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
| Autor: | Jan Rydström, Nikolay B. Pestov, Mikhail I. Shakhparonov, Tatyana V. Korneenko, M. B. Kostina, Anna Katarina Tigerström, Maxim V Egorov |
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| Rok vydání: | 2004 |
| Předmět: |
Calmodulin
Calmodulin binding domain Recombinant Fusion Proteins Genetic Vectors Molecular Sequence Data Calcium-Transporting ATPases Chromatography Affinity Protein structure Affinity chromatography Escherichia coli NADP Transhydrogenases Humans Amino Acid Sequence Cloning Molecular G alpha subunit chemistry.chemical_classification biology Cell Membrane Membrane Proteins Molecular biology Fusion protein Protein Structure Tertiary Amino acid Protein Subunits Biochemistry Membrane protein chemistry biology.protein Calmodulin-Binding Proteins Biotechnology |
| Zdroj: | Protein Expression and Purification. 36:31-39 |
| ISSN: | 1046-5928 |
| DOI: | 10.1016/j.pep.2004.03.002 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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