Construction of human insulinogen C peptide high-yield strains

A technology of human insulin and high-efficiency cells, which is applied in the direction of fungi, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of increasing separation and purification steps, reducing the yield of human proinsulin C-peptide, and achieving low cost Effect

Active Publication Date: 2009-03-11
TIBET JINKE GRP +2
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  • Summary
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  • Description
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AI Technical Summary

Problems solved by technology

The advantage of this method is that a high-expression engineering strain of a fusion protein of poly(individual) human proinsulin C-peptide series can be obtained in Escherichia coli or yeast, and the disadvantage is that expensive dual proteases (trypsin and carboxypeptidase B) are required for cleavage , while increasing the separation and purification steps and being affected by the efficiency of double protease digestion, ultimately reducing the yield of human proinsulin C-peptide

Method used

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  • Construction of human insulinogen C peptide high-yield strains
  • Construction of human insulinogen C peptide high-yield strains
  • Construction of human insulinogen C peptide high-yield strains

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example 1

[0038] Example 1 The construction process of the recombinant expression vector containing human proinsulin C peptide

[0039] Chemical synthesis of human proinsulin C-peptide gene: According to the amino acid sequence of human proinsulin C-peptide (SEQ ID NO: 2), two complementary nucleotide sequences were synthesized.

[0040] The forward nucleotide sequence contains the following sequence (5'→3'): gaggccgaagatta cag gtaggacaagttgagttgggaggaggcccaggggcaggctcgctacagcccttggccttggaaggctcactgcag;

[0041]The reverse nucleotide sequence contains the following sequence (5'→3'):

[0042] ctgcagtgagccttccaaggccaagggctgtagcgagcctgcccctggggcctcctcccaactcaacttgtcctacctgtaaatcttcggcctc.

[0043] Restriction endonuclease EcoR I was designed at the 5' end of the forward and reverse nucleotide sequences.

[0044] The human proinsulin C-peptide gene is obtained by annealing the two complementary nucleotide sequences. The above-mentioned nucleic acid product and expression vector pPIC3K we...

example 2

[0047] Example 2 Shake flask expression of human proinsulin C-peptide situation

[0048] Pick a single colony from the solid plate, inoculate into a 250mL shake flask containing 25mL BMGY, grow overnight at 30°C, 220rpm to OD 600 =4.

[0049] Inoculate the above 25mL culture solution into 1LBMGY, and continue to amplify to the cell concentration OD 600 =6.

[0050] Centrifuge at 5000rpm for 5min at room temperature to harvest the cells. Suspend the bacteria with fresh sterile BMMY to the OD of the bacteria solution 600 =1.0, 30°C, 220rpm induced expression of human proinsulin C-peptide. Samples were taken every 24 hours and supplemented with 100% methanol to a final concentration of 1.5%. After 120 hours of induction culture, the cells were harvested by centrifugation. The bacteria were analyzed by protein electrophoresis, and the results were as follows: figure 2 As shown, the results show that: human proinsulin C-peptide was expressed in Pichia pastoris.

[0051] Sh...

example 3

[0052] Example 3 Fermentative expression of human proinsulin C-peptide

[0053] Pick a single colony from the solid plate, inoculate into a 250mL shake flask containing 25mL BMGY, grow overnight at 30°C, 220rpm to OD 600 =4.

[0054] Inoculate the above 25mL culture solution into 250mL BMGY, and continue to amplify to the cell concentration OD 600 =6, inoculate the culture solution into 2L BSM fermentation medium, adjust pH=4.0-7.5, temperature at 28°C, DO=40%, rotating speed 500rpm, after cultivating for a period of time, DO rises rapidly to more than 100%, indicating After the consumption of glycerol in the medium was completed, 50% glycerol was added slowly, the glycerol flow rate was 30mL / h, and the DO was maintained above 35%, and the glycerol supplementation time was 4h.

[0055] Methanol induction stage: After stopping glycerol supplementation, DO rises rapidly to above 100%. After starvation for a period of time, methanol is slowly supplemented. The methanol flow rat...

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Abstract

The invention discloses a method for expressing human proinsulin C peptide by yeast pichia. The method comprises the following steps: firstly, human proinsulin C peptide genes are synthesized; secondly, the genes are recombined with yeast pichia expression vectors, and the recombinant expression vectors obtained are transferred into the yeast pichia; and thirdly, an engineering strain for expressing the human proinsulin C peptide in high-efficiency cells is obtained through screening of a large number of transformants.

Description

technical field [0001] The invention belongs to the production method of recombinant polypeptide medicine in the field of medicine. Background technique [0002] Human proinsulin C-peptide is the connecting peptide between chain A and chain B in human proinsulin, containing 31 amino acids. Human proinsulin is enzymatically hydrolyzed to form equimolar insulin and C-peptide. Insulin is used to control blood sugar in diabetic patients. C-peptide was once considered to have no clinical value. The latest research proves that C-peptide can reduce the most serious complications of diabetic patients, such as diabetic nephropathy, diabetic retinopathy and diabetic neuropathy. The combination of insulin and C-peptide can effectively control blood sugar and reduce the occurrence of diabetic complications. Since human proinsulin C-peptide has great therapeutic value and economic value, countries are now vigorously developing its production technology and preclinical research, and it h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/17C12N15/81C12N1/19C12P21/04C12R1/84
Inventor 王福清郑权王秀云
Owner TIBET JINKE GRP
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