Purification of recombinant proteins based on the interaction between a phenothiazine-derivatized column and a calmodulin fusion tail

A method to purify proteins by fusing them to the Ca2+-dependent protein ca lmodulin is described by using glutathione-S-transferase (GST) from Schisto soma japonicum as a model. Glutathione-S-transferase was genetically fused to calmodulin (CaM). The designed GST-CaM fusion protein has a selective fa ctor Xa cleavage site located between the C-terminus of GST and the N-termi nus of CaM. The recombinant fusion protein was expressed in Escherichia col i, and the crude cell extract was loaded onto a phenothiazine affinity colu mn in the presence of Ca2+. Calmodulin was used as an affinity tail to enab le binding of the fusion protein to the phenothiazine column. Removal of Ca 2+ with a calcium-complexing solution causes elution of the fusion protein. The GST-CaM fusion protein was then digested with factor Xa, and the targe t protein GST was isolated. The purity of the isolated GST was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).