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Purification of a r...
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.
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Egorov, Maxim V (author)
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- TIGERSTRÖM, ANNA KATARINA, 1976 (author)
- Gothenburg University,Göteborgs universitet,Institutionen för kemi,Department of Chemistry,University of Gothenburg
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Pestov, Nikolay B (author)
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Korneenko, Tatyana V (author)
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Kostina, Maria B (author)
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Shakhparonov, Mikhail I (author)
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- Rydström, Jan, 1943 (author)
- Gothenburg University,Göteborgs universitet,Institutionen för kemi,Department of Chemistry,University of Gothenburg
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(creator_code:org_t)
- Elsevier BV, 2004
- 2004
- English.
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In: Protein expression and purification. - : Elsevier BV. - 1046-5928 .- 1096-0279. ; 36:1, s. 31-9
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http://dx.doi.org/10...
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Abstract
Subject headings
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- 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.
Subject headings
- NATURVETENSKAP -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)
Keyword
- Amino Acid Sequence
- Calcium-Transporting ATPases
- chemistry
- genetics
- Calmodulin
- chemistry
- Calmodulin-Binding Proteins
- chemistry
- genetics
- Cell Membrane
- metabolism
- ultrastructure
- Chromatography
- Affinity
- methods
- Cloning
- Molecular
- Escherichia coli
- chemistry
- enzymology
- Genetic Vectors
- genetics
- Humans
- Membrane Proteins
- chemistry
- genetics
- isolation & purification
- Molecular Sequence Data
- NADP Transhydrogenase
- chemistry
- genetics
- isolation & purification
- Protein Structure
- Tertiary
- Protein Subunits
- chemistry
- genetics
- isolation & purification
- Recombinant Fusion Proteins
- chemistry
- genetics
- isolation & purification
- Chromatography
Publication and Content Type
- ref (subject category)
- art (subject category)
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