AFFINITY-CHROMATOGRAPHY OF RECOMBINANT PEPTIDES PROTEINS BASED ON A CALMODULIN FUSION TAIL

An affinity chromatography system has been developed for the separatio n of recombinant fusion proteins based on the Ca2+-dependent binding o f calmodulin (CaM) to the drug phenothiazine. Specifically, in the pre sence of Ca2+, a recognition site for phenothiazine is exposed on calm odulin, allowing the binding of this drug to CaM, Upon removal of Ca2 with EGTA, the conformation of calmodulin changes, and the phenothiaz ine-CaM complex dissociates. This Ca2+-dependent binding has been expl oited in the development of a fusion tail approach for the affinity pu rification of recombinant proteins and peptides. Protein A (ProtA) was employed as a model protein to demonstrate the advantages of this app roach. In particular, the developed affinity chromatography system was used to isolate several ProtA-CaM fusion proteins. These recombinant fusion proteins were expressed in Escherichia coli and Saccharomyces c erevisiae from appropriately designed plasmids. Four different plasmid s (two each for the bacteria and yeast) were used that encoded the fus ion of CaM to the immunoglobulin-binding portion of protein A. After e xpression of the fusion protein, the crude cell lysates were loaded on to the phenothiazine affinity column in the presence of a Ca2+-contain ing buffer. Upon elution with an EGTA buffer, the ProtA-CaM fusion pro tein was purified, as confirmed by SDS-PAGE electrophoresis and Wester n blot analysis.