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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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  • Abstract
  • Description
  • Claims
  • Application Information

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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 Saccharomyces cerevisiae: (1) The expression vector of Saccharomyces cerevisiae is unstable in passage; (2) The secreted and expressed products are often hyperglycosylated, and the core oligosaccharide chain of the glycoprotein contains α-1,3 glycan linkages Head, it will increase the antigenicity of the product, which is not conducive to treatment
(3) Saccharomyces cerevisiae produces ethanol during large-scale fermentation, making it difficult to carry out high-density fermentation culture
However, due to the process of expressing heterologous proteins in Pichia pastoris, there are many rate-limiting steps that limit the secretion of heterologous proteins

Method used

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  • Method for increasing output of insulin precursors

Examples

Experimental program
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Effect test

Embodiment 1

[0055] Embodiment 1: Containing the construction of insulin precursor (PI) genetically engineered bacteria

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

[0057] (2) Gel-recover the PI fragment and pPIC9K fragment after double enzyme digestion, respectively, connect the PI and pPIC9K after gel recovery, and connect overnight at 16°C;

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

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

[0060] (5) Pick the correct bacterial strains verified by colony PCR and culture them overnight in 25mL / 250mL liquid LB ...

Embodiment 2

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

[0066] (1) Pick a single colony of the verified recombinant strain CL001 in 25mL / 250mL liquid YPD medium, culture at 30°C, 200r / min for 20h, and transfer 0.5mL of the above bacterial liquid to 25mL YPD liquid medium , 30°C, 200r / min culture for 8h (about OD600=1.3-1.5), prepare Pichia pastoris (CL001) competent;

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

[0068] (3) Pichia pastoris (CL001) transformed with SacI-digested plasmid pPIC9K-PI was competent. After electroporation, 1 mL, 1 mol / L sorbitol was added to incubate for 1 h, and 200 μL of the liqui...

Embodiment 3

[0081] Embodiment 3: Contain the construction of SNAREs recombinant bacterial strain

[0082] (1) Extract the genome of baker's yeast, inoculate a single colony of baker's yeast into a 50mL / 500mLYPD conical flask, culture at 30°C to the logarithmic phase, and extract the genome according to the Tiangen yeast genome extraction kit;

[0083] (2) using the yeast genome as a template to amplify the components SNC2 and Sso2 in SNAREs;

[0084] (3) After the amplified target fragment was verified to be correct by agarose gel electrophoresis, it was ligated with pMD19-T carrier, and ligated 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 spread on ampicillin-resistant plate, and incubate in an incubator at 37°C upside down for 8h;

[0086] (5) Re-stretch the grown single colony on the ampicillin-resistant plate, incubate it upside down in a 37°C incubator for 8 hours, and perform c...

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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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