Biotinylation of the Fc gamma receptor ectodomains by mammalian cell co-transfection: application to the development of a surface plasmon resonance-based assay

摘要

We here report the production of four biotinylated Fc gamma receptor (Fc gamma R) ectodomains and their subsequent stable capture on streptavidin-biosensor surfaces. For receptor biotinylation, we first describe an in-cell protocol based on the co-transfection of two plasmids corresponding to one of the Fc gamma R ectodomains and the BirA enzyme in mammalian cells. This strategy is compared with a standard sequential in vitro enzymatic biotinylation with respect to biotinylation level and yield. Biotinylated Fc gamma R ectodomains that have been prepared with both strategies are then compared by analytical ultracentrifugation and surface plasmon resonance (SPR) analyses. Overall, we demonstrate that in-cell biotinylation is an interesting alternative to standard biotinylation protocol, as it requires less purification steps while yielding higher titers.