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

作者：WEIERGRABER O; HEMMANN U; KUSTER A; MULLERNEWEN G; SCHNEIDER J; ROSEJOHN S; KURSCHAT P; BRAKENHOFF JPJ; HART MHL; STABEL S; HEINRICH PC

来源：European Journal of Biochemistry, 1995, 234(2): 661-669.

DOI：10.1111/j.1432-1033.1995.661_b.x

• INTERLEUKIN-6
• SOLUBLE INTERLEUKIN-6 RECEPTOR
• BACULOVIRUS SYSTEM
• PLASMA CLEARANCE

The extracellular domain of the human interleukin-6 (IL-6) receptor, comprising 339 amino acids following the signal peptide, has been expressed in baculovirus-infected insect cells (Sf158). When the soluble receptor secreted into the culture medium was purified by affinity chromatography, 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 peptide N-glycosidase F resulted in an increase in electrophoretic mobility and a decrease in the apparent molecular mass from 48 kDa to about 41-44 kDa. As expected, the soluble human IL-6 receptor bound human I-125-labeled IL-6 with low affinity (K-d = 500 pM). Furthermore, the binding of soluble human IL-6 receptor to immobilized IL-6 was studied using real-time interaction analysis. The recombinant soluble receptor showed biological activity on HepG2 cells stably transfected with a cDNA coding for IL-6 (HepG2-IL-6 cells). Haptoglobin mRNA synthesis was induced by the soluble IL-6 receptor at concentrations as low as 10 ng/ml. Five monoclonal antibodies were generated. Two groups of antibodies were identified mapping to amino acids 1-67 and 68-143 of the soluble IL-6 receptor, respectively. The plasma clearance of soluble I-125-labeled IL-6 receptor in the absence and presence of IL-6 was studied in rats as a model system. The kinetics was biphasic. Soluble IL-6 receptor/IL-6 complexes were cleared more rapidly than the soluble receptor alone. Intravenously injected soluble I-125-labeled IL-6 receptor, as well as complexes with IL-6, rapidly accumulated in liver and to a lesser extent in skeletal muscle, skin and kidneys. Subsequently, the radioactivity appeared in the gut content.

• 出版日期1995-12-1