Yokohama Research Center, JNC Corporation, 5-1 Okawa, Kanazawa-ku, Yokohama, 236-8605, Japan.
Yokohama Research Center, JNC Corporation, 5-1 Okawa, Kanazawa-ku, Yokohama, 236-8605, Japan.
Protein Expr Purif. 2020 Jul;171:105615. doi: 10.1016/j.pep.2020.105615. Epub 2020 Feb 28.
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).
海兔发光蛋白是一种活性氧敏感的发光蛋白，由脱辅基蛋白（apoPholasin）和一个未知的发色团组成。我们选择了经过优化的人源密码子 apoPholasin 基因，通过瞬时转染在哺乳动物细胞中表达，并用抗重组 apoPholasin 抗体进行检测。我们首次发现分泌到培养基中的 apoPholasin 可以催化腔肠素（CTZ，一种荧光素）的氧化，产生持续的发光。使用冷诱导系统，apoPholasin 与谷胱甘肽 S-转移酶（GST-apoPholasin）的融合蛋白可以在细菌细胞中以可溶形式成功表达。纯化的 GST-apoPholasin 与 CTZ 也具有发光活性，在 461nm 处显示出生物发光发射峰，在 365nm 光下产物显示出紫色蓝色荧光。出乎意料的是，CTZ 的主要氧化产物被鉴定为腔肠胺（CTM），而不是腔肠酰胺（CTMD）。
