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

Sakihama T, Sato T, Iwanari H, Kitamura T, Sakaguchi S, Kodama T, Hamakubo T - PLoS ONE (2008)

Bottom Line: Finally we isolated CD2 cDNA from a cDNA expression library by magnetic separation using CD58-displaying BV and anti-gp64 antibody.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Medicine, The University of Tokyo, Tokyo, Japan. sakihama@med.rcast.u-tokyo.ac.jp

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.

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Expression cloning of human CD2 by using CD58-displaying BV as the probe.(A) Enrichment of human CD2-positive cells from BaF/3 cells transfected with a human T cell cDNA library. The staining of cells with an FITC-labeled anti humanCD2 monoclonal antibody is shown. The thin line indicates cells incubated without anti-CD2 antibody. (B) Binding of anti-human CD2 antibody and CD58-displaying BV to BaF/3 cells isolated by magnetic sorting with CD58-BV. After the 3rd magnetic sorting and subcloning, cells were stained with an FITC-labeled anti-human CD2 monoclonal antibody and CD58-BV plus biotinylated anti-gp64 antibody and PE-streptavidin. Staining of a representative of three clones is shown.
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pone-0004024-g005: Expression cloning of human CD2 by using CD58-displaying BV as the probe.(A) Enrichment of human CD2-positive cells from BaF/3 cells transfected with a human T cell cDNA library. The staining of cells with an FITC-labeled anti humanCD2 monoclonal antibody is shown. The thin line indicates cells incubated without anti-CD2 antibody. (B) Binding of anti-human CD2 antibody and CD58-displaying BV to BaF/3 cells isolated by magnetic sorting with CD58-BV. After the 3rd magnetic sorting and subcloning, cells were stained with an FITC-labeled anti-human CD2 monoclonal antibody and CD58-BV plus biotinylated anti-gp64 antibody and PE-streptavidin. Staining of a representative of three clones is shown.

Mentions: Our results prompted us to explore the possibility of utilizing BV displaying membrane proteins as the probe for expression cloning of receptor (or ligand) cDNA. To this end, we transfected BaF/3 cells with a human T cell cDNA library using a retroviral transduction system. Cells bound to CD58-displaying BV were selected with an anti-viral gp64 antibody plus secondary antibody-coated magnetic beads. After magnetic sorting of the BV-bound cells three times, cells expressing human CD2 were enriched (Fig. 5A). The recovered cells were cloned by limiting dilution. PCR of cloned BaF/3 cell genomic DNA with retroviral vector-derived primers amplified the human CD2 cDNA sequence. Furthermore, flowcytometric analysis confirmed that these clones expressed human CD2 and that CD58-BV bound to these cells (Fig. 5B).


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

Sakihama T, Sato T, Iwanari H, Kitamura T, Sakaguchi S, Kodama T, Hamakubo T - PLoS ONE (2008)

Expression cloning of human CD2 by using CD58-displaying BV as the probe.(A) Enrichment of human CD2-positive cells from BaF/3 cells transfected with a human T cell cDNA library. The staining of cells with an FITC-labeled anti humanCD2 monoclonal antibody is shown. The thin line indicates cells incubated without anti-CD2 antibody. (B) Binding of anti-human CD2 antibody and CD58-displaying BV to BaF/3 cells isolated by magnetic sorting with CD58-BV. After the 3rd magnetic sorting and subcloning, cells were stained with an FITC-labeled anti-human CD2 monoclonal antibody and CD58-BV plus biotinylated anti-gp64 antibody and PE-streptavidin. Staining of a representative of three clones is shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2602974&req=5

pone-0004024-g005: Expression cloning of human CD2 by using CD58-displaying BV as the probe.(A) Enrichment of human CD2-positive cells from BaF/3 cells transfected with a human T cell cDNA library. The staining of cells with an FITC-labeled anti humanCD2 monoclonal antibody is shown. The thin line indicates cells incubated without anti-CD2 antibody. (B) Binding of anti-human CD2 antibody and CD58-displaying BV to BaF/3 cells isolated by magnetic sorting with CD58-BV. After the 3rd magnetic sorting and subcloning, cells were stained with an FITC-labeled anti-human CD2 monoclonal antibody and CD58-BV plus biotinylated anti-gp64 antibody and PE-streptavidin. Staining of a representative of three clones is shown.
Mentions: Our results prompted us to explore the possibility of utilizing BV displaying membrane proteins as the probe for expression cloning of receptor (or ligand) cDNA. To this end, we transfected BaF/3 cells with a human T cell cDNA library using a retroviral transduction system. Cells bound to CD58-displaying BV were selected with an anti-viral gp64 antibody plus secondary antibody-coated magnetic beads. After magnetic sorting of the BV-bound cells three times, cells expressing human CD2 were enriched (Fig. 5A). The recovered cells were cloned by limiting dilution. PCR of cloned BaF/3 cell genomic DNA with retroviral vector-derived primers amplified the human CD2 cDNA sequence. Furthermore, flowcytometric analysis confirmed that these clones expressed human CD2 and that CD58-BV bound to these cells (Fig. 5B).

Bottom Line: Finally we isolated CD2 cDNA from a cDNA expression library by magnetic separation using CD58-displaying BV and anti-gp64 antibody.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Medicine, The University of Tokyo, Tokyo, Japan. sakihama@med.rcast.u-tokyo.ac.jp

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.

Show MeSH
Related in: MedlinePlus