Probing Surface Membrane Receptors Using Engineered Bacteriophage Bioconjugates

Abstract

Specific recognition of ligands by surface receptors of eukaryotic cells is a fundamental process in sensing of the exogenous environment, including cell-to-cell communication. These interactions are therefore widely probed in both basic studies and drug development to enhance or interrupt them. Here, we designed a high-throughput publicly available platform for visualization and selection of eukaryotic cells according to the specificity of surface-exposed receptors by consolidation of phage display and flow cytometry techniques. Polypeptide ligands for membrane receptors are incorporated into every copy of p3 protein of M13K07 bacteriophage, which is intracellularly biotinylated to further accept PE-Cy7-labled streptavidin. Transgenic antigen-specific B-cells expressing membrane-tethered lymphoid B-cell receptor in a single-chain format interacted with engineered bacteriophages exposing the polypeptide ligand with an unprecedented selectivity of 97% and a false-positive detection value of 2.0%. Multivalent binding of the phage bioconjugates with the receptor provided significantly better specificity and sensitivity allowing application of engineered bacteriophage bioconjugates at a concentration 3 orders of magnitude lower in comparison with synthetic biotinylated peptide. We suggest that the platform described in this work may be applied either for routine staining or characterization of orphan membrane receptors exposed on the surface of living mammalian cells in their native environment.

Exhaustive Search of the Receptor Ligands by the CyCLOPS (Cytometry Cell-Labeling Operable Phage Screening) Technique

“…The binding of bacteriophages to the eukaryotic cells was detected by incubation with the anti-HA-PE-Cy7 ( Figure 2 A) or anti-flag-PE ( Figure 2 B) fluorescent antibodies, which was followed by flow cytometry. Similar to the results obtained previously [ 30 ], usage of the hyperphage and pHen2-based vector allowed detecting 97.0% of antigen-specific cells with a 2% false-positive signal level, which corresponded to a control experiment with Raji-FL cells stained with bacteriophages that carried an irrelevant peptide P#2. The application of the fADL-1e-produced bacteriophages with either HA- or 3xFLAG-tag at a range of concentrations from 2 × 10 11 to 5 × 10 12 phage particles per mL allowed us to confidently select 98.5 ± 0.4% of Raji-FL cells with a 0.2% false-positive signal level.…”

“…We assembled fADL-1e-flag-linker-p3 (Addgene ID: 139441) and fADL-1e-HA-linker-p3 (Addgene ID: 139440) vectors that contain p3-N-terminally fused cluster encoding the 3xFLAG or HA (hemagglutinin) epitopes for detection, serine-glycine linkers for the conformational flexibility, and Nco I and Nhe I restriction sites for the subsequent cloning of the oligonucleotides coding for the custom peptide ligands ( Figure 1 A). As a model for the ligand–receptor interaction, we used transgenic Raji-FL cells that express membrane-tethered BCR (B-cell receptor) in a single-chain format, derived from a patient with follicular lymphoma cells, and its peptide ligand P#1 (CILDLPKFC) [ 30 , 32 ] cloned into the phage vector (P#1-HA-p3-fADL-1e or P#1-flag-p3-fADL-1e). Our data suggest that the fADL-1e-based phage vector increased bacteriophage yield by an order of magnitude in comparison with the pHen2-based phagemid vector and hyperphage infection ( Figure 1 B).…”

“…Further, we tested if a fADL-1e- or pHen2-based protocol generates functional phage particles that can be used for the CyCLOPS technique. The ligand–receptor interaction was studied following the previously developed methodology [ 30 ]. Raji cells that express membrane-tethered BCR in a single-chain format (Raji-FL) were stained with its BCR peptide ligand (P#1) exposed on the surface of filamentous bacteriophages produced via phage fADL-based or phagemid pHen2-based vectors.…”

“…In our recent studies, we showed the possibility to select the antigen-specific B cells using a combination of the engineered bacteriophage bioconjugates and flow cytometry technique with an unprecedented selectivity reaching 97% [ 30 , 31 ]. This approach allows us to screen thousands of ligands in a single experiment.…”

“…In this study, we compared the efficiency of phage- and phagemid-based systems for the selection of antigen-specific cells. Similar to the previously published pHen2-based phagemid constructs [ 30 , 31 ], we created a publicly available fADL-1e-based phage vector, containing a cassette for the integration of ligand and a detection tag. Here, we show that the developed system is not only highly efficient in selecting the antigen-specific cells using flow cytometry, but is also beneficial for identifying ligands of the peptide-HLA-TCR trimolecular complexes.…”