Tetrahymena thermophila is a ciliated protist that has played pivotal roles in biological discovery. Functional studies of Tetrahymena proteins have largely relied on gene knockouts. Because protein depletion upon knockout typically spans multiple cell cycles, compensatory mechanisms can confound phenotypic interpretation. To instead enable rapid and acute protein depletion, we modified and adapted the Trim-Away system for use in Tetrahymena (Tet Trim-Away). Trim-Away is based on the E3 ubiquitin ligase, TRIM21, that binds to antibody-bound proteins and targets them for proteasome mediated degradation. Here, Trim-Away was modified with a fusion of the N-terminal RBCC (RING, B-box, coiled-coil) domains of TRIM21 with an -mCherry (mCh) nanobody sequence that recognizes endogenously tagged mCh proteins of interest (NbmCh). Expression of the RBCC:NbmCh degron, which is controlled by an inducible promotor, promotes rapid target protein depletion within 30 minutes and can be sustained for weeks. Tet Trim-Away is reversible, functions against targets in multiple cellular compartments, and produces loss-of-function phenotypes in Tetrahymena cells.
Dholakia, G. L., Stemm-Wolf, A. J., Anzar, A., Turkewitz, A., Pearson, C. G.
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