Recombinant soluble human interleukin-6 receptor. Expression in Escherichia coli, renaturation and purification.
The recombinant soluble human interleukin-6 receptor (srhIL-6R) was expressed in Escherichia coli as a non-glycosylated protein comprising the first 339 amino acids after the signal peptide. The protein accumulated within the cells as insoluble protein aggregates (inclusion bodies). After solubilization, 10% of the denatured srhIL-6R could be renaturated by an in vitro folding procedure using L-arginine and the glutathione-redox system. The native receptors were purified to near homogeneity by affinity chromatography on an IL-6-Sepharose column. The functional features of the recombinant soluble receptor were further analysed. A part of the extracellular domain (amino acids 145-345) of the human interleukin-6 receptor (IL-6R) was expressed in E. coli and the purified protein was used to raise antibodies in rabbits. Characterization of the antiserum obtained indicated that an epitope of 13 amino acids close to the transmembrane region is needed for recognition by the antibodies. Since the antiserum obtained did not interfere with IL-6 binding, it could be used to establish a cell-free IL-6-binding assay, In this assay, the srhIL-6R bound IL-6 with an affinity of Kd = 1.5 nM as measured by Scatchard-plot analysis. When 125I-IL-6 was chemically cross-linked to the purified srhIL-6R and analyzed by SDS/PAGE, several 125I-IL-6-containing bands were detected, indicating the possible existence of a multimeric structure of the natural IL-6/IL-6R complex. The srhIL-6R was shown to exhibit biological activity, i.e. it stimulated acute-phase protein synthesis in the recently established human hepatoma cell line HepG2-IL-6 which does not express the IL-6-binding subunit of the IL-6R complex on the cell surface.
Stoyan
T
T
Institut für Biochemie der RWTH-Aachen, Germany.
Michaelis
U
U
Schooltink
H
H
Van Dam
M
M
Rudolph
R
R
Heinrich
P C
PC
Rose-John
S
S
D000906:Antibodies / Q000276:immunology; D001120:Arginine / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D005978:Glutathione / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017510:Protein Folding; D011971:Receptors, Immunologic / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D019947:Receptors, Interleukin-6; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D014407:Tumor Cells, Cultured; D000514:alpha 1-Antichymotrypsin / Q000096:biosynthesis
D000906:Antibodies; D011971:Receptors, Immunologic; D019947:Receptors, Interleukin-6; D011994:Recombinant Proteins; D000514:alpha 1-Antichymotrypsin; D001120:Arginine; D005978:Glutathione