The Expression And Purification Of Protein CBP60g In Escherichia Coli And Insect Cells Systems

Plants defend against adverse environmental factors by regulating intracellular calcium concentration.Calmodulin is one of the most important calcium receptors.The change of the binding conformation of Ca2+-CaM complex with calmodulin binding protein participates in the response of plants to biotic and abiotic stresses.As an important member of the CBPs protein family,CBP60g plays an important role in disease resistance,drought resistance,positive regulation of ABA and SA mediated signaling pathways,and negative regulation of anthocyanin synthesis.In this study,soluble expression and purification of Arabidopsis CBP60g protein in Escherichia coli expression system and insect cell expression system were attempted,in order to provide a basis for further analysis of its three-dimensional structure.The specific results are as follows:1.Analysis and prediction of the amino acid sequence of CBP60g showed that it contained 11 glycosylation sites,76 phosphorylation sites and no transmembrane domain.2.Arabidopsis CBP60g gene was amplified by PCR using wild Arabidopsis cDNA as template.The full length of the gene was 1689 bp.3.The expression vectors were constructed by TEV-LIC technology and transformed into BL21(DE3).Induced by EPTG at different concentrations and detected by SDS-PAGE,it was found that the target protein CBP60g existed mainly in the form of inclusion bodies.4.The baculovirus transfer vector was successfully constructed and transformed into E.coli DH10Bac.The recombinant baculovirus shuttle vector was obtained and transfected into insect cells to express the protein.The size of the target protein detected by SDS-PAGE was about 75 KDa,slightly larger than that predicted by SDS-PAGE.After anion exchange purification,the protein concentration was calculated to be 996.5ng/mL according to the established protein standard curve.Western Blotting validation showed that the purified protein was CBP60g.