Multi-Pass Proteins Displayed on Virus-Like Particles Are Bioactive in Binding to Specific Antibodies and Ligands

Multi-pass proteins are integral membrane proteins containing two or more helical transmembrane domains (TMDs) that traverse the cell membrane lipid bilayer. These proteins, including G protein-coupled receptors (GPCRs), transporters, ion-channels and Claudin family proteins, regulate nearly every aspect of human physiology and diseases. They are one of the largest drug target families. GPCRs have 7-TMDs, glucose transporters have 12-TMDs and Claudins have 4-TMDs.  Given the structure complexity, it is very difficult to produce recombinant proteins for research and drug discovery. We have developed an advanced platform to display multi-pass proteins with 4-12 TMDs in the natural membrane lipid bilayer on virus-like particles (VLPs) as antigen for antibody screening/characterization and receptor/ligand interaction analysis. We developed a platform to enhance multi-pass membrane protein expression on the VLP surface and properly embed it in the natural lipid bilayer of VLP nanoparticles. The target is co-expressed with a lentiviral structure protein using HEK293 cells. The purified VLP-nanoparticles are tested for size using dynamic light scattering. The target proteins were analyzed by multiple tests including antibody and ligand binding assays. In this project, we use two 4-TMD Claudin family proteins (Claudin 18.2 and Claudin 6), three 7-TMD GPCR chemokine receptors (CXCR4, CXCR5 and CX3CR1), and one 12-TMD glucose transporter GLUT1 as examples to generate target-expressing VLPs.  The Claudin 18.2, Claudin 6, CXCR4, CXCR5 and GLUT1 VLPs could each bind potently to its specific antibody. The chemokine receptor VLPs could interact with their ligands as measured by ELISA. CXCR4-VLP could bind to its chemokine ligand CXCL12 and HIV-1 envelope glycoprotein gp120. CXCR5-VLP could bind to its chemokine ligand CXCL13. Conclusions: The multi-pass membrane proteins can be properly displayed on VLPs. These Claudin family VLPs, GPCR-VLPs, transporter and ion-channel VLPs can be used to evaluate receptor/ligand interactions and as antigen or immunogen for drug discovery.