A human dipeptidyl peptidase III modified by site-directed mutagenesis

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

Active Publication Date: 2018-10-30
JINAN UNIVERSITY
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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 human dipeptidyl peptidase III modified by site-directed mutagenesis
  • A human dipeptidyl peptidase III modified by site-directed mutagenesis
  • A human dipeptidyl peptidase III modified by site-directed mutagenesis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Synthesis of wtDPP and humDPP

[0025] The present invention takes the dipeptidyl peptidase gene (NCBI database AJ271216.1) sequence of Homo sapiens 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 wtDPP gene was synthesized by artificial total synthesis.

[0026] The present invention takes the dipeptidyl peptidase gene (AJ271216.1) sequence of Homo sapiens as a reference, the 560th amino acid is substituted with alanine (Alanine, ALa, A), and the 562nd amino acid is substituted with lysine ( Lysine, Lys, K) substitution, the 564th 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 the restriction enzyme cutting site BamHI. ...

Embodiment 2

[0029] Example 2: Construction of recombinant plasmids wtDPP and humDPP 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 wtDPP and humDPP obtained in Example 1 were respectively cloned into pHIL-S1 to construct the expression plasmid pHIL-S1 -wthDPP and the expression plasmid pHIL-S1-humDPP, 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 humDPP is as follows: EcoRI+BamHI double-digestion of the plasmid pHIL-S1 and the target fragment humDPP, the digested products were subjected to 0.8% agarose gel electrophoresis, and the gel was cut and recovered. Ligation of plasmid pHIL-S1 and humDPP was performed using T4 DNA ligase. CaCl 2 Prepare E.coli DH5α competent cells, transform DH5α competent...

Embodiment 3

[0034] Example 3: Expression of recombinant humDPP and recombinant wthDPP

[0035] Expression of recombinant humDPP: Recombinant plasmid pHIL-S1-humDPP 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-humDPP and Plasmid pHIL-S1. According to the protoplast method in the Pichia Expression Kit (Invitrogen Inc., U.S.) manual, Pichia hominis GS115 was transformed, and Mut + Turn. Using methanol as the sole carbon source to induce expression of recombinant bacteria (operated according to the PichiaExpression Kit manual), the SDS-PAGE electrophoresis analysis of the culture solution showed that after the transformants were induced to express, the supernatant of the culture solution showed obvious target protein bands, while The control bacteria transformed with an empty plasmid without the target gene were induced under the same conditions for 96 hours, and no target p...

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Abstract

The invention relates to human dipeptidyl peptidase III reconstructed by site-directed mutagenesis. The human dipeptidyl peptidase III reconstructed by site-directed mutagenesis is characterized in that the human dipeptidyl peptidase III reconstructed by site-directed mutagenesis is a mutant produced by making a plurality of amino acid substitutions in human dipeptidyl peptidase III having an amino acid sequence of SEQ ID NO.1, and the amino acid substitutions comprise substitutions of the No.560, No.562 and No.564 sites, so that the human dipeptidyl peptidase III reconstructed by site-directed mutagenesis is an enzyme having an oxygenolysis function for 6-methoxy difuran coumarin. The human dipeptidyl peptidase III reconstructed by site-directed mutagenesis can be applied for preparing a product for eliminating 6-methoxy difuran coumarin in feed, additives of the feed, or food and additives of the food, and can be applied for preparing medicines preventing diseases induced by the 6-methoxy difuran coumarin.

Description

technical field [0001] The present invention relates to a dipeptidyl peptidase III, in particular to a human-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 human, dipeptidase III from rabbit, etc., are a group of molecules containing special The metalloprotease of 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 ca...

Claims

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Application Information

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IPC IPC(8): C12N9/48C12N15/57C12N15/63A61K38/48A61P35/00A23K20/189A23L29/00
CPCA61K38/00C12N9/485C12Y304/14004
Inventor 刘大岭姚冬生虞晓丹谢春芳
Owner JINAN UNIVERSITY
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