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Reconstitution of blue fluorescent protein from recombinant apoaequorin and synthetic coelenteramide |
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| Authors: | Satoshi Inouye Takamitsu Hosoya |
| Institution: | a Yokohama Research Center, Chisso Corporation, 5-1 Okawa, Kanazawa-ku, Yokohama 236-8605, Japan b Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan |
| Abstract: | Blue fluorescent protein of aequorin (BFP) is a complex of Ca2+-bound apoaequorin with coelenteramide and is a bifunctional protein, which shows blue fluorescence and the luminescence activity like a luciferase. To reconstitute synthetic BFP (syn-BFP) from apoaequorin and coelenteramide, we established new synthetic route of coelenteramide and prepared highly purified recombinant aequorin using the histidine-tagged secretion system in Escherichia coli cells. As a result, we succeeded in reconstituting syn-BFP quantitatively and the fluorescence and luminescence properties of syn-BFP were identical to that of BFP obtained from aequorin. |
| Keywords: | Apoaequorin apoprotein of aequorin BFP blue fluorescent protein of aequorin gFP greenish fluorescent protein syn-BFP blue fluorescent protein reconstituted from apoaequorin and coelenteramide in the presence of calcium syn-gFP greenish fluorescent protein reconstituted from apoaequorin and coelenteramide in the presence of EDTA EDTA ethylenediaminetetraacetic acid DTT dithothreitol rlu relative light units MALDI-TOF-MS Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis |
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