School of Biochemistry, University of Bristol, Bristol, UK.
School of Biochemistry, University of Bristol, Bristol, UK; BrisSynBio, University of Bristol, Bristol, UK.
J Mol Biol. 2019 Apr 5;431(8):1689-1699. doi: 10.1016/j.jmb.2019.03.007. Epub 2019 Mar 13.
Protein translocation is a fundamental process in biology. Major gaps in our understanding of this process arise due the poor sensitivity, low time resolution and irreproducibility of translocation assays. To address this, we applied NanoLuc split-luciferase to produce a new strategy for measuring protein transport. The system reduces the timescale of data collection from days to minutes and allows for continuous acquisition with a time resolution in the order of seconds, yielding kinetics parameters suitable for mechanistic elucidation and mathematical fitting. To demonstrate its versatility, we implemented and validated the assay in vitro and in vivo for the bacterial Sec system and the mitochondrial protein import apparatus. Overall, this technology represents a major step forward, providing a powerful new tool for fundamental mechanistic enquiry of protein translocation and for inhibitor (drug) screening, with an intensity and rigor unattainable through classical methods.
蛋白质易位是生物学中的一个基本过程。由于易位测定的灵敏度差、时间分辨率低和重现性差，我们对这一过程的理解存在很大差距。为了解决这个问题，我们应用 NanoLuc 分裂荧光酶来产生一种新的测量蛋白质运输的策略。该系统将数据采集的时间尺度从几天缩短到几分钟，并允许以秒为单位的连续采集，产生适合于机制阐明和数学拟合的动力学参数。为了证明其多功能性，我们在体外和体内对细菌 Sec 系统和线粒体蛋白导入装置实施和验证了该测定。总的来说，这项技术是向前迈出的一大步，为蛋白质易位的基础机制研究和抑制剂（药物）筛选提供了一个强大的新工具，其强度和严格性是经典方法无法达到的。
