CLONING AND EXPRESSION IN MURINE ERYTHROLEUKEMIA-CELLS - THE SOLUBLE FORMS OF THE TYPE-I AND TYPE-II TUMOR-NECROSIS-FACTOR RECEPTORS FUSED TO AN IMMUNOGENIC AFFINITY TAG

We have cloned, expressed, and purified the extracellular domains of t ypes I and II human tumor necrosis factor receptors. Both proteins wer e expressed in and secreted by murine erythroleukemia cells under the control of the human beta-globin promoter placed downstream from the h uman globin locus control region. Secretion of both proteins was direc ted by the respective tumor necrosis factor receptor signal sequence. Each tumor necrosis factor receptor extracellular domain was expressed as a chimeric protein, fused to a carboxy terminal flexible peptide l inker and an antigenic affinity tag. Secretion of both proteins into t he growth medium in a hollow fiber bioreactor was achieved. A monoclon al antibody generated against the affinity tag allowed the purificatio n of both proteins. These were isolated as biologically active product s in that they bound human tumor necrosis factor-alpha in a I-125-radi oiodinated ligand binding assay. The two proteins also bound tumor nec rosis factor-alpha at approximately equimolar ratios as demonstrated b y BIAcore sensorgram analysis. (C) 1994 Academic Press, Inc.