Development of a New DHFR-Based Destabilizing Domain with Enhanced Basal Turnover and Applicability in Mammalian Systems.

ABSTRACT: Destabilizing domains (DDs) are an attractive strategy allowing for positive post-transcriptional small molecule-regulatable control of a fusion protein's abundance. However, in many instances, the currently available DDs suffer from higher-than-desirable basal levels of the fusion protein. Accordingly, we redesigned the E. coli dihydrofolate reductase (ecDHFR) DD by introducing a library of ∼1200 random ecDHFR mutants fused to YFP into CHO cells. Following successive rounds of fluorescence-activated cell sorting, we identified six new ecDHFR DD clones with significantly enhanced proteasomal turnover in the absence of a stabilizing ligand, trimethoprim (TMP). One of these clones, designated as "C12", contained four unique missense mutations (W74R/T113S/E120D/Q146L) and demonstrated

SUBMITTER: Nakahara E 

PROVIDER: S-EPMC11212008 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature