Control of transcription factor activity and osteoblast differentiation in mammalian cells using an evolved small-molecule-dependent intein.
Authors | |
Keywords | |
Abstract | Inteins are naturally occurring protein elements that catalyze their own excision from within a larger protein together with the ligation of the flanking "extein" sequences. Previously we reported the directed evolution of an intein-based molecular switch in which intein splicing in yeast cells was made dependent on the cell-permeable small molecule 4-hydroxytamoxifen (4-HT). Here we show that these evolved inteins are effective means of rendering protein function and biological signaling pathway activation dependent on 4-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 to control in mammalian cells the function of Gli1 and a truncated form of Gli3, two transcriptional mediators of the Hedgehog signaling pathway. Finally, we show that a complex biological process such as osteoblast differentiation can be made dependent on 4-HT using the evolved intein system. Our findings suggest that evolved 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 mammalian systems. |
Year of Publication | 2006
|
Journal | J Am Chem Soc
|
Volume | 128
|
Issue | 27
|
Pages | 8939-46
|
Date Published | 2006 Jul 12
|
ISSN | 0002-7863
|
DOI | 10.1021/ja062980e
|
PubMed ID | 16819890
|
PubMed Central ID | PMC2519127
|
Links | |
Grant list | R01 NS033642 / NS / NINDS NIH HHS / United States
R01GM065400 / GM / NIGMS NIH HHS / United States
R37 NS033642 / NS / NINDS NIH HHS / United States
R01 GM065400 / GM / NIGMS NIH HHS / United States
NS033642 / NS / NINDS NIH HHS / United States
R01 GM065400-07 / GM / NIGMS NIH HHS / United States
|