Overexpression, Purification, and Use of a Soluble Human Interleukin-4 Receptor α-chain/Igγ1 Fusion Protein for Ligand Binding Studies

Abstract

The pleiotropic cytokine IL-4 transmits cellular signals mainly via the IL-4 receptor complex, with the α-chain as the high affinity binding subunit. Here we describe the overexpression of a soluble IL-4R α-chain (sIL-4R) as a fusion to immunoglobulin γ1heavy chain, consisting of the H–CH2–CH3domains, in baby hamster kidney cells. The dimeric fusion protein named sIL-4R:Eγ1was 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:Eγ1fusion 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 (konand koff) for binding of IL-4 or the antagonistic mutant IL-4Y124Dto the sIL-4R:Eγ1fusion 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 konrate and the relatively slow koffrate 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-4Y124D(IC50=1.1nM), thus offering a simple alternative for initial characterization of IL-4 mutants.