(485ar) Cell-Free Protein Synthesis and Artificial Post-Translation Modification of Virus-Like Particles

Virus-like particles (VLPs) have received considerable attention for vaccination, drug delivery, gene therapy and material science applications. VLP-based vaccines have repeatedly demonstrated high responses from low doses due to the strong antigenic nature of their repeated ordered surface. Examples of VLP-based vaccines include the Human Papillomavirus vaccine, Gardasil, approved by the FDA in 2006 and the Hepatitis B vaccine. By genetically and/or chemically modifying VLPs, VLPs have been used as efficient cell-specific drug/gene delivery devices and self-assembling nanostructure templates. Also, by chemically attaching antigens to the surface of VLPs, low-dose vaccines have been developed, such as the nicotine decorated Qb bacteriophage VLP vaccine developed by Cytos Biotechnology. However, these next generation vaccination, material science, and drug delivery applications are limited by chemical linkage reactions which are not site specific. Although lysines and cysteines among others amino acids can be targeted, the multiplicity of lysines, cysteines, or other amino acids on the surface of a VLP monomer make site-specific linkage to the VLP difficult. To overcome this limitation, we have developed an Escherichia coli-based cell-free protein synthesis system for the production of self-assembling VLPs with site-specifically incorporated unnatural amino acids. We have demonstrated that by incorporating unnatural amino acids with unique functionality we can site-specifically perform post-translation modification on VLPs with >95% attachment efficiencies.