Yi Li, Taft Joseph M, Li Qing, Gebhard Mark C, Georgiou George, Iverson Brent L
Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, TX, 78712, USA,
Methods Mol Biol. 2015;1319:81-93. doi: 10.1007/978-1-4939-2748-7_5.
There is significant interest in engineering proteases with desired proteolytic properties. We describe a high-throughput fluorescence-activated cell sorting (FACS) assay for detecting altered proteolytic activity of protease in yeast, at the single cell level. This assay relies on coupling yeast endoplasmic reticulum (ER) retention, yeast surface display, and FACS analysis. The method described here allows facile screening of large libraries, and of either protease or substrate variants, including the screening of protease libraries against substrate libraries. We demonstrate the application of this technique in the screening of libraries of Tobacco Etch Virus protease (TEV-P) for altered proteolytic activities. In addition, the generality of this method is also validated by other proteases such as human granzyme K and the hepatitis C virus protease, and the human Abelson tyrosine kinase.
人们对设计具有所需蛋白水解特性的蛋白酶有着浓厚兴趣。我们描述了一种高通量荧光激活细胞分选（FACS）测定法，用于在单细胞水平检测酵母中蛋白酶的蛋白水解活性变化。该测定法依赖于酵母内质网（ER）保留、酵母表面展示和FACS分析的结合。本文所述方法允许轻松筛选大型文库以及蛋白酶或底物变体，包括针对底物文库筛选蛋白酶文库。我们展示了该技术在筛选烟草蚀纹病毒蛋白酶（TEV-P）文库以寻找蛋白水解活性变化方面的应用。此外，该方法的通用性也通过其他蛋白酶如人颗粒酶K、丙型肝炎病毒蛋白酶和人阿贝尔森酪氨酸激酶得到了验证。
