Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA.
Appl Biochem Biotechnol. 2012 Mar;166(5):1340-54. doi: 10.1007/s12010-011-9522-6. Epub 2012 Jan 21.
Development of endoproteases, programmed to promote degradation of peptides or proteins responsible for pathogenic states, represents an attractive therapeutic strategy, since such biocatalytic agents could be directed against a potentially unlimited repertoire of extracellular proteinaceous targets. Difficulties associated with engineering enzymes with tailor-made substrate specificities have, however, hindered the discovery of proteases possessing both the efficiency and selectivity to act as therapeutics. Here, we disclose a genetic system, designed to report on site-specific proteolysis through the survival of a bacterial host, and the implementation of this method in the directed evolution of proteases with a non-native substrate preference. The high sensitivity potential of this system was established by monitoring the activity of the Tobacco Etch Virus protease (TEV-Pr) against co-expressed substrates of various recognition level and corroborated by both intracellular and cell-free assays. The genetic selection system was then used in an iterative mode with a library of TEV-Pr mutants to direct the emergence of proteases favoring a nominally poor substrate of the stringently selective protease. The retrieval of mutant enzymes displaying enhanced proteolytic properties against the non-native sequence combined with reduced recognition of the cognate hexapeptide substrate demonstrates the potential of this system for evolving proteases with improved or completely unprecedented properties.
开发靶向负责致病状态的肽或蛋白质降解的内肽酶代表了一种有吸引力的治疗策略，因为这类生物催化试剂可以针对潜在的无限数量的细胞外蛋白质靶标。然而，与具有定制底物特异性的酶工程相关的困难阻碍了发现具有作为治疗剂的效率和选择性的蛋白酶。在这里，我们揭示了一种遗传系统，该系统旨在通过细菌宿主的存活来报告特定部位的蛋白水解，并在具有非天然底物偏好的蛋白酶的定向进化中实施该方法。该系统的高灵敏度潜力通过监测烟草蚀纹病毒蛋白酶 (TEV-Pr) 对各种识别水平的共表达底物的活性来建立，并通过细胞内和无细胞测定得到证实。然后，该遗传选择系统以迭代模式与 TEV-Pr 突变体文库一起使用，以引导出现有利于严格选择性蛋白酶的名义上较差底物的蛋白酶。检索显示对非天然序列具有增强的蛋白水解特性且对同源六肽底物的识别降低的突变酶表明，该系统具有进化出具有改进或完全前所未有的特性的蛋白酶的潜力。
