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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

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

Maxim V Egorov, Anna Tigerström, Nikolay B Pestov, Tatyana V Korneenko, Maria B Kostina, Mikhail I Shakhparonov, Jan Rydström

Protein Expr Purif. 2004 Jul;36(1):31-9.

PMID: 15177281

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR42412783	Calmodulin, His•Tag Human, Recombinant			Price

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