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.
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.
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.
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.
Intellectual Property Status: Patent(s) Pending