Cloning, expression, purification and functional characterization of full-length and truncated forms of aroA CP4 gene from genetically modified maize

Development of genetically modified (GM) food has generated worldwide public controversy over food safety issues. To assess the food safety aspects and potential risk of horizontal gene transfer (HGT) from GM plants and food derived thereof, we have chosen aroA CP4 coding EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) from GM maize. During the study of possible HGT of aroA CP4 gene from GM food, we have discovered and characterized truncated form of aroA CP4 within the cloning experiments. Comparison of properties of recombinant E. colt strains with both forms of CP4 EPSPS gene were performed on DNA and protein levels. We report on cloning, expression, purification and enzyme activity studies of both genes. The specific activities of both enzymes were indistinguishable indicating that the truncated CP4 EPSPS enzyme retained the activity of a full-length CP4 EPSPS. The in vitro growth test showed that both forms of aroA CP4 genes grew well at concentrations of 10-40 mmo.l(-1) glyphosate and the in vivo complementation test revealed uniform growth. The results indicate that both full-length and truncated aroA CP4 have similar characteristics (full length CP4 EPSPS specific activity 0.063 +/- 0.01 mu mol.min(-1).mg(-1); truncated CP4 EPSPS specific activity 0.055 +/- 0.01 mu mol.min(-1).mg(-1)). These results should also be taken into account in risk analysis of possible unintended effects from GM plants.