title: Purification of recombinant membrane proteins tagged with calmodulin-binding domains by affinity chromatography on calmodulin-agarose: example of nicotinamide nucleotide transhydrogenase.
abstract: This protocol describes affinity purification of bacterially expressed, recombinant membrane proteins fused with calmodulin-binding domains. As exemplified by the Escherichia coli nicotinamide nucleotide transhydrogenase, this method allows isolation of the protein fusions in a single chromatography step using elution with the calcium chelating agent EDTA and, unlike purification of His-tagged proteins on nickel chelate, it is not sensitive to the presence of strong reducing agents (e.g., DTT). Our protocol involves disruption of host bacteria by sonication, sedimentation of membranes by differential centrifugation, solubilization of membrane proteins and affinity chromatography on calmodulin-agarose. To achieve maximum purity and yield, the use of a combination of non-ionic and anionic detergents is suggested. Purification takes two working days, with an overnight wash of the column to increase the purity of the product.
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, Moscow 117997, Russia.
authors.forename: Nikolay B
authors.initials: NB
authors.lastname: Pestov
authors.forename: Jan
authors.initials: J
authors.lastname: Rydström
mesh_terms: D002147:Calmodulin; D002148:Calmodulin-Binding Proteins / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D008565:Membrane Proteins / Q000302:isolation & purification*; D009250:NADP Transhydrogenases / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification*
chemical_list: D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D009250:NADP Transhydrogenases