Cysteine protease domain (CPD) derives from multifunctional autoprocessing repeats-in-toxin (MARTX) of Vibrio cholerae. The domain is responsible for the virulence of the toxin, as it cleaves on multiple points, thus activating other domains, such as the actin crosslinking and Rho-inactivating domains. For its activation, CPD needs to be induced by inositol hexakisphosphate (IP6), present in the eukaryotic cell cytosol. As CPD specifically cleaves at the site in its own amino acid sequence, it is possible to use it as a self-cleaving protein tag. Its advantages are: eliminating the need to use commercial proteases, the ability to induce the cleavage on histidine trap and the low cost of using IP6.
As part of our research, we used bacteria Escherichia coli strain BL21[DE3] to express an EGFP protein with C-end CPD fusion with a hexahistidine tag. Through test expression, we determined that protein folding, when induced with IPTG, is best at 18 ⁰C. We applied the same conditions for large-scale expression. We isolated the recombinant protein by hexahistidine tag, activated it with different concentrations of IP6 and monitored if self-cleavage was successful. We observed the cleavage in free solution and while bonded to activated nickel-agarose beads. We confirmed that premature activation and unspecific cleavage is not present. The best results were achieved with two-hour incubation on activated nickel-agarose beads with 500 μM IP6 at 18 ⁰C. To further characterize the protein, after purifying it with size exclusion chromatography, we performed several crystallization screens and crystallized inactive CPD in the form of EGFP-CPD-His6 protein.
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