Detection of CP4 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) in soybean food products by affinity chromatography

Abstract

The Round-up Ready£¿ soybean, which has the tolerance against an herbicide, glyphosate, is widely used for soybean processing. Recently, detection of genetically modified (GM) food becomes more important because of mandatory labeling. In general, there are two quantitation methods of genetically modified soybean in soybean food products; one is based on PCR analysis at DNA level and the other is an immunoassay for the CP4 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) at protein level. However, these two methods have limitations when GM soybean is processed to Korean traditional foods. Therefore, development of a new detection method is needed for soybean food products. Affinity chromatography could be a possibility as an alternative detection method at protein level using the shikimate 3-phosphate as a ligand. Especially, because CP4 EPSPS is intact after the heat-treatment for 90 min at 95 ¡ÆC, tofu can be applied onto affinity chromatography. An affinity chromatography column was made by immobilizing shikimate 3-phosphate to the beaded agarose gel through the carbodiimide condensation. Since the shikimate 3- phosphate was not available commercially, it was synthesized enzymatically by shikimate kinase II and purified by ion exchange chromatography. The protein samples isolated from GM soybean were applied to an affinity chromatography column and CP4 EPSPS was analyzed by SDS-PAGE. It seemed that CP4 EPSPS was adsorbed onto affinity chromatography column. However, Because shikimate 3- phosphate was purified with low level, the efficiency of affinity chromatography column was not good. To prepare a large quantity of shikimate 3-phoshate, an affinity chromatography can be a successful detection method for CP4 EPSPS from GM soybeans and its food products. For the better analysis of CP4 EPSPS, the changes of ligand immobilizing method and protein elution condition should be considered.