Chemical Control of Protease Activity and Gene Expression Using a Biotin-Released Inhibitory Domain

Chemical tools that enable precise temporal control of protein function are valuable reagents for probing dynamic processes within live cells. Here, we introduce the biotin unblocking of the StrepTactin steric block (BUSS) system, a novel chemogenetic tool that enables precise temporal control of protein activity and interactions using biotin. BUSS leverages the small StrepTagII (STII) peptide to flank target domains, blocking their function by binding to StrepTactin, with this steric block rapidly released upon biotin addition. Unlike existing systems, which rely on larger protein tags that can disrupt sensitive proteins, BUSS uses a smaller, minimally disruptive tag compatible with a wide range of target proteins. We demonstrate the versatility of the BUSS system for chemical control over diverse cellular processes, including protein localization, protease activity, and gene expression. With its compact design and broad utility, BUSS offers a powerful approach for manipulating protein functions in living cells.