Instituto de Biotecnología, CICVyA, Instituto Nacional de Tecnología Agropecuaria, Las Cabañas y Los Reseros, Hurlingham, Buenos Aires, Argentina.
Insect Mol Biol. 2011 Oct;20(5):675-85. doi: 10.1111/j.1365-2583.2011.01100.x.
Insect genomics is a growing area of research. To exploit fully the genomic data that are being generated, high-throughput systems for the functional characterization of insect proteins and their interactomes are required. In this work, a Gateway-compatible vector set for expression of fluorescent fusion proteins in insect cells was developed. The vector set was designed to express a protein of interest fused to any of four different fluorescent proteins [green fluorescent protein (GFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and mCherry] by either the C-terminal or the N-terminal ends. Additionally, a collection of organelle-specific fluorescent markers was assembled for colocalization with fluorescent recombinant proteins of interest. Moreover, the vector set was proven to be suitable for simultaneously detecting up to three proteins by multiple labelling. The use of the vector set was exemplified by defining the subcellular distribution of Mal de Río Cuarto virus (MRCV) outer coat protein P10 and by analysing the in vivo self-interaction of the MRCV viroplasm matrix protein P9-1 in Förster resonance energy transfer (FRET) experiments. In conclusion, we have developed a valuable tool for high-throughput studies of protein subcellular localization that will aid in the elucidation of the function of newly described insect and virus proteins.
昆虫基因组学是一个不断发展的研究领域。为了充分利用正在生成的基因组数据，需要高通量系统来对昆虫蛋白及其互作组进行功能特征分析。在这项工作中，我们开发了一套适用于昆虫细胞中荧光融合蛋白表达的 Gateway 兼容载体集。该载体集旨在通过 C 端或 N 端融合表达目标蛋白与四种不同荧光蛋白（绿色荧光蛋白（GFP）、青色荧光蛋白（CFP）、黄色荧光蛋白（YFP）和 mCherry）之一。此外，还组装了一套细胞器特异性荧光标记物，用于与感兴趣的荧光重组蛋白进行共定位。此外，该载体集被证明适合通过多重标记同时检测多达三种蛋白。该载体集的使用通过定义 Mal de Río Cuarto 病毒（MRCV）外壳蛋白 P10 的亚细胞分布和分析 MRCV 质粒体基质蛋白 P9-1 在Förster 共振能量转移（FRET）实验中的体内自相互作用得到了例证。总之，我们开发了一种用于蛋白质亚细胞定位高通量研究的有价值的工具，这将有助于阐明新描述的昆虫和病毒蛋白的功能。
