Ferguson B, Rosenberg M, Krippl B
J Biol Chem. 1986 Nov 5;261(31):14760-3.
Human adenovirus 2/5 E1A proteins were used to evaluate protoplast fusion as a method of transferring functional proteins into mammalian cells. Both the E1A 13 and 12 S mRNA products expressed in Escherichia coli are shown to activate in trans adenovirus gene expression following transfer into monkey kidney cells by protoplast fusion. Approximately 20% of the recipient mammalian cells exhibited positive nuclear E1A-specific immunofluorescence following fusion with protoplasts containing E1A protein. E. coli-expressed E1A protein was modified post-translationally in Vero cells following protoplast fusion, as evidenced by its shift in sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility. These results establish protoplast fusion as a simple rapid method for examining the functional activity, intracellular distribution, and post-translational modification of E. coli-expressed proteins in intact mammalian cells.
人腺病毒2/5 E1A蛋白被用于评估原生质体融合作为一种将功能性蛋白转移到哺乳动物细胞中的方法。在大肠杆菌中表达的E1A 13S和12S mRNA产物，经原生质体融合转移到猴肾细胞后，均显示出能反式激活腺病毒基因表达。与含有E1A蛋白的原生质体融合后，约20%的受体哺乳动物细胞呈现出阳性的核E1A特异性免疫荧光。原生质体融合后，大肠杆菌表达的E1A蛋白在Vero细胞中发生了翻译后修饰，这可通过其在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳迁移率上的变化得以证明。这些结果确立了原生质体融合是一种用于检测完整哺乳动物细胞中大肠杆菌表达蛋白的功能活性、细胞内分布及翻译后修饰的简单快速方法。
