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Method for preparing recombinant bovine enterokinase catalytic subunit protein

A kinase catalytic subunit and protein technology, applied in the field of genetic engineering, can solve problems such as limiting wide application, and achieve the effects of simple production process, significant application value, and high protein yield

Active Publication Date: 2016-06-29
北京世纪伟信医药科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Naturally extracted enterokinase is easily mixed with other types of proteases, which limits its wide application in genetic engineering

Method used

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  • Method for preparing recombinant bovine enterokinase catalytic subunit protein
  • Method for preparing recombinant bovine enterokinase catalytic subunit protein
  • Method for preparing recombinant bovine enterokinase catalytic subunit protein

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Embodiment Construction

[0025] The method for preparing the catalytic subunit protein of recombinant bovine enterokinase comprises the following steps:

[0026] (1) In order to improve the activity and stability of the recombinant protein, based on the crystal structure of bovine enterokinase and the gene EKL of the catalytic subunit of bovine enterokinase as a template, the mutant protein EKLM was designed, and the mutation sites included C112S, S121P and Y175R. The full-length nucleotide sequence of the mutated EKLM is shown in SEQ ID NO.1.

[0027] (2) In order to increase the protein yield, the codons unfavorably expressed by Escherichia coli in the mutant EKLM were replaced to obtain the optimized gene sequence EKLMO. The nucleotide sequence of EKLMO is shown in SEQ ID NO.2.

[0028] (3) For the convenience of subsequent purification, the cleavage site sequence DDDDK of bovine enterokinase itself was added to the 5' end of EKLMO, and four consecutive histidine tag sequences HIS were added to the...

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Abstract

The invention relates to preparation methods for recombinant protein in the field of genetic engineering, in particular to a preparation method for recombinant bovine enterokinase catalytic subunit protein. The preparation method comprises the steps that (1) a mutant EKLM is constructed on a gene EKL of a bovine enterokinase catalytic subunit, wherein a nucleotide sequence of the mutant EKLM is shown as SEQIDNO.1; (2) codons, which are not beneficial to expression, of colibacillus in the mutant EKLM are replaced, so that an optimized gene sequence EKLMO is obtained, wherein a nucleotide sequence of the EKLMO is shown as SEQIDNO.2. On the basis of the preparation method for the recombinant bovine enterokinase catalytic subunit protein, the activity of the bovine enterokinase catalytic subunit can be effectively maintained, and the protein yield is quite high. In addition, according to the method, the production process is simple, and product quality is stable. The post-purification protein can be used for bioengineering or pharmaceutical production or the like, thereby having significant application value.

Description

technical field [0001] The invention relates to a method for preparing a recombinant protein in the field of genetic engineering, in particular to a method for preparing a recombinant bovine enterokinase catalytic subunit protein. Background technique [0002] Enterokinase (Enterokinase, abbreviated as EK) is a heterodimeric serine protease present in the duodenum of mammals. Its natural protein molecular weight is 150kDa, which is composed of a heavy chain of 115kDa and a light chain of 35kDa, at pH 4.5-9.5 and temperature 4-45 O Both C can specifically hydrolyze protein substrates. The sequence of the substrate protein recognized by enterokinase is DDDDK, which can cleave at the C-terminus of lysine residues. Such enzyme cutting advantages make it an ideal tool enzyme in fusion protein expression system. Naturally extracted enterokinase is easily mixed with other types of proteases, which limits its wide application in genetic engineering. Therefore, the preparation of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/48C12N15/70
CPCC12N9/6424C12N15/70C12Y304/21009
Inventor 杨霞
Owner 北京世纪伟信医药科技有限公司
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