Abstract 1851 Isolating the C. elegans 26S proteasome for LOCKR optimization

The ability to inactivate gene function is essential for understanding normal development and the development of disease. The Latching Orthogonal Cage and Key pRoteins (LOCKR) technology uses orthogonal Switches and Keys to control gene expression at the protein level. The Switch functions to cage a functional protein sequence, such as a degron (degronSwitch), in the off or "locked" state. Upon Key addition, the Switch is turned on or "unlocked," which exposes the degron, promoting the proteasomal degradation of the Switch and any proteins fused to it. To date, degronSwitches have been successfully designed to induce protein degradation in yeast and mammalian cell cultures, but have not yet been used in an animal model. In addition, yeast and mammalian degronSwitches utilized different LOCKR designs for optimal function, emphasizing the need to optimize the Switch and Key constructs for use in other organisms. We set out to optimize the LOCKR system for use in C. elegans. To this end, we will conduct in vitro experiments to assess the interactions of different Switch and Key designs, benefiting from isolated C. elegans 26S proteasome. To isolate the 26S proteasome from C. elegans, we pursued two techniques: 1) affinity purification of proteasomal subunits following the addition of a purification tag using CRISPR-Cas9 and 2) ion-exchange and hydrophobic interaction chromatography. For affinity purification, we engineered C-terminal 3xFLAG tags for CRISPR-Cas9 modification of two genes in the 19S regulatory particle (rpn-1, rpn-11) and one gene in the 20S core particle (pbs-4). We report the successful construction of CRISPR-Cas9 gRNAs for rpn-1, rpn-11, and pbs-4, which will allow for 26S proteasome isolation in the presence of ATP. We also report our initial experiments to isolate the 26S proteasome from C. elegans using chromatography. We anticipate that isolated proteasome will be useful to optimize the LOCKR system and in future experiments that explore proteasome function in C. elegans. This work is supported by the Wabash College Haines Biochemistry Fund, the Wabash College Treves Biology Fund, and NSF Grant MCB-2146714.