Expression In E.coli And Preparation Of The Standard Tracing Protein CP4-EPSPS

EPSPS (5-enolpyruvyl-shikimate-3-phosphate synthase, EPSPS: EC 2.5.1.19) is a key enzyme in shikimate pathway that catalyzes the PEP (phosphoenolpyruvate type pyruvate) and SP3 (shikimic acid -3-phosphate) generated EPSP (5-enolacetone shikimic acid-3-phosphoric acid), provide the precursors for aromatic amino acids generating. EPSPS exists widely in microbes and higher plants. EPSPS is also the target enzyme of glyphosate herbicide, since this reason the research related to the EPSPS has been widely carried out. CP4 EPSPS is from Agrobacterium tumefaciens (Agrobacterium sp.) isolated in the high concentration of glyphosate condition, exhibited very high resistance to the glyphosate. So this gene was extensively used in transgenic study. By 2010, including soybeans, corn, canola, cotton, sugar beets and alfalfa, nearly 89.3 million hectares GM crops contain the herbicide-resistant gene were planted. Facing the fact that more and more crops and food containing CP4-EPSPS gene importing to our country, established scientific detection method is particularly urgent. Although there are several products can detect the GMO crops on the market today, but different manufacturers use different techniques and methods to determine the expression of the target crops. Those methods use different measurement standards, resulting in inaccurate and lack of determination results comparability, which has brought risks to the bio-safety regulation, legal, trade and other economic and social condition.In order to obtain a stable source and higher purity CP4-EPSPS protein, this study constructed the bacterial CP4-EPSPS expression vector, and the first time this gene was expressed in E. coli BL21 (transtta). Sonication the expression cell, it was found that the recombinant protein did not form inclusion bodies in the cytoplasm but exists in a soluble form solvated in the disruption supernatant. This property brought a great convenience to the downstream protein purification work. After two-step nickel-affinity chromatography, the CP4-EPSPs concentration can reach up to 99.9%, which can meet the standard certifier.To verify the purification of the CP4-EPSPS protein as the feasibility of reference materials, we construct the plant expression vector of CP4-EPSPS. Through tissue culturation, we get 49 strains positive tobaccos, after verification with Elisa, there are 16 plants showed an obvious expression. Making the purification of CP4-EPSPS protein as standard substances, diluted into different concentrations, we draw a slandered observation curve. Take three high expression tobaccos for the detection, using the standard curve we have detected expression level of these three tobaccos.In conclusion, using this protein expression and purification systems, we can scale up and standardized to generate the CP4-EPSPS protein. This can in timely provide the course for the Beijing Polytechnic University and the National Measurement Institute and other cooperative units, in order to carry out further research work.