Simplified, Enhanced Protein Purification Using an
Inducible, Autoprocessing Enzyme Tag
Aimee Shen1, Patrick J. Lupardus2, Montse Morell1, Elizabeth L. Ponder1, A. Masoud Sadaghiani3, K.
Christopher Garcia2,4, Matthew Bogyo1*
1Department of Pathology, Stanford School of Medicine, Stanford, California, United States of America, 2Department of Molecular and Cellular Physiology, Stanford
School of Medicine, Stanford, California, United States of America, 3Department of Systems and Chemical Biology, Stanford School of Medicine, Stanford, California,
United States of America, 4Howard 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, self-
cleaving 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 InsP6to 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.
Citation: Shen A, Lupardus PJ, Morell M, Ponder EL, Sadaghiani AM, et al. (2009) Simplified, Enhanced Protein Purification Using an Inducible, Autoprocessing
Enzyme Tag. PLoS ONE 4(12): e8119. doi:10.1371/journal.pone.0008119
Editor: Wenqing Xu, University of Washington, United States of America
Received October 8, 2009; Accepted November 5, 2009; Published December 2, 2009
Copyright: ? 2009 Shen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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 Dean’s 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.
* E-mail: email@example.com
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 . 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 .
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 .
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 endopro-
teases are costly, often exhibit poor solubility, and require the
inclusion of additional chromatography steps to remove the
To circumvent these disadvantages, we have developed an on-
bead cleavage purification system in which a site-specific affinity-
tagged 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) . 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 . Second, the
CPD is inducible, as it is specifically activated by the eukaryotic-
specific small molecule inositol hexakisphosphate (InsP6) [5,6].
Since InsP6is absent from bacterial cells [7,8], full-length CPD-
His6fusion proteins can be purified from bacterial lysates in a
protease-inactive form using imidzaole affinity chromatography
(IMAC). Addition of InsP6to an immobilized, C-terminally His6-
tagged 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
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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 one-
step purification systems [9,10] with many of the advantages of
affinity tags  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
individual research labs and high-throughput structural and
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-CPDSalIvector carries the P2-P1 residues of the native CPD
(Ala-Leu, respectively) and the P4-P3 residues encoded by the SalI
site (Val-Asp, respectively) (Figures 1 and 2). The P1 residue refers
to the amino acid N-terminal to the scissile bond, while the residue
N-terminally adjacent to the P1 residue is termed P2, and so on.
When InsP6is added to induce CPD-mediated autocleavage of the
fusion protein, the untagged target protein is released from the
resin and carries four additional C-terminal residues (Val-Asp-Ala-
Leu); the His6-tagged CPD remains bound to the resin (Figure 1).
The Val-Asp-Ala-Leu C-terminal addition can be reduced to two
amino acids (Glu-Leu) by cloning into the SacI site, or to a single
amino acid (Leu) by cloning into the BamHI site and adding a Leu
codon to the 39 cloning primer (Figure 2).
To demonstrate the feasibility of this system, we first expressed
and purified green fluorescent protein (GFP) as a fusion to CPD-
His6using IMAC. As anticipated, addition of increasing amounts
of InsP6stimulated the release of GFP from the Ni2+-NTA resin in
a dose-dependent manner (Figures 3A and B), while the His6-
tagged CPD remained bound to the Ni2+-NTA agarose beads
(bead eluate, Figure 3A).
The CPD Cleaves Exclusively at the Fusion Protein
We have previously shown that V. cholerae CPD is positioned to
undergo autocleavage at a proximal N-terminal leucine and that it
exhibits significantly reduced transcleavage efficiency [4,5], which
should limit its ability to cleave target proteins at heterologous sites.
Indeed, mutation of the P1 Leu to an Ile residue is sufficient to
prevent CPD-mediated transcleavage, a finding that is explained by
the observation that the P1 Leu residue fits snugly into the S1
substrate binding pocket in the crystal structure of the P1 Leu aza-
epoxide inhibitor modified V. cholerae CPD . Nevertheless, since
other site-specific proteases used to remove fusion tags have been
observed to cleave target proteins at secondary sites , we
examined whether the CPDwould spuriously cleavetarget proteins.
Specifically, we tested whether the CPD would cleave an
intrinsically disordered protein after Leu residues within the target
protein. We used the intracellular domain (ICD) of the cytokine
receptor gp130 as a test substrate, since it is unstructured in solution
by NMR  and contains multiple Leu residues that might serve
as cleavage substrates . The ICD-CPD-His6fusion protein was
expressed and purified from E. coli lysates using IMAC, and CPD-
mediated cleavage of the immobilized fusion protein was activated
by InsP6addition. As shown in Figure 4, autoprocessing occurred
exclusively at the ICD-CPD interdomain junction, with a single
protein equivalenttothe sizeofHis6-tagged ICDbeingreleasedinto
the supernatant fraction. These results strongly suggest that the
CPD will not promiscuously cleave target proteins.
