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.
Artikel i vetenskaplig tidskrift, 2004

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.

Chromatography

genetics

isolation & purification

Escherichia coli

chemistry

metabolism

Humans

Genetic Vectors

chemistry

chemistry

Recombinant Fusion Proteins

ultrastructure

genetics

Protein Structure

Amino Acid Sequence

enzymology

NADP Transhydrogenase

Molecular

genetics

genetics

Protein Subunits

Calmodulin

isolation & purification

genetics

Cloning

chemistry

isolation & purification

chemistry

methods

Calmodulin-Binding Proteins

Calcium-Transporting ATPases

chemistry

isolation & purification

Affinity

chemistry

genetics

Tertiary

Cell Membrane

Membrane Proteins

genetics

chemistry

Molecular Sequence Data

Författare

Maxim V Egorov

ANNA KATARINA TIGERSTRÖM

Göteborgs universitet

Nikolay B Pestov

Tatyana V Korneenko

Maria B Kostina

Mikhail I Shakhparonov

Jan Rydström

Göteborgs universitet

Protein Expression and Purification

1046-5928 (ISSN) 1096-0279 (eISSN)

Vol. 36 1 31-9

Ämneskategorier

Biokemi och molekylärbiologi

DOI

10.1016/j.pep.2004.03.002

PubMed

15177281