Method for preparing enzyme through coexpression of recombinant protease and molecular chaperone

A molecular chaperone and protease technology, applied in the field of recombinant protein expression, can solve problems such as difficult to obtain soluble expression of protease

Inactive Publication Date: 2011-11-23
上海近岸科技有限公司
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Problems solved by technology

[0004] Experiments show that the genes encoding formaldehyde dehydrogenase and formaldehyde oxide dismutase are constructed in the pET expression vector (containing T7 promoter), so that the protease does not contain a fusion tag or contains a fu

Method used

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  • Method for preparing enzyme through coexpression of recombinant protease and molecular chaperone
  • Method for preparing enzyme through coexpression of recombinant protease and molecular chaperone
  • Method for preparing enzyme through coexpression of recombinant protease and molecular chaperone

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Embodiment

[0028] 1. Gene source

[0029] Protease sequences are available via the Uniprot protein database under accession numbers E4RD31, B7V5W2, Q52078. Perform reverse transcription on the amino acid sequence of the protease to obtain the gene nucleotide (DNA) sequence, and optimize the codon preferred by Escherichia coli for the DNA sequence (codon optimization is completed on the following website: http: / / www.jcat.de / ) . Gene synthesis codon-optimized gene sequence, sequenced correctly, to obtain the target gene. The amino acid sequence whose sequence number is E4RD31 is shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.2; the amino acid sequence whose sequence number is B7V5W2 is shown in SEQ ID NO.3, and its nucleotide sequence The sequence is shown in SEQ ID NO.4; the amino acid sequence of Q52078 is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.6.

[0030] 2. Construction of expression vector pCold recombinant plasmid

[0031]...

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Abstract

The invention discloses a method for preparing enzyme through coexpression of recombinant protease and molecular chaperone. In the method, recombinant plasmids for encoding formaldehyde dehydrogenase and formaldehyde superoxide dismutase are respectively converted into expression strains containing molecular chaperone coexpression plasmids, so that protease and the molecular chaperone are co-expressed and soluble recombinant expression of the protease (comprising histidine tag or not comprising the histidine tag) is achieved. The formaldehyde dehydrogenase, the formaldehyde superoxide dismutase and the histidine tag are subjected to fusion expression, soluble recombinant protein is subjected to affinity purification by the histidine tag to form the protein with the purity of over 90 percent, and about 1 milligram of protease can be obtained when 1 gram of thalli are obtained averagely. An enterokinase cutting sequence is introduced between the histidine tag and the protease, the tag can be removed through enzyme cutting, and amino acid carrier residue is absent. By the method, the soluble expression of the protease in an Escherichia coli expression system can be promoted, and massive recombinant protease can be efficiently prepared through purification.

Description

technical field [0001] The invention relates to a method for expressing a recombinant protein, in particular to a method for co-expressing a recombinant protease and a molecular chaperone to prepare an enzyme. Background technique [0002] Formaldehyde dehydrogenase (Glutathione-independent formaldehyde dehydrogenase) (gene: fdhA) proteases from the two species of Pseudomonas putida and Pseudomonas aeruginosa have 86% identical amino acid sequences. Its properties are similar to type III alcohol dehydrogenase (Alcoholdehydrogenase, ADH). The recombinant expression information about this type of aldehyde dehydrogenase in the literature is very limited. In the report (Biochemistry 39, 10720-10729, 2000), pKK223-3 (Tacpromoter, i.e. Tac promoter) expression vector and Escherichia coli TG1 expression strain were used Soluble expression of aldehyde dehydrogenase from Pseudomonas putida was obtained. Purified by Mimetic Blue-2 affinity column (using the binding properties of ald...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/70C12R1/19
Inventor 冉晓园闫敏刘程王爽
Owner 上海近岸科技有限公司
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