Potomac Affinity Proteins, North Potomac, MD, USA.
Institute for Bioscience and Biotechnology Research, Rockville, MD, USA.
Commun Biol. 2021 Mar 5;4(1):299. doi: 10.1038/s42003-021-01818-7.
We describe the design, kinetic properties, and structures of engineered subtilisin proteases that degrade the active form of RAS by cleaving a conserved sequence in switch 2. RAS is a signaling protein that, when mutated, drives a third of human cancers. To generate high specificity for the RAS target sequence, the active site was modified to be dependent on a cofactor (imidazole or nitrite) and protease sub-sites were engineered to create a linkage between substrate and cofactor binding. Selective proteolysis of active RAS arises from a 2-step process wherein sub-site interactions promote productive binding of the cofactor, enabling cleavage. Proteases engineered in this way specifically cleave active RAS in vitro, deplete the level of RAS in a bacterial reporter system, and also degrade RAS in human cell culture. Although these proteases target active RAS, the underlying design principles are fundamental and will be adaptable to many target proteins.
我们描述了经过工程改造的枯草杆菌蛋白酶的设计、动力学特性和结构，这些蛋白酶通过切割开关 2 中的保守序列来降解 RAS 的活性形式。RAS 是一种信号蛋白，当其发生突变时，会导致三分之一的人类癌症。为了提高对 RAS 靶序列的特异性，活性位点被修饰为依赖辅因子（咪唑或亚硝酸盐），并且蛋白酶亚位点被工程改造以在底物和辅因子结合之间建立连接。活性 RAS 的选择性蛋白水解来自两步过程，其中亚位点相互作用促进辅因子的有效结合，从而实现切割。以这种方式工程改造的蛋白酶可特异性地在体外切割活性 RAS，耗尽细菌报告系统中 RAS 的水平，并且还可在人细胞培养物中降解 RAS。尽管这些蛋白酶靶向活性 RAS，但基本的设计原则是通用的，并且可以适用于许多靶蛋白。
