Method used for selective modification of protein carbon terminal carboxyl groups with polyethylene glycol

A polyethylene glycol and selective technology, applied in the field of protein chemistry, can solve the problems of protein molecular cross-linking and poor selectivity, and achieve the effect of overcoming poor selectivity and realizing selective modification

Inactive Publication Date: 2014-03-12
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method overcomes the poor selectivity existing in the traditional protein carboxyl modification, and the problem of prone to protein intramolecular and intermolecular crosslinking

Method used

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  • Method used for selective modification of protein carbon terminal carboxyl groups with polyethylene glycol
  • Method used for selective modification of protein carbon terminal carboxyl groups with polyethylene glycol
  • Method used for selective modification of protein carbon terminal carboxyl groups with polyethylene glycol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The C-terminal carboxyl group selective modification of embodiment 1 recombinant staphylokinase

Embodiment approach

[0026] Replace staphylokinase into 0.05mol / L MES buffer (pH 5.5), then add EDC and cystamine to react at 4°C for 3 hours, wherein the molar ratio of EDC and cystamine to staphylokinase is 2:2:1, Then add TCEP for reduction reaction, react at 4°C for 2 hours, and finally react with PEG-mal with a molecular weight of 20kDa, the molar ratio of staphylokinase and polyethylene glycol modifier is 1:4, and react at 4°C for 8 hours. After separation and purification of the above-mentioned PEG-modified products, they were analyzed by Superdex 200 gel filtration column and SDS-PAGE electrophoresis.

[0027] result

[0028] Such as figure 1 As shown, a single characteristic elution peak of staphylokinase (SAK) appears on the gel filtration column. After introducing sulfhydryl groups on the staphylokinase molecule, the characteristic elution peak position of thiolated staphylokinase (SAK-SH) is basically No change occurred. After binding PEG molecules, the position of the elution peak ...

Embodiment 2

[0030] The PEG modification site identification of embodiment 2 staphylokinase single modification product

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Abstract

The invention relates to a polyethylene glycol (PEG) modification method. The method is capable of realizing selective modification of carbon terminal carboxyl groups of proteins and polypeptides, and each protein molecule and polypeptide molecule is connected with one PEG molecule. According to the method, staphylokinase is used as a model protein. The polyethylene glycol (PEG) modification method comprises following main steps: (1) under slightly acidic conditions (pH 5.0), carboxyl groups of staphylokinase and amino groups of cystamine are connected; and (2) reduction of disulfide bonds of cystamine is realized using a reducing agent, and generated sulfhydryl groups are reacted with methoxy polyethylene glycol-maleinimide (mPEG-Mal). pKa value of the carbon terminal carboxyl groups of staphylokinase is 2.1 to 2.4, pKa value of side chain carboxyl groups of aspartic acid is 3.7 to 4.0, and pKa value of side chain carboxyl groups of glutamic acid is 4.2 to 4.5, so that under the conditions with a pH value of 5.0, PEG is capable of realizing selective modification of the carbon terminal carboxyl groups of staphylokinase. The method is mainly used for PEG selective modification of the carbon terminal carboxyl groups of the proteins and the polypeptides; a novel PEG modification method is provided; and an advantage of the method is that uniformity of modification sites of the PEG modification product is excellent.

Description

technical field [0001] The invention belongs to the fields of protein chemistry and biomedicine, and relates to a modification method for selectively modifying protein C-terminal carboxyl groups with polyethylene glycol (PEG). Background technique [0002] Biopharmaceuticals developed using technologies such as molecular biology, structural biology, molecular immunology, genetic engineering, protein engineering, and monoclonal antibodies can make it possible to treat various diseases. Among them, gene recombinant protein and polypeptide drugs are the most prominent class of biological drugs. Since the first genetically engineered drug, recombinant human insulin, was launched in 1982, a large number of gene-recombined protein and peptide drugs have been used in clinical research and have achieved great success. Compared with traditional small-molecule chemical drugs, biopharmaceuticals are identical or nearly identical to endogenous proteins in terms of molecular structure a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/107C12N9/96C12N9/48
Inventor 胡涛王俊苏志国马光辉安红波
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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