title: Purification of a recombinant membrane protein tagged with a calmodulin-binding domain: properties of chimeras of the Escherichia coli nicotinamide nucleotide transhydrogenase and the C-terminus of human plasma membrane Ca2+ -ATPase.
abstract: A Ca2+ -dependent calmodulin-binding peptide (CBP) is an attractive tag for affinity purification of recombinant proteins, especially membrane proteins, since elution is simply accomplished by removing/chelating Ca2+. To develop a single-step calmodulin/CBP-dependent purification procedure for Escherichia coli nicotinamide nucleotide transhydrogenase, a 49 amino acid large CBP or a larger 149 amino acid C-terminal fragment of human plasma membrane Ca2+ -ATPase (hPMCA) was fused C-terminally to the beta subunit of transhydrogenase. Fusion using the 49 amino acid fragment resulted in a dramatic loss of transhydrogenase expression while fusion with the 149 amino acid fragment gave a satisfactory expression. This chimeric protein was purified by affinity chromatography on calmodulin-Sepharose with mild elution with EDTA. The purity and activity were comparable to those obtained with His-tagged transhydrogenase and showed an increased stability. CBP-tagged transhydrogenase contained a 4- to 10-fold higher amount of the alpha subunit relative to the beta subunit as compared to wild-type transhydrogenase. To determine whether the latter was due to the CBP tag, a double-tagged transhydrogenase with both an N-terminal 6x His-tag and a CBP-tag, purified by using either tag, gave no significant increase in purity as compared to the single-tagged protein. The reasons for the altered subunit composition are discussed. The results suggest that, depending on the construct, the CBP-tag may be a suitable affinity purification tag for membrane proteins in general.
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, 117871 Moscow.
authors.forename: Maxim V
authors.initials: MV
authors.lastname: Egorov
authors.forename: Anna
authors.initials: A
authors.lastname: Tigerström
authors.forename: Nikolay B
authors.initials: NB
authors.lastname: Pestov
authors.forename: Tatyana V
authors.initials: TV
authors.lastname: Korneenko
authors.forename: Maria B
authors.initials: MB
authors.lastname: Kostina
authors.forename: Mikhail I
authors.initials: MI
authors.lastname: Shakhparonov
authors.forename: Jan
authors.initials: J
authors.lastname: Rydström
mesh_terms: D000595:Amino Acid Sequence; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics*; D002147:Calmodulin / Q000737:chemistry; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009250:NADP Transhydrogenases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D008565:Membrane Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D009250:NADP Transhydrogenases; D000252:Calcium-Transporting ATPases