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Method for purifying gene-recombinant insulin precursor

A technology of insulin precursor and gene recombination, which is applied in the direction of insulin, chemical instruments and methods, peptide preparation methods, etc. It can solve the problems that the fractionation medium cannot be applied to large-scale production, the pigment cannot be completely removed, and it is unfavorable for large-scale production. , to achieve the effect of low cost, high speed, high purity and recovery rate

Active Publication Date: 2010-09-29
YICHANG HEC CHANGJIANG PHARMA CO LTD
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AI Technical Summary

Problems solved by technology

The decolorization and separation methods reported in the literature generally use HPLC, which is not conducive to large-scale production
However, the method of cation exchange chromatography cannot completely remove the pigments in the Pichia fermented liquid. Although hydrophobic chromatography can remove most of the pigments, the exchange capacity is small, or the binding force is too weak, or the activity recovery rate is low. Insufficient low, so its application is limited
Gel filtration can also remove pigments well, but there is a medium used for fractionation that is limited by the amount of sample loaded and cannot be applied to large-scale production

Method used

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  • Method for purifying gene-recombinant insulin precursor

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Comparison scheme
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Embodiment

[0022] 1. After passing the pretreated powdered activated carbon through a 60-mesh sieve, weigh 20 grams and add it to 2 liters of fermentation broth. Stir with a glass rod for 10 minutes, then centrifuge in a high-speed centrifuge at 8,000 rpm for 20 minutes, and collect 1 L of supernatant.

[0023] 2. Weigh 30 grams of pretreated granular activated carbon (20mm×40mm), and put it on a 20cm×2.6cm glass column. After washing with 1% acetic acid aqueous solution, the collected supernatant was introduced into the column with a pump, and the flow rate was controlled at 3 mL / min. After the sample was loaded, the sample was also ejected from the activated carbon column with 1% acetic acid aqueous solution. Collect 1.2L of penetrating fluid.

[0024] 3 Cationic filler sp550EC, take 100mL and put it on a 20cm×2.6cm glass column.

[0025] The balance solution is pH4.0, 50mmolNaAc-HAC

[0026] The washing solution is pH4.0, 50mmolNaAc-HAC+0.1mol / L NaCl+40% ethanol

[0027] The elue...

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Abstract

A process for purifying the genetically recombined insulin precursor features that the activated carbon as adsorbent and cationic chromatography are used to remove the pigment from fermented liquid and purify the active protein. Its recovery rate is more than 85% and its purity is more than 95%.

Description

technical field [0001] The invention belongs to a method for producing insulin, and mainly relates to the expression of genetically recombined insulin precursor protein in Pichia pastoris. Background technique [0002] Extracting natural insulin from animals and plants cannot meet the clinical needs. It has been reported to express recombinant insulin using hosts such as Escherichia coli, Saccharomyces cerevisiae, and Hansenula, but the yield is low (<0.5mg / mL). The artificially synthesized exogenous porcine insulin gene is expressed in hosts such as Escherichia coli and Saccharomyces cerevisiae; it has the disadvantages of easy formation of inclusion bodies, low expression yield and more miscellaneous proteins. [0003] Using Pichia pastoris as an expression system overcomes the above shortcomings. Using Pichia pastoris as a host can greatly increase the production of recombinant insulin (≥1.0 mg / mL), and has a good application prospect. However, a large amount of pigm...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/14C07K14/62C12N1/19
Inventor 周红谢俊杰张富权葛小荆鲁斌
Owner YICHANG HEC CHANGJIANG PHARMA CO LTD
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