Department of Protein Science, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH - Royal Institute of Technology, Stockholm, Sweden.
Biotechnol J. 2023 Nov;18(11):e2200625. doi: 10.1002/biot.202200625. Epub 2023 Jul 28.
Due to their ability to catalytically cleave proteins and peptides, proteases present unique opportunities for the use in industrial, biotechnological, and therapeutic applications. Engineered proteases with redesigned substrate specificities have the potential to expand the scope of practical applications of this enzyme class. We here apply a combinatorial protease engineering-based screening method that links proteolytic activity to the solubility and correct folding of a fluorescent reporter protein to redesign the substrate specificity of tobacco etch virus (TEV) protease. The target substrate EKLVFQA differs at three out of seven positions from the TEV consensus substrate sequence. Flow cytometric sorting of a semi-rational TEV protease library, consisting of focused mutations of the substrate binding pockets as well as random mutations throughout the enzyme, led to the enrichment of a set of protease variants that recognize and cleave the novel target substrate.
由于其催化切割蛋白质和肽的能力，蛋白酶为工业、生物技术和治疗应用提供了独特的机会。具有重新设计的底物特异性的工程蛋白酶有可能扩大这类酶的实际应用范围。在这里，我们应用一种组合蛋白酶工程筛选方法，将蛋白水解活性与荧光报告蛋白的可溶性和正确折叠联系起来，重新设计烟草蚀纹病毒（TEV）蛋白酶的底物特异性。目标底物 EKLVFQA 在七个位置中有三个与 TEV 共有底物序列不同。半理性 TEV 蛋白酶文库的流式细胞术分选，包括底物结合口袋的焦点突变以及整个酶的随机突变，导致一组能够识别和切割新型靶标底物的蛋白酶变体的富集。
