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Biotechnol Lett. 2013 Mar;35(3):383-8. doi: 10.1007/s10529-012-1090-5. Epub 2012 Nov 10.

New strategy for specific activation of recombinant microbial pro-transglutaminase by introducing an enterokinase cleavage site.

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1
Department of Bioengineering, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, People's Republic of China. wangkuntwt@163.com

Abstract

Recombinant microbial transglutaminase (rMTG) is usually expressed as a soluble zymogen (pro-rMTG) in heterologous expression systems but proteolytic activation of the inactive pro-rMTG is essential. Instead of screening proteases for activating pro-rMTG, we examined an alternative method by introducing a specific cleavage site of enterokinase between the pro-peptide and mature rMTG, generating three pro-rMTG variants (Pro-mrMTG, Pro-m-rMTG and mPro-rMTG). Pro-mrMTG and Pro-m-rMTG were activated by enterokinase without degrading mature rMTG. The activation productivity of Pro-m-rMTG by enterokinase reached 92 % after 22 h activation, while the activation productivity of Pro-rMTG activated by trypsin was 47 %. MALDI-MS analysis revealed that the pro-peptide including the cleavage site was specifically removed from Pro-m-rMTG after activation. This methodology has the potential to be applied in rMTG production by incorporating highly specific cleavage sites of other proteases.

PMID:
23143177
DOI:
10.1007/s10529-012-1090-5
[Indexed for MEDLINE]
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