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Production and purification of recombinant goldfish (Carassius auratus) prolactin in Escherichia coli.

by Cheung Yeuk Siu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 141-153). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Abstract --- p.ii / 摘要 --- p.iv / List of abbreviations --- p.v / Table of contents --- p.viii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Prolactin (PRL) --- p.1 / Chapter 1.1.1 --- General introduction --- p.1 / Chapter 1.1.2 --- Genomic organization of teleost PRL gene --- p.2 / Chapter 1.1.3 --- Conserved domains of fish PRL --- p.3 / Chapter 1.1.4 --- Structure of teleost PRL --- p.6 / Chapter 1.1.5 --- Tissue sources of PRL --- p.8 / Chapter 1.2 --- Prolactin receptor (PRLR) --- p.9 / Chapter 1.2.1 --- Tissue distribution in teleosts --- p.9 / Chapter 1.2.2 --- Receptor structure and multiple forms of PRLR --- p.11 / Chapter 1.2.3 --- Possible action mechanisms of PRL --- p.14 / Chapter 1.3 --- Control of PRL release --- p.17 / Chapter 1.4 --- Biological functions of PRL in vertebrates --- p.19 / Chapter 1.4.1 --- Biological effects on teleosts --- p.19 / Chapter 1.4.1.1 --- Osmoregulatory roles --- p.20 / Chapter 1.4.1.2 --- Non-osmoregulatory roles --- p.29 / Chapter 1.5 --- Aim of the present study --- p.32 / Chapter Chapter 2 --- "Recombinant goldfish (Carassius auratus) prolactin: subcloning, expression, purification and refolding of the recombinant protein" / Chapter 2.1 --- Introduction --- p.35 / Chapter 2.2 --- Materials --- p.38 / Chapter 2.3 --- Methods --- p.48 / Chapter 2.3.1 --- Subcloning of the gfPRL cDNA --- p.48 / Chapter 2.3.1.1 --- PCR cloning of gfPRL cDNA --- p.48 / Chapter 2.3.1.2 --- DNA sequencing of the subcloned fragment --- p.51 / Chapter 2.3.1.3 --- Subcloning of the gfPRL cDNA fragment into the expression vector --- p.52 / Chapter 2.3.2 --- Expression and purification of rgfPRL --- p.53 / Chapter 2.3.2.1 --- Transformation of pRSETA/gfPRL into BL21(DE3)pLysS cells --- p.53 / Chapter 2.3.2.2 --- Prokaryotic expression of rgfPRL --- p.53 / Chapter 2.3.2.3 --- Affinity purification of rgfPRL --- p.55 / Chapter 2.3.2.4 --- Western blot analysis of the purified rgfPRL --- p.57 / Chapter 2.3.2.5 --- Protein concentration determination of the rgfPRL --- p.59 / Chapter 2.3.3 --- Protein refolding --- p.60 / Chapter 2.4 --- Results --- p.61 / Chapter 2.4.1 --- Subcloning and DNA sequencing of the gfPRL --- p.61 / Chapter 2.4.2 --- Expression and purification of rgfPRL --- p.63 / Chapter 2.4.2.1 --- Prokaryotic expression of rgfPRL --- p.63 / Chapter 2.4.2.2 --- Affinity purification of rgfPRL --- p.66 / Chapter 2.4.2.3 --- Western blot analysis of the purified rgfPRL --- p.68 / Chapter 2.4.2.4 --- Protein concentration determination of the rgfPRL --- p.68 / Chapter 2.4.3 --- Protein refolding --- p.70 / Chapter 2.5 --- Discussion --- p.72 / Chapter Chapter 3 --- Production of polyclonal antibodies against rgfRPL / Chapter 3.1 --- Introduction --- p.81 / Chapter 3.2 --- Materials --- p.82 / Chapter 3.3 --- Methods --- p.84 / Chapter 3.3.1 --- Immunization of rabbits --- p.84 / Chapter 3.3.2 --- Collection of the polyclonal antisera --- p.85 / Chapter 3.3.3 --- Purification of IgG from the polyclonal antisera --- p.86 / Chapter 3.3.4 --- Enzyme linked immunosorbent assay (ELISA) --- p.87 / Chapter 3.3.5 --- Western blot analysis for cross-reactivity --- p.88 / Chapter 3.4 --- Results --- p.90 / Chapter 3.4.1 --- Isolation and purification of IgG from the polyclonal antisera --- p.90 / Chapter 3.4.2 --- ELISA --- p.93 / Chapter 3.4.3 --- Western blot analysis for cross-reactivity --- p.96 / Chapter 3.5 --- Discussion --- p.98 / Chapter Chapter 4 --- Isolation of native PRL from goldfish pituitaries / Chapter 4.1 --- Introduction --- p.100 / Chapter 4.2 --- Materials --- p.101 / Chapter 4.3 --- Methods --- p.103 / Chapter 4.3.1 --- Alkaline extraction --- p.103 / Chapter 4.3.2 --- Size exclusion chromatography --- p.104 / Chapter 4.3.3 --- Anion exchange chromatography --- p.104 / Chapter 4.4 --- Results --- p.106 / Chapter 4.4.1 --- Size exclusion chromatography --- p.106 / Chapter 4.4.2 --- Anion exchange chromatography --- p.109 / Chapter 4.4.3 --- SDS-PAGE analysis and immuno-detection of the purified protein --- p.112 / Chapter 4.5 --- Discussion --- p.114 / Chapter Chapter 5 --- Receptor binding assays / Chapter 5.1 --- Introduction --- p.115 / Chapter 5.2 --- Materials --- p.117 / Chapter 5.3 --- Methods --- p.119 / Chapter 5.3.1 --- Gill membrane preparation --- p.119 / Chapter 5.3.2 --- Radioactive labelling of the primary ligand --- p.120 / Chapter 5.3.3 --- Determination of the percentage of 125I incorporation and specific radioactivity of the radioligand --- p.121 / Chapter 5.3.4 --- Membrane protein dependence assay --- p.122 / Chapter 5.3.5 --- Receptor binding study using rgfPRL --- p.124 / Chapter 5.4 --- Results --- p.125 / Chapter 5.4.1 --- Radioactive labelling of the primary ligand --- p.125 / Chapter 5.4.2 --- Determination of the percentage of 125I incorporation and specific radioactivity of the radioligand --- p.127 / Chapter 5.4.3 --- Membrane protein dependence assay --- p.129 / Chapter 5.4.4 --- Receptor binding study using rgfPRL --- p.131 / Chapter 5.5 --- Discussion --- p.133 / Chapter Chapter 6 --- General discussion and conclusion --- p.136 / References --- p.141

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_323158
Date January 2000
ContributorsCheung, Yeuk Siu., Chinese University of Hong Kong Graduate School. Division of Biochemistry.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xii, 153 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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