Overexpression, purification, and use of a soluble human interleukin-4 receptor alpha-chain/Ig gamma 1 fusion protein for ligand binding studies. Characterization of ligand binding to soluble IL-4 receptor alpha-chain by surface plasmon resonance measurements and by microtiter-plate-based ELISA with biotinylated IL-4.
The pleiotropic cytokine IL-4 transmits cellular signals mainly via the IL-4 receptor complex, with the alpha-chain as the high affinity binding subunit. Here we describe the overexpression of a soluble IL-4R alpha-chain (sIL-4R) as a fusion to immunoglobulin gamma 1 heavy chain, consisting of the H-CH2-CH3 domains, in baby hamster kidney cells. The dimeric fusion protein named sIL-4R:E gamma 1 was purified from culture supernatant by protein-A affinity chromatography, yielding up to 10 mg/l homogenous protein which was highly stable. The antibody-like features of the sIL-4R:E gamma 1 fusion protein allowed immobilization on a biosensor matrix for surface plasmon resonance measurements by direct amine coupling as well as immobilization on microtiter plates coated with protein A for displacement binding. Kinetic parameters (kon and koff) for binding of IL-4 or the antagonistic mutant IL-4(Y124D) to the sIL-4R:E gamma 1 fusion protein on the chip as determined with the BIAcore instrument showed a high affinity binding with KD = 239 +/- 35 pM and KD=148 +/- 33 pM, respectively. The extremely high kon rate and the relatively slow koff rate for both ligands highlighted the limits of the BIAcore technology. The binding affinity as calculated in displacement binding studies with biotinylated IL-4 was similar for IL-4 and IL-4(Y124D) (IC50=1.1nM), thus offering a simple alternative for initial characterization of IL-4 mutants.
Seipelt
I
I
Department of Cancer Research, ASTA Medica AG, Frankfurt/Main, Germany.
Hoffmann
S H
SH
Schmidt
J
J
Engels
J W
JW
Beckers
T
T
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015374:Biosensing Techniques; D019921:Biotinylation; D002460:Cell Line; D002469:Cell Separation; D002999:Clone Cells; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation / Q000379:methods; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics*; D015847:Interleukin-4 / Q000378:metabolism*; D007668:Kidney / Q000166:cytology; D008024:Ligands; D011233:Precipitin Tests; D011485:Protein Binding / Q000235:genetics; D019948:Receptors, Interleukin-4 / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D017186:Titrimetry
D007074:Immunoglobulin G; D008024:Ligands; D019948:Receptors, Interleukin-4; D011993:Recombinant Fusion Proteins; D015847:Interleukin-4