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Method for producing recombined human proinsulin

A technology of human insulin and original protein, applied in the field of genetic engineering, which can solve the problems of low yield of active products and increased post-processing complexity

Inactive Publication Date: 2006-12-06
SHANGHAI NEWSUMMIT BIOPHARMA
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] Proinsulin and its analog genes have been expressed in many expression systems such as Escherichia coli, Bacillus subtilis, yeast and mammalian cells, and some of them have relatively high expression levels, but there are various problems. It’s okay, but the expression products often form inclusion bodies, which is beneficial to the high-efficiency expression and stability of the products, but these products need to be denatured to restore their natural structure and activity, which increases the complexity of post-processing, and to Low yield of final active product

Method used

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  • Method for producing recombined human proinsulin
  • Method for producing recombined human proinsulin

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Embodiment 1

[0052] The acquisition of embodiment 1 fusion gene and the construction of expression plasmid

[0053] The gene sequence of the human proinsulin molecule (INS) of the novel N-terminal leader sequence was synthesized from the whole gene, the gene was cloned into pUC19 and sequenced and verified, and then the fusion sequence of the fusion protein α-INS was constructed in vitro by molecular biological methods ( See Figure 1), using the plasmid pUC19 / rINS containing the rINS coding sequence as a template, the INS gene that can be fused with the Saccharomyces cerevisiae α-factor leader peptide sequence in the correct frame was obtained by PCR amplification. At the same time, using the plasmid pPIC9K (purchased from Invitrogen) containing the Saccharomyces cerevisiae α-factor leader peptide coding sequence as a template, the α-factor leader peptide sequence was amplified by PCR. The purified above two PCR products were respectively digested with restriction enzyme XhoI, and the two ...

Embodiment 2

[0055] Example 2 Screening of Highly Expressed Recombinant Human Proinsulin Engineering Strain

[0056] The constructed recombinant plasmid pGAPZA / α-INS was prepared in large quantities, linearized, electroporated and transformed into host cell P. pastoris GS115, coated with YPDS plates containing different concentrations of the antibiotic Zeocin to screen high-resistance positive clones, and a small shaker expression experiment was performed The engineered yeast strain (containing α-INS) with high expression of recombinant human proinsulin was screened.

Embodiment 3

[0057] Embodiment 3 selects suitable expression vector

[0058] strain

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Abstract

This invention provides a method for producing recombinant human proinsulin from N-end leader peptide, and relevant expression vector. The method comprises: (1) fusing yeast alpha-factor leader peptide sequence and the human proinsulin N-end leader peptide, and introducing into a yeast constitutive expression vector; (2) transferring into Pichia pastoris cells to realize constitutive and secretory expression; (3) fermenting and purifying. The recombinant human proinsulin has such advantages as high stability, high activity and simple production. The method has such advantages as high expression level, high purification yield, and high efficiency.

Description

technical field [0001] The invention relates to the technical field of genetic engineering. More specifically, the present invention provides a human proinsulin molecule (INS) with a novel N-terminal leader sequence, a method for producing human proinsulin using the molecule, and the construction of related expression vectors and engineered cells, human insulin The original expression and purification process. Background technique [0002] Insulin is a protein hormone secreted by the pancreas that lowers blood sugar. The phases of insulin secretion under normal physiological conditions are: postprandial supplemental secretion and basal secretion. [0003] Insulin is secreted by pancreatic B cells. Islet B cells first synthesize a macromolecular pre-proinsulin, then process it into 86-peptide proinsulin, and then hydrolyze it to become insulin. [0004] Mature insulin consists of two polypeptide chains, A and B, with a total of 51 amino acids and a molecular weight of abo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/17C07K14/62C12N15/63C12N15/81C12P21/02
Inventor 孙九如任军黄阳滨杜碧金祈晖
Owner SHANGHAI NEWSUMMIT BIOPHARMA
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