Patent ID: 7541450

Claim:
A method of producing and selecting a ligand-dependent intein, the method comprising the steps of: (a) providing a polynucleotide encoding a hybrid protein comprising a single polypeptide having a target protein with a ligand-dependent intein embedded in the sequence of said target protein, wherein said hybrid protein has the structure: target(N)-Intein(N)-ligand binding domain-Intein(C)-target(C); (b) mutating the sequence of the polynucleotide encoding the hybrid protein to generate a plurality of mutated polynucleotides; (c) transforming the polynucleotides produced in step (b) into cells; (d) selecting those cells having ligand-dependent intein activity based on the activity of the ligand-dependent intein in the presence of an appropriate ligand that binds the ligand binding domain, wherein a positive selection is based on the ability of the ligand-binding intein to excise itself from the target protein upon ligand binding and produce a functional target protein; and (e) optionally isolating the polynucleotides produced from the cells identified in step (d).