We report that the N- and C-terminal splicing domains of the intein found in the dnaE gene of Synechocystis sp. PCC6803 (Ssp DnaE intein) are capable of association in vivo and in vitro, even with key splicing residues changed to alanine (Cys(1), Asn(159), and Cys(+1) to Ala). These studies utilized the herbicide resistant form of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Salmonella typhimurium and an Escherichia coli strain with the EPSPS gene deleted from its genome (E. coli strain ER2799). EPSPS was mapped to identify potential split sites using a facile Tn7 linker scanning procedure. Forty positions were found to tolerate a five amino acid insertion while 21 sites did not, as assayed by the rescue of growth of E. coli strain ER2799. Further characterization of these sites by inserting a full length 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 intein, respectively. Expression of the EPSPS-intein fusions from separate DNA molecules conferred resistance to the herbicide glyphosate, indicating that the intein splicing domains were bringing the EPSPS fragments together to generate activity. As a control the split EPSPS without the intein-affinity domain did not allow cell growth. The use of an intein as an in vivo affinity domain was termed intein-mediated protein complementation (IPC). Intein fragment assembly was verified in vitro by immobilizing the C-terminal splicing domain of the Ssp DnaE intein on a resin and demonstrating that the 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 gene fragments from separate DNA locations, such as the nuclear and chloroplast genome, and using the split intein to generate protein activity.
3-Phosphoshikimate 1-Carboxyvinyltransferase Alkyl and Aryl Transferases