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Transglutaminase mutant as well as coding gene and application thereof

A technology of transglutaminase and mutant, applied in the field of genetic engineering

Active Publication Date: 2020-11-17
ANHUI MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing studies on the thermostability of transglutaminase mainly focus on single-point mutations, and there are few studies on combinatorial mutations. There is no report on protein degradation in the recombinant MTG secreted by Pichia pastoris. Is it possible to reduce the degradation of secreted proteins? It will affect the enzyme activity and thermal stability, what is the mechanism of the influence, these problems have not been reported

Method used

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  • Transglutaminase mutant as well as coding gene and application thereof
  • Transglutaminase mutant as well as coding gene and application thereof
  • Transglutaminase mutant as well as coding gene and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1 Obtaining of transglutaminase mutant gene

[0051] Table 1 takes the gene (mtg1) of the artificially synthesized Mut1 as the mutant construction primer of the template

[0052]

[0053] Mut1 gene (mtg 1 ) was synthesized by Nanjing GenScript Biotechnology Co., Ltd., mtg 1 The 5' end of the gene introduces an XhoI site (CTCGAG) and a Kex2 endopeptidase recognition site (aaaaga), and a NotI site (GCGGCCGC) and a 6×His tag sequence (ATGGTGATGGTGATGATG) are introduced at the 3' end. The tag sequence is mainly Facilitates the purification of recombinant proteins. Synthetic mtg 1 The gene was cloned into the pUC57 vector, and the mtg was 993bp.

[0054] Mut2, Mut3 gene (mtg 2 , mtg 3 ) is based on pUC57-mtg1 as a template, using Max Super-Fidelity DNAPolymerase amplifies the target plasmid, and the amplified product of the target plasmid is digested with DpnI, Transformation was carried out directly after recombination circularization, and the specif...

Embodiment 2

[0055] Embodiment 2 Construction of transglutaminase mutant recombinant vector

[0056] Respectively use restriction endonucleases XhoI and NotI to double-enzyme-cut the mutant plasmid and expression vector pPICza, recover the enzyme-digested products from the gel, ligate the recovered products with ligase, transfer to E.coli TOP10 competent cells, and spread evenly to a concentration of 50 μg / mL Zeocin-resistant LLB plate, cultivate overnight at 37°C, and grow single clones. The correctly sequenced plasmid was named pPICZα-mtg1, and the other three were numbered sequentially. The sequenced correct mutant recombinant vector was transformed into expression host pPIC9k-pro / GS115 (expression leader peptide pro peptide or PRO).

Embodiment 3

[0057] Embodiment 3 Construction of Transglutaminase Mutant Recombinant Strain

[0058] The three successfully constructed mutant recombinant vectors pPICZα-mtg-mut were linearized with SpeI and electrotransformed into pPIC9k-pro / GS115 (prepared competent in advance) to obtain recombinant yeast strain GS115 (pro / mtg-mut).

[0059] Pick the strain GS115(pro / mtg-mut) containing the recombinant plasmid, inoculate it into a small culture tube of 4mL BMGY medium, place it at 28°C and culture it on a shaker at 200rpm for 48h; then centrifuge the culture solution at 3000g for 5min, discard the supernatant, The pellet was resuspended with 4 mL of BMMY medium containing 1.0% methanol, and placed at 25° C. and 200 rpm for induction culture for 48 h. Finally, the supernatant was taken to detect the enzyme activity. The results of small shake tube induced expression showed that the activity of the small tube supernatant of the three mutants was higher than that of the wild enzyme.

[00...

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Abstract

The invention relates to the field of gene engineering, in particular to a transglutaminase mutant as well as an encoding gene and application thereof. The method comprises the following steps: carrying out S2P, S23V, Y24N, R215A, K294L, and S2P, S23V, Y24N, R215A, H289Y, or S23V, Y24N, R215A, K269D and H289Y site-specific mutagenesis on a wild enzyme with an amino acid sequence as shown in SEQ IDNO: 1 to obtain the mutant. The specific activities of the three mutant enzymes are 1.9 times, 2.4 times and 1.4 times of those of wild enzymes MTG-WT respectively, and the thermal stability of the three mutant enzymes is superior to that of the wild enzymes.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a transglutaminase mutant and its coding gene and application. Background technique [0002] Transglutaminase (Transglutaminase, TG) can catalyze the acyl transfer reaction between the γ-carboxamide group of the glutamine residue in the protein peptide chain and various acyl acceptors, and is an effective protein cross-linking agent. TG is widely distributed in the human body, mammals, plants and microorganisms, etc. Among them, microbial transglutaminase (Microbial Transglutaminase, MTG) has low protein molecular weight, non-Ca 2+ The superior enzymatic properties such as dependence and wide substrate range have a wide range of application values ​​in the food industry, biomedicine, tissue engineering, site-directed protein cross-linking, and the construction of homologous antibody-drug conjugates. For some applications, including thermoplastic processing of extrusion and hea...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/81C12N1/19C12R1/84
CPCC12N9/1044C12Y203/02013C12N15/815
Inventor 宋小平王雅洁王蔷蔡晶晶
Owner ANHUI MEDICAL COLLEGE