(794h) Synthesis of Highly Stable Self-Assembled Virus-Like Particles

Virus-like particles (VLPs) mimic the structural skeleton of real viruses, but are non-infectious as they lack the viral genome. They are typically stable over wide ranges of pH, temperature, and solvent and have repetitive surfaces that can display a high density of intrinsic or attached molecules. Because of these unique features, VLPs are particularly attractive as vaccines as they offer in vivo stability, trafficking to lymph nodes, and stimulation of B and T cell responses to displayed epitopes.

   In this study, we describe Escherichia coli-based cell-free protein synthesis (CFPS) as a method to rapidly produce Hepatitis B core protein (HBc) VLPs that display unnatural amino acids as unique conjugation sites. However, repeated observations indicated that these nanoparticles were at least partially disassembled by the click conjugation conditions. We therefore took advantage of the repetitive and modular nature of VLPs to introduce 240 new disulfide bonds to totally stabilize the nanoparticles with covalent connections. VLPs have been successfully produced in different expression host systems (bacteria, yeast, insect, plant and mammalian), but CFPS enables rapid production of VLPs at high yields (~0.5 g/L) in a few hours. This CFPS technology has been shown to be scalable from the microliter to the liter scale. Taking advantage of the open CFPS environment and post-expression processing, the virus capsid coat proteins self-assemble into stable VLPs under reducing conditions and can then be cross-linked using a mild oxidizing agent.