Ruiz-Ferrer Virginia, Goytia Elisa, Martínez-García Belén, López-Abella Dionisio, López-Moya Juan José
Departamento de Biología de Plantas, Centro de Investigaciones Biológicas, (CIB, CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain.
J Gen Virol. 2004 Jan;85(Pt 1):241-249. doi: 10.1099/vir.0.19557-0.
Tobacco etch potyvirus (TEV) is transmitted by aphids in a non-persistent manner with the assistance of a virus-encoded protein known as helper component (HC-Pro). To produce a biologically active form of recombinant TEV HC-Pro protein, heterologous expression in the methylotrophic yeast Pichia pastoris was used. A cDNA encoding the TEV HC-Pro region, fused to a Saccharomyces cerevisiae alpha-mating factor secretory peptide coding region, was inserted into the P. pastoris genome using a modified version of the pPIC9 vector. The expressed TEV HC-Pro protein was obtained directly from culture medium of recombinant yeast colonies; it was able to interact with TEV particles in a protein overlay binding assay, and also to assist aphid transmission of purified TEV particles to plants using the aphid Myzus persicae as vector. Our results indicate that P. pastoris provides a rapid and low-cost heterologous expression system that can be used to obtain biologically active potyvirus HC-Pro protein for in vitro transmission assays.
烟草蚀纹马铃薯Y病毒（TEV）通过蚜虫以非持久性方式传播，在一种名为辅助成分（HC-Pro）的病毒编码蛋白的协助下进行传播。为了产生具有生物活性形式的重组TEV HC-Pro蛋白，采用了在甲基营养型酵母毕赤酵母中进行异源表达的方法。将编码TEV HC-Pro区域的cDNA与酿酒酵母α-交配因子分泌肽编码区域融合，使用改良版的pPIC9载体插入毕赤酵母基因组。表达的TEV HC-Pro蛋白直接从重组酵母菌落的培养基中获得；它能够在蛋白质印迹结合试验中与TEV颗粒相互作用，并且还能够以桃蚜作为载体，协助将纯化的TEV颗粒传播到植物上。我们的结果表明，毕赤酵母提供了一种快速且低成本的异源表达系统，可用于获得具有生物活性的马铃薯Y病毒HC-Pro蛋白，用于体外传播试验。
