Cloning, expression and in vitro characterization of muTNFR-Fc. (a) Schematic representation of the cloning strategy of muTNFR-Fc. muTNFR was directly fused to the Fc fragment (hinge, CH2 and CH3 regions) of murine IgG1, containing a signal sequence (SS) for secretion of the protein at the N-terminus. (b) Schematic representation of the formation of a muTNFR-Fc dimer trough disulfide bridges in the hinge region. (c) SDS-PAGE analysis of purified muTNFR-Fc. M, molecular weight marker; NR, nonreducing conditions; R, reducing conditions. (d) Size exclusion chromatography (SEC200) of covalent homodimeric muTNFR-Fc. (e) Bioactivity assay of muTNFR-Fc. muTNFR-Fc inhibited tumor necrosis factor (TNF)-induced killing of mouse fibroblasts with a half-maximal inhibitory concentration of 0.1 nM (mean ± standard deviation, n = 3).
Doll et al. Arthritis Research & Therapy 2013 15:R138 doi:10.1186/ar4319