A yeast dipeptidyl peptidase III engineered by site-directed mutagenesis

一种二肽基肽酶、定点突变的技术，应用在肽酶、酶、酵素等方向

Active Publication Date: 2016-08-24
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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  • A yeast dipeptidyl peptidase III engineered by site-directed mutagenesis
  • A yeast dipeptidyl peptidase III engineered by site-directed mutagenesis
  • A yeast dipeptidyl peptidase III engineered by site-directed mutagenesis

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 Saccharomyces cerevisiae S288c 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 Saccharomyces cerevisiae S288c as a reference, the 570th amino acid is substituted with alanine (Alanine, ALa, A), and the 572nd amino acid is substituted with lysine (Lysine , Lys, K) substitution, the 574th amino acid is replaced by 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 EcoRI, ( GGATCC ) is th...

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 the expression plasmid pHIL-S1 -wtyDPP and the expression plasmid 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, s...

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 Pichia Expression Kit (Invitrogen Inc., U.S.) manual, and screen Mut + Turn. Using methanol as the only carbon source to induce expression of recombinant bacteria (operated according to the Pichia Expression Kit manual), the analysis of the culture solution by SDS-PAGE electrophoresis showed that after the transformant was induced to express, the supernatant of the culture solution showed an obvious band of the target protein. While the control bacteria transformed with an empty plasmid containing no target gene were induced for 96 hours under the same conditions, no targe...

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Abstract

The invention relates to a yeast dipeptidyl peptidase III transformed by site-directed mutation. The yeast dipeptidyl peptidase III transformed by site-directed mutagenesis according to the present invention is characterized in that it is produced from yeast dipeptidyl peptidase III derived from Saccharomyces cerevisiae S288c with the amino acid sequence of SEQ ID NO.1. A mutant produced by amino acid substitution, the amino acid substitution comprising substitutions at positions 570, 572, and 574, so that the yeast dipeptidyl peptidase III transformed by site-directed mutation is p-6-methoxybisfuranocoumarin Enzymes with oxidative decomposition. The yeast dipeptidyl peptidase III transformed by site-directed mutation of the present invention can be applied to the preparation of feed and its additives or food and its additives to eliminate 6-methoxydifuranocoumarin products, and can also be applied to the preparation of preventive Drugs for diseases induced by 6‑methoxydifuranocoumarins.

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, EC 3.4.14.4), 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 special The metalloprotease of the HEXXGH zinc finger structure has 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 DPP I to DPP IV according to their subcellular localization and the sensitivity of specific non-nuclear inhibitors. DPP III ...

Claims

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Application Information

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Patent Type & Authority Patents(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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