[Fusion expression, purification and bioactivity assay of CpTI in Escherichia coli].
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.
Yang
Li-Chen
LC
Plant Genetic Manipulation Laboratory, Institute of Genetics, Chinese Academy of Sciences, Beijing 100101, China.
Chang
Tuan-Jie
TJ
Chen
Wan-Xin
WX
Yang
Xiao-Guang
XG
Zhu
Zhen
Z
D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D014361:Trypsin Inhibitors