Oriented immobilization of antibodies with GST-fused multiple Fc-specific B-domains on a gold surface.
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.
Ha
Tai Hwan
TH
BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, 305-333 Korea.
Jung
Sun Ok
SO
Lee
Jeong Min
JM
Lee
Kun Yeong
KY
Lee
Yan
Y
Park
Jong Sang
JS
Chung
Bong Hyun
BH
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
D000906:Antibodies; D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D011993:Recombinant Fusion Proteins; D006046:Gold; D005982:Glutathione Transferase