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Site-directed mutagenesis modified saccharomyces cerevisiae dipeptidyl peptidase III

A technology of dipeptidyl peptidase and site-directed mutagenesis, applied in the direction of peptidase, enzyme, enzyme, etc.

Active Publication Date: 2016-03-02
GUANGDONG GENUIZYMES ANIMAL HEALTH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are very few biological enzymes that have been found to decompose 6-methoxydifuranocoumarin

Method used

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  • Site-directed mutagenesis modified saccharomyces cerevisiae dipeptidyl peptidase III
  • Site-directed mutagenesis modified saccharomyces cerevisiae dipeptidyl peptidase III
  • Site-directed mutagenesis modified saccharomyces cerevisiae dipeptidyl peptidase III

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Synthesis of wtyDPP and myDPP

[0025] The present invention takes the dipeptidyl peptidase gene (NCBI database NM_001183312) sequence of SaccharomycescerevisiaeS288c as a reference, and adds 5'-GTC at the 5' end GAATTC -3' join 3'- at the 3' end CCTAGG GAC-5', ( GAATTC ) is the restriction enzyme cutting site EcoRI, ( GGATCC ) is the restriction enzyme cutting site BamHI. The wtyDPP gene was synthesized by artificial total synthesis.

[0026] The present invention takes the dipeptidyl peptidase gene (NM_001183312) sequence of SaccharomycescerevisiaeS288c as a reference, the amino acid at position 570 is replaced with alanine (Alanine, ALa, A), and the amino acid at position 572 is replaced with lysine (Lysine, Lys , K) substitution, the amino acid at position 574 is replaced with histidine (Histidine, His, H), and 5'-GTC is added at the 5' end GAATTC -3' join 3'- at the 3' end CCTAGG GAC-5', ( GAATTC ) is the restriction enzyme cutting site Eco...

Embodiment 2

[0029] Example 2: Construction of recombinant plasmids wtyDPP and myDPP expression plasmids

[0030] Gene cloning was carried out according to conventional methods (Sambrook, et al. 2001, Molecular Cloning A Laboratory Manual. Cold Spring Harbor Laboratory Press. USA), and the wtyDPP and myDPP obtained in Example 1 were respectively cloned into pHIL-S1 to construct expression plasmids pHIL-S1-wtyDPP and expression plasmids pHIL-S1 -myDPP, the cloned target gene was identified by enzyme digestion and sequencing.

[0031] specific methods:

[0032] The construction process of the recombinant plasmid pHIL-S1 containing myDPP is as follows: EcoRI+BamHI double-digestion plasmid pHIL-S1 and the target fragment myDPP, and the digested product was subjected to 0.8% agarose gel electrophoresis, and the gel was cut and recovered. Ligation of plasmid pHIL-S1 and myDPP was performed using T4 DNA ligase. CaCl 2 Prepare E.coli DH5α competent cells, transform DH5α competent cells, screen ...

Embodiment 3

[0034] Example 3: Expression of recombinant myDPP and recombinant wtyDPP

[0035] Expression of recombinant myDPP: Recombinant plasmid pHIL-S1-myDPP and plasmid pHIL-S1 were digested with SacI, the digested products were subjected to 0.8% agarose gel electrophoresis, and the linear recombinant plasmids pHIL-S1-myDPP and Plasmid pHIL-S1. Transform Pichia yeast GS115 according to the protoplast method in the PichiaExpressionKit (InvitrogenInc., U.S.) manual, and screen Mut + Turn. In the induced expression of recombinant bacteria using methanol as the sole carbon source (operated according to the PichiaExpressionKit manual), the SDS-PAGE electrophoresis analysis of the culture solution showed that after the transformant was induced to express, the supernatant of the culture solution showed an obvious target protein band, and the transformation The control bacteria without the empty plasmid containing the target gene were induced under the same conditions for 96 hours, and no t...

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Abstract

The invention relates to a site-directed mutagenesis modified saccharomyces cerevisiae dipeptidyl peptidase III. The site-directed mutagenesis modified saccharomyces cerevisiae dipeptidyl peptidase III is characterized in that the site-directed mutagenesis modified saccharomyces cerevisiae dipeptidyl peptidase III is a mutant generated by substitution of multiple amino acids in saccharomyces cerevisiae dipeptidyl peptidase III derived from saccharomyces cerevisiae S288c and having an amino acid sequence being SEQ ID NO.1, substitution of the multiple amino acids refers to substitution of a 570th site, a 572th site and a 574th site, and the site-directed mutagenesis modified saccharomyces cerevisiae dipeptidyl peptidase III is modified into an enzyme with an oxygenolysis effect on 6-methoxy di-furocoumarin. The site-directed mutagenesis modified saccharomyces cerevisiae dipeptidyl peptidase III can be applied to preparation of products for removing the 6-methoxy di-furocoumarin from feedstuffs, additives of the feedstuffs, foods and additives of the foods and can be further applied to preparation of medicines for preventing diseases induced by the 6-methoxy di-furocoumarin.

Description

technical field [0001] The present invention relates to a dipeptidyl peptidase III, in particular to a yeast-derived dipeptidyl peptidase III modified by site-directed mutation. Background technique [0002] Dipeptidase III (DPP S III, EC3.4.14.4), such as dipeptidase III from yeast, dipeptidase III from human, dipeptidase III from rat, dipeptidase III from rabbit, etc., are a group of molecules containing A metalloprotease with a special HEXXGH zinc finger structure, a peptidase that hydrolyzes the amino terminal of the polypeptide chain and cuts off the dipeptide. DPPIII is related to the metabolism of important physiologically active peptides such as enkephalin, angiotensin II, angiotensin III and melanin in terms of physiological function. DPP S Existing in various mammalian tissues, dipeptidyl peptidases are divided into different types DPPI-DPPIV according to their subcellular localization and the sensitivity of specific non-nuclear inhibitors. DPPIII can selective...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/48C12N15/57A23K20/189A23L5/20A61K38/48A61P35/00
CPCA61K38/00C12N9/485C12Y304/14004A61K38/48A23L5/20A61P35/00A23K20/189C12N9/48C12N1/185C12R2001/85
Inventor 刘大岭姚冬生吴曦阳谢春芳
Owner GUANGDONG GENUIZYMES ANIMAL HEALTH CO LTD
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