Process for obtaining insulin and insulin derivatives having correctly bonded crystine bridges

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: 2004-03-24
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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  • Process for obtaining insulin and insulin derivatives having correctly bonded crystine bridges
  • Process for obtaining insulin and insulin derivatives having correctly bonded crystine bridges

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Embodiment 1 (comparative example, prior art)

[0095] Genetically modified E. coli cells were fermented (EP 0 489 780) to produce a fusion protein having the following amino acid sequence.

[0096] Proinsulin sequence 1 (SEQ ID NO.: 4):

[0097] Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg Phe Val Asn Gln HisLeu

[0098] Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly GluArg

[0099]Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp LeuGln

[0100] Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser LeuGln

[0101] Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly Ile Val GluGln

[0102] Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr CysAsn

[0103] Proinsulin sequence 1 corresponds to formula II, where

[0104] X is the C-peptide of human insulin (SEQ ID NO.: 3)

[0105] Y is threonine (B30),

[0106] R 1 is phenylalanine (B1),

[0107] R 2 is a peptide with 10 amino acid residues,

[0108] R 3 For asparagine (A21) and

[0109] A2-A20 ...

Embodiment 2

[0135] Embodiment 2 (method of the present invention)

[0136] A fusion protein having the amino acid sequence shown in Example 1 (proinsulin sequence 1. SEQ ID NO.: 4) was prepared by fermenting genetically modified Escherichia coli cells (EP 0 489 780).

[0137] The expressed fusion protein with proinsulin sequence 1 aggregated in E. coli cells to form inclusion bodies. After the fermentation culture is over, the cells are separated by centrifugation, the cells are broken by conventional high-pressure homogenization, and finally the inclusion bodies of the released fusion protein are separated by centrifugation.

[0138] 5 kg of cysteine ​​hydrochloride hydrate was added to the fusion protein aqueous suspension containing 40 kg of fusion protein (as determined by lyophilized samples).

[0139] The suspension containing proinsulin sequence 1 (the concentration of the fusion protein containing insulin determined by HPLC is 50%) was dissolved in 550 L of urea solution with a p...

Embodiment 3

[0144] Embodiment 3 (comparative embodiment, prior art)

[0145] A fusion protein with the following amino acid sequence was prepared by fermentation of genetically modified E. coli cells (EP 0 489 780)

[0146] Proinsulin sequence 2 (SEQ ID NO.: 5):

[0147] Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg Phe Val Asn Gln HisLeu

[0148] Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly GluArg

[0149] Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp LeuGln

[0150] Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser LeuGln

[0151] Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly Ile Val GluGln

[0152] Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr CysGly

[0153] Proinsulin sequence 2 corresponds to formula II, where

[0154] X is the C-peptide of human insulin (SEQ ID NO.: 3)

[0155] Y is threonine (B30),

[0156] R 1 is phenylalanine (B1),

[0157] R 2 is a peptide with 10 amino acid residues,

[0158] R 3 for glycine (A21) and

[0159] A2-...

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PUM

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Abstract

The present invention relates to an improved process for obtaining a precursor of an insulin or insulin derivative having correctly bonded cystine bridges in the presence of cysteine or cysteine hydrochloride and chaotropic auxiliary.

Description

[0001] This application is a divisional application with the filing date of August 17, 1998, the application number of 98117909.6, and the title of the invention entitled "Improved Method for Obtaining Insulin Precursor with Correctly Bonded Cystine Bonds". technical field [0002] The present invention relates to an improved process for obtaining precursors of insulin or insulin derivatives with properly bonded cystine bonds in the presence of cysteine ​​or cysteine ​​hydrochloride and a chaotropic agent. The present invention also relates to a method for obtaining insulin or insulin derivatives from the aforementioned precursors. Background technique [0003] Human insulin protein has two chains of amino acids 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 biologically active human insulin, the A...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A61K38/00A61K38/28A61P5/50C07K1/02C07K1/113C07K1/12C07K1/14C07K14/575C07K14/62C12N1/21C12N15/00C12P21/02C12R1/19
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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