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

Abstract：

: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.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Glynou K,Ioannou PC,Christopoulos TK

doi

10.1016/s1046-5928(02)00614-9

keywords:

subject

Has Abstract

pub_date

2003-02-01 00:00:00

pages

384-90

issue

2

eissn

1046-5928

issn

1096-0279

pii

S1046592802006149

journal_volume

27

pub_type

杂志文章

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