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General Methods to Conditionally Regulate Protein Function
Author(s) -
Wandless Thomas J
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.200.2
Subject(s) - dihydrofolate reductase , fusion protein , microbiology and biotechnology , mutant , chemistry , biology , synthetic biology , computational biology , biochemistry , gene , recombinant dna
Advances in our understanding of complex, sometimes interconnected, signaling networks will likely require the implementation of tunable perturbation techniques that enable sensitivity analyses of these circuits. We have developed a general experimental technique in which the stability of a specific protein is regulated by a synthetic small molecule. Mutants of the E. coli dihydrofolate reductase (ecDHFR) protein were engineered to be degraded when expressed in mammalian cells. Trimethoprim (TMP) is a high‐affinity ligand for ecDHFR that stabilizes fusion proteins in a predictable, dose‐dependent manner, allowing investigators to tunably regulate genetically engineered alleles of any protein of interest. DD‐regulated proteins are degraded very rapidly upon withdrawal of TMP, and protein levels in the absence of TMP are barely detectable. The ability of TMP to cross the blood‐brain barrier enables the tunable regulation of proteins expressed in cells found within the mammalian central nervous system.