Fusion Expression, Purification and Bioactivity Assay of CpTI in Escherichia coli
CpTI蛋白在大肠杆菌中的高效融合表达及其纯化与活性测定

CpTI (Cowpea Trypsin Inhibitor) is a widely used insect resistance gene in the plant genetic engineering for its high insecticidal activity and the minimal ability of the insects to evolve resistance to it. To facilitate the safety assessment of genetically modified foods (GMFs) with CpTI protein, we need to produce gram quantities of this protein in microbes. With the pGEX fusion expression system, we expressed the GST CpTI protein in E.coli BL21, which accounted for approximately 40% of germ proteins. By Glutathione Sephrose 4B affinity chromatography, GST CpTI was obtained with the purity up to 90%. Overnight incubate the fusion proteins with Thrombin protease, we got the CpTI proteins cleavage of GST tag. Both of the GST CpTI and CpTI proteins showed notable trypsin inhibitor activity. Immunization of rabbits with purified fusion protein generated high titer antibodies (>20000), measuring by ELISA. Western Blotting also showed specific Ag Ab binding band between the antiserum and the CpTI proteins no matter in the whole supersonic germ proteins or purified from the column. All these made a good ground for the further safety assessment of CpTI protein.