(755e) A Cell-Free Approach to Optimized Production and Self-Assembly of Novel Monodisperse Virus-Based Nanoparticles

Virus-like particles (VLPs) are composed of viral proteins that assemble into highly symmetrical nanoparticles similar to viruses but do not contain the infectious genome.  A wide variety of VLPs have been discovered and engineered for diverse applications such as drug/gene delivery, vaccine development, hydrogen storage, MRI imaging, and biosensing.  A particularly attractive VLP for the above applications is the Qbeta VLP which is composed of 180 copies of the coat protein.  The Qbeta VLP is very stable under typically bio-incompatible conditions due to disulfide bonds which covalently cross-link the protein monomers of the VLP together.  Herein, we present the use of a cell-free protein synthesis system to directly control Qbeta VLP protein production for optimal VLP production and self-assembly.  Cell-free protein synthesis is an in vitro expression system wherein cytotoxic proteins can be produced at high yields and the DNA expression template concentrations can be directly controlled to optimize the relative expression rates of multiple proteins.  Using this system we expressed at high yields the cytotoxic A2 protein which is incorporated into the Qbeta virus such that only a single protein is inserted into each virus.  Due to its cytotoxic nature, the crystal structure for the A2 protein has not been solved and the mechanism and interactions of its insertion into the Qbeta virus is not well understood.  However, by coexpressing the Qbeta coat protein and A2 protein a novel VLP which incorporated the A2 protein was found to self-assemble.  We also found that the relative expression rate and concentrations of the A2 protein relative to the coat protein had a strong impact on A2 incorporation efficiency and VLP self-assembly efficiency.  This novel VLP would be useful for applications where a single unique attachment site on a nanoparticle is desired.