Fusion of Target Proteins to the CPD Can Increase Their
Expression and Purity
We noticed that the expression of the ICD-CPD-His6fusion
protein was at least three-fold higher than the ICD-His6protein in
Figure 1. CPD fusion protein purification system. (A) Schematic
of target protein purification using the CPD, described in detail in the
text. (B) Schematic of CPD fusion protein. The P4 and P3 residues Val
and Asp, respectively, encoded by the SalI site, and the remaining P2-
P49 residues contained within the CPD are shown. Prime positions refer
to residues C-terminal to the autocleavage site, which is demarcated as
a black vertical line. The composition of residues appended to the C-
terminus of target proteins following autoprocessing can vary between
one and four residues as described in Figure 2. At present, the CPD
system functions as a C-terminal fusion to target proteins and thus
complements existing methods in which the affinity tag can only be
applied as an N-terminal fusion .
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E. coli lysates (Figure 4, compare + lanes). This result suggested
that the CPD might generally enhance target protein expression
and/or solubility levels. To test this hypothesis, we compared the
expression and solubility of CPD fusions to several other target
proteins carrying either a His6-tag and/or GST-fusion tag
(Figures 5-8 and Table 1). In all cases, the presence of the CPD-
His6fusion tag increased the expression and solubility of target
proteins. For example, fusion of the CPD-His6tag to biotin ligase
(BirA) from E. coli (BirA-CPD-His6) raised BirA expression levels
by three-fold over the GST-BirA construct  (Figure 5 and
The CPD purification system also enhanced the expression and
purity of a previously uncharacterized SUMO/Sentrin-specific
peptidase 1 (SENP1) from the parasitic pathogen Plasmodium
falciparum, the causative agent of malaria (Figure 6) . Although
PfSENP1 carrying an N-terminal His6-tagged can be readily
expressed and purified from E. coli, a number of contaminating
bands are present, and the N-terminal His6-tag must be removed by
the addition of thrombin followed by multiple chromatography
steps (Table 2). In contrast, when PfSENP1 is expressed as a fusion
to CPD-His6 and released as untagged PfSENP1 upon InsP6
addition, only one minor contaminant co-purifies with PfSENP1
(Figure 6B). This variant is easily removed using gel filtration
chromatography (Figure 6C), and the untagged PfSENP1 is of
sufficient purity that we have used it to obtain diffraction-quality
crystals (E. Ponder, unpublished results). Notably, although the
heterologous expression of P. falciparum proteins in E. coli is typically
challenging, we haveobserved that thissystem canenhance the
expression and purification of other parasite proteins from P.
falciparum and a related apicomplexan parasite Toxoplasma gondii.
Figure 2. Schematic of pET-CPD expression vectors. Bent arrow, T7 promoter, Oval (RBS), ribosome binding site, green rectangle, target
protein, grey rectangle, CPD, V. cholerae MARTX (aa. 3440–3650), darker grey rectangle, DP1-CPD, V. cholerae MARTX (aa. 3442–3650), darkest grey
rectangle, DP29-CPD, V. cholerae MARTX (aa. 3444–3650), black rectangle, His6-tag, white rectangle, HA-tag. The dotted vertical line and arrow
indicate the CPD cleavage site. Residues added onto the C-terminus of the target protein following CPD-mediated cleavage, and the relevant
restriction sites are shown (residues encoded by the restriction sites that are appended to the C-terminus of target proteins are underlined). The
composition of the residues added to the C-terminus of the target protein can be varied depending on the cloning site and pET-CPD vector used. It
should be noted that the P1 Leu shown for pET22b-CPDBamHI-Leumust be encoded in the 39 cloning primer of the target gene (i.e. add a Leu codon to
the end of the target insert). Both pET22b and pET28a vector backbones were used to construct the CPD expression vectors.
One-Step Affinity Purification
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Figure 3. Purification of GFP using the CPD-His6tag. (A) SDS-PAGE analysis of GFP purification using Coomassie stain. GFP-CPD-His6bound to
Ni2+-NTA resin was incubated with increasing amounts of InsP6for 2 hrs at 4uC. GFP released into the supernatant was collected (InsP6supernatant);
Ni2+-bound proteins were then eluted from the resin by the addition of 200 mM imidazole (Imidazole elution). Collected fractions were analyzed by
SDS-PAGE. (B) Visual analysis of GFP released into the supernatant fraction upon InsP6addition to immobilized GFP-CPD-His6fusion protein.
