Protocol for Efficient Cell-Free Synthesis of Cowpea Chlorotic Mottle Virus-Like Particles Containing Heterologous...

We report a protocol for efficient cell-free synthesis of cowpea chlorotic mottle virus (CCMV)-like particles containing a broad range of lengths and sequences of RNA. Our protocol starts with a purified stock of wild-type CCMV (protocols for harvesting and purifying the virus are detailed elsewhere) and features three basic steps: disassembly of the CCMV and purification of the capsid protein (CP) from the viral RNA; coassembly of the purified CP and an RNA of choice; and characterization of the assembly products. We highlight several key factors that increase the yield of the assembly reaction: the CP should be uncleaved and sufficiently free of viral RNA; the length of the RNA should be between about 100 and 4000 nucleotides; and the stoichiometry of CP and RNA should be 6-1 by mass. Additionally, we point out that separating the assembly reaction into multiple steps-by successively lowering the ionic strength and then the pH of the assembly buffers-results in the highest yields of well-formed, nuclease-resistant, CCMV-like particles. Finally, we describe methods for characterizing the assembly products using native agarose gel electrophoresis and negative-stain transmission electron microscopy.