UC Riverside

Bridging effector cells to immunoglobulin G (IgG) antibodies, Fc gamma receptors (FcγRs) initiate and regulate humoral immunity and thus their ectodomains are important as therapeutics and biomedical reagents. This study aims to produce fully biotinylated, N-glycosylated, and disulfide bond formed FcγRs in mammalian cells. Several low-affinity FcγRs were the targets, including FcγRIIA and FcγRIIIA of activation functions and FcγRIIB of inhibition functions. In vivo biotinylation in 293 cells was achieved by co-expression of AviTag fused FcγRs and E. coli biotin ligase (BirA). To facilitate biotinylation, endoplasmic reticulum retention signal DYKDEL was fused with BirA. Both stable and transient transfections were studied and the ratio of FcγR and BirA plasmids during co-transfection was optimized as well. Produced FcγRs were tested by SDS-PAGE, streptavidin pulldown assays, and binding kinetic analysis. Results indicated that FcγRIIIA(F176), FcγRIIIA(V176) and FcγRIIB produced from the associated stably transfected cell lines exhibited expected disulfide and glycosylation profiles with complete biotinylation at yields of 15.3, 9.1, and 25.7 µg purified receptor ectodomains per mL culture. And transient transfection led production of 7.0 and 3.6 µg purified FcγRIIA(H131) and FcγRIIA(R131) per mL culture with high quality in terms of biotinylation efficiency, disulfide bond formation, and glycosylation. Produced FcγRs demonstrated their expected binding affinities towards IgG1 trastuzumab consistent with values reported in literatures. This work provided high quality reagents for engineering endeavor of antibody Fc fragments, and the methods demonstrated here can be applied for in vivo biotinylation of other important glycoproteins in mammalian cells.