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Method for obtaining insulin or insulin derivatives with correctly bonded cystine bonds

An insulin derivative, cystine bond technology, applied in microorganism-based methods, biochemical equipment and methods, insulin, etc., can solve problems such as loss

Inactive Publication Date: 2012-02-08
SANOFI AVENTIS DEUT GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the subsequent purification process to obtain the final product, there will also be a large loss

Method used

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  • Method for obtaining insulin or insulin derivatives with correctly bonded cystine bonds
  • Method for obtaining insulin or insulin derivatives with correctly bonded cystine bonds
  • Method for obtaining insulin or insulin derivatives with correctly bonded cystine bonds

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Example 1 (comparative example, prior art)

[0096] The genetically modified E. coli cells were fermented (EP 0 489 780) to prepare a fusion protein with the following amino acid sequence.

[0097] Proinsulin sequence 1 (SEQ ID NO.: 4):

[0098] Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg Phe Val Asn Gln HisLeu

[0099] Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly GluArg

[0100] Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp LeuGln

[0101] Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser LeuGln

[0102] Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly Ile Val GluGln

[0103] Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr CysAsn

[0104] Proinsulin sequence 1 corresponds to formula II, where

[0105] X is the C-peptide of human insulin (SEQ ID NO.: 3)

[0106] Y is Threonine (B30),

[0107] R 1 Is phenylalanine (B1),

[0108] R 2 Is a peptide with 10 amino acid residues,

[0109] R 3 Is asparagine (A21) and

[0110] A2-A20 is the amino acid sequence of human...

Embodiment 2

[0136] Example 2 (Method of the present invention)

[0137] By fermenting the genetically modified E. coli cells (EP0489780), a fusion protein having the amino acid sequence shown in Example 1 (proinsulin sequence 1. SEQ ID NO.: 4) was prepared.

[0138] The expressed fusion protein with proinsulin sequence 1 aggregated in E. coli cells to form inclusion bodies. After the fermentation and culture, the cells are separated by centrifugation, and the cells are broken by conventional high-pressure homogenization, and finally the released inclusion bodies of the fusion protein are separated by centrifugation.

[0139] 5 kg of cysteine ​​hydrochloride hydrate was added to an aqueous suspension of fusion protein containing 40 kg of fusion protein (determined by freeze-dried samples).

[0140] The suspension containing proinsulin sequence 1 (the concentration of the insulin-containing fusion protein is determined to be 50% by HPLC) was dissolved in 550 L of urea solution with a pH of 10.2 and...

Embodiment 3

[0145] Example 3 (comparative example, prior art)

[0146] By fermenting the genetically modified E. coli cells (EP0489780), a fusion protein with the following amino acid sequence was prepared

[0147] Proinsulin sequence 2 (SEQ ID NO.: 5):

[0148] Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg Phe Val Asn Gln HisLeu

[0149] Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly GluArg

[0150] Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp LeuGln

[0151] Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser LeuGln

[0152] Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly Ile Val GluGln

[0153] Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr CysGly

[0154] Proinsulin sequence 2 corresponds to formula II, where

[0155] X is the C-peptide of human insulin (SEQ ID NO.: 3)

[0156] Y is Threonine (B30),

[0157] R 1 Is phenylalanine (B1),

[0158] R 2 Is a peptide with 10 amino acid residues,

[0159] R 3 Is glycine (A21) and

[0160] A2-A20 is the amino acid sequence of human insu...

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Abstract

A process for obtaining insulin or insulin derivatives with properly bonded cystine bonds from a precursor of insulin or insulin derivatives in the presence of cysteine ​​or cysteine ​​hydrochloride and a chaotropic agent undergoes a folding process in the presence of , after which insulin or insulin derivatives are obtained from said precursor by enzymatic cleavage with trypsin or tryptase and optionally additionally with carboxypeptidase B, followed by Purification is carried out on adsorbent resins.

Description

[0001] This application is a divisional application of the application date of August 17, 1998, the application number is 03152330.7, and the title of the invention is "method for obtaining insulin or insulin derivative with correctly bonded cystine bond". Technical field [0002] The present invention relates to an improved method for obtaining insulin or insulin derivative precursors with correctly bonded cystine bonds in the presence of cysteine ​​or cysteine ​​hydrochloride and a chaotropic auxiliary. The present invention also relates to a method for obtaining insulin or an insulin derivative from the aforementioned precursor. Background technique [0003] Human insulin protein has two amino acid chains with a total of 51 amino acid residues. The two amino acid chains contain 6 cysteine ​​residues, and every two cysteine ​​residues are connected to each other by disulfide bonds. In human insulin with biological activity, the two chains of A and B are connected by two cystine...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/02C07K14/62C12N15/09A61K38/00A61K38/28A61P5/50C07K1/02C07K1/113C07K1/12C07K1/14C07K14/575C12N1/21C12N15/00C12R1/19
CPCC07K14/62A61K38/00A61P3/10A61P5/50C07K1/12C07K1/14C07K1/02
Inventor F-J·鲁伯洛德R·凯勒
Owner SANOFI AVENTIS DEUT GMBH
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