用杆状病毒表达系统表达小鼠cFos蛋白及其特性:与共表达cJun蛋白形成功能性AP-1复合物的能力

C M Corvello, R Metz, R Bravo, M C Armelin
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引用次数: 0

摘要

原癌基因c-fos和c-jun的产物在细胞生长控制、分化和恶性转化中起重要作用。纯化的致癌蛋白是细胞生长控制研究中必不可少的工具。在这里,我们描述了使用杆状病毒表达系统在Sf9昆虫细胞中产生小鼠cFos和cJun癌蛋白。将小鼠c-fos cDNA亚克隆到两种不同杆状病毒表达载体pVL1392和pVLMH6中,分别在昆虫细胞中生成非融合和his融合的cFos癌蛋白。产物经免疫荧光、免疫印迹、免疫沉淀鉴定,并能与体外翻译的JunB蛋白结合形成AP-1复合物。利用pVLMH6杆状病毒载体在昆虫细胞中也产生了His-cJun融合蛋白。凝胶阻滞实验表明,昆虫细胞中cFos和cJun的共表达产生了功能性AP-1复合物。这为利用昆虫细胞进行不同Fos/Jun AP-1复合物的结构和功能研究提供了可能。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Expression and characterization of mouse cFos protein using the baculovirus expression system: ability to form functional AP-1 complex with coexpressed cJun protein.

The products of the proto-oncogenes c-fos and c-jun play important roles in cell growth control, differentiation, and malignant transformation. Purified oncogenic proteins are essential tools in cell growth control studies. Here we describe the production of mouse cFos and cJun oncoproteins in Sf9 insect cells using the baculovirus expression system. The mouse c-fos cDNA was subcloned into two different baculovirus expression vectors, namely pVL1392 and pVLMH6, to generate, respectively, nonfusion and His-fusion cFos oncoproteins in insect cells. The products were characterized by immunofluorescence, immunoblotting, immunoprecipitation, and ability to bind to the in vitro translated JunB protein to form the AP-1 complex. A His-cJun fusion protein was also produced in insect cells using the pVLMH6 baculovirus vector. Coexpression of cFos and cJun in insect cells yielded functional AP-1 complexes as judged from gel retardation assays. The results point to the possibility of using insect cells for structural and functional studies of different Fos/Jun AP-1 complexes.

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