A simple detection method for low-affinity membrane protein interactions by baculoviral display.
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.


RESULTS
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.
Sakihama
Toshiko
T
Department of Molecular Biology and Medicine, The University of Tokyo, Tokyo, Japan. sakihama@med.rcast.u-tokyo.ac.jp
Sato
Takato
T
Iwanari
Hiroko
H
Kitamura
Toshio
T
Sakaguchi
Shimon
S
Kodama
Tatsuhiko
T
Hamakubo
Takao
T
D000818:Animals; D000954:Antigens, Surface / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism*; D018801:CD2 Antigens / Q000378:metabolism; D023201:CD40 Ligand / Q000378:metabolism; D018968:CD58 Antigens / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D019151:Peptide Library; D011485:Protein Binding; D018411:Spodoptera; D013379:Substrate Specificity
D000954:Antigens, Surface; D018801:CD2 Antigens; D018968:CD58 Antigens; D008565:Membrane Proteins; D019151:Peptide Library; D023201:CD40 Ligand