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Enzyme digestion transformation method of insulin glargine precursor

A technology of insulin glargine and its precursor, which is applied in the direction of fermentation, etc., to achieve the effects of high enzyme digestion yield, increased insulin glargine content, and easy subsequent purification

Inactive Publication Date: 2013-03-27
鲁南新时代生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports on the enzymatic digestion process and parameters of recombinant insulin glargine in the published literature

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Enzymatic conversion of insulin glargine precursor

[0026] At room temperature, dissolve 1 g of insulin glargine precursor (produced by Escherichia coli through recombinant DNA technology) in 100 ml of 20 mM ammonium bicarbonate buffer, pH 8.0, so that the protein concentration of the precursor is about 10 g / L. Control the temperature of the solution at 4°C, then add bovine trypsin to make enzyme: substrate = 1:1000, and start the enzyme cleavage reaction. The reaction temperature was maintained at 4°C.

[0027] After 5 hours, 6 mol / L hydrochloric acid solution was added to adjust the pH to 3.5 to stop the reaction. Take 10 μl of the digestion solution and analyze it by HPLC. According to the peak area of ​​insulin glargine, calculate the amount of insulin glargine actually obtained after enzyme digestion according to the peak area normalization method M 2 , so the enzymatic cleavage yield of insulin glargine is calculated to be about 86%.

Embodiment 2

[0028] Example 2 Enzymatic conversion of insulin glargine precursor

[0029] At room temperature, dissolve 1 g of insulin glargine precursor (produced by Escherichia coli through recombinant DNA technology) in 200 ml of 100 mM ammonium bicarbonate buffer, pH 10, so that the protein concentration of the precursor is about 5 g / L. Control the temperature of the solution at 25°C, then add TPCK-treated bovine trypsin to make enzyme:substrate = 1:10000, start the enzyme cleavage reaction, and keep the reaction temperature at 25°C.

[0030] After 30 hours, 6 mol / L hydrochloric acid solution was added to adjust the pH to 3.5 to stop the reaction. Take 10 μl of enzyme digestion solution and analyze and detect it by HPLC, calculate the amount of insulin glargine actually obtained after enzyme digestion according to the peak area of ​​insulin glargine 2 , so the enzymatic cleavage yield of insulin glargine is calculated to be about 83%.

Embodiment 3

[0031] Example 3 Enzymatic conversion of insulin glargine precursor

[0032] At room temperature, dissolve 1 g of insulin glargine precursor (produced by Escherichia coli through recombinant DNA technology) in 200 ml of 20 mM Tris-CL buffer, pH 9.0, so that the protein concentration of the precursor is about 5 g / L. Control the temperature of the solution at 4°C, then add porcine trypsin to make enzyme:substrate = 1:10000, start the enzyme cleavage reaction, and keep the reaction temperature at 4°C.

[0033] After 30 hours, 6 mol / L hydrochloric acid solution was added to adjust the pH to 3.5 to stop the reaction. Take 10 μl of enzyme digestion solution and analyze and detect it by HPLC, calculate the amount of insulin glargine actually obtained after enzyme digestion according to the peak area of ​​insulin glargine 2 , so the enzymatic cleavage yield of insulin glargine is calculated to be about 87%.

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PUM

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Abstract

The invention discloses an enzyme digestion transformation method of an insulin glargine precursor. The method comprises the following steps: dissolving the insulin glargine precursor in a buffer solution with pH of 8-11; controlling a temperature of a reaction system at 0-37 DEG C; adding trypsin according to a mass ratio of trypsin to insulin glargine precursor being 1:1000-10000; and reacting for 2-40 h. The enzyme digestion transformation method provided by the invention can significantly increase the amount of produced insulin glargine, while significantly reducing by-product, has high enzyme yield and easy purification of the enzyme digestion solution, and is suitable for industrial production.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a method for enzymatic cleavage and conversion of insulin glargine precursor, in particular to a method for enzymatic cleavage of insulin glargine precursor by using trypsin to convert it into insulin glargine. Background technique [0002] Glycine-arginine insulin (glycine-arginine insulin) is a long-acting insulin biological product obtained through genetic recombination technology and used to treat type Ⅰ and Ⅱ diabetes mellitus. , thus reducing the solubility, forming insulin glargine precipitates, delaying the body's absorption, and prolonging the time of its hypoglycemic effect. Recombinant insulin glargine is a very convenient and effective drug for the treatment of type I and type II diabetes. The trade name of recombinant insulin glargine produced by Aventis abroad is Lantus, and the trade name of recombinant insulin glargine injection produced by Gan & Lee Pharmac...

Claims

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Application Information

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IPC IPC(8): C12P21/06
Inventor 赵志全熊继元于锋臣
Owner 鲁南新时代生物技术有限公司
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