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

Part of the Methods in Molecular Biology book series (MIMB,volume 1776)

Abstract

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.

Key words

  • CCMV
  • Virus-like particle
  • Cell-free synthesis
  • Self-assembly
  • Heterologous RNA packaging

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Acknowledgments

Much of the original work that serves as the basis for this protocol was performed in close collaboration with Mauricio Comas-Garcia and Ruben Cadena-Nava, and was supported by the National Science Foundation grants CHE 0714411 and 1051507.

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Correspondence to Rees F. Garmann .

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Garmann, R.F., Knobler, C.M., Gelbart, W.M. (2018). Protocol for Efficient Cell-Free Synthesis of Cowpea Chlorotic Mottle Virus-Like Particles Containing Heterologous RNAs. In: Wege, C., Lomonossoff, G. (eds) Virus-Derived Nanoparticles for Advanced Technologies. Methods in Molecular Biology, vol 1776. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7808-3_17

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  • DOI: https://doi.org/10.1007/978-1-4939-7808-3_17

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