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 supp lied with RNA extracted from HepG2 cells, the system synthesized liver specific proteins such as albumin and apolipoprotein B-100. Significa nt amounts of microsomes were also detected in the lysate by measuring NADH-cytochrome c reductase activity and ultracentrifugation. Proteas e protection assays showed the capability of the HepG2 lysate to trans locate newly-synthesized proteins such as apolipoprotein AI, albumin, and apoB into the microsomes as they were protected from digestion wit h exogenously added protease K, but not protected in the presence of p rotease K and Triton X-100. The system also proved to be very active t oward translation of exogenous mRNAs as evidenced by efficient transla tion of brome mosaic virus RNA. The HepG2 translation-translocation sy stem appears to provide a unique homologous system for studies on the biogenesis of liver specific proteins, particulary apoB(100). (C) 1996 Academic Press, Inc.