Abstract
CRISPR-Cas systems employ diverse and often multimeric CRISPR-associated (Cas) protein effector complexes to mediate antiviral defense. The elucidation of the mechanistic details and the protein interaction partners requires production of recombinant Cas proteins. However, these proteins are often produced as inactive inclusion bodies. Here, we present a detailed protocol for the isolation and purification of insoluble Cas proteins. Guidelines for their solubilization via co-reconstitution strategies and procedures to upscale the production of soluble multimeric Cas protein complexes are provided.
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Acknowledgement
The authors thank Patrick Dennis for critical reading of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (DFG, FOR1680) and the Max Planck Society.
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Plagens, A., Randau, L. (2015). In Vitro Co-reconstitution of Cas Protein Complexes. In: Lundgren, M., Charpentier, E., Fineran, P. (eds) CRISPR. Methods in Molecular Biology, vol 1311. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2687-9_2
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DOI: https://doi.org/10.1007/978-1-4939-2687-9_2
Publisher Name: Humana Press, New York, NY
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