SOLUBLE HUMAN INTERLEUKIN-6 RECEPTOR - EXPRESSION IN INSECT CELLS, PURIFICATION AND CHARACTERIZATION

The extracellular domain of the human interleukin-6 (IL-6) receptor, c omprising 339 amino acids following the signal peptide, has been expre ssed in baculovirus-infected insect cells (Sf158). When the soluble re ceptor secreted into the culture medium was purified by affinity chrom atography, using IL-6 immobilized on Sepharose, 6 mg soluble receptor was isolated from 1 l conditioned medium of Sf158 suspension cultures. A molar absorption coefficient of 9.3 x 10(4) l . mol(-1). cm(-1) was calculated from the ultraviolet spectrum of the soluble IL-6 receptor . After SDS/PAGE and silver staining, an apparent molecular mass of 48 kDa was estimated for the purified protein. Deglycosylation with pept ide N-glycosidase F resulted in an increase in electrophoretic mobilit y and a decrease in the apparent molecular mass from 48 kDa to about 4 1-44 kDa. As expected, the soluble human IL-6 receptor bound human I-1 25-labeled IL-6 with low affinity (K-d = 500 pM). Furthermore, the bin ding of soluble human IL-6 receptor to immobilized IL-6 was studied us ing real-time interaction analysis. The recombinant soluble receptor s howed biological activity on HepG2 cells stably transfected with a cDN A coding for IL-6 (HepG2-IL-6 cells). Haptoglobin mRNA synthesis was i nduced by the soluble IL-6 receptor at concentrations as low as 10 ng/ ml. Five monoclonal antibodies were generated. Two groups of antibodie s were identified mapping to amino acids 1-67 and 68-143 of the solubl e IL-6 receptor, respectively. The plasma clearance of soluble I-125-l abeled IL-6 receptor in the absence and presence of IL-6 was studied i n rats as a model system. The kinetics was biphasic. Soluble IL-6 rece ptor/IL-6 complexes were cleared more rapidly than the soluble recepto r alone. Intravenously injected soluble I-125-labeled IL-6 receptor, a s well as complexes with IL-6, rapidly accumulated in liver and to a l esser extent in skeletal muscle, skin and kidneys. Subsequently, the r adioactivity appeared in the gut content.