Intracellular detection and evolution of site-specific proteases using a genetic selection system.
Intracellular detection and evolution of site-specific proteases using a genetic selection system.
复制标题
使用遗传选择系统进行位点特异性蛋白酶的细胞内检测和进化。
DOI：
10.1007/s12010-011-9522-6
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发表时间：
2012
影响因子：
3
通讯作者：
Savinov,SergeyN
中科院分区：
文献类型：
--
作者：
Verhoeven,KathrynD;Altstadt,OlviaC;Savinov,SergeyN
内切蛋白酶的开发，被编程为促进负责致病状态的肽或蛋白质的降解，代表了一种有吸引力的治疗策略，因为这种生物催化剂可以针对潜在无限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具有作为治疗剂的效率和选择性的蛋白酶的发现。在这里，我们公开了一种遗传系统，旨在通过细菌宿主的存活来报告位点特异性蛋白水解，以及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是通过监测烟草蚀纹病毒蛋白酶（TEV-Pr）针对不同识别水平的共表达底物的活性而建立的，并通过细胞内和无细胞测定得到证实。然后，遗传选择系统以迭代模式与 TEV-Pr 突变体文库一起使用，以指导蛋白酶的出现，有利于严格选择性蛋白酶的名义上较差的底物。对非天然序列表现出增强的蛋白水解特性以及对同源六肽底物的识别减少的突变酶的检索证明了该系统在进化具有改进或完全前所未有的特性的蛋白酶方面的潜力。
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.