In vitro translation and translocation of apolipoprotein B in a cell-free system from HepG2 cells

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.