Expression, purification and characterization of a functional extracellular domain of porcine FcγRII

FcγRs are involved in regulating a multitude of innate and adaptive immune responses, which makes them attractive targets for the development of novel immunotherapeutic approaches. In this report, we describe a simple method for the production of a large quantity of recombinant porcine FcγRII. The extracellular domain of the porcine FcγRII (poFcγRII) gene was constructed and cloned into the Escherichiacoli expression vector pET-28a. The recombinant protein was expressed at high level in E. coil BL21 (DE3) and existed mainly as inclusion bodies. The inclusion bodies were solubilized in 6 M guanidine hydrochloride and purified by Ni-chelation, and refolded by rapid dilution. After purification and renaturation, the recombinant soluble protein (rsFcγRII) coated on high-binding ELISA plates, showed concentration dependent binding of porcine IgG and the binding of porcine IgG to the surface bound rsFcγRII was inhibited in a dose-dependent manner by soluble rsFcγRII itself. Then by the inhibition assay we evaluated the effectiveness of the rsFcγRII in inhibiting the IgG binding to the whole molecule of poFcγRII expressed on the Marc-145 cell surface, the rsFcγRII inhibited the binding of porcine IgG to the transfected Marc-145 cell’s surface, with an IC50 value of 0.87 μM, demonstrating that rsFcγRII manifests the similar specificity as native poFcγRII. The method for highly efficient production of biologically active poFcγRII may be employed for both basic research and potential clinical applications.