Clinically approved chemical-controlled suppression of protein expression in BmN cells

Abstract

 Rapid control of protein abundance is crucial in both basic and applied research. Small molecule-controlled protein expression is promising tools for such rapid control. However, concerns arise regarding the residual chemicals in cells treated with these molecules, especially when recombinant proteins are intended for medical purposes. The small molecule-assisted shut-off (SMASh) utilizes inhibitors against hepatitis C virus (HCV) non-structural protein 3 protease, enabling use of the Pharmaceuticals and Medical Devices Agency (PMDA)-approved, less concerning chemicals for the target protein control. In this study, we developed SMASh-tagged enhanced green fluorescent protein (EGFP) reporters to evaluate the efficacy of SMASh and confirmed its functionality in the Bombyx mori-derived ovary cell line, BmN. Upon comparing degron efficiencies, we discovered that the HCV-derived degron in the SMASh tag was more effective at degrading the fused EGFP than the commonly used PEST sequence of mouse ornithine decarboxylase. The activity of the SMASh tag was quantitatively tunable within the range of 10⁻³ to 1 mM of Asunaprevir in BmN cells. Higher concentrations, such as 5 µM, were toxic to the cells. We also revealed the irreversibility of SMASh-mediated gene suppression in BmN cells. Our findings pave the way for achieving rapid and robust suppression of target protein expression with PMDA-approved drugs in silkworm biology.