Protein trans-splicing and cyclization by a naturally split intein from the dnaE gene of Synechocystis species PCC6803

A naturally occurring split intein from the dnaE gene of Synechocystis sp. PCC6803 (Ssp DnaE intein) has been shown to mediate efficient in vivo and i n vitro transsplicing in a foreign protein context. A cis-splicing Ssp DnaE intein construct displayed splicing activity similar to the trans-splicing form, which suggests that the Nand C-terminal intein fragments have a high affinity interaction. An in vitro trans-splicing system was developed that used a bacterially expressed N-terminal fragment of the Ssp DnaE intein an d either a bacterially expressed or chemically synthesized intein C-termina l fragment. Unlike artificially split inteins, the Ssp DnaE intein fragment s could be reconstituted in vitro under native conditions to mediate splici ng as well as peptide bond cleavage. This property allowed the development of an on-column trans-splicing system that permitted the facile separation of reactants and products. Furthermore, the trans-splicing activity of the Ssp DnaE intein was successfully applied to the cyclization of proteins in vivo, Also, the isolation of the unspliced precursor on chitin resin allowe d the cyclization reaction to proceed in vitro. The Ssp DnaE intein thus re presents a potentially important protein for in vivo and in vitro protein m anipulation.