Available Technology
Small-molecule-triggered intein splicing as a universal switch for protein activation
Technology:
Small-molecule-triggered inteins
Markets Addressed
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.
Innovations and Advantages
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.
Additional Information
Intellectual Property Status: Issued U.S. patent nos.: 7,192,739; 7,541,450
Publications:
Directed Evolution of a Small-Molecule-Triggered Intein with Improved Splicing Properties in Mammalian Cells, Peck, SH; Chen, I; Liu, DR. Chemistry & Biology 18 (5), 619-630 (2011); PMID: 21609843
Directed Evolution of Ligand Dependence: Small Molecule-Activated Protein Splicing , Buskirk, AR; Ong, Y-C; Gartner, ZJ; Liu, DR. Proc. Natl. Acad. Sci. USA 101, 10505-10510 (2004); PMID: 15247421
Related technology:
An in vivo selection system for evolving the specificity of endonucleases and inteins
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Inventor(s):
Buskirk, Allen R.
Liu, David R.
Peck, Sun H.
Categories:
For further information, please contact:
Vivian Berlin, Director of Business Development
(617) 496-0474
Reference Harvard Case #2334