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Method for producing recombined insulin human

A technology of recombinant human insulin and human insulin, which is applied to the preparation methods of peptides, insulin, chemical instruments and methods, etc., can solve the problems of complex production process, and achieve the effect of simple process and lower production cost.

Active Publication Date: 2008-05-07
JIANGSU WANBANG BIOPHARMLS +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Therefore, the technical problem to be solved by the present invention is to provide a preparation method for expressing recombinant human insulin in Escherichia coli to overcome the technical defects of using the toxic and harmful substance CNBr and complex production process in the prior art

Method used

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  • Method for producing recombined insulin human
  • Method for producing recombined insulin human
  • Method for producing recombined insulin human

Examples

Experimental program
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Embodiment 1

[0028] The preparation of embodiment 1 recombinant human insulin

[0029] Step 1: Cell disruption and inclusion body collection

[0030] Add 1 liter of TE buffer solution [5mM EDTA; 50mM Tris-HCl (pH9.0)] for every 100g of wet bacteria, mix well, crush in a homogenizer at 700-750 bar, until the bacteria are completely broken, use a centrifuge Centrifuge to collect the precipitate. The obtained precipitates are mainly inclusion bodies, which are washed three times as follows to remove most of the impurities: 1 L of STET buffer [5mM EDTA; 50mM Tris-HCl (pH 9.0); Triton-X100, 3% (V / V) per 100g of precipitate ] and mix well, and stir at room temperature for 30 minutes; centrifuge again to collect the precipitate; the inclusion bodies after washing are detected by SDS-PAGE, see attached figure 1 As shown, it can be seen that its components are less, the purity is higher, and inclusion body dissolution can be performed.

[0031] Step 2: Inclusion Body Solubilization

[0032]Add ...

Embodiment 2

[0042] The preparation of embodiment 2 recombinant human insulin

[0043] Proinsulin S-sulfonate purified by SP column was diluted with 50mM Gly buffer solution (pH11.5) containing 1M urea to make the protein concentration reach 0.75mg / ml, degas immediately, fill with nitrogen, and add the prepared mercaptoethanol solution, so that the molar ratio of its concentration to proinsulin is 1:3, and stirred at 4°C for 12 hours; through this step of folding, at least 60% of proinsulin S-sulfonate can be folded into the correct conformation. Other operations are the same as in Example 1. The purity of the insulin reaches over 99.0%, and the SDS-PAGE test has the same molecular weight as the insulin standard. Has the same RP-HPLC retention characteristics as human insulin standards.

Embodiment 3

[0044] Embodiment 3 Redo the preparation of human insulin

[0045] Proinsulin S-sulfonate purified by SP column was diluted with 50mM Gly buffer solution (pH11.5) containing 1M urea to make the protein concentration reach 1.0mg / ml, immediately degassed, filled with nitrogen, and added to the prepared A solution of mercaptoethanol so that the molar ratio of its concentration to proinsulin was 1:6 was stirred at 4°C for 12 hours.

[0046] Others are the same as implementation 1. The purity of the insulin reaches over 99.0%, and the SDS-PAGE detection has the same molecular weight as the insulin standard product, and has the same RP-HPLC retention characteristics as the human insulin standard product.

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Abstract

The invention discloses a preparation method of recombinant human insulin, which relates to the protein polypeptide type medicine field. The technical problem needed to be solved by the invention aims at providing a preparation method for Escherichia coli to express the recombinant human insulin in order to overcome the technical disadvantages in the prior art of using the poisonous and harmful substance CNBr and having a complex production process. The preparation method for the recombinant human insulin of the invention comprises the steps of: thallus crashing, inclusion body dissolving, proinsulin sulfonating, purifying proinsulin, and proinsulin renaturation, enzyme cutting transmission, etc., and the preparation method is characterized in that during the inclusion body dissolving process, the -SH of proinsulin is transformed into -SSO3<->, and then human insulin is obtained through purifying the sulfonated proinsulin, reduction renaturation and enzyme cutting transmission by positive ion, and then through the negative ion exchange layer purification. The preparation method of the recombinant human insulin of the invention does not use the CNBr and has simple technique with high human insulin yield coefficient and purity.

Description

technical field [0001] The invention relates to the field of protein and polypeptide drugs, in particular to a preparation method of recombinant human insulin. Background technique [0002] Diabetes is the third leading cause of death after cardiovascular and tumors. There are 200 million diabetic patients in the world today, and about 6 million new cases are added every year, and 3.2 million people die from diabetes and related complications every year (2006 Report of the American Diabetes Annual Meeting, June 2009). Insulin is a specific drug for the treatment of diabetes, especially in the treatment of patients with type I diabetes and type II diabetes, and there is no other drug that can replace it. Insulin has been successfully used clinically for more than 80 years. With the development of biotechnology, recombinant human insulin has gradually replaced animal insulin extracted from animal viscera. With the expansion of the population of diabetic patients and the div...

Claims

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Application Information

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IPC IPC(8): C07K14/62C07K1/14
Inventor 王伟刚曹韫旭文良柱
Owner JIANGSU WANBANG BIOPHARMLS
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