Purification process for expressing tandem protein by pichia pastoris

A technology of protein and protein precipitation, which is applied in the field of purification process of expressing tandem protein in Pichia pastoris, which can solve the problems of high price, complicated method and low expression level

Inactive Publication Date: 2020-07-03
SHANGHAI XINSHENGYUAN BIOLOGICAL MEDICAL +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the expression level is low, the method is complicated, and Escherichia coli must use a large amount of casein hydrol...

Method used

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  • Purification process for expressing tandem protein by pichia pastoris
  • Purification process for expressing tandem protein by pichia pastoris
  • Purification process for expressing tandem protein by pichia pastoris

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Embodiment 1

[0107] Embodiment 1 provides a method for purifying recombinant human proinsulin C-peptide tandem protein to obtain recombinant human proinsulin C-peptide stock solution, the specific steps are as follows:

[0108] Step (1): centrifuging the fermentation broth containing the recombinant human proinsulin C-peptide tandem protein to obtain the supernatant of the fermentation broth.

[0109] Equipment: Centrifuge Model: SORVALL RC12BP

[0110] Fermentation broth: fermented by genetically engineered Pichia pastoris strains (containing multiple human proinsulin C-peptide genes in tandem) to produce a fermentation broth containing recombinant human proinsulin C-peptide tandem proteins (using the pilot test in Example 5 on page 11 of the Chinese patent CN1298742C specification Fermentation method), the fermented liquid volume is 200L.

[0111] Parameter conditions: centrifugation speed 4500rpm; time 20min; centrifugation temperature 4°C.

[0112] Collection of fermentation centrifu...

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Abstract

The invention relates to a purification process for expressing tandem protein by pichia pastoris, and particularly provides a purification method for recombinant human proinsulin C peptide tandem protein. The purification method comprises the following steps of: (1) centrifuging fermentation liquor to obtain fermentation centrifugal supernatant; (2) adding electrolyte for salting out to obtain precipitated protein; (3) adding a cosolvent to obtain a precipitated protein complex solution; (4) purifying the precipitated protein complex solution through first column chromatography; (5) adding trypsin and carboxypeptidase B for double enzyme digestion to obtain enzyme digestion liquor; (6) sequentially performing reverse-phase ultrafiltration and normal-phase ultrafiltration on the enzyme digestion liquor to obtain ultrafiltrate; (7) purifying the ultrafiltrate through second column chromatography; and (8) performing normal-phase ultrafiltration to obtain recombinant human proinsulin C peptide stock solution. The method can be used for preparing recombinant human proinsulin C peptide with high purity and high yield.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to a purification process for expressing tandem proteins from Pichia pastoris. Background technique [0002] Since the 1990s, proinsulin C-peptide (referred to as C-peptide) has been paid more and more attention. C-peptide is a polypeptide composed of 31 amino acids secreted by pancreatic beta cells. A research team led by Swedish scientists Johansson and Professor Waren[1-2] found that C-peptide can improve the long-term complications of diabetes (including diabetic nephropathy). [0003] Proinsulin C-peptide is mainly obtained by chemical synthesis and genetic engineering methods. Chemical synthesis has been gradually replaced by genetic engineering because of high cost, low yield, long production cycle and environmental pollution. At present, the most promising method is to directly express proinsulin C-peptide by genetic engineering. A method for expressing proinsulin C-pep...

Claims

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Application Information

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IPC IPC(8): C07K14/62C12P21/06C07K1/30C07K1/16C07K1/34C07K1/36
CPCC07K14/62C12P21/06
Inventor 贾伟平李校堃程萍倪晶
Owner SHANGHAI XINSHENGYUAN BIOLOGICAL MEDICAL
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