title: Oriented immobilization of antibodies with GST-fused multiple Fc-specific B-domains on a gold surface.
abstract: We have constructed a novel platform for the oriented buildup of immunoglobulins on a gold surface for a surface plasmon resonance imaging microarray. To this end, genetically engineered glutathione S-transferase proteins bearing one, two, and three Fc-specific B-domains in protein G from Streptococci (GST-GB1, -GB2, and -GB3, respectively) were produced. In order to tether these GST-GBx proteins specifically, a novel glutathione-derivatized ligand (LA-GSH) was also synthesized from a biaminated tri(ethylene glycol) backbone. Each end of the backbone was further functionalized with a maleimide group for a glutathione modification and a lipoic acid for surface immobilization. The glutathione ligand demonstrated a negligible nonspecific protein adsorption toward other spectator proteins while showing a strong specific association toward GST-GBx proteins. This Fc-specific surface exhibited at least a 2-fold enhancement in the immunoglobulin density (from human and mouse) with its antigen capture capability totally conserved compared to a covalently tethered GBx proteins. A single antibody tethered on the GST-GB3 is estimated to capture two antigens (enhanced green fluorescent protein), and this antigen capture ratio seems to be the most efficient value ever observed.
authors.affiliation: BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, 305-333 Korea.
authors.forename: Tai Hwan
authors.initials: TH
authors.lastname: Ha
authors.forename: Sun Ok
authors.initials: SO
authors.lastname: Jung
authors.forename: Jeong Min
authors.initials: JM
authors.lastname: Lee
authors.forename: Kun Yeong
authors.initials: KY
authors.lastname: Lee
authors.forename: Yan
authors.initials: Y
authors.lastname: Lee
authors.forename: Jong Sang
authors.initials: JS
authors.lastname: Park
authors.forename: Bong Hyun
authors.initials: BH
authors.lastname: Chung
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics; D006046:Gold / Q000737:chemistry*; D006801:Humans; D051379:Mice; D040081:Protein Array Analysis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D020349:Surface Plasmon Resonance / Q000379:methods*; D013499:Surface Properties
chemical_list: D000906:Antibodies; D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D011993:Recombinant Fusion Proteins; D006046:Gold; D005982:Glutathione Transferase