A novel cell-free translation/glycosylation system prepared from insect cells

A cell-free translation/glycosylation system derived from lepidopteran (Sf2 1) cells, which are widely used to express high yields of foreign active pr oteins that have post-translational modifications, was constructed. The ins ect cell extract was prepared using a Mini-Bomb cell disruption chamber by nitrogen pressure treatment, which stably retains translational and post-tr anslational components. The gp120 mRNA was transcribed from the human immun odeficiency virus type-1 envelope glycoprotein gp120 gene with T7 RNA polym erase. When the gp120 mRNA was translated in the insect cell-free system, g p120 having a molecular mass of 100 kDa was detected by Western blot analys is. Synthesized gp120 and gp120 expressed in the intracellular fraction of recombinant-baculovirus-infected Sf21 cells had the same molecular mass, an d they both had reduced mobility compared with gp120 secreted by recombinan t baculovirus-infected Sf21 cells. In contrast, the 56-kDa gp120 protein, w hich corresponds to the poly-peptide backbone of gp120, was synthesized in wheat germ and rabbit reticulocyte systems. The molecular mass of synthesiz ed gp120 decreased from 100 kDa to 61 kDa after endoglycosidase H treatment , indicating that synthesized gp120 had been glycosylated with N-linked oli gosaccharides. Furthermore, glycosylated gp120 was bound to human CD4 molec ules expressed on the surface of quail cells. These results revealed that t he insect cell-free system can synthesize gp120 that is folded in the prope r conformation to provide a CD4-binding domain.