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Preparation of Recombinant Human Insulin and C Peptide by Transgenic Yeast Pichia Pastoris GS115

Author: LengXue
Tutor: ChenJinChun
School: Beijing University of Chemical Technology
Course: Biochemical Engineering
Keywords: Recombinant human insulin C-peptide Pichia pastoris Separation and purification
CLC: TQ467
Type: Master's thesis
Year: 2006
Downloads: 131
Quote: 1
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Abstract


Insulin effects of drugs for the treatment of diabetes , C-peptide is the peptide of the connection between the insulin A and B chains . Relationship of the research structure and biological function of insulin , in the past people have always thought that C-peptide biological activity , only play a role in the insulin disulfide bonds correctly paired , but in recent years a lot of research confirms the C-peptide has multiple biological role . Pichia yeast strain expressing recombinant human insulin and C-peptide building to be completed by the laboratory . This paper has constructed strains secretion expression of high - density fermentation and purification process of recombinant human proinsulin analogues of recombinant human insulin and C-peptide after digestion . Thesis, investigated three different formulations of the medium , the medium most suitable for the growth of this strain were identified ; simultaneously optimized fermentation conditions , and inducing conditions affecting the secretion expression when induced pH of 5 , the methanol concentration is maintained at 1% (NH 4 ) 2 SO 4 concentration 9G / L PTM added amount of 2mL / L, glycerol concentration 40 g / L , target protein production . Repeatedly 10L fermenter amplification experiments, the target protein production in shake flask cultures of 1.5-2 times . Closure target protein using hollow fiber ultrafiltration , can remove more than 50% of the contaminating proteins , but can also be the fermentation broth to desalting and preliminary concentrated final yield is substantially maintained at between 30 % to 50% , and on the activity destruction; paper of SP Sepharose Fast Flow strong cation exchange column CM Sepharose Fast Flow weak cation -exchange column , DEAE - Sepharose Fast Flow weak anion exchange column adsorption of the target protein to inspect to determine the optimum of each exchange column pH value and elution concentration of NaCl ; wherein a cation exchange column SP-FF adsorbed target protein effect

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