SPR-based immunocapture approach to creating an interfacial sensing architecture: mapping of the MRS18-2 binding site on retinoblastoma protein |
| |
Authors: | Boris Snopok Mariya Yurchenko Laszlo Szekely George Klein Elena Kashuba |
| |
Institution: | (1) V. Lashkaryov Institute of Semiconductor Physics, NASU, Prospekt Nauki 41, 03028 Kiev-28, Ukraine;(2) R. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NASU, Ul. Vasylkivska 45, 03022 Kiev-22, Ukraine;(3) Microbiology and Tumor Biology Center, Karolinska Institute, Box 280, 17177 Stockholm, Sweden;(4) IRIS (Center for Integrative Recognition in the Immune System), Karolinska Institute, 17177 Stockholm, Sweden |
| |
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. |
| |
Keywords: | Surface plasmon resonance GST fusion proteins Interfacial complex Protein– protein interaction Epstein– Barr virus |
本文献已被 PubMed SpringerLink 等数据库收录! |
|