Institute of Biophysics, Federal Research Center "Krasnoyarsk Science Center SB RAS", 660036 Krasnoyarsk, Russia.
School of Fundamental Biology and Biotechnology, School of Non-Ferrous Metals and Material Science, Siberian Federal University, pr. Svobodny 79, 660041 Krasnoyarsk, Russia.
Int J Mol Sci. 2023 Jan 21;24(3):2144. doi: 10.3390/ijms24032144.
Ca-triggered coelenterazine-binding protein (CBP) is a natural form of the luciferase substrate involved in the Renilla bioluminescence reaction. It is a stable complex of coelenterazine and apoprotein that, unlike coelenterazine, is soluble and stable in an aquatic environment and yields a significantly higher bioluminescent signal. This makes CBP a convenient substrate for luciferase-based in vitro assay. In search of a similar substrate form for the luciferase NanoLuc, a furimazine-apoCBP complex was prepared and verified against furimazine, coelenterazine, and CBP. Furimazine-apoCBP is relatively stable in solution and in a frozen or lyophilized state, but as distinct from CBP, its bioluminescence reaction with NanoLuc is independent of Ca. NanoLuc turned out to utilize all the four substrates under consideration. The pairs of CBP-NanoLuc and coelenterazine-NanoLuc generate bioluminescence with close efficiency. As for furimazine-apoCBP-NanoLuc pair, the efficiency with which it generates bioluminescence is almost twice lower than that of the furimazine-NanoLuc. The integral signal of the CBP-NanoLuc pair is only 22% lower than that of furimazine-NanoLuc. Thus, along with furimazine as the most effective NanoLuc substrate, CBP can also be recommended as a substrate for in vitro analytical application in view of its water solubility, stability, and Ca-triggering "character".
钙触发腔肠素结合蛋白（CBP）是参与海肾荧光素酶生物发光反应的荧光素酶底物的天然形式。它是腔肠素和脱辅基蛋白的稳定复合物，与腔肠素不同，它在水相环境中是可溶和稳定的，并产生显著更高的生物发光信号。这使得 CBP 成为基于荧光素酶的体外测定的方便底物。在寻找与荧光素酶 NanoLuc 相似的底物形式时，制备了一种荧光素-apoCBP 复合物，并针对荧光素、腔肠素和 CBP 对其进行了验证。荧光素-apoCBP 在溶液中和冷冻或冻干状态下相对稳定，但与 CBP 不同，其与 NanoLuc 的生物发光反应不依赖于 Ca。事实证明，NanoLuc 可以利用所有这四种考虑的底物。CBP-NanoLuc 和腔肠素-NanoLuc 对产生的生物发光效率非常接近。对于荧光素-apoCBP-NanoLuc 对，其产生生物发光的效率比荧光素-NanoLuc 低近两倍。CBP-NanoLuc 对的积分信号仅比荧光素-NanoLuc 低 22%。因此，除了作为最有效的 NanoLuc 底物的荧光素之外，CBP 还可以因其水溶性、稳定性和 Ca 触发“特性”而被推荐为体外分析应用的底物。
