Recombinant soluble human interleukin-5 (hIL-5) receptor molecules. Cross-linking and stoichiometry of binding to IL-5.
Recombinant soluble human interleukin-5 receptor alpha (shIL-5R alpha) has been expressed in COS-1 cells and in baculovirus-infected cells. The protein was purified from the supernatant by chromatography on concanavalin A-Sepharose, MonoQ, and a final gel filtration step. A chimeric fusion receptor protein (hIL-5R alpha-h gamma 3) was constructed by fusion of the cDNA corresponding to the shIL-5R alpha to the cDNA corresponding to the Fc part of the human IgG C gamma 3 chain, and was expressed in baculovirus-infected insect cells. The chimeric receptor was secreted as a disulfide-linked homodimer, and was purified by protein G affinity chromatography. In a solid-phase binding assay the shIL-5R alpha and the bivalent hIL-5R alpha-h gamma 3 were found to bind hIL-5 with a similar affinity, corresponding to the membrane-bound, low affinity hIL-5R alpha. SDS-polyacrylamide gel electrophoresis of shIL-5R alpha cross-linked to radiolabeled hIL-5, suggested that one shIL-5R alpha molecule binds to one hIL-5 homodimer molecule. Gel filtration studies of the complex formed between the shIL-5R alpha and hIL-5 pointed toward the same stoichiometry of binding. The formation of such a complex could be observed by electrophoresis in native gels. Immunoaffinity chromatography using a non-neutralizing monoclonal antibody directed against hIL-5, followed by size column chromatography, allowed the purification of the complex. The data obtained from the amino acid analysis of the constituents of the complex blotted from an SDS-polyacrylamide gel, and from the amino acid composition of the complex blotted from a native polyacrylamide gel, provided direct evidence that the shIL-5R alpha binds the hIL-5 dimer in a 1:1 ratio.
Devos
R
R
Roche Research Gent, F. Hoffmann-La Roche & Co., Ltd., Belgium.
Guisez
Y
Y
Cornelis
S
S
Verhee
A
A
Van der Heyden
J
J
Manneberg
M
M
Lahm
H W
HW
Fiers
W
W
Tavernier
J
J
Plaetinck
G
G
D000818:Animals; D002460:Cell Line; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics; D015848:Interleukin-5 / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D009036:Moths; D011971:Receptors, Immunologic / Q000378:metabolism*; D018123:Receptors, Interleukin; D053649:Receptors, Interleukin-5; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012995:Solubility
D007074:Immunoglobulin G; D015848:Interleukin-5; D008024:Ligands; D011971:Receptors, Immunologic; D018123:Receptors, Interleukin; D053649:Receptors, Interleukin-5; D011993:Recombinant Fusion Proteins