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Method for purifying non-labeled CRM197 protein by using IMAC chromatography

A CRM197, chromatographic purification technology, applied in the field of genetic engineering, can solve problems such as changing protein spatial structure, protein activity and immunogenicity, and achieve the effect of increasing binding capacity

Active Publication Date: 2021-09-17
CHENGDU KANGHUA BIOLOGICAL PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The interaction strength of immobilized metal ions depends on the type, quantity and spatial distribution of amino acid side chains. Usually, 6 His are added to the C-terminus or N-terminus of the protein during design to enhance the binding ability to metal ions. However, this design will inevitably change the spatial structure of the protein and affect the activity and immunogenicity of the protein.

Method used

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  • Method for purifying non-labeled CRM197 protein by using IMAC chromatography
  • Method for purifying non-labeled CRM197 protein by using IMAC chromatography
  • Method for purifying non-labeled CRM197 protein by using IMAC chromatography

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Embodiment 1

[0033] (1) The present invention analyzes and optimizes the CRM197 sequence, replaces rare codons with commonly used codons in Escherichia coli, and balances the ratio and distribution of the four bases A, T, C, and G. Insert BamHI and NcoI restriction sites at both ends of the CRM197 sequence, respectively.

[0034] (2) After the optimized and synthesized CRM197 gene was double-digested with BamHI and NcoI, it was connected to the expression vector pET28a(+) that was also double-digested with BamHI and NcoI, and transformed into E. coli competent cell DH5α, cultured at 37°C overnight. The next day, single clones were picked in 5 mL LB (Kana) liquid medium, cultured at 37°C, 220 r / min for 12 hours, plasmids were extracted, BamHI and NcoI double enzyme digestion was performed to identify the insertion of the target gene, and sent for sequencing. The correctly sequenced plasmid was named pET28a(+)-CRM197.

[0035] (3) The pET28a(+)-CRM197 plasmid was transformed into Escherich...

Embodiment 2

[0044] (1) The present invention analyzes and optimizes the CRM197 sequence, replaces rare codons with commonly used codons in Escherichia coli, and balances the ratio and distribution of the four bases A, T, C, and G. Insert BamHI and NcoI restriction sites at both ends of the CRM197 sequence, respectively.

[0045] (2) After the optimized and synthesized CRM197 gene was double-digested with BamHI and NcoI, it was connected to the expression vector pET28a(+) that was also double-digested with BamHI and NcoI, and transformed into E. coli competent cell DH5α, cultured at 37°C overnight. The next day, single clones were picked in 5 mL LB (Kana) liquid medium, cultured at 37°C, 220 r / min for 12 hours, plasmids were extracted, BamHI and NcoI double enzyme digestion was performed to identify the insertion of the target gene, and sent for sequencing. The correctly sequenced plasmid was named pET28a(+)-CRM197.

[0046] (3) The pET28a(+)-CRM197 plasmid was transformed into Escherich...

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Abstract

The invention discloses a method for purifying non-labeled CRM197 protein by using IMAC chromatography. The method comprises the following steps of: (1) treating a non-labeled CRM197 protein inclusion body obtained by an escherichia coli expression system with a denaturant; (2) filtering, then loading a sample on an IMAC chromatographic column, eluting with an eluent, and collecting an elution peak; (3) renaturing with a renaturation solution, and performing ultrafiltration concentration; and (4) then performing molecular sieve chromatography or hydrophobic chromatography to obtain the CRM197 protein. According to the method disclosed by the invention, a His label does not need to be added, and only the protein needs to be denatured, so that a protein structure is opened, His inside the protein structure is exposed, and the binding capacity with metal ions is increased; and then the target protein is separated from impurities according to appropriate elution steps. The technical scheme is particularly suitable for purification of the inclusion body, and a sample with the purity of more than 90% can be obtained by one-step chromatography.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a method for purifying non-labeled CRM197 protein by using IMAC chromatography. Background technique [0002] Diphtheria toxin (DT) is an exotoxin synthesized by β phage tox gene after lysogenization of Bacillus diphtheriae β phage. The full length of the DT molecule is 533aa, the molecular weight is 58330D, Cys186 and Cys201 form a disulfide bond, and the stalk-shaped ring TL1 (187-200aa) in between is rich in Arg and is easily cut by trypsin, making DT an A linked by a disulfide bond Fragment (1-193aa) and Fragment B (194-533aa). [0003] Diphtheria toxin mutant (Cross reacting material, CRM197) is a mutant that loses the toxicity of diphtheria toxin. It is a mutation of a base G to A in the nucleotide sequence of wild-type DT, resulting in the 52nd amino acid Gly becomes Glu. Structurally, CRM197 has a complete DT functional structure. However, experiments ha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/34C07K1/22
CPCC07K14/34C07K1/22Y02A50/30
Inventor 魏鑫侯文礼黄杰孙俊陈邱李松赖艺薛冰冯晓
Owner CHENGDU KANGHUA BIOLOGICAL PROD