One-step purification and refolding of recombinant photoprotein aequorin by immobilized metal-ion affinity chromatography

A hexahistidine tag was fused to the N-terminus of apoaequorin. A suitable vector encoding the fusion protein was constructed and used for transformation of Escherichia coli JM109 cells. Apoaequorin was overexpressed under the control of tac promoter. It was found, however, that most of the protein...

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Bibliographic Details
Published in:Protein expression and purification 2003-02, Vol.27 (2), p.384-390
Main Authors: Glynou, Kyriaki, Ioannou, Penelope C, Christopoulos, Theodore K
Format: Article
Language:English
Subjects:
Aequorin
Aequorin - chemistry
Aequorin - isolation & purification
Aequorin - metabolism
Bioluminescence
Calcium - metabolism
Chromatography, Affinity
DNA - metabolism
Electrophoresis, Agar Gel
Electrophoresis, Polyacrylamide Gel
Escherichia coli - metabolism
Immobilized metal ion-affinity chromatography
Kinetics
Plasmids - metabolism
Promoter Regions, Genetic
Protein Folding
Recombinant Proteins - chemistry
Recombinant Proteins - isolation & purification
Time Factors
Urea - pharmacology
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Summary:A hexahistidine tag was fused to the N-terminus of apoaequorin. A suitable vector encoding the fusion protein was constructed and used for transformation of Escherichia coli JM109 cells. Apoaequorin was overexpressed under the control of tac promoter. It was found, however, that most of the protein existed in the form of inclusion bodies. Inclusion bodies were solubilized with urea, followed by purification and refolding of (His) 6-apoaequorin in a single chromatographic step by immobilized metal-ion affinity chromatography using Ni 2+–nitrilotriacetic acid agarose. The purity, as determined by SDS–PAGE analysis, was greater than 80%. The yield was 0.7–1 mg apoaequorin from a 50 ml bacterial culture. The kinetics of light emission of purified aequorin upon addition of Ca 2+ was typical of the commercial aequorin. The luminescence of the purified aequorin was a linear function of its concentration extending over six orders of magnitude. As low as 0.5 attomoles purified aequorin gave a signal-to-noise ratio of 1.8.
ISSN:1046-5928
1096-0279
DOI:10.1016/S1046-5928(02)00614-9