Production of cyclic peptides and proteins in vivo

Combinatorial libraries of synthetic and natural products are an important source of molecular information for the interrogation of biological targets . Methods for the intracellular production of libraries of small, stable mo lecules would be a valuable addition to existing library technologies by co mbining the discovery potential inherent in small molecules with the large library sizes that can be realized by intracellular methods. We have explor ed the use of split inteins (internal proteins) for the intracellular catal ysis of peptide backbone cyclization as a method for generating proteins an d small peptides that are stabilized against cellular catabolism. The DnaE split intein from Synechocystis sp. PCC6803 was used to cyclize the Escheri chia coil enzyme dihydrofolate reductase and to produce the cyclic, eight-a mino acid tyrosinase inhibitor pseudostellarin F in bacteria. Cyclic dihydr ofolate reductase displayed improved in vitro thermostability, and pseudost ellarin F production was readily apparent in vivo through its inhibition of melanin production catalyzed by recombinant Streptomyces antibioticus tyro sinase. The ability to generate and screen for backbone cyclic products in vivo is an important milestone toward the goal of generating intracellular cyclic peptide and protein libraries.