biostudies-literatureNakahara ENEI NIH HHSNational Eye InstituteChan Zuckerberg InitiativeRoger and Dorothy Hirl Research Fund2877-2889https://www.ebi.ac.uk/biostudies/studies/S-EPMC11212008biostudies-literatureUnknown17(10)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 <i>E. coli</i> 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 ACS chemical biologyDevelopment of a New DHFR-Based Destabilizing Domain with Enhanced Basal Turnover and Applicability in Mammalian Systems.PMC11212008R21 EY033522R01 EY027785EY0304132018-191983EY027785P30 EY030413Collier GEHulleman JDNakahara EMullapudi VJoachimiak LAfalseDevelopment of a New DHFR-Based Destabilizing Domain with Enhanced Basal Turnover and Applicability in Mammalian Systems.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 <i>E. coli</i> 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 2022-01-01T00:00:00Z2022 Oct2026-05-29T15:09:06.326Z2026-04-08T05:06:44.667ZS-EPMC112120083612292810.1021/acschembio.2c00518