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Recombinant production method of microbial transglutaminase

A technology of transglutaminase and production method, which is applied in the field of protein and enzyme engineering, and can solve the problems of cumbersome steps, great influence of medicinal protein modification, strong affinity and the like

Pending Publication Date: 2022-07-19
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods require post-treatment of the purified protein (inclusion body or zymogen), which is cumbersome and uneconomical. At the same time, the leader peptide Pro has a strong affinity with the mature MTG protein, which is difficult to remove during purification and reduces the enzyme activity.
Although leader peptide residues have little impact on the food industry application of MTG, they have a greater impact on its application in pharmaceutical protein modification

Method used

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  • Recombinant production method of microbial transglutaminase
  • Recombinant production method of microbial transglutaminase
  • Recombinant production method of microbial transglutaminase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Construction of transglutaminase recombinant expression host and protein expression and purification

[0042] (1) Construction of MTG recombinant plasmid

[0043] Connect 6 histidine purification tags to the C-terminus of mature MTG, which is convenient for the subsequent separation and purification of mature MTG. The amino acid sequence of MTG and the His6 tag are connected by G-S-L-E tetrapeptide, denoted as MEG-His6. The amino acid sequence of MEG-His6 is as follows As shown in SEQ ID NO.1, the coding gene was designed according to the above-mentioned amino acid sequence, and the nucleotide sequence of the coding gene was shown in SEQ ID NO.2. The nucleotide sequence was optimized by codons and was suitable for recombinant expression in Escherichia coli. Synthesize MTG-His6 encoding gene; when artificially synthesizing MTG-His6 encoding gene, NcoI and BglII restriction endonuclease sites were added at both ends of the nucleotide sequence;

[0044] The art...

Embodiment 2

[0054] Example 2: Optimization of "molecular chaperone" Trx-Pro stripping conditions and acquisition of highly active transglutaminase

[0055] The primary purified protein obtained in Example 1 was subjected to secondary purification treatment using different treatment conditions, the protein concentration was determined using an ultra-micro spectrophotometer, and the protein composition was analyzed using SDS-PAGE. Enzyme Activity Detection Method" to measure the specific activity of transglutaminase.

[0056] (1) Screening of treatment reagents

[0057] Use dimethyl sulfoxide as a solvent to dissolve the small molecule treatment reagent, wherein the small molecule treatment reagent includes fluorescein isothiocyanate, 3-(4-hydroxyphenyl) propionate N-hydroxysuccinimide ester, 4-benzene ureaazole, acetate-N-succinimidyl ester, 4-phenyl-1,2,4-triimidazole-3,5-dione, 4-(aminomethyl)phenol, N-acetyl-L Tyrosine, glycyl-L-tyrosine hydrate, 4-hydroxybenzyl alcohol, L-tyrosine, 3...

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Abstract

The invention relates to a recombinant production method of microbial transglutaminase. The recombinant production method comprises the following steps: respectively constructing recombinant plasmids from a mature MTG coding gene and a leading peptide Pro coding gene, transforming the recombinant plasmids into the same host cell for co-expression, and primarily purifying to obtain the active microbial transglutaminase. The MTG leading peptide Pro and Trx protein are fused and then expressed into a'molecular chaperone 'Trx-Pro, MTG is assisted in correct folding when co-expressed with mature MTG protein, damage of MTG to recombinant expression host cells is inhibited, effective soluble expression and one-step simple purification of microbial transglutaminase are achieved, preliminarily purified protein is obtained after simple purification of the one-step method, and the Trx-Pro-Trx-Pro protein is obtained. The recombinant transglutaminase with high purity and high activity is obtained by further treatment with a reaction reagent and efficient stripping of a molecular chaperone Trx-Pro, and has important value in the aspects of medicinal protein modification and the like.

Description

technical field [0001] The invention relates to a recombinant production method of microbial transglutaminase, belonging to the technical field of protein and enzyme engineering. Background technique [0002] Transglutaminase (TGase), also known as transglutaminase, EC number 2.3.2.13; catalyzes γ-carboxamide (acyl donor) and lysine of glutamine residues in proteins or peptide chains The ε-amino group (acyl acceptor) of the residue undergoes an acyl transfer reaction, releasing a molecule of ammonia to form a protein polymer linked by isopeptide bonds. TGase widely exists in various organisms, including mammals, plants and microorganisms, and has important biological functions. Because of its protein cross-linking properties, it can be applied to the modification of protein-containing foods; at the same time, because long-chain primary amines and other chemical molecules similar to the lysine side chain structure can also be used as acyl acceptors of TGase, so it also has a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N9/10C12N1/21C12R1/19
CPCC12N15/70C12N9/1044C12Y203/02013C07K2319/35C07K2319/21C12N2800/22
Inventor 刘现伟李子涛刘静郑照萱武小聪邢爽
Owner SHANDONG UNIV
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