Recombinant production of active microbial transglutaminase in E. coli using self-cleavable zymogen with mutated propeptide
| Autor: | Ryo Sato, Noriho Kamiya, Hiromasa Taniguchi, Ryutaro Ariyoshi, Kosuke Minamihata |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
0106 biological sciences
medicine.medical_treatment 01 natural sciences law.invention 03 medical and health sciences Residue (chemistry) Bacterial Proteins law 010608 biotechnology Zymogen Escherichia coli TEV protease medicine Protein precursor 030304 developmental biology 0303 health sciences Enzyme Precursors Transglutaminases Protease biology Tobacco etch virus Chemistry biology.organism_classification Fusion protein Recombinant Proteins Streptomyces Glutamine Biochemistry Mutation Recombinant DNA Biotechnology |
| DOI: | 10.1101/2020.02.25.965541 |
| Popis: | Microbial transglutaminase from Streptomyces mobaraensis (MTG) has been widely used in food industry and also in research and medical applications, since it can site-specifically modify proteins by the cross-linking reaction of glutamine residue and the primary amino group. The recombinant expression system of MTG in E. coli provides better accessibility for the researchers and thus can promote further utilization of MTG. Herein, we report production of active and soluble MTG in E. coli by using a chimeric protein of tobacco etch virus (TEV) protease and MTG zymogen. A chimera of TEV protease and MTG zymogen with native propeptide resulted in active MTG contaminated with cleaved propeptide due to the strong interaction between the propeptide and catalytic domain of MTG. Introduction of mutations of K9R and Y11A to the propeptide facilitated dissociation of the cleaved propeptide from the catalytic domain of MTG and active MTG without any contamination of the propeptide was obtained. The specific activity of the active MTG was 22.7±2.6 U/mg. The successful expression and purification of active MTG by using the chimera protein of TEV protease and MTG zymogen with mutations in the propeptide can advance the use of MTG and the researches using MTG mediated cross-linking reactions. |
| Databáze: | OpenAIRE |
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