Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 53, Issue 2, Pages 384-389Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2006.12.014
Keywords
secretion; expression vector; bioluminescence; histidine-tag; aequorin
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A novel histidine-tagged secretion vector in Escherichia coli was constructed and large amounts of highly pure clytin, a calcium-binding photoprotein, was prepared. The histidine-tagged apoclytin expressed into the periplasmic space in E coli was purified by nickel chelate affinity chromatography. Recombinant clytin was regenerated from apoclytin by incubation with coelenterazine in the presence of dithiothreitol and then purified by anion-exchange chromatography and hydrophobic chromatography. The yield of recombinant clytin was 20 mg from 2 L of cultured cells with purity greater than 95%. Luminescence properties of recombinant clytin were identical to that of native clytin (phialidin). The Ca2+ sensitivity of recombinant clytin is lower than that of aequorin and clytin is suited for measuring higher concentration of Ca2+. Semi-synthetic clytins were also prepared with coelenterazine analogues, and the initial intensity, luminescence capacity and half decay time were characterized. (C) 2006 Elsevier Inc. All rights reserved.
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