Multivalent display of membrane proteins using HIV-1 Gag VLPs

Background Membrane proteins are increasingly becoming attractive targets for biotherapeutics, but their production is challenging due to extraction with detergents often resulting in low yields and purity, and potential changes in conformation. In this study, we evaluated HIV-1 Gag Virus Like Particles (VLPs) for the display of heterologous membrane proteins in their native conformation and lipid environment to serve as reagents for antibody discovery. Methods We have developed a scalable workflow to purify and characterize the VLPs pseudotyped with recombinant membrane proteins. Furthermore, we engineered a dual snorkel–sortase tag for enzymatic, site specific biotin conjugation to enable binding studies. The particle quality was assessed by differential light scattering, analytical HPLC, nanoparticle tracking, and cryo-EM. The antigen presence and display was determined by BLI, Dot -blot, Western blot, and LC-MS/MS. Surface plasmon resonance (SPR) was used to measure binding kinetics with an internal biotherapeutic. Results Our workflow yields pure, stable, and monodisperse VLPs that present recombinant membrane proteins in their native conformation and lipid environment. Site specific biotinylation via the dual snorkel\x{2013}sortase tag did not alter particle size, purity, or antigen expression/ conformation. Biotinylated, membrane protein pseudotyped VLPs were compatible with SPR and enabled kinetic measurement for the tested biotherapeutic. Conclusions VLP display of membrane proteins combined with precise surface biotinylation provides a robust platform for displaying membrane antigens in their native conformation and supports antibody discovery against challenging membrane targets.