Protein trans-splicing to produce herbicide-resistant acetolactate synthase.
Protein splicing in trans has been demonstrated both in vivo and in vitro by biochemical and immunological analyses, but in vivo production of a functional protein by trans-splicing has not been reported previously. In this study, 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 herbicide-resistant ALSII was fused in frame to DnaE intein segments capable of promoting protein splicing in trans and was expressed from two compatible plasmids as two unlinked fragments. Cotransformation of E. coli with the two plasmids led to production of a functional enzyme that conferred herbicide resistance to the host E. coli cells. These results demonstrate the feasibility of expressing functional genes from two unlinked DNA loci and provide a model for the design of nontransferable transgenes in plants.
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New England Biolabs, Inc., Beverly, Massachusetts 01915, USA.
Ghosh
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Paulus
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D000095:Acetolactate Synthase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000458:Cyanobacteria / Q000235:genetics / Q000378:metabolism; D004258:DNA Polymerase III / Q000235:genetics* / Q000378:metabolism; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D006540:Herbicides / Q000494:pharmacology*; D015151:Immunoblotting; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020040:Trans-Splicing; D003313:Zea mays / Q000201:enzymology
D006540:Herbicides; D011993:Recombinant Fusion Proteins; D000095:Acetolactate Synthase; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III