A novel yellowish-green fluorescent protein from the marine copepod, Chiridius poppei, and its use as a reporter protein in HeLa cells
A novel yellowish-green fluorescent protein from the marine copepod, Chiridius poppei, and its use as a reporter protein in HeLa cells
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DOI：
10.1016/j.gene.2005.11.031
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发表时间：
2006-05-10
期刊：
影响因子：
3.5
通讯作者：
Mizuno, H
中科院分区：
文献类型：
--
作者：
Masuda, H;Takenaka, Y;Mizuno, H
通过表达克隆，从深海（底栖）桡足类甲壳类 Chiridius poppei 的 cDNA 文库中分离出了编码一类新型 GFP 样蛋白的甲壳类基因。在pBluescript II载体中构建cDNA文库，并使用非UV透照仪进行筛选，获得阳性克隆。该克隆由 781 bp 的 cDNA 片段和 660 bp 开放阅读框组成，编码 219 个氨基酸的多肽，计算分子量为 24.7 kDa。该蛋白在大肠杆菌中过表达，通过阴离子交换和尺寸排阻色谱纯化至均质。蛋白质 CpYGFP 的激发和发射最大值分别为 507 和 517 nm。 CpYGFP 在溶液中以二聚体形式存在，可以在 HeLa 细胞中单独表达或作为融合蛋白表达。使用 CpYGFP-肌动蛋白和 DsRed2-Nuc 进行的双标记实验证明了 CpYGFP 作为肌动蛋白亚细胞定位报告基因的有用性。 (c) 2006 Elsevier B.V. 保留所有权利。
A crustacean gene, encoding for a new class of GFP-like protein, has been isolated from a cDNA library of the deep-sea (benthic) copepod crustacean, Chiridius poppei, by expression cloning. The cDNA library was constructed in a pBluescript II vector and screened using a non-UV transilluminator, obtaining a positive clone. The clone consisted of a 781 -bp fragment of cDNA with a 660-bp open reading frame, which encoded for a 219-amino acid polypeptide with a calculated molecular mass of 24.7 kDa. The protein was overexpressed in Escherichia coli, purified to homogeneity by anion-exchange and size-exclusion chromatographies. The protein, CpYGFP, had excitation and emission maxima at 507 and 517 nm, respectively. CpYGFP existed as a dimer in solution and could be expressed either alone or as a fusion protein in HeLa cells. Dual labeling experiments carried out with CpYGFP-actin and DsRed2-Nuc demonstrated the usefulness of CpYGFP as a reporter in the subcellular localization of actin. (c) 2006 Elsevier B.V. All rights reserved.