Expression and functional characterization of recombinant tributyltin-binding protein type 2.
Expression and functional characterization of recombinant tributyltin-binding protein type 2.
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DOI：
10.2131/jts.38.885
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发表时间：
2013-12
期刊：
影响因子：
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通讯作者：
Hina Satone;Eriko Akahoshi;A. Nakamura;J. M. Lee;Masato Honda;Y. Shimasaki;S. Kawabata;T. Kusakabe;Y. Oshima
中科院分区：
文献类型：
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作者：
Hina Satone;Eriko Akahoshi;A. Nakamura;J. M. Lee;Masato Honda;Y. Shimasaki;S. Kawabata;T. Kusakabe;Y. Oshima
三丁基锡结合蛋白（TBT-bps）是从牙鲆（Paralichthys olivaceus）血液中分离得到的鱼类脂质运载蛋白的一个成员，具有结合和解毒三丁基锡的功能。在本研究中，我们构建了一个杆状病毒-家蚕表达系统，并获得了重组TBT-bp 2（rTBT-bp 2; 31.5 kDa）从家蚕幼虫的血淋巴注射含有TBT-bp 2基因的重组杆状病毒。研究了rTBT-bp 2的结合潜力，并与先前可用的重组TBT-bp 1（rTBT-bp 1）的结合潜力进行了比较。rTBT-bp 2和rTBT-bp 1均与脂质运载蛋白的典型荧光配体DAUDA结合，解离常数分别为0.97和1.75 µM。Hill系数表明rTBT-bp 2可能具有多个结合位点和较强的负协同作用。这些结果表明，由8个特异性β-折叠组成的脂质运载蛋白的典型中央空腔在rTBT-bp 2中是保守的，就像在rTBT-bp 1中一样，尽管rTBT-bp 2在TBT结合中具有与rTBT-bp 1不同的作用。在竞争试验中，rTBT-bp 2显示出与TBT的指数结合亲和力，抑制常数为0.29 µM，表明TBT与rTBT-bp 2的中心配体口袋结合。然而，三种脂肪酸对rTBT-bp 2没有表现出任何亲和力。TBT-bps作为鱼类脂质运载蛋白的内源性功能及其对外源性物质的应答功能有待进一步研究。
Tributyltin-binding proteins (TBT-bps) are members of the fish lipocalins that were isolated from the blood of Japanese flounder (Paralichthys olivaceus) and function in the binding and detoxification of TBT. In this study, we constructed a baculovirus-silkworm expression system and obtained recombinant TBT-bp2 (rTBT-bp2; 31.5 kDa) from the hemolymph of silkworm larvae injected with a recombinant baculovirus containing the TBT-bp2 gene. The binding potential of rTBT-bp2 was investigated and compared to that of the previously available recombinant TBT-bp1 (rTBT-bp1). Both rTBT-bp2 and rTBT-bp1 bound to DAUDA, a typical fluorescent ligand of lipocalins, with dissociation constants of 0.97 and 1.75 µM, respectively. The Hill coefficient value indicated that rTBT-bp2 may have multiple binding sites and strong negative cooperativity. These results suggest that the typical central cavity of lipocalins composed of eight specific β-sheets is conserved in rTBT-bp2, as it is in rTBT-bp1, although rTBT-bp2 has different effects than rTBT-bp1 in TBT binding. In a competition assay, rTBT-bp2 displayed exponential binding affinity to TBT with an inhibition constant of 0.29 µM, demonstrating that TBT binds to the central ligand pocket of rTBT-bp2. However, three fatty acids did not show any affinity to rTBT-bp2. Further studies are required to elucidate the endogenous function of TBT-bps as fish lipocalins and their function in responding to xenobiotics.