Pholasin is a reactive oxygen-sensitive photoprotein that consists of an apoprotein (apoPholasin) and an unknown chromophore. The preferred human codon-optimized apoPholasin gene was transiently expressed in mammalian cells and apoPholasin was detected using an anti-recombinant apoPholasin antibody. For the first time, we found that apoPholasin secreted into the culture medium could catalyze the oxidation of coelenterazine (CTZ, a luciferin) to produce continuous luminescence. The fusion protein of apoPholasin and glutathione S-transferase (GST-apoPholasin) was successfully expressed as a soluble form in bacterial cells using the cold induction system. The purified GST-apoPholasin also had luminescence activity with CTZ, showing the bioluminescence emission peak at 461 nm, and the resultant product showed purple blue fluorescence under 365 nm light. Unexpectedly, the main oxidation product of CTZ was identified as coelenteramine (CTM), not coelenteramide (CTMD).

中文翻译：

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重组载脂蛋白的表达，纯化和鉴定。

荧光素是一种对氧敏感的活性光蛋白，由载脂蛋白（apoPholasin）和未知的发色团组成。优选的人密码子优化的载脂蛋白基因在哺乳动物细胞中瞬时表达，并且使用抗重组载脂蛋白抗体检测载脂蛋白。我们首次发现分泌到培养基中的载脂蛋白可以催化腔肠素（CTZ，一种荧光素）的氧化以产生连续的发光。使用冷诱导系统，载脂蛋白与谷胱甘肽S-转移酶（GST-载脂蛋白）的融合蛋白成功地以可溶形式表达在细菌细胞中。纯化的GST-载脂蛋白也具有CTZ的发光活性，在461 nm处显示出生物发光峰，所得产物在365nm光下显示紫蓝色荧光。出乎意料的是，CTZ的主要氧化产物被鉴定为腔肠素（CTM），而不是腔肠酰胺（CTMD）。