Engineered reversible inhibition of SpyCatcher reactivity enables rapid generation of bispecific antibodies.

ABSTRACT: The precise regulation of protein function is essential in biological systems and a key goal in chemical biology and protein engineering. Here, we describe a straightforward method to engineer functional control into the isopeptide bond-forming SpyTag/SpyCatcher protein ligation system. First, we perform a cysteine scan of the structured region of SpyCatcher. Except for two known reactive and catalytic residues, none of these mutations abolish reactivity. In a second screening step, we modify the cysteines with disulfide bond-forming small molecules. Here we identify 8 positions at which modifications strongly inhibit reactivity. This inhibition can be reversed by reducing agents. We call such a reversibly inhibitable SpyCatcher "SpyLock". Using "BiLockCatcher", a genetic fusion of wild-ty

SUBMITTER: Hentrich C 

PROVIDER: S-EPMC11251281 | biostudies-literature | 2024 Jul

REPOSITORIES: biostudies-literature