Deng Yibing, Zheng Bin, Liu Yutong, Shi Shengchao, Nie Jingyuan, Wu Tao, Zheng Peng
State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University.
State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University;
J Vis Exp. 2020 Feb 5(156). doi: 10.3791/60774.
Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled protein polymerization process is always a challenge. Here, we have developed an enzymatic methodology for constructing polymerized protein step by step in a rationally-controlled sequence. In this method, the C-terminus of a protein monomer is NGL for protein conjugation using OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases) 1) while the N-terminus was a cleavable TEV (tobacco etch virus) cleavage site plus an L (ENLYFQ/GL) for temporary N-terminal protecting. Consequently, OaAEP1 was able to add only one protein monomer at a time, and then the TEV protease cleaved the N-terminus between Q and G to expose the NH2-Gly-Leu. Then the unit is ready for next OaAEP1 ligation. The engineered polyprotein is examined by unfolding individual protein domain using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Therefore, this study provides a useful strategy for polyprotein engineering and immobilization.
近年来，化学和生物共轭技术发展迅速，使得蛋白质聚合物的构建成为可能。然而，可控的蛋白质聚合过程一直是一个挑战。在此，我们开发了一种酶促方法，用于以合理控制的顺序逐步构建聚合蛋白。在这种方法中，蛋白质单体的C末端是用于使用OaAEP1（水线草天冬酰胺内肽酶）进行蛋白质共轭的NGL 1），而N末端是一个可切割的TEV（烟草蚀纹病毒）切割位点加上一个L（ENLYFQ/GL）用于临时N末端保护。因此，OaAEP1一次只能添加一个蛋白质单体，然后TEV蛋白酶在Q和G之间切割N末端以暴露NH2-Gly-Leu。然后该单元准备好进行下一次OaAEP1连接。使用基于原子力显微镜的单分子力谱（AFM-SMFS）展开单个蛋白质结构域来检查工程化多蛋白。因此，本研究为多蛋白工程和固定化提供了一种有用的策略。
