Thermus thermophilus CRISPR Cas6 Heterologous Expression and Purification.

Abstract

The CRISPR-Cas system of Thermus thermophilus has emerged as a potent biotechnological tool, particularly its Cas6 endonuclease, which plays a crucial role in CRISPR RNA (crRNA) maturation. This protocol details a robust and reproducible method for the high-level expression and purification of recombinant T. thermophilus Cas6 proteins (Cas6-1 and Cas6-2) in E. coli. We describe a streamlined approach encompassing plasmid construction using seamless assembly, optimized bacterial heterologous expression, and multi-step purification leveraging affinity and size-exclusion chromatography. The protocol outlines the generation of both His-tagged and GST-tagged Cas6 variants, enabling flexibility in downstream applications. Key steps, including primer design, PCR optimization, competent cell transformation, and chromatography strategies, are meticulously detailed with critical parameters and troubleshooting guidance to ensure experimental success and high yields of highly pure and active T. thermophilus Cas6 proteins. This protocol is useful for researchers requiring purified T. thermophilus Cas6 for structural studies, biochemical characterization, and the development of CRISPR-based biotechnological tools. Key features • Robust method for expressing and purifying Thermus thermophilus Cas6 proteins in E. coli. • Seamless assembly cloning and dual affinity tagging system: Offers options for both His-tag and GST-tag purification strategies for increased versatility. • Applicable for diverse heterologous expression and purification of well-folding thermostable proteins in mesophilic host chassis cells [E. coli BL21(DE3)].