title: SPR-based immunocapture approach to creating an interfacial sensing architecture: Mapping of the MRS18-2 binding site on retinoblastoma protein.
abstract: Biosensor technologies based on optical readout are widely used in protein-protein interaction studies. Here we describe a fast and simple approach to the creation of oriented interfacial architectures for surface plasmon resonance (SPR) transducers, based on conventional biochemical procedures and custom reagents. The proposed protocol permits the oriented affinity-capture of GST fusion proteins by a specific antibody which is bound to protein A, which in turn has been immobilized on the transducer surface (after the surface has been modified by guanidine thiocyanate). The applicability of the method was demonstrated by studying the interaction between retinoblastoma tumor suppressor protein (pRb) and MRS18-2 proteins. The formation of the pRb-MRS18-2 protein complex was examined and the pRb binding site (A-box-spacer-B-box) was mapped. We have also shown that MRS18-2, which was detected as the Epstein-Barr virus-encoded EBNA-6 binding partner using the yeast two-hybrid system, binds to pRb in GST pull-down assays.
authors.affiliation: V. Lashkaryov Institute of Semiconductor Physics, NASU, Prospekt Nauki 41, 03028 Kiev-28, Ukraine.
authors.forename: Boris
authors.initials: B
authors.lastname: Snopok
authors.forename: Mariya
authors.initials: M
authors.lastname: Yurchenko
authors.forename: Laszlo
authors.initials: L
authors.lastname: Szekely
authors.forename: George
authors.initials: G
authors.lastname: Klein
authors.forename: Elena
authors.initials: E
authors.lastname: Kashuba
mesh_terms: D000906:Antibodies / Q000276:immunology; D001665:Binding Sites / Q000276:immunology; D045744:Cell Line, Tumor; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D004854:Herpesvirus 4, Human; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D020349:Surface Plasmon Resonance / Q000379:methods*; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D000906:Antibodies; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; D014764:Viral Proteins; D005982:Glutathione Transferase