We have developed a novel one-step purification system that accelerates untagged recombinant protein purification from bacterial systems. By directly fusing an affinity-tagged, site-specific protease to a target protein, the CPD system ensures rapid and efficient removal of the fusion tag in a cost-effective manner. As a result, the CPD system overcomes many of the disadvantages associated with the exogenous addition of site-specific proteases, like thrombin and TEV protease, to remove fusion tags. These disadvantages can include their expense, generally low activity 
, sensitivity to buffer conditions, and cleavage of target proteins at spurious sites 
. In contrast, the CPD rapidly completes tag removal within two hours of addition (-), since the CPD is present at a 1
1 ratio to the target protein and poised to undergo the autocleavage reaction 
. Furthermore, the responsiveness of the protease specifically to InsP6
provides the user with complete control over the timing and conditions of fusion tag removal, while the autoprocessing nature of the CPD confers a high degree of specificity to fusion tag removal 
. Specifically, the protease is poised to undergo autocleavage upon InsP6
addition and exhibits poor transcleavage efficiency, as evidenced by the lack of CPD-mediated cleavage within any of the target proteins tested (–), including an intrinsically unstructured protein ().
While purification systems based on fusing a protease to target proteins have previously been developed 
, our demonstration that the CPD can enhance the expression, solubility, and stability of target proteins (–) suggests that the CPD system likely represents an improvement over existing methods like the intein-chitin-binding-domain (CBD) 
one-step purification systems 
. Although these self-cleaving systems simplify the purification of well-expressed proteins, the large size of the intein-CBD fusion tag can decrease target protein solubility 
, while sortase-His6
fusion tags do not increase target protein solubility 
. Furthermore, unlike self-cleaving elastin-like polypeptide (ELP) tags 
, CPD fusion proteins do not need to be subjected to temperature cycles, pH shifts, or high salt concentrations, a feature that is critical for the purification of intractable proteins. Based on the properties reported here, the CPD could replace the intein-tag in the self-cleaving-ELP system and potentially improve the solubility of ELP-tagged proteins while retaining their self-cleavability 
Indeed, a considerable strength of this method is that the CPD remains active over a wide range of conditions. CPD-mediated cleavage is complete within 1–2 hrs at temperatures between 4°C and 37°C, requires only micromolar concentrations of the small molecule InsP6 (an abundant and inexpensive reagent), and occurs efficiently both in the presence of standard protease inhibitor cocktails and in the absence of salt. This latter property carries the additional advantage of allowing the user to determine the buffer system in which to elute the target protein, eliminating the need for desalting or buffer exchange steps that can reduce protein yields. In addition, we have created a number of vector backbones that can be used to vary the residues that are appended to the target protein following CPD-mediated cleavage, which can range from a single amino acid residue to an HA epitope tag (). Thus, the CPD system allows for considerable flexibility in optimizing purification procedures, as is often necessary for uncharacterized target proteins.
This versatility, combined with our observation that it can improve the solubility and integrity of difficult-to-express proteins ( to ), suggests that it will have widespread utility in biological research. The simplicity of this system will also make it amenable for large-scale proteomic, structural genomic, and commercial applications by eliminating the cost and complexity associated with exogenous site-specific proteases, potentially permitting its use in robotic systems for constructing protein arrays for screening purposes.