Department of Integrated OMICS for Biomedical Science, College of Life science and Biotechnology, Yonsei University, Seoul, Korea.
Present affiliation: Department of Chemistry and Biochemistry, Knoebel Institute for Healthy Aging, University of Denver, Denver, Colorado.
Biotechnol Bioeng. 2019 Mar;116(3):490-502. doi: 10.1002/bit.26879. Epub 2019 Jan 4.
Transglutaminase (TGase) induces the cross-linking of proteins by catalyzing an acyl transfer reaction. TGase is a zymogen, activated by the removal of its pro-region. Because the pro-region is crucial for folding and inhibition of the TGase activity, the recombinant expression of the mature TGase (mTGase) without the pro-region, usually results in inactive inclusion bodies or low protein yield. Here, Streptomyces netropsis TGase was fused with Escherichia coli lysyl-tRNA synthetase (LysRS), as a module with chaperoning activity in an RNA dependent manner (chaperna). The TGase activity from purified fusion protein induced via the removal of LysRS by tev protease in vitro. Moreover, active mTGase was produced in E. coli via an intracellular cleavage system, wherein LysRS-mTGase was cleaved by the coexpressed tev protease in vivo. The results suggest that LysRS essentially mimics pro-region, which exerts a dual function-folding of TGase into active conformation and keeping it as dormant state-in an RNA-dependent manner. Thus, trans-acting RNAs, prompt the cis-acting chaperone function of LysRS, while being mechanistically similar to the intramolecular chaperone function of the pro-region. These results could be implemented and extended for the folding of "difficult-to-express" recombinant proteins, by harnessing the chaperna function.
转谷氨酰胺酶（TGase）通过催化酰基转移反应诱导蛋白质的交联。TGase 是一种酶原，通过去除其前导区而被激活。由于前导区对于折叠和抑制 TGase 活性至关重要，因此通常情况下，不含有前导区的成熟 TGase（mTGase）的重组表达会导致无活性的包涵体或低蛋白产量。在这里，链霉菌 netropsis TGase 与大肠杆菌赖氨酸-tRNA 合成酶（LysRS）融合，作为一种具有 RNA 依赖性伴侣活性的模块（chaperna）。通过 TEV 蛋白酶体外去除 LysRS 后，从纯化的融合蛋白中诱导的 TGase 活性。此外，通过在体内共表达的 TEV 蛋白酶，在大肠杆菌中产生了活性 mTGase。结果表明，LysRS 本质上模拟了前导区，以 RNA 依赖性方式发挥双重功能——将 TGase 折叠成活性构象并使其处于休眠状态。因此，反式作用 RNA 促使 LysRS 的顺式作用伴侣功能，而在机制上类似于前导区的分子内伴侣功能。通过利用 chaperna 功能，可以将这些结果实施和扩展到“难以表达”的重组蛋白的折叠中。
