RRC ID 43535
著者 Sakihama T, Sato T, Iwanari H, Kitamura T, Sakaguchi S, Kodama T, Hamakubo T.
タイトル A simple detection method for low-affinity membrane protein interactions by baculoviral display.
ジャーナル PLoS One
Abstract BACKGROUND:Membrane protein interactions play an important role in cell-to-cell recognition in various biological activities such as in the immune or neural system. Nevertheless, there has remained the major obstacle of expression of the membrane proteins in their active form. Recently, we and other investigators found that functional membrane proteins express on baculovirus particles (budded virus, BV). In this study, we applied this BV display system to detect interaction between membrane proteins important for cell-to-cell interaction in immune system.
METHODOLOGY/PRINCIPAL FINDINGS:We infected Sf9 cells with recombinant baculovirus encoding the T cell membrane protein CD2 or its ligand CD58 and recovered the BV. We detected specific interaction between CD2-displaying BV and CD58-displaying BV by an enzyme-linked immunosorbent assay (ELISA). Using this system, we also detected specific interaction between two other membrane receptor-ligand pairs, CD40-CD40 ligand (CD40L), and glucocorticoid-induced TNFR family-related protein (GITR)-GITR ligand (GITRL). Furthermore, we observed specific binding of BV displaying CD58, CD40L, or GITRL to cells naturally expressing their respective receptors by flowcytometric analysis using anti-baculoviral gp64 antibody. Finally we isolated CD2 cDNA from a cDNA expression library by magnetic separation using CD58-displaying BV and anti-gp64 antibody.
CONCLUSIONS:We found the BV display system worked effectively in the detection of the interaction of membrane proteins. Since various membrane proteins and their oligomeric complexes can be displayed on BV in the native form, this BV display system should prove highly useful in the search for natural ligands or to develop screening systems for therapeutic antibodies and/or compounds.
巻・号 3(12)
ページ e4024
公開日 2008-1-1
DOI 10.1371/journal.pone.0004024
PMID 19107192
PMC PMC2602974
MeSH Animals Antigens, Surface / genetics Antigens, Surface / isolation & purification Antigens, Surface / metabolism Baculoviridae / genetics Baculoviridae / metabolism* CD2 Antigens / metabolism CD40 Ligand / metabolism CD58 Antigens / metabolism Cells, Cultured Cloning, Molecular / methods Enzyme-Linked Immunosorbent Assay Humans Membrane Proteins / genetics Membrane Proteins / isolation & purification* Membrane Proteins / metabolism* Mice Models, Biological Peptide Library* Protein Binding Spodoptera Substrate Specificity
IF 2.74
引用数 15
WOS 分野 BIOCHEMISTRY & MOLECULAR BIOLOGY
リソース情報
ヒト・動物細胞 BaF/3(RCB0805)