28 / Chapter 1.5.6 --- Expression of BACE in E.coli --- p.29 / Chapter 1.5.7 --- Expression of BACE in mammalian cells --- p.30 / Chapter 1.6 --- Objectives of the present study --- p.32 / Chapter Chapter 2 --- Materials and Methods --- p.34 / Chapter 2.1 --- Recombinant DNA techniques --- p.34 / Chapter 2.1.1 --- Amplification of genes by PCR techniques --- p.34 / Chapter 2.1.2 --- Agarose gel electrophoresis --- p.34 / Chapter 2.1.3 --- Extraction of DNA from agarose gel --- p.35 / Chapter 2.1.4 --- Digestion of various vectors and inserts --- p.36 / Chapter 2.1.5 --- Ligation of DNA fragments --- p.36 / Chapter 2.1.6 --- Preparation of Escherichia coli competent cells --- p.37 / Chapter 2.1.7 --- Bacterial transformation --- p.38 / Chapter 2.1.8 --- Minipreparation of plasmid DNA --- p.38 / Chapter 2.1.9 --- Large scale preparation of plasmid DNA --- p.39 / Chapter 2.1.10 --- Strain storage and revival --- p.40 / Chapter 2.1.11 --- Plasma DNA purification by High Pure plasmid isolation kit --- p.41 / Chapter 2.1.12 --- DNA sequencing --- p.42 / Chapter 2.1.13 --- Quantitation of DNA by spectrophotometric method --- p.43 / Chapter 2.2 --- Prokaryotic protein expression --- p.43 / Chapter 2.2.1 --- Selection of appropriate clones for recombinant protein expression using conventional method --- p.43 / Chapter 2.2.2 --- Selection of appropriate clones for recombinant protein expression using modified method --- p.44 / Chapter 2.2.3 --- Large -scale expression of recombinant human BACE protein using modified method --- p.45 / Chapter 2.2.4 --- Preparation of inclusion body from the bacterial expression culture --- p.46 / Chapter 2.2.5 --- Refolding of human BACE --- p.47 / Chapter 2.2.6 --- Purification of recombinant human BACE by immobilized metal ion affinity chromatography (IMAC) --- p.47 / Chapter 2.2.7 --- Protein concentration determination --- p.48 / Chapter 2.2.8 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.48 / Chapter 2.2.9 --- Western blotting --- p.50 / Chapter 2.2.10 --- Plasmid stability test --- p.50 / Chapter 2.3 --- Mammalian cell expression --- p.51 / Chapter 2.3.1 --- Transient transfection --- p.51 / Chapter 2.3.2 --- Measuring transfection efficiency --- p.52 / Chapter 2.3.3 --- Stable transfection --- p.52 / Chapter 2.3.4 --- Preparation of membrane extracts from CHO cells --- p.53 / Chapter 2.4 --- HPLC analysis --- p.53 / Chapter 2.4.1 --- Preparation of samples --- p.53 / Chapter 2.4.2 --- Reverse phase HPLC --- p.54 / Chapter 2.5 --- Fluorometric assay --- p.54 / Chapter 2.6 --- Immunohistochemistry --- p.55 / Chapter 2.7 --- Reagents and buffers --- p.55 / Chapter 2.7.1 --- Medium for bacterial culture --- p.56 / Chapter 2.7.2 --- Reagents for preparation of plasmid DNA --- p.56 / Chapter 2.7.3 --- Buffers for agarose gel electrophoresis --- p.57 / Chapter 2.7.4 --- Buffers for SDS-PAGE --- p.58 / Chapter 2.7.5 --- Buffer for purification of protein --- p.60 / Chapter 2.7.6 --- Buffer for Western Blotting --- p.61 / Chapter 2.7.7 --- Culturing medium of CHO cells --- p.62 / Chapter 2.7.8 --- Solutions for estimating transfection efficiency --- p.63 / Chapter 2.7.9 --- Reagents for HPLC --- p.64 / Chapter 2.7.10 --- Reagents for fluorometric assays --- p.65 / Chapter 2.7.11 --- Reagents for Immunohistochemistry --- p.66 / Chapter Chapter 3 --- Results --- p.67 / Chapter 3.1 --- Expression of BACE in E. coli --- p.67 / Chapter 3.1.1 --- Cloning of truncated human and mouse BACE into pRSET --- p.67 / Chapter 3.1.2 --- Expression of BACE in BL21(DE3)LysS cells --- p.70 / Chapter 3.1.2.1 --- Expression of truncated mouse and human BACEin BL21(DE3)LysS cells using conventional method --- p.70 / Chapter 3.1.2.2 --- Expression of truncated mouse and human BACEin BL21(DE3)LysS cells using modified method --- p.72 / Chapter 3.1.3 --- Analysis of BACE activity of purified recombinant proteins --- p.76 / Chapter 3.1.3.1 --- Fluorometric analysis --- p.76 / Chapter 3.2 --- Expression of BACE in mammalian cells --- p.81 / Chapter 3.2.1 --- "Cloning of full length mouse and human BACE into pCDNA3, pCDNA4HisMax" --- p.81 / Chapter 3.2.2 --- Transient transfection --- p.84 / Chapter 3.2.2.1 --- Western blot analysis --- p.86 / Chapter 3.2.2.2 --- Fluorometric analysis --- p.88 / Chapter 3.2.2.3 --- HPLC --- p.91 / Chapter 3.2.3 --- Stable transfection --- p.100 / Chapter 3.2.3.1 --- Western blot analysis --- p.101 / Chapter 3.2.3.2 --- Fluorometric analysis --- p.103 / Chapter 3.2.3.3 --- HPLC --- p.105 / Chapter 3.2.3.4 --- Immunohistochemistry --- p.112 / Chapter Chapter 4 --- Discussion --- p.115 / References --- p.126 / Appendix --- p.i / Chapter A1 --- Vector circle map --- p.i / Chapter A1-1 --- Vector circle map of pBluescript II- --- p.i / Chapter A1-2 --- Vector circle map of pCDNA3 --- p.ii / Chapter A1-3 --- Vector circle map of pCDNA4HisMax --- p.iii / Chapter A1-4 --- Vector circle map of pRSET --- p.iv / Chapter A2 --- Primer lists --- p.v / Chapter A3 --- Chemical structure of fluorophore and quench used in fluorometric assay --- p.vi
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_324046 |
| Date | January 2002 |
| Contributors | Chung, Wilson., 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, xix, 149, vi 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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