Expression of the grass carp growth hormone: gene in Escherichia coli.

• Other Authors: Chinese University of Hong Kong Graduate School. Division of Biochemistry.
• Format: Others
• Language: English
• Published: Chinese University of Hong Kong 1993
• Subjects: Ctenopharyngodon idella; Escherichia coli > Genetics; Molecular cloning; Nucleotide sequence; Somatomedin
• Online Access: http://library.cuhk.edu.hk/record=b5887816; http://repository.lib.cuhk.edu.hk/en/item/cuhk-319204

by Pong Tsang Wai Hai. === Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. === Includes bibliographical references (leaves 98-105). === Acknowledgements --- p.i === Abstract --- p.ii === Abbreviations --- p.v === Chapter Chapter 1 --- Introduction === Chapter 1.1 --- Biological functions and structure of GH --- p.1 === Chapter 1.2 --- Application of recombinant GH --- p.2 === Chapter 1.3 --- Expression of eukaryotic gene in E.coli --- p.4 === Chapter 1.4 --- Methods for increasing expression of a cloned gene --- p.6 === Chapter 1.4.1 --- Changing the 5' end codons of the cDNA to E.coli preferred codons --- p.6 === Chapter 1.4.2 --- Optimization of distance between SD sequence and the initiation codons --- p.6 === Chapter 1.4.3 --- "Construction of a short ""dummy"" cistron at the 5' end of the cloned gene to improve attachment of ribosome" --- p.7 === Chapter 1.4.4 --- Increasing the copy number of recombinant expression plasmid --- p.8 === Chapter 1.4.5 --- Optimizing high density cell expression --- p.9 === Chapter 1.5 --- Quantitating the expression of cloned gene --- p.10 === Chapter 1.6 --- Inclusion bodies formation --- p.11 === Chapter 1.7 --- The purification of eukaryotic polypeptides synthesized as inclusion bodies --- p.12 === Chapter 1.7.1 --- Solubilization of the inclusion bodies --- p.13 === Chapter 1.7.2 --- Refolding the polypetides and disulfide bond formation --- p.13 === Chapter 1.8 --- Expression of secreted recombinant protein --- p.14 === Chapter 1.9 --- Purpose of present study --- p.15 === Chapter Chapter 2 --- Materials and Methods === Chapter 2.1 --- General techniques --- p.16 === Chapter 2.1.1 --- Chemical Synthesis of DNA linkers and primers --- p.16 === Chapter 2.1.2 --- Manipulation of DNA --- p.16 === Chapter 2.1.3 --- Electro-elution of DNA from Agarose Gel --- p.17 === Chapter 2.1.4 --- Preparation of Competent Cells and Transformation --- p.18 === Chapter 2.1.5 --- Screening of the Expressed Clones --- p.19 === Chapter 2.1.6 --- Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.21 === Chapter 2.1.7 --- Western blot analysis --- p.21 === Chapter 2.2 --- Purification procedures --- p.23 === Chapter 2.2.1 --- Growing up the cells in large scale --- p.23 === Chapter 2.2.2 --- Harvesting of cells from large scale culture --- p.23 === Chapter 2.2.3 --- Sonication of the cells --- p.24 === Chapter 2.2.4 --- Washing of the inclusion body --- p.24 === Chapter 2.2.5 --- Solubilization of the inclusion bodies --- p.25 === Chapter 2.2.6 --- Renaturation of r-gcGH --- p.26 === Chapter 2.2.6.1 --- Step down dilution mehtod --- p.26 === Chapter 2.2.6.2 --- Rapid dilution method --- p.26 === Chapter 2.2.7 --- Separation by reverse phase chromatography --- p.27 === Chapter 2.2.7.1 --- Octadodecylsilica (ODS) column separation --- p.27 === Chapter 2.2.7.2 --- Fast performance Liquid Chromatography(FPLC) --- p.28 === Chapter 2.3 --- Characterization methods --- p.29 === Chapter 2.3.1 --- Radioimmunoassay --- p.29 === Chapter 2.3.1.1 --- Iodination of r-gcGH --- p.29 === Chapter 2.3.1.2 --- Binding assay --- p.31 === Chapter 2.3.2 --- Preparation of anti-r-gcGH serum --- p.32 === Chapter 2.3.3 --- Determination of amino acid composition and N-terminal sequence of r-gcGH --- p.32 === Chapter Chapter 3 --- Results === Chapter 3.1 --- Recombinant plasmids construction --- p.34 === Chapter 3.1.1 --- Basic construction of plasmid producing gcGH in E.coli --- p.34 === Chapter 3.1.2 --- N-terminal modification of gcGH cDNA --- p.38 === Chapter 3.1.3 --- Constuction of a short 'dummy' cistron at the 5'end of gcGH cDNA --- p.40 === Chapter 3.1.4 --- Optimization of distance between ribosomal binding site and initiation codon --- p.42 === Chapter 3.1.5 --- Increasing expression level by increasing plasmid copy number --- p.44 === Chapter 3.1.6 --- Optimizing the high density expression by changing the promoter --- p.48 === Chapter 3.1.7 --- Construction of excretion plasmid for gcGH production from E. coli --- p.48 === Chapter 3.2 --- Quantitation and qualitation of the expressed protein --- p.51 === Chapter 3.3 --- Effect of IPTG on induction of r-gcGH in pp5 --- p.57 === Chapter 3.4 --- Stability of overproducing strain pp5 during continuous culture --- p.59 === Chapter 3.5 --- Stability of overproducing strain ppADH4 during continuous culture --- p.61 === Chapter 3.6 --- "Optimization of culture condition for high level expression strains,pp5 and ppADH4" --- p.64 === Chapter 3.7 --- Purification of r-gcGH --- p.67 === Chapter 3.7.1 --- Distribution of r-gcGH as Soluble and insoluble protein in E. coli --- p.67 === Chapter 3.7.2 --- Isolation and cleaning of the inclusion bodies --- p.69 === Chapter 3.7.3 --- Solubilization and renaturation of r-gcGH --- p.71 === Chapter 3.7.4 --- Purification of r-gcGH by chromatography --- p.73 === Chapter 3.8 --- Characterization of r-gcGH --- p.78 === Chapter 3.8.1 --- Amino acid composition and N-terminal sequence determination --- p.78 === Chapter 3.8.2 --- Immunological property of r-gcGH --- p.81 === Chapter 3.8.3 --- Physical Property of r-gcGH --- p.84 === Chapter 3.8.4 --- Stability of r-gcGH --- p.84 === Chapter 3.9 --- Expression and purification of r-gcGH in excretion vector ppSP14 --- p.86 === Chapter Chapter 4 --- Discussion === Chapter 4.1 --- Evaluation of expression strains --- p.88 === Chapter 4.1.1 --- Strain pKgcGH2 --- p.88 === Chapter 4.1.2 --- Strain pKgcGH2-17 --- p.88 === Chapter 4.1.3 --- Strain pSD78 --- p.89 === Chapter 4.1.4 --- "Strains pLl,pL2 and pL4" --- p.90 === Chapter 4.1.5 --- "Strains pp5,pplA,pp2I and pp4Q" --- p.90 === Chapter 4.1.6 --- Strain ppADH4 --- p.91 === Chapter 4.1.7 --- Strain ppSP14 --- p.91 === Chapter 4.2 --- Disulfide bond formation during refolding process --- p.92 === Chapter 4.2.1 --- Renaturaion in the presence of L-arginine and thiol reagent in oxidized form --- p.93 === Chapter 4.2.2 --- Renaturation in the presence of thiol reagent and 3M guanidine hydrochloride --- p.94 === Chapter 4.3 --- Stability of the r-gcGH --- p.94 === Chapter 4.4 --- Further studies --- p.96 === References --- p.98