Directed evolution of ligand dependence: Small-molecule-activated protein splicing

Allen R. Buskirk, Yi Ching Ong, Zev J. Gartner, David R. Liu

Research output: Contribution to journalArticlepeer-review


Artificial molecular switches that modulate protein activities in response to synthetic small molecules would serve as tools for exerting temporal and dose-dependent control over protein function. Self-splicing protein elements (inteins) are attractive starting points for the creation of such switches, because their insertion into a protein blocks the target protein's function until splicing occurs. Natural inteins, however, are not known to be regulated by small molecules. We evolved an intein-based molecular switch that transduces binding of a small molecule into the activation of an arbitrary protein of interest. Simple insertion of a natural ligand-binding domain into a minimal intein destroys splicing activity. To restore activity in a ligand-dependent manner, we linked protein splicing to cell survival or fluorescence in Saccharomyces cerevisiae. Iterated cycles of mutagenesis and selection yielded inteins with strong splicing activities that highly depend on 4-hydroxytamoxifen. Insertion of an evolved intein into four unrelated proteins in living cells revealed that ligand-dependent activation of protein function is general, fairly rapid, dose-dependent, and posttranslational. Our directed-evolution approach therefore evolved small-molecule dependence in a protein and also created a general tool for modulating the function of arbitrary proteins in living cells with a single cell-permeable, synthetic small molecule.

Original languageEnglish (US)
Pages (from-to)10505-10510
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number29
StatePublished - Jul 20 2004
Externally publishedYes

ASJC Scopus subject areas

  • General


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