Engineering subtilisin proteases that specifically degrade active RAS

• DOI: 10.1038/s42003-021-01818-7
• Publication Date: 2021-03-05T11:02:36Z
• Authors: Yingwei Chen; Eric A. Toth; Biao Ruan; Eun Jung Choi; Richard Simmerman; Yihong Chen; Yanan He; Ruixue Wang; Raquel Godoy-Ruiz; Harlan King; Gregory Custer; D. Travis Gallagher; David A. Rozak; Melani Solomon; Silvia Muro; David J. Weber; John Orban; Thomas R. Fuerst; Philip N. Bryan
• Keywords: Subtilisin; Cleave; Protein Engineering; Proteolysis; Cleavage (geology)

Abstract We describe the design, kinetic properties, and structures of engineered subtilisin proteases that degrade active form RAS by cleaving a conserved sequence in switch 2. is signaling protein that, when mutated, drives third human cancers. To generate high specificity for target sequence, site was modified to be dependent on cofactor (imidazole or nitrite) protease sub-sites were create linkage between substrate binding. Selective proteolysis arises from 2-step process wherein sub-site interactions promote productive binding cofactor, enabling cleavage. Proteases this way specifically cleave vitro, deplete level bacterial reporter system, also cell culture. Although these RAS, underlying design principles are fundamental will adaptable many proteins.