title: Biotinylation of the Fcγ receptor ectodomains by mammalian cell co-transfection: application to the development of a surface plasmon resonance-based assay.
abstract: We here report the production of four biotinylated Fcγ receptor (FcγR) 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 FcγR 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 FcγR 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. Finally, biotin-tagged FcγRs produced with the in-cell approach are successfully applied to the development of SPR-based assays to evaluate the impact of the glycosylation pattern of monoclonal antibodies on their interaction with CD16a and CD64. In that endeavor, we unambiguously observe that highly galactosylated trastuzumab (TZM-gal), non-glycosylated trastuzumab (TZM-NG), and reference trastuzumab are characterized by different kinetic profiles upon binding to CD16a and CD64 that had been captured at the biosensor surface via their biotin tag. More precisely, while TZM-NG binding to CD16a was not detected, TZM-gal formed a more stable complex with CD16a than our reference TZM. In contrast, both glycosylated TZM bound to captured CD64 in a stable and similar fashion, whereas the interaction of their non-glycosylated form with CD64 was characterized by a higher dissociation rate.
authors.affiliation: Department of Chemical Engineering, Groupe de Recherche en Sciences et Technologies Biomédicales, Bio-P2 Research Unit, École Polytechnique de Montréal, P.O. Box 6079, succ. Centre-ville, Montreal, QC, Canada, H3C 3A7.|Human Health Therapeutics Portfolio, National Research Council Canada, Montreal, QC, Canada, H4P 2R2.
authors.forename: July
authors.initials: J
authors.lastname: Dorion-Thibaudeau
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council Canada, Montreal, QC, Canada, H4P 2R2.
authors.forename: Gilles
authors.initials: G
authors.lastname: St-Laurent
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council Canada, Montreal, QC, Canada, H4P 2R2.|Département de biochimie et médecine moléculaire, Faculté de médecine, Université de Montréal, Montreal, QC, Canada, H3C 3 J7.
authors.forename: Céline
authors.initials: C
authors.lastname: Raymond
authors.affiliation: Department of Chemical Engineering, Groupe de Recherche en Sciences et Technologies Biomédicales, Bio-P2 Research Unit, École Polytechnique de Montréal, P.O. Box 6079, succ. Centre-ville, Montreal, QC, Canada, H3C 3A7.
authors.forename: Gregory
authors.initials: G
authors.lastname: De Crescenzo
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council Canada, Montreal, QC, Canada, H4P 2R2.|Département de biochimie et médecine moléculaire, Faculté de médecine, Université de Montréal, Montreal, QC, Canada, H3C 3 J7.
authors.forename: Yves
authors.initials: Y
authors.lastname: Durocher
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D019921:Biotinylation; D016466:CHO Cells; D003412:Cricetulus; D005690:Galactose / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D017452:Receptors, IgG / Q000737:chemistry*; D019809:Streptavidin / Q000737:chemistry*; D020349:Surface Plasmon Resonance; D014162:Transfection; D000068878:Trastuzumab / Q000737:chemistry / Q000378:metabolism*
chemical_list: D017452:Receptors, IgG; D019809:Streptavidin; D000068878:Trastuzumab; D005690:Galactose
keywords: BirA enzyme; FcγR; biotin acceptor peptide (BAP); glycosylation; monoclonal antibody; surface plasmon resonance