Conditional control of protein function

Deciphering the myriad ways in which proteins interact with each other to give rise to complex behaviors that define living systems is a significant challenge. Genetic analyses have provided many insights into the functions of proteins encoded by specific genes; however, it can be difficult to study essential genes, and many biological processes occur on a fast timescale that precludes study using genetic methods. For these reasons and others, it is often desirable to target proteins directly rather than the genes that encode them. Our goal is the development of destabilizing domains that, when fused to a gene of interest, can be used to degrade proteins of interest. These chimeras would then be stabilized through addition of a cell‐permeable small‐molecule ligand, thus restoring function to the protein of interest. Such destabilizing domains were identified through error prone PCR and subsequent FACS‐based screening of the libraries in the presence and absence of the small molecule. Destabilizing domains identified show differences in protein levels in the presence and absence of ligand of up to two orders of magnitude, with ligand concentrations as low as 5 nM giving 50% rescue. Degradation of the chimeras is rapid, with protein levels becoming negligible within 4 hours of ligand removal. These destabilizing domains elicit conditional protein degradation of a variety of different classes of proteins, with protein function being restored upon addition of the ligand. This technology will allow investigation of the function of many unknown genes, providing greater understanding of complex biological systems. This work is funded by NIH GM068589.