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Ultrasonic assisted enzyme method-based enterokinase extraction process

An auxiliary enzyme, enterokinase technology, applied in the direction of enzymes, peptidases, hydrolases, etc., can solve problems such as disulfide bond mismatches, and achieve high specificity.

Inactive Publication Date: 2017-09-19
RUGAO YONGXING CASING
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Problems solved by technology

[0007] Since the medaka enterokinase light chain contains 9 cysteine ​​(Cys) residues, among which 4 pairs of disulfide bonds are formed, and there is a free Cys residue that does not participate in the pairing, so it is very easy to form disulfide during recombinant expression Bond mismatch, resulting in isomers

Method used

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Embodiment

[0024] The construction of killifish enterokinase mutant was searched for the killifish enterokinase sequence (MN_001104898) in GenBank, and the sequence was optimized by using the codon preference of Escherichia coli, and the nucleoside encoding the novel killifish enterokinase light chain (C112S) was designed The acid sequence, the 112th Cys of the killifish enterokinase light chain is replaced by serine (Ser), its nucleotide sequence is shown in the sequence table SEQ ID NO: 1, its nucleotide is the same as the wild type sequence in the killifish body, black The bottom region is the modified gene after optimization according to the codon bias of E. coli, and the selective mutation site is marked with a black triangle. The artificially synthesized killifish enterokinase light chain gene length.

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Abstract

The invention discloses an ultrasonic assisted enzyme method-based enterokinase extraction process belonging to the field of biological pharmacy and basic scientific research. A novel recombinant killifish enterokinase light chain disclosed by the invention can be obtained through simple separation and purification steps; by simple renaturation and purification, fast, simple, stable, high-activity and high-specificity recombinant killifish enterokinase light chain proteins can be obtained. The enterokinase is applicable to research and application in the fields of biomedical engineering, gene engineering, biochemistry, molecular biology and the like.

Description

technical field [0001] The invention belongs to the technical field of biotechnology and relates to an ultrasonic-assisted enzymatic method for extracting enterokinase. Background technique [0002] The expression methods of exogenous proteins can be divided into direct expression and fusion expression. Among them, the fusion expression method can often achieve high-efficiency expression of exogenous target proteins, and is conducive to separation and purification. However, sometimes the target protein cannot be used in the form of a fusion protein, so it is necessary to design a specific protease cleavage site between the target protein and the tag protein, and use a unique protease cleavage to obtain the complete target protein. Currently, the common tool proteases include enterokinase, factor Xa or thrombin, among which enterokinase is most commonly used because it can completely remove the N-terminal sequence of the target protein. (Gasparian, M. et al. Expression, puri...

Claims

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Application Information

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IPC IPC(8): C12N9/64C12N15/57C12N15/70
CPCC12N9/6408C12Y304/21009
Inventor 费苹陈银芳费正明陈明全
Owner RUGAO YONGXING CASING
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