Simplified, Enhanced Protein Purification Using an Inducible, Autoprocessing Enzyme Tag

Autoprocessing Enzyme Tag. PLoS ONE 4(12): e8119. doi:10.1371/journal.pone.0008119 Simplified, Enhanced Protein Purification Using an Inducible, Autoprocessing Enzyme Tag Aimee Shen 0 Patrick J. Lupardus 0 Montse Morell 0 Elizabeth L. Ponder 0 A. Masoud Sadaghiani 0 K. Christopher Garcia 0 Matthew Bogyo 0 Wenqing Xu, University of Washington, United States of America 0 1 Department of Pathology, Stanford School of Medicine, Stanford, California, United States of America, 2 Department of Molecular and Cellular Physiology, Stanford School of Medicine, Stanford, California, United States of America, 3 Department of Systems and Chemical Biology, Stanford School of Medicine, Stanford, California, United States of America, 4 Howard Hughes Institute, Stanford School of Medicine , Stanford, California , United States of America We introduce a new method for purifying recombinant proteins expressed in bacteria using a highly specific, inducible, selfcleaving protease tag. This tag is comprised of the Vibrio cholerae MARTX toxin cysteine protease domain (CPD), an autoprocessing enzyme that cleaves exclusively after a leucine residue within the target protein-CPD junction. Importantly, V. cholerae CPD is specifically activated by inositol hexakisphosphate (InsP6), a eukaryotic-specific small molecule that is absent from the bacterial cytosol. As a result, when His6-tagged CPD is fused to the C-terminus of target proteins and expressed in Escherichia coli, the full-length fusion protein can be purified from bacterial lysates using metal ion affinity chromatography. Subsequent addition of InsP6 to the immobilized fusion protein induces CPD-mediated cleavage at the target protein-CPD junction, releasing untagged target protein into the supernatant. This method condenses affinity chromatography and fusion tag cleavage into a single step, obviating the need for exogenous protease addition to remove the fusion tag(s) and increasing the efficiency of tag separation. Furthermore, in addition to being timesaving, versatile, and inexpensive, our results indicate that the CPD purification system can enhance the expression, integrity, and solubility of intractable proteins from diverse organisms. - Funding: This work was supported by a Burroughs Wellcome Foundation grant and NIH grants R01 AI078947 and R01 EB005011 to M.B., the Damon Runyon Cancer Research Fellowship to P.J.L., a Keck Foundation and Howard Hughes Medical Institute grant to K.C.G., a Stanford Deans Fellowship to A.S., and a Beautriu de Pino s of Agaur Fellowship to M.M. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: A.S., P.J.L., K.C.G., and M.B. are listed as inventors on a provisional patent application describing the CPD purification system technology. This patent will not alter the authors adherence to PLoS ONE policies on sharing data and materials. Materials and information associated with the authors publication will be freely available to those as requested for the purpose of academic, non-commercial research. The availability of simple, reliable, and cost-effective methods for recombinant protein purification is critical for the work of high throughput structural and proteomic centers and many individual researchers alike. While the addition of affinity tags such as poly-His and glutathione transferase (GST) to target proteins has greatly simplified purification strategies, it is often difficult to obtain soluble recombinant protein [1]. As a result, intractable affinity-tagged target proteins are often fused to small proteins such as NusA and SUMO to improve their solubility, expression, and stability [2]. Since these tags can alter the biological activity of target proteins and interfere with protein crystallization studies, many biological and biomedical applications require that the tag be removed from the target protein. Most commonly used methods involve the addition of exogenous site-specific proteases to cleave the affinity tag off the target protein at engineered sites [2]. Unfortunately, high levels of endoprotease must often be applied for extended periods of time, and this can result in undesirable cleavages within the target protein. Furthermore, these endoproteases are costly, often exhibit poor solubility, and require the inclusion of additional chromatography steps to remove the exogenous protease. To circumvent these disadvantages, we have developed an onbead cleavage purification system in which a site-specific affinitytagged protease is fused directly to the target protein. This approach condenses affinity purification, cleavage, and tag separation into a single step, simplifying protein purification procedures and increasing purification yields. The key element of this purification method is the Vibrio cholerae MARTX toxin cysteine protease domain (CPD) [3]. The CPD exhibits several properties that facilitate its development into an inducible, autocleaving protease tag. First, the CPD is a highly specific protease that cleaves exclusively after Leu residues [4]. Second, the CPD is inducible, as it is specifically activated by the eukaryoticspecific small molecule inositol hexakisphosphate (InsP6) [5,6]. Since InsP6 is absent from bacterial cells [7,8], full-length CPDHis6 fusion proteins can be purified from bacterial lysates in a protease-inactive form using imidzaole affinity chromatography (IMAC). Addition of InsP6 to an immobilized, C-terminally His6tagged fusion protein induces autoprocessing at the P1 Leu cleavage site (P1 refers to the residue N-terminal to the scissile bond), which is located at the target protein-CPD junction (Figure 1). This processing event releases the untagged target protein into the supernatant, while the C-terminally His6-tagged CPD remains immobilized on the Ni2+-NTA resin. Third, as an autoprocessing enzyme, the CPD exhibits poor transcleavage efficiency [4,5]. This property should limit fusion protein cleavage to the CPD-target protein junction and permit the high fidelity removal of the His6-CPD tag from the target protein. In this report, we demonstrate using a variety of target proteins that this novel purification system combines the simplicity of onestep purification systems [9,10] with many of the advantages of affinity tags [2] in that it can increase the expression, solubility, and integrity of target proteins. Thus, this method facilitates the rapid purification of both soluble and intractable, recombinant, untagged proteins, suggesting that it will have widespread utility in Development of the One-Step CPD Purification System In order to produce CPD fusion proteins, we first constructed CPD expression vectors (pET-CPD expression vectors) using the pET expression vector backbone. DNA encoding the CPD was cloned into the SalI restriction site (Figure 2) such that the fusion protein produced upon IPTG induction of E. coli harboring the pET-CPDS (...truncated)