Limits...
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.

Show MeSH

Related in: MedlinePlus

Specific binding of ligand-displaying BV to cells expressing receptor.(A) Schematic view. X corresponds to CD2 (for B), CD40 (for C), or GITR (for D). Y corresponds to CD58 (for B), CD40L (for C), or GITRL (for D). (B) CD58-displaying BV binds to CD2-positive (top), but not to CD2-negative Jurkat cells (bottom). Left: binding of an anti-CD2 monoclonal antibody (clone RPA-2.10) plus a PE-anti mouse Ig-κ chain antibody. Right: binding of CD58-displaying BV plus an anti-gp64 antibody (clone A0505A) and a PE-anti mouse Ig-κ chain antibody. The thin line indicates cells incubated with the 2nd antibody only. (C) CD40-dependent binding of CD40L-displaying BV to mouse splenic B cells. BALB/c mouse splenocytes were incubated with CD40L-displaying BV and a biotinylated anti-gp64 monoclonal antibody (clone B8147A), and then were stained with FITC-labeled streptavidin and a PE-labeled anti-B220 antibody. CD40L-BV binding to the B cells (shown in the center lower panel) was blocked by pre-incubation with an anti-mouse CD40 monoclonal antibody (clone HM40-3) (the right upper panel) but not by control hamster IgG (right lower panel). (D) Binding of GITRL-displaying BV to GITR-positive cells. GITR-expressing mouse T cell hybridoma 18.3.5 cells were incubated with GITRL-displaying BV (right) or wild-type BV (left). Binding of BV was detected with a biotinylated anti-gp64 monoclonal antibody B8147A and PE-streptavidin. The thin line indicates cells incubated without BV.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2602974&req=5

pone-0004024-g004: Specific binding of ligand-displaying BV to cells expressing receptor.(A) Schematic view. X corresponds to CD2 (for B), CD40 (for C), or GITR (for D). Y corresponds to CD58 (for B), CD40L (for C), or GITRL (for D). (B) CD58-displaying BV binds to CD2-positive (top), but not to CD2-negative Jurkat cells (bottom). Left: binding of an anti-CD2 monoclonal antibody (clone RPA-2.10) plus a PE-anti mouse Ig-κ chain antibody. Right: binding of CD58-displaying BV plus an anti-gp64 antibody (clone A0505A) and a PE-anti mouse Ig-κ chain antibody. The thin line indicates cells incubated with the 2nd antibody only. (C) CD40-dependent binding of CD40L-displaying BV to mouse splenic B cells. BALB/c mouse splenocytes were incubated with CD40L-displaying BV and a biotinylated anti-gp64 monoclonal antibody (clone B8147A), and then were stained with FITC-labeled streptavidin and a PE-labeled anti-B220 antibody. CD40L-BV binding to the B cells (shown in the center lower panel) was blocked by pre-incubation with an anti-mouse CD40 monoclonal antibody (clone HM40-3) (the right upper panel) but not by control hamster IgG (right lower panel). (D) Binding of GITRL-displaying BV to GITR-positive cells. GITR-expressing mouse T cell hybridoma 18.3.5 cells were incubated with GITRL-displaying BV (right) or wild-type BV (left). Binding of BV was detected with a biotinylated anti-gp64 monoclonal antibody B8147A and PE-streptavidin. The thin line indicates cells incubated without BV.

Mentions: Next, we attempted to utilize the ligand protein-displaying BV as a tool to detect receptor expression on the cell surface. To this end, we detected the binding of BV to cells by flowcytometer using an antibody specific to baculoviral envelope protein gp64 and a fluorochrome-conjugated secondary antibody (illustrated in Fig. 4A). As shown in Fig. 4B, CD58-displaying BV bound to CD2-positive, but not CD2-negative Jurkat cells, a human T cell leukemia cell line. We also observed the binding of mouse CD40L-displaying BV to mouse splenic B cells, which express CD40. This binding was blocked by an anti-mouse CD40 monoclonal antibody (Fig. 4C). Furthermore, GITRL-displaying BV bound to GITR-expressing T cell hybridoma 18.3.5 cells (Fig. 4D).


