EXPRESSION OF BIOLOGICALLY-ACTIVE MOUSE AND HUMAN CD95 APO-1/FAS LIGAND IN THE BACULOVIRUS SYSTEM/

CD95L (CD95/APO-1/Fas ligand) is a type II transmembrane glycoprotein that induces apoptosis in sensitive target cells. CD95L can be proteol ytically cleaved from the membrane by a metalloprotease and occurs in a soluble form. Thus CD95L may act as a cytotoxic effector molecule at a distance from the producer cell. In order to develop an expression system yielding large quantities of CD95L, we expressed mouse and huma n CD95L tagged with a RAG sequence in insect cells (Sf9) infected with recombinant baculovirus. CD95L expressed by Sf9 cells was detected wi th rabbit antibodies directed against the carboxy-terminal region of C D95L (which is highly conserved between mouse and human CD95L) and wit h an anti-FLAG monoclonal antibody. Immunoblotting showed that recombi nant mouse and human CD95L expression was associated with the presence of 40 kDa and 32-33 kDa proteins. CD95L released into the supernatant of infected Sf9 cells specifically induced apoptosis in sensitive tar get cells, thus indicating that recombinant mouse and human CD95L were functional. The presence of the amino-terminal FLAG sequence did not modify this biological activity. Infection of Sf9 cells with recombina nt baculoviruses may thus provide an efficient system for the expressi on of biologically active recombinant CD95L.