Mohammadi A, Theriault A, Adeli K
Department of Chemistry and Biochemistry, University of Windsor, Ontario, Canada.
Biochem Biophys Res Commun. 1996 Nov 21;228(3):852-8. doi: 10.1006/bbrc.1996.1743.
An mRNA-dependent cell-free system has been developed from HepG2 cells by hydrolysis of endogenous mRNA with micrococcal nuclease. When supplied with RNA extracted from HepG2 cells, the system synthesized liver specific proteins such as albumin and apolipoprotein B100. Significant amounts of microsomes were also detected in the lysate by measuring NADH-cytochrome c reductase activity and ultracentrifugation. Protease protection assays showed the capability of the HepG2 lysate to translocate newly-synthesized proteins such as apolipoprotein Al, albumin, and apoB into the microsomes as they were protected from digestion with exogenously added protease K, but not protected in the presence of protease K and Triton X-100. The system also proved to be very active toward translation of exogenous mRNAs as evidenced by efficient translation of brome mosaic virus RNA. The HepG2 translation-translocation system appears to provide a unique homologous system for studies on the biogenesis of liver specific proteins, particulary apoB100.
通过用微球菌核酸酶水解内源性mRNA，从肝癌细胞系HepG2中开发出了一种依赖mRNA的无细胞系统。当提供从HepG2细胞中提取的RNA时，该系统能合成肝脏特异性蛋白，如白蛋白和载脂蛋白B100。通过测量NADH-细胞色素c还原酶活性和超速离心，在裂解物中也检测到了大量微粒体。蛋白酶保护试验表明，HepG2裂解物有能力将新合成的蛋白质，如载脂蛋白A1、白蛋白和载脂蛋白B转运到微粒体中，因为它们在添加外源蛋白酶K时能免受消化，但在蛋白酶K和Triton X-100存在时则不受保护。该系统对外源mRNA的翻译也非常活跃，如通过雀麦花叶病毒RNA的有效翻译所证明。HepG2翻译转运系统似乎为研究肝脏特异性蛋白，特别是载脂蛋白B100的生物发生提供了一个独特的同源系统。
