In Vitro Co-reconstitution of Cas Protein Complexes

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.