Figure 4. The CPD does not cleave within an intrinsically unstructured protein. gp130 intracellular domain (ICD)-CPD-His6or gp130(ICD)-
His6bound to Ni2+-NTA resin was incubated with 100 mM InsP6for 2 hr at room temperature; the resin was washed four times, followed by elution of
Ni2+-bound proteins by 200 mM imidazole. Purification fractions were analyzed by SDS-PAGE followed by Coomassie staining. CL, cleared lysate, FT,
flowthrough, IP6, elution from InsP6incubation.
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Fusion of Target Proteins to the CPD Can Improve Their
Stability and Solubility
In addition to augmenting the expression of target proteins,
CPD-His6 fusions protected target proteins from proteolytic
degradation. This effect was observed when the CRAC-activation
domain (CAD) of the ER calcium sensor STIM1 was fused to the
CPD (Figure 7). CAD is a small 107 aa polypeptide that activates
Ca2+release-activated Ca2+(CRAC) channels by binding to the
CRAC channel protein Orai1 . Until now, large-scale
expression and purification of this important regulatory domain
has proven difficult due to its apparent instability even when fused
to GST (Figure 7). However, using the CPD system, we were able
to obtain significant quantities of a CAD-containing polypeptide
(CAD128), which has subsequently been used in high-throughput
screens for Orai1-CAD binding partners (A.M. Sadaghiani).
Finally, the CPD purification system also increased the solubility
of difficult-to-express proteins. Fusion of the mouse macrophage
metalloelastase (MMP12) to CPD-His6facilitated its purification
from the soluble fraction of E. coli lysates, whereas His6-tagged
MMP12 remained largely insoluble (Figure 8A). The currently used
method for purification of His6-tagged MMP12 is a laborious
procedure that requires solubilization of MMP12 inclusion bodies,
refolding over multipledays,followed byanionandcationexchange
chromatography . The CPD purification system dramatically
purify and analyze MMP12 mutant proteins.
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
[2,18], 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 (Figures 3-8),
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 InsP6provides 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 [4,5].
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 (Figures 3–8), including an intrinsically
unstructured protein (Figure 4).
While purification systems based on fusing a protease to target
proteins have previously been developed [9,10], our demonstra-
tion that the CPD can enhance the expression, solubility, and
stability of target proteins (Figures 4–8) suggests that the CPD
system likely represents an improvement over existing methods like
the intein-chitin-binding-domain (CBD) [9,10] and sortase-His6
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-His6fusion 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
Figure 5. The CPD improves biotin ligase expression relative to a GST tag. SDS-PAGE analysis of purifications of biotin ligase (BirA, 35 kDa)
fused to either CPD-His6or GST-His6using Coomassie stain. His6-tagged proteins bound to the Ni2+-NTA resin were incubated with 50 mM InsP6for
1 hr at room temperature, and the resin was washed three times, followed by elution of Ni2+-bound proteins by 200 mM imidazole. CL, cleared lysate,
+, IPTG induced culture, FT, flowthrough, IP6, elution from InsP6incubation.
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cleavage is complete within 1–2 hrs at temperatures between 4uC
and 37uC, 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
Figure 6. Comparison of His6-tag removal from Plasmodium falciparum SENP1 using thrombin relative to the CPD. (A) Coomassie stain
of SDS-PAGE analysis of P. falcipaurm SENP1 (PfSENP1, 25 kDa) purified using either the CPD-His6or His6-affinity tags. PfSENP1-CPD-His6or His6-
PfSENP1 bound to the Ni2+-NTA resin was incubated with 100 mM InsP6for 2 hr at room temperature; the resin was washed three times, and wash
fractions were collected. Ni2+-bound proteins were eluted by adding 200 mM imidazole. +, IPTG induced culture, CL, cleared lysate, E, imidazole
elution prior to InsP6addition, IP6, elution following InsP6incubation. (B) UV trace PfSENP1 further purified by gel filtration chromatography following
His6-tag removal. Inset, Coomassie stain of gel filtration fractions of PfSENP1 purifications. Thrombin refers to PfSENP1 purified following thrombin-
mediated removal of the N-terminal His6-tag, while InsP6refers to InsP6-induced, CPD-mediated removal of the C-terminal CPD-His6-tag. The residues
added to the resulting PfSENP1 protein are shown: GSHM is added to the N-terminus of PfSENP1 following thrombin cleavage, while VDAL is added
to the C-terminus of PfSENP1 following InsP6-activated CPD cleavage). (C) Coomassie stain of SDS-PAGE analyses of fractions taken during His6-
PfSENP1 purification prior to thrombin incubation (–), following 12 hr thrombin incubation (+), and following subtractive IMAC to remove uncleaved
His6-PfSENP1 (Ni2+-NTA). The yield of PfSENP1 diminished with each experimental manipulation.
