Efficient silkworm expression of human GPCR (nociceptin receptor) by a Bombyx mori bacmid DNA system
Efficient silkworm expression of human GPCR (nociceptin receptor) by a Bombyx mori bacmid DNA system
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DOI：
10.1016/j.bbrc.2009.05.063
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发表时间：
2009-07-31
影响因子：
3.1
通讯作者：
Maenaka, Katsumi
中科院分区：
文献类型：
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作者：
Kajikawa, Mizuho;Sasaki, Kaori;Maenaka, Katsumi
鸟嘌呤核苷酸结合蛋白(G蛋白)偶联受体(GPCRs)通常通过杆状病毒表达载体系统(BEVS)表达。我们最近利用家蚕核型多角体病毒(BmNPV)的杆状病毒系统建立了一种新的BEVS，该系统直接适用于家蚕蛋白质的表达。在这里，我们报告了第一个通过简单注射BmNPV bacmidDNA在家蚕中表达GPCRs的例子。人伤害素受体及其与抑制性G蛋白α亚单位(G(I)α)的融合蛋白在家蚕幼虫脂肪体和BmNPV病毒组分中均得到了成功表达。其产量远高于Sf9细胞。包括伤害素受体融合在内的微体组分在伤害素的刺激下显示出与[S-35]GTP-S结合的活性，这些微体组分只需经过离心步骤即可制备。因此，这种快速的方法简单易用，表达水平高，将成为人类GPCR生产的重要工具。(C)2009 Elsevier Inc.保留所有权利。
Guanine nucleotide-binding protein (G protein) coupled receptors (GPCRs) are frequently expressed by a baculovirus expression vector system (BEVS). We recently established a novel BEVS Using the bacmid system of Bombyx mori nucleopolyhedrovirus (BmNPV), which is directly applicable for protein expression in silkworms. Here, we report the first example of GPCR expression in silkworms by the simple injection of BmNPV bacmid DNA. Human nociceptin receptor, an inhibitory GPCR, and its fusion protein with inhibitory G protein alpha Subunit (G(i)alpha) were both successfully expressed in the fat bodies of silkworm larvae as well as in the BmNPV viral fraction. Its yield was much higher than that from Sf9 cells. The microsomal fractions including the nociceptin receptor fusion, which are easily prepared by only centrifugation steps, exhibited [S-35]GTP gamma S-binding activity upon specific stimulation by nociceptin. Therefore, this rapid method is easy-to-use and has a high expression level, and thus will be an important tool for human GPCR production. (C) 2009 Elsevier Inc. All rights reserved.