Mariani S M, Matiba B, Sparna T, Krammer P H
Division of Immunogenetics, German Cancer Research Center, Heidelberg, Germany.
J Immunol Methods. 1996 Jun 14;193(1):63-70. doi: 10.1016/0022-1759(96)00051-8.
CD95L (CD95/APO-1/Fas ligand) is a type II transmembrane glycoprotein that induces apoptosis in sensitive target cells. CD95L can be proteolytically 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 human CD95L tagged with a FLAG sequence in insect cells (Sf9) infected with recombinant baculovirus. CD95L expressed by Sf9 cells was detected with rabbit antibodies directed against the carboxy-terminal region of CD95L (which is highly conserved between mouse and human CD95L) and with an anti-FLAG monoclonal antibody. Immunoblotting showed that recombinant 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 target 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 recombinant baculoviruses may thus provide an efficient system for the expression of biologically active recombinant CD95L.