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)

Specific binding of ligand-displaying BV to cells expressing receptor.(A) Schematic view. X corresponds to CD2 (for B), CD40 (for C), or GITR (for D). Y corresponds to CD58 (for B), CD40L (for C), or GITRL (for D). (B) CD58-displaying BV binds to CD2-positive (top), but not to CD2-negative Jurkat cells (bottom). Left: binding of an anti-CD2 monoclonal antibody (clone RPA-2.10) plus a PE-anti mouse Ig-κ chain antibody. Right: binding of CD58-displaying BV plus an anti-gp64 antibody (clone A0505A) and a PE-anti mouse Ig-κ chain antibody. The thin line indicates cells incubated with the 2nd antibody only. (C) CD40-dependent binding of CD40L-displaying BV to mouse splenic B cells. BALB/c mouse splenocytes were incubated with CD40L-displaying BV and a biotinylated anti-gp64 monoclonal antibody (clone B8147A), and then were stained with FITC-labeled streptavidin and a PE-labeled anti-B220 antibody. CD40L-BV binding to the B cells (shown in the center lower panel) was blocked by pre-incubation with an anti-mouse CD40 monoclonal antibody (clone HM40-3) (the right upper panel) but not by control hamster IgG (right lower panel). (D) Binding of GITRL-displaying BV to GITR-positive cells. GITR-expressing mouse T cell hybridoma 18.3.5 cells were incubated with GITRL-displaying BV (right) or wild-type BV (left). Binding of BV was detected with a biotinylated anti-gp64 monoclonal antibody B8147A and PE-streptavidin. The thin line indicates cells incubated without BV.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004024-g004: Specific binding of ligand-displaying BV to cells expressing receptor.(A) Schematic view. X corresponds to CD2 (for B), CD40 (for C), or GITR (for D). Y corresponds to CD58 (for B), CD40L (for C), or GITRL (for D). (B) CD58-displaying BV binds to CD2-positive (top), but not to CD2-negative Jurkat cells (bottom). Left: binding of an anti-CD2 monoclonal antibody (clone RPA-2.10) plus a PE-anti mouse Ig-κ chain antibody. Right: binding of CD58-displaying BV plus an anti-gp64 antibody (clone A0505A) and a PE-anti mouse Ig-κ chain antibody. The thin line indicates cells incubated with the 2nd antibody only. (C) CD40-dependent binding of CD40L-displaying BV to mouse splenic B cells. BALB/c mouse splenocytes were incubated with CD40L-displaying BV and a biotinylated anti-gp64 monoclonal antibody (clone B8147A), and then were stained with FITC-labeled streptavidin and a PE-labeled anti-B220 antibody. CD40L-BV binding to the B cells (shown in the center lower panel) was blocked by pre-incubation with an anti-mouse CD40 monoclonal antibody (clone HM40-3) (the right upper panel) but not by control hamster IgG (right lower panel). (D) Binding of GITRL-displaying BV to GITR-positive cells. GITR-expressing mouse T cell hybridoma 18.3.5 cells were incubated with GITRL-displaying BV (right) or wild-type BV (left). Binding of BV was detected with a biotinylated anti-gp64 monoclonal antibody B8147A and PE-streptavidin. The thin line indicates cells incubated without BV.
Mentions: Next, we attempted to utilize the ligand protein-displaying BV as a tool to detect receptor expression on the cell surface. To this end, we detected the binding of BV to cells by flowcytometer using an antibody specific to baculoviral envelope protein gp64 and a fluorochrome-conjugated secondary antibody (illustrated in Fig. 4A). As shown in Fig. 4B, CD58-displaying BV bound to CD2-positive, but not CD2-negative Jurkat cells, a human T cell leukemia cell line. We also observed the binding of mouse CD40L-displaying BV to mouse splenic B cells, which express CD40. This binding was blocked by an anti-mouse CD40 monoclonal antibody (Fig. 4C). Furthermore, GITRL-displaying BV bound to GITR-expressing T cell hybridoma 18.3.5 cells (Fig. 4D).

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