New strategy for specific activation of recombinant microbial pro-transglutaminase by introducing an enterokinase cleavage site
New strategy for specific activation of recombinant microbial pro-transglutaminase by introducing an enterokinase cleavage site
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通过引入肠激酶裂解位点特异性激活重组微生物转谷氨酰胺酶原的新策略
DOI：
10.1007/s10529-012-1090-5
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发表时间：
2013-03-01
影响因子：
2.7
通讯作者：
Pan, Li
中科院分区：
文献类型：
--
作者：
Wang, Kun;Wang, Bin;Pan, Li
在异源表达系统中，重组微生物谷氨酰胺转氨酶(RMTG)通常以可溶性酶原(ProrMTG)的形式表达，但非活性的prorMTG的蛋白水解性是必需的。我们研究了另一种方法，即在前肽和成熟的rMTG之间引入肠激酶的特定切割位点，产生三种前rMTG变异体(Pro-mrMTG、Pro-m-rMTG和MPRO-rMTG)，而不是筛选激活PRO-rMTG的蛋白酶。肠激酶可激活Pro-mrMTG和Pro-m-rMTG，但不降解成熟的rMTG。肠激酶法激活Pro-m-rMTG的活化率为92.0%，胰酶激活的Pro-rMTG的活化率为47.0%。MALDI-MS分析表明，Pro-m-rMTG被激活后，包括切割位点在内的前肽被特异性地移除。这种方法通过结合其他蛋白酶的高度特异性的裂解位点，有可能应用于rMTG的生产。
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.