Polygene coexpression system and production method containing disulfide-bond functional protein

A co-expression, multi-gene technology, applied in the field of soluble active protein, can solve the problem of low yield and high

Inactive Publication Date: 2012-08-22
EAST CHINA UNIV OF SCI & TECH
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Problems solved by technology

At present, although it has been reported that the co-expression or fusion expression of the target protein and disulfide bond isomerase PDI has produced a soluble functional target protein [M.Ostermeier et al., J Biol Chem, 1996, 271 (18): 10616-10622], but

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  • Polygene coexpression system and production method containing disulfide-bond functional protein
  • Polygene coexpression system and production method containing disulfide-bond functional protein
  • Polygene coexpression system and production method containing disulfide-bond functional protein

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Embodiment Construction

[0039]The first aspect of the present invention is to provide genes related to enzymes or chaperones that are co-transcribed and translated with the target protein gene in a prokaryotic cell system to promote the correct folding of the target protein to form a soluble functional protein, or two or three Gene nucleotide sequences of fusion proteins composed of these related enzymes or chaperone proteins. Due to the degeneracy of the codons of the gene nucleotide sequence encoding the protein (that is, some amino acids can have two, or three, or four codons, which are called the degenerate codons of the amino acids), the present invention includes All degenerate nucleotide sequences encoding the related enzymes or chaperone proteins containing such degenerate codons in the gene can produce proteins with the same amino acid sequence after transcription and translation.

[0040] The term "target protein" used herein refers to a protein containing disulfide bonds, especially a prot...

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Abstract

The invention provides a polygene coexpression system which comprises an expression vector of disulfide-bond target protein coding genes, and one or more than one expression vector of coding genes of an enzyme and/or chaperonin which has the effect of promoting the functionalization of disulfide-bond target protein; the coding genes of the enzyme and/or chaperonin are constructed in one reading frame of the expression vector in a form of fusion genes, or two or more than two of the coding genes of the enzyme and/or chaperonin are respectively constructed in different reading frames of the expression vector; the coding genes of the target protein are constructed in an expression vector which contains different resistant genes from the expression vector of the enzyme and/or chaperonin. The invention also provides a preparation method of the polygene coexpression system, and a method for preparing soluble functional target protein by using the preparation method. The polygene coexpression system of the invention enables the expressed disulfide bond-containing protein to form correct disulfide bonds, to fold into correct spatial conformation, and thus to dissolve in a cracking supernatant with maintained corresponding biological functions or activity.

Description

technical field [0001] The invention relates to the fields of gene technology and biochemistry, and relates to expressing eukaryotic disulfide bond proteins in prokaryotic bacteria to form correct disulfide bonds, fold into correct conformations, and become soluble active proteins. Specifically, genes such as homologous sulfhydryl oxidase, disulfide bond isomerase, peptidyl-prolyl cis-trans isomerase, chaperone protein and disulfide bond protein genes that can promote the correct formation of disulfide bonds were used in prokaryotic cells A method for co-expressing these related enzyme proteins and chaperones and disulfide bond proteins extracellularly to produce soluble active disulfide bond proteins and related substances. The invention also relates to the technical field of renaturation of inclusion body protein. Background technique [0002] The advantages of recombinant expression of eukaryotic proteins with Escherichia coli (E.coli.) prokaryotic system are low cost an...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/62C12P21/00C07K2/00C07K19/00
Inventor 杨弋郑文云张文耀
Owner EAST CHINA UNIV OF SCI & TECH
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