Method for increasing output of insulin precursors

A technology of insulin precursor and carrier, applied in the field of bioengineering, can solve problems such as excessive glycosylation, increase product antigenicity, difficult high-density fermentation culture, etc., and achieve the effect of increasing expression

Active Publication Date: 2017-05-24
JIANGNAN UNIV
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  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Escherichia coli and Saccharomyces cerevisiae have some problems as the expression system for producing human insulin, such as the problems in the expression system of Escherichia coli: (1) Since Escherichia coli can produce more proteases of its own, it is particularly easy to degrade small proteins similar to the precursor of human insulin; (2) The overexpression product will be produced in the cytoplasm in the form of inclusion bodies, which requires complex denaturation and renaturation processes before it can be converted into active insulin, increasing the difficulty and complexity of subsequent processing
Problems in the expression system of Sac

Method used

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  • Method for increasing output of insulin precursors
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  • Method for increasing output of insulin precursors

Examples

Experimental program
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Example Embodiment

[0055] Example 1: Construction of genetically engineered bacteria containing insulin precursor (PI)

[0056] (1) Digest the synthesized PI and Pichia pastoris expression vector pPIC9K with EcoRI and NotI respectively, and digest at 37°C for 2 hours;

[0057] (2) Recover the PI fragment and pPIC9K fragment after double digestion by gel respectively, connect the recovered PI and pPIC9K, and connect overnight at 16°C;

[0058] (3) Transform the overnight ligation product into JM109 competent, add 1mL liquid LB medium, incubate at 37°C, 200rpm for 2h, then spread ampicillin resistant plate, incubate upside down in 37°C incubator for 8h;

[0059] (4) Re-streak the single colony grown in the previous step on the ampicillin plate, invert it in the 37°C incubator for 8 hours, and perform colony PCR verification;

[0060] (5) Pick out the correct strains verified by colony PCR and culture them in 25mL / 250mL liquid LB medium containing ampicillin at 37°C and 200r / min overnight;

[0061] (6) Extrac...

Example Embodiment

[0065] Example 2: Construction of recombinant strains containing different copy numbers of insulin precursor genes

[0066] (1) Select the correct recombinant strain CL001, pick a single colony in 25mL / 250mL liquid YPD medium, culture at 30℃, 200r / min for 20h, transfer 0.5mL of the above bacterial liquid to 25mL YPD liquid medium , 30℃, 200r / min culture for 8h (approximately OD600=1.3-1.5) to prepare Pichia pastoris (CL001) competence;

[0067] (2) Inoculate the strain containing the expression vector pPIC9K-PI into 25mL / 250mL liquid LB medium containing ampicillin, cultivate overnight at 37°C, 200r / min, extract the plasmid from the overnight cultured strain, and then use SacI single enzyme digestion, 37 After digestion at ℃ for 2h, use PCR product purification column for column recovery;

[0068] (3) Pichia pastoris (CL001) transformed with the plasmid pPIC9K-PI after digestion with SacI, add 1 mL of 1 mol / L sorbitol and incubate for 1 h after electric shock, and apply 200 μL of li...

Example Embodiment

[0081] Example 3: Construction of recombinant strain containing SNAREs

[0082] (1) Extract baker's yeast genome, inoculate a single colony of baker's yeast in a 50mL / 500mLYPD Erlenmeyer flask, cultivate to logarithmic phase at 30°C, and extract the genome according to the Tiangen Yeast Genome Extraction Kit;

[0083] (2) Using yeast genome as template, amplify the components SNC2 and Sso2 in SNAREs;

[0084] (3) After verifying that the amplified target fragment is correct by agarose gel electrophoresis, connect to pMD19-T carrier and connect overnight at 16°C;

[0085] (4) Transform the ligation product into JM109 competent, add 1mL liquid LB medium, incubate at 37°C, 200r / min for 2h, then apply to ampicillin resistant plate, incubate upside down in 37°C incubator for 8h;

[0086] (5) Re-streak the grown single colony on the ampicillin resistant plate, invert it in the 37°C incubator for 8 hours, and then perform colony PCR verification;

[0087] (6) Send the verified strains for seque...

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Abstract

The invention discloses a method for increasing output of insulin precursors, and belongs to the technical field of biological engineering. The method comprises the following steps of connecting an artificially synthesized insulin precursor and a pichia pastoris expression carrier pPIC9K by dual-enzyme digestion through a molecular biological method, so as to build a recombination strain; performing G418 resistance screening, so as to obtain high-copying recombination type insulin precursor-containing engineering bacteria P.pastoris GS115-PI (CL012), and coexpressing SNC2 and Sso2 genes in the recombination strain. The method has the advantages that by utilizing high-density fermentation, the output of the insulin precursor of the strain is 78mg/L, and compared with the strain CL012, the output is increased by 47%.

Description

technical field [0001] The invention relates to a method for increasing the yield of insulin precursor, which belongs to the technical field of bioengineering. Background technique [0002] In recent years, with the development of social economy and the improvement of residents' living standards in various countries in the world, the incidence and prevalence of diabetes have been increasing year by year, becoming a major social problem that threatens people's health. Insulin drugs are specific and necessary drugs for the treatment of diabetes, but the contradiction between limited production and huge demand urgently requires us to find effective strategies to increase insulin production as soon as possible. [0003] At present, more people use Escherichia coli and yeast expression system to ferment and produce recombinant human insulin precursor, and then process it into active recombinant human insulin. For example: Eli Lilly uses Escherichia coli to produce recombinant hu...

Claims

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Application Information

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IPC IPC(8): C07K14/62C12N15/17C12N1/19C12N15/81C12P21/02C12R1/84
CPCC07K14/62C12N15/815C12N2800/102
Inventor 吴静梁晨晨
Owner JIANGNAN UNIV
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