Continuous-exchange cell-free protein-synthesizing system: Synthesis of HIV-1 antigen Nef

HIV-1 antigen Nef, Green Fluorescent Protein (GFP), and the fusion protein GFP-Nef ("Green Fluorescent Nef") were synthesized in bacterial cell-free s ystem with continuous supply of substrates and continuous removal of low-mo lecular-weight products through a dialysis membrane during incubation, the so-called continuous-exchange cell-free (CECF) system. The identity of synt hesized proteins was confirmed by electrophoretic mobility, Western blottin g, and GFP fluorescence. The system produced several nanomoles (hundreds of mug) of each protein per mi of the reaction mixture. Construction of GFP-f used proteins is considered as a general strategy for visualization and mon itoring of cell-free production of the proteins that have no easily testabl e functions. (C) 2001 Academic Press.