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Small-molecule-triggered intein splicing as a universal switch for protein activation

Researchers in the laboratory of Professor David Liu have used directed evolution techniques to evolve an intein-based molecular switch that transduces binding of a small molecule into the activation of an arbitrary protein of interest. To create the switch, the researchers replaced the dispensable homing endonuclease domain of the RecA intein with the ligand-binding domain of the human estrogen receptor (ER) to yield an (N)-interin-ER-intein(C) fusion. This chimeric intein was then fused into a number of protein (extein) contexts to facilitate rounds of positive (negative) selection for ligand-dependent splicing in the presence (absence) of the synthetic small molecule 4-hydroxytamoxifen. The switch was shown to be effective in multiple protein contexts in both yeast and mammalian cells. The researchers recently reported second-generation evolved inteins that exhibit substantially improved splicing yields and kinetics over previously reported first generation inteins.

These new ligand-dependent inteins represent effective and broadly applicable tools for the small-molecule-triggered, posttranslational modulation of protein activities in living systems including mammalian cells, and for the in vivo activation of intein-fused therapeutic proteins.



Intein Evolution Approach. Overview of the directed evolution strategy used to isolate improved small-molecule-dependent inteins.

Intellectual Property Status: Patent(s) Pending

Applications

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 known as inteins, which are able to catalyze their excision out of a single polypeptide and leave behind precisely ligated flanking sequences (exteins), 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, and because they are able to rapidly splice out of a wide variety of extein contexts. Natural inteins, however, are not known to be regulated by small molecules.

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