Protein trans-splicing to produce herbicide-resistant acetolactate synthase

Protein splicing in trans has been demonstrated both in vivo and in vitro b y biochemical and immunological analyses, but in vivo production of a funct ional protein by trans-splicing has not been reported previously. In this s tudy, we used the DnaE intein from Synechocystis sp. strain PCC6803, which presumably reconstitutes functional DnaE protein by trans-splicing in vivo, to produce functional herbicide-resistant acetolactate synthase II (ALSII) from two unlinked gene fragments in Escherichia coli. The gene for herbici de-resistant ALSII was fused in frame to DnaE intein segments capable of pr omoting protein splicing in trans and was expressed from two compatible pla smids as two unlinked fragments. Cotransformation of E. coli with the two p lasmids led to production of a functional enzyme that conferred herbicide r esistance to the host E. coli cells. These results demonstrate the feasibil ity of expressing functional genes from two unlinked DNA loci and provide a model for the design of nontransferable transgenes in plants.