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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 (Figure 2).
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
(Figures 5 to 8), 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.
Materials and Methods
Bacterial Growth Condition
Overnight bacterial strains were grown at 37uC in Luria-
Bertrani (LB) broth. Antibiotics were used at 100 mg/mL
carbenicillin for pET22b vectors expressed in E. coli.
Primers used are listed in Table S1; strains constructed are listed
in Table S2 in the Supporting Information. For construction of
pET-CPDSalIvectors, DNA encoding Vibrio cholerae MARTX toxin
amino acids 3440-3650 from Vibrio cholerae N16961 was PCR
amplified from genomic DNA using primers #1 and #2. The
resulting PCR fragment was cloned into the SalI and XhoI sites of
the pET22b and pET28a expression vectors, respectively (Nova-
gen). For construction of the pET-CPDSacIvector, DNA encoding
Vibrio cholerae MARTX toxin amino acids 3442-3650 from Vibrio
cholerae N16961 was PCR amplified from genomic DNA using
primers #3 and #2, and the resulting PCR fragment was cloned
into the SacI and XhoI sites of pET22b. To construct the pET-HA-
CPDSalIvectors, DNA encoding the HA epitope tag was added to
the 59 end of primer #4, and PCR amplification using primers #4
and #2 was used to fuse the HA tag directly to amino acid 3440 of
V. cholerae MARTX CPD. The resulting PCR fragment was cloned
into the SalI and XhoI sites of the pET22b and pET28a expression
vectors, respectively. For construction of the pET-CPDBamHI-Leu
vector, DNA encoding Vibrio cholerae MARTX toxin amino acids
3444-3650 from Vibrio cholerae N16961 was PCR amplified from
genomic DNA using primers #5 and #2, and the resulting PCR
fragment was cloned into the BamHI and XhoI sites of pET22b.
For construction of the pET-CPDBamHIvector, DNA encoding
Vibrio cholerae MARTX toxin amino acids 3440-3650 from Vibrio
cholerae N16961 was PCR amplified from genomic DNA using
primers #6 and #2, and the resulting PCR fragment was cloned
into the BamHI and XhoI sites of pET22b.
The pET22b-GFP-CPD construct was cloned by PCR
amplifying GFP from pEGFPN3 (Clontech) using primers #7
and #8. To construct the pET22b-gp130(ICD)-CPD vector,
amino acids 642-918 of gp130 corresponding to the intracellular
domain were PCR amplified using primers #9 and #10 and
pET21a-gp130(ICD) as a template. The pET22b-BirA-CPD
vector was constructed by PCR amplifying the birA gene from a
pGEX4T1-BirA template using primers #9 and #10. The
pET22b-STIM1(CAD)-CPD plasmid was constructed by PCR
amplifying DNA encoding amino acids 342–469 of STIM1 using
pGEX6-CAD128 as a template and primers #13 and #14. The
pET22b-mMMP12-CPD construct was constructed by PCR
amplifying the catalytic domain of mouse MMP12 (amino acids
29–267) using pET41a-mMMP12 as a template using primers
#15 and #16. In all cases, the resulting PCR products were
cloned into the NdeI and SalI sites of pET22b-CPDSalI.
Protein Expression and Purification
For purification of His6-tagged CPD fusion proteins, overnight
cultures of the appropriate strain were diluted 1:500 into 1 L 2YT
Figure 7. The CPD can improve the stability of target proteins. The Crac activation domain (CAD128) of STIM1 (amino acids 342–469, 14 kDa)
was expressed in E. coli fused to either CPD-His6or GST-His6. Asterisks indicate GST-STIM1(CAD)-His6derived degradation products. His6-tagged
proteins bound to the Ni2+-NTA resin were incubated with 50 mM InsP6for 1 hr at room temperature, and the resin was washed three times, followed
by elution of Ni2+-bound proteins by 200 mM imidazole. CL, cleared lysate, +, IPTG induced culture, FT, flowthrough, IP6, elution from InsP6
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media and grown shaking at 37uC. When an OD600of 0.6 was
reached, IPTG was added to 250 mM, and cultures were grown
for 3-4 hrs at 30uC. Cultures were pelleted, resuspended in 25 mL
lysis buffer [500 mM NaCl, