Expression of recombinant apopholasin using a baculovirus-silkworm multigene expression system and activation via dehydrocoelenterazine
Expression of recombinant apopholasin using a baculovirus-silkworm multigene expression system and activation via dehydrocoelenterazine
复制标题
使用杆状病毒-家蚕多基因表达系统表达重组阿波拉辛并通过脱氢腔肠素激活
DOI：
10.1016/j.bmcl.2020.127177
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发表时间：
2020
影响因子：
2.7
通讯作者：
Masaki Kuse
中科院分区：
文献类型：
--
作者：
Maiko Moriguchi;RyoTakahashi;Bubwoong Kang;Masaki Kuse
Pholasin是来自发光双壳类软体动物Pholas dactylus的发光蛋白。尽管负责软体动物发光特性的辅基（发色团）的化学结构仍然未知，但研究表明脱氢腔肠素（DCL）的存在增加了发光，并且DCL的二硫苏糖醇加合物从Pholasin®中分离。迄今为止，我们的研究一直集中在使用DCL激活Pholasin®的天然脱辅基蛋白apopholasin。本研究利用杆状病毒-家蚕多基因表达系统表达重组脱辅基酶。此外，报道了使用Flag®-亲和柱纯化脱辅基酶，使用DCL活化脱辅基酶，以及通过添加过氧化物酶-过氧化氢混合物引发其发光特性。重组脱辅基酶经DCL激活后可产生过氧化物酶-H_2O_2依赖性发光。
Pholasin is a photoprotein derived from the glowing bivalve mollusk,Pholas dactylus. Even though the chemical structure of the prosthetic group (chromophore) responsible for the light emission character of the mollusk remains unknown, research has shown that the presence of dehydrocoelenterazine (DCL) increased light emission and that the dithiothreitol adduct of DCL was isolated from Pholasin®. To date, our research has been focused on activating apopholasin, the naturally occurring apoprotein of Pholasin®, using DCL. In the current study, the expression of recombinant apopholasin via a baculovirus–silkworm multigene expression system is reported. Additionally, the purification of apopholasin using a Flag®-affinity column, the activation of apopholasin using DCL, and the initiation of its luminescent character through the addition of a peroxidase–hydrogen peroxide mixture are reported. The peroxidase–H2O2-dependent luminescence was observed from the recombinant apopholasin activated with DCL.