Expression, purification and characterization of a functional extracellular domain of porcine FcgammaRII.
FcgammaRs 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 FcgammaRII. The extracellular domain of the porcine FcgammaRII (poFcgammaRII) 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 6M guanidine hydrochloride and purified by Ni-chelation, and refolded by rapid dilution. After purification and renaturation, the recombinant soluble protein (rsFcgammaRII) coated on high-binding ELISA plates, showed concentration dependent binding of porcine IgG and the binding of porcine IgG to the surface bound rsFcgammaRII was inhibited in a dose-dependent manner by soluble rsFcgammaRII itself. Then by the inhibition assay we evaluated the effectiveness of the rsFcgammaRII in inhibiting the IgG binding to the whole molecule of poFcgammaRII expressed on the Marc-145 cell surface, the rsFcgammaRII inhibited the binding of porcine IgG to the transfected Marc-145 cell's surface, with an IC(50) value of 0.87 microM, demonstrating that rsFcgammaRII manifests the similar specificity as native poFcgammaRII. The method for highly efficient production of biologically active poFcgammaRII may be employed for both basic research and potential clinical applications.
Tian
Xiaohui
X
Key Laboratory of Animal Immunology of the Ministry of Agriculture, Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhenzhou 450002, China.
Wang
Aiping
A
Qiao
Songlin
S
Zhang
Gaiping
G
Xi
Jun
J
Li
Xuewu
X
Liu
Yunchao
Y
Xu
Yuqing
Y
D000818:Animals; D000918:Antibody Specificity; D002460:Cell Line; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D007074:Immunoglobulin G / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D017452:Receptors, IgG / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013552:Swine
D007074:Immunoglobulin G; D017452:Receptors, IgG; D011994:Recombinant Proteins