title: New strategy for specific activation of recombinant microbial pro-transglutaminase by introducing an enterokinase cleavage site.
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.
authors.affiliation: Department of Bioengineering, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, People's Republic of China. wangkuntwt@163.com
authors.forename: Kun
authors.initials: K
authors.lastname: Wang
authors.forename: Bin
authors.initials: B
authors.lastname: Wang
authors.forename: Hui-Lin
authors.initials: HL
authors.lastname: Yang
authors.forename: Li
authors.initials: L
authors.lastname: Pan
mesh_terms: D004765:Enteropeptidase / Q000378:metabolism*; D007700:Kinetics; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D011503:Transglutaminases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D011498:Protein Precursors; D011994:Recombinant Proteins; D011503:Transglutaminases; D004765:Enteropeptidase