Herbicide resistance from a divided EPSPS protein: the split SynechocystisDnaE intein as an in vivo affinity domain

We report that the N- and C-terminal splicing domains of the intein found i n the dnaE gene of Synechocystis sp. PCC6803 (Ssp DnaE intein) are capable of association in vivo and in vitro, even with key splicing residues change d to alanine (Cys(1), Asn(159), and Cys(+1) to Ala). These studies utilized the herbicide resistant form of 5-enolpyruvylshikimate-3-phosphate synthas e (EPSPS) from Salmonella typhimurium and an Escherichia coli strain with t he EPSPS gene deleted from its genome (E. coli strain ER2799). EPSPS was ma pped to identify potential split sites using a facile Tn7 linker scanning p rocedure. Forty positions were found to tolerate a five amino acid insertio n while 21 sites did not, as assayed by the rescue of growth of E. coli str ain ER2799. Further characterization of these sites by inserting a full len gth Ssp DnaE intein identified residue 235 of EPSPS as the optimal position . The EPSPS gene was then divided into amino acids 1-235 and 236-427 which were fused to residues 1-123 and 124-159 of a splicing defective Ssp DnaE i ntein, respectively. Expression of the EPSPS-intein fusions from separate D NA molecules conferred resistance to the herbicide glyphosate, indicating t hat the intein splicing domains were bringing the EPSPS fragments together to generate activity. As a control the split EPSPS without the intein-affin ity domain did not allow cell growth. The use of an intein as an in vivo af finity domain was termed intein-mediated protein complementation (TPC). Int ein fragment assembly was verified in vitro by immobilizing the C-terminal splicing domain of the Ssp DnaE intein on a resin and demonstrating that th e N-terminal 235 amino acids of EPSPS only bound to the resin when fused to the N-terminal splicing domain of the Ssp DnaE intein. As chloroplast DNA is not transmitted by pollen in plants such as corn and soybean, transgene spread via pollen may be controlled in the future by expressing inactive ge ne fragments from separate DNA locations, such as the nuclear and chloropla st genome, and using the split intein to generate protein activity. (C) 200 1 Elsevier Science B.V. All rights reserved.