| Abstract
| - Inteins are naturally occurring protein elements that catalyze their own excision from within alarger protein together with the ligation of the flanking “extein” sequences. Previously we reported the directedevolution of an intein-based molecular switch in which intein splicing in yeast cells was made dependenton the cell-permeable small molecule 4-hydroxytamoxifen (4-HT). Here we show that these evolved inteinsare effective means of rendering protein function and biological signaling pathway activation dependent on4-HT in mammalian cells. We have characterized the generality, speed, and dose dependence of ligand-induced protein splicing in murine NIH3T3 cells and in human HEK293 cells. Evolved inteins were used tocontrol in mammalian cells the function of Gli1 and a truncated form of Gli3, two transcriptional mediatorsof the Hedgehog signaling pathway. Finally, we show that a complex biological process such as osteoblastdifferentiation can be made dependent on 4-HT using the evolved intein system. Our findings suggest thatevolved small-molecule-dependent inteins may serve as a general means of achieving gene-specific, dose-dependent, post-translational, and small-molecule-induced control over protein activity in mammaliansystems.
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