(324e) Escherichia Coli-Based Cell-Free Protein Synthesis of Virus-like Particles

Over the past two decades virus-like particles (VLPs) have gained great interest. The major thrust of VLP research has been the development of effective vaccines without the risk of disease from attenuated pathogens. Antigen epitopes fused or chemically linked to VLPs have been shown to generate immune responses from both B and T cells. Also, phase I and II human trials by Merck Research Laboratories with Human papillomavirus derived VLPs showed them to be effective as a vaccine without serious side effects. Other human trials with VLPs from the Norwalk virus, parvovirus and HIV have also been successful. The majority of VLPs have been synthesized in insect cells using the baculovirus expression system or with mammalian expression systems. We propose an E. coli-based cell-free protein synthesis system as a potential technology for more efficient, cost-effective, and higher-throughput production of VLPs. The cell-free system allows greater control over the assembly environment for optimization and study of VLP assembly. The cell-free environment is also more adaptable, providing the opportunity to fold VLPs that normally would not assemble in vivo. Currently, we have demonstrated feasibility by producing MS2 bacteriophage capsid protein at high expression levels in our cell-free system and verifying self-assembly into VLPs at greater than 70% efficiency. Work is continuing in order to demonstrate efficient linkage to the VLP using classical protein linking chemistries. Also, unnatural amino acid chemistries which would provide more specificity and are particularly accessible in our cell-free system are being explored. Although these VLPs produced using cell-free technology could serve as general scaffolds for vaccine production, other applications are rapidly developing. With the rise of virus-based templating of single crystal nanostructures, VLPs could be used with linker chemistries to create cost-effective complex nanostructure devices and matrixes. Also, VLPs could be designed for efficient drug delivery. By masking the VLP from the immune system with polyethylene glycol and also projecting epitopes specific to specific cell receptors, we could potentially increase a drug's effective half-life while efficiently targeting the drug. Using the E. coli-based cell-free technology we seek to engineer the production of complex VLPs for use in the above applications.