title: Affinity chromatography of recombinant peptides/proteins based on a calmodulin fusion tail.
abstract: An affinity chromatography system has been developed for the separation of recombinant fusion proteins based on the Ca(2+)-dependent binding of calmodulin (CaM) to the drug phenothiazine. Specifically, in the presence of Ca2+, a recognition site for phenothiazine is exposed on calmodulin, allowing the binding of this drug to CaM. Upon removal of Ca2+ with EGTA, the conformation of calmodulin changes, and the phenothiazine--CaM complex dissociates. This Ca(2+)-dependent binding has been exploited in the development of a fusion tail approach for the affinity purification of recombinant proteins and peptides. Protein A (ProtA) was employed as a model protein to demonstrate the advantages of this approach. 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 cerevisiae from appropriately designed plasmids. Four different plasmids (two each for the bacteria and yeast) were used that encoded the fusion of CaM to the immunoglobulin-binding portion of protein A. After expression of the fusion protein, the crude cell lysates were loaded onto the phenothiazine affinity column in the presence of a Ca(2+)-containing buffer. Upon elution with an EGTA buffer, the ProtA--CaM fusion protein was purified, as confirmed by SDS-PAGE electrophoresis and Western blot analysis.
authors.affiliation: Department of Chemistry, University of Kentucky, Lexington 40506-0055, USA.
authors.forename: N G
authors.initials: NG
authors.lastname: Hentz
authors.forename: V
authors.initials: V
authors.lastname: Vukasinovic
authors.forename: S
authors.initials: S
authors.lastname: Daunert
mesh_terms: D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli; D010640:Phenothiazines / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D013205:Staphylococcal Protein A / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D002147:Calmodulin; D010640:Phenothiazines; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; C014867:trifluoromethylphenothiazine; D002118:Calcium