Recombinant human proinsulin transpeptidation method and application in recombinant human proinsulin downstream purification thereof

A technology for recombinant human insulin and insulin, which is applied in the biological field, can solve the problems of many by-products, complexity, and increasing the difficulty of insulin purification, and achieve the effects of improving production efficiency, shortening time, and solving the long time of transpeptide reaction

Inactive Publication Date: 2013-01-09
上海泰龙生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the transpeptidation reaction is relatively complicated and takes a long time, the process of transpeptidation usually brings some by-products that are difficult to remove. amino acid threonine), which will increase the difficulty of insulin purification
The existing recombinant human insulin has long transpeptide reaction time and many by-products in the process of separation and purification

Method used

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  • Recombinant human proinsulin transpeptidation method and application in recombinant human proinsulin downstream purification thereof
  • Recombinant human proinsulin transpeptidation method and application in recombinant human proinsulin downstream purification thereof
  • Recombinant human proinsulin transpeptidation method and application in recombinant human proinsulin downstream purification thereof

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Embodiment 1

[0038] Embodiment 1 Purification of recombinant human insulin

[0039] Process steps:

[0040] A, Purification of proinsulin expressed by Pichia pastoris by SP cation exchange chromatography

[0041] 4 liters of recombinant human proinsulin yeast fermentation broth (prepared according to the method described in Kjeldsen T, Pettersson AF, Hach M: Secretory expression and characterization of insulin in Pichia pastoris. Biotechnol Appl Biochem 1999, 29: 79-86), centrifuged at 7000RPM Collect the supernatant. SP Sepharose FF (purchased by GE) was packed with 200 ml of XK50 / 30 chromatographic column. First, the SP column was equilibrated for 5 column volumes with 25 mmol acetic acid-sodium acetate pH3.5 buffer solution. After the equilibrium, the pH of the centrifuged supernatant was adjusted to 3.5. After loading the sample, wash the column with the equilibration solution to the baseline, about 3 column volumes. Sodium dihydrogen phosphate / disodium hydrogen phosphate buffer sol...

Embodiment 2

[0073] Embodiment 2 Purification of recombinant human insulin

[0074] Process steps:

[0075] A, Purification of proinsulin expressed by Pichia pastoris by SP cation exchange chromatography

[0076] 4 liters of recombinant human proinsulin yeast fermentation broth (prepared according to the method described in Kjeldsen T, Pettersson AF, Hach M: Secretory expression and characterization of insulin in Pichia pastoris. Biotechnol Appl Biochem 1999, 29: 79-86), centrifuged at 7000RPM Collect the supernatant. SP Sepharose FF (purchased by GE) was packed with 200 ml of XK50 / 30 chromatographic column. First, the SP column was equilibrated for 5 column volumes with 25 mmol acetic acid-sodium acetate pH3.5 buffer solution. After the equilibrium, the pH of the centrifuged supernatant was adjusted to 3.5. After loading the sample, wash the column with the equilibration solution to the baseline, about 3 column volumes. Sodium dihydrogen phosphate / disodium hydrogen phosphate buffer sol...

Embodiment 3

[0096] Embodiment 3 Purification of recombinant human insulin

[0097] Process steps:

[0098] A, Purification of proinsulin expressed by Pichia pastoris by SP cation exchange chromatography

[0099] 4 liters of recombinant human proinsulin yeast fermentation broth (prepared according to the method described in Kjeldsen T, Pettersson AF, Hach M: Secretory expression and characterization of insulin in Pichia pastoris. Biotechnol Appl Biochem 1999, 29: 79-86), centrifuged at 7000RPM Collect the supernatant. SP Sepharose FF (purchased by GE) was packed with 200 ml of XK50 / 30 chromatographic column. First, the SP column was equilibrated for 5 column volumes with 25 mmol acetic acid-sodium acetate pH3.5 buffer solution. After the equilibrium, the pH of the centrifuged supernatant was adjusted to 3.5. After loading the sample, wash the column with the equilibration solution to the baseline, about 3 column volumes. Sodium dihydrogen phosphate / disodium hydrogen phosphate buffer solut...

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Abstract

The invention provides a recombinant human proinsulin transpeptidation method and an application in recombinant human proinsulin downstream purification thereof. The recombinant human proinsulin transpeptidation method is characterized by carrying out transpeptidation reaction on purified recombinant human proinsulin to obtain recombinant human insulin, wherein the reaction system of the transpeptidation reaction comprises 10-30g/L of recombinant human proinsulin, 100-200ml/L of dimethyl sulfoxide (DMSO), 63-103g/L of o-tert-butyl threonine tert-butyl ester, 600-800ml/L of 1,4-butanediol, 2-6g/L of trypsin, 0.2-100mmol/L of Ca<2+>, 0.2-100mmol/L of Mg<2+>, and water, and the pH value is 6.5-7.0. The reaction conditions of the transpeptidation reaction comprise a temperature of 27-47 DEG C, and a stirring time of 2-10 h. According to the invention, the recombinant human proinsulin transpeptidation method disclosed herein can be applied in downstream purification of recombinant human proinsulin. The recombinant human proinsulin transpeptidation method disclosed herein shortens the time of the transpeptidation reaction, raises the production efficiency, and can reduce by-products.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to the downstream purification of recombinant human insulin, and more specifically, the present invention relates to a recombinant human proinsulin transpeptide method and its application in the downstream purification of recombinant human insulin. Background technique [0002] Diabetes is the third leading cause of death in the world after tumors and cardiovascular diseases. There are more than 200 million people with diabetes in the world today. Insulin is a specific drug for treating diabetes, especially for the treatment of type I diabetes and late type II diabetes There is no other drug that can replace it. Insulin has been in clinical application for more than 80 years. With the development of biotechnology, recombinant human insulin has gradually replaced animal insulin. It is of practical significance to improve production efficiency and improve product quality in the p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/06C07K14/62C07K1/16C12R1/84
CPCY02P20/55
Inventor 郭颀然许必雄
Owner 上海泰龙生物医药科技有限公司
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