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Applied and Environmental Microbiology, March 2001, p. 1025-1029, Vol. 67, No. 3
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.3.1025-1029.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Protein trans-Splicing To Produce Herbicide-Resistant Acetolactate Synthase

Luo Sun,1 Inca Ghosh,1 Henry Paulus,2 and Ming-Qun Xu1,*

New England Biolabs, Inc., Beverly, Massachusetts 01915,1 and Boston Biomedical Research Institute, Watertown, Massachusetts 024722

Received 24 August 2000/Accepted 13 November 2000

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.

* Corresponding author. Mailing address: New England Biolabs, Inc., 32 Tozer Road, Beverly, MA 01915. Phone: (978) 927-5054. Fax: (978) 921-1350. E-mail: xum{at}neb.com.

Applied and Environmental Microbiology, March 2001, p. 1025-1029, Vol. 67, No. 3
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.3.1025-1029.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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Copyright © 2001 by the American Society for Microbiology. All rights reserved.