OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy
OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy
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DOI：
10.3791/60774
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发表时间：
2020-02-01
影响因子：
1.2
通讯作者：
Zheng, Peng
中科院分区：
文献类型：
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作者：
Deng, Yibing;Zheng, Bin;Zheng, Peng
近年来，化学和生物缀合技术得到了迅速发展，并允许构建蛋白质聚合物。然而，受控的蛋白质聚合过程始终是一个挑战。在这里，我们已经开发了一种酶促方法，用于在合理控制的序列中逐步构建聚合蛋白质。在该方法中，蛋白质单体的C-末端是NGL，用于使用OaAEP 1（Oldenlandia affinis天冬酰胺酰内肽酶）1）进行蛋白质缀合，而N-末端是可裂解的TEV（烟草蚀纹病毒）裂解位点加上L（ENLYFQ/GL），用于暂时的N-末端保护。因此，OaAEP 1一次只能添加一个蛋白单体，然后TEV蛋白酶切割Q和G之间的N-末端以暴露NH 2-Gly-Leu。然后，该单元准备好进行下一个OaAEP 1连接。利用基于原子力显微镜的单分子力光谱（AFM-SMFS），通过展开单个蛋白质结构域来检查工程化的多聚蛋白。因此，本研究为多聚蛋白的工程化和固定化提供了一个有用的策略。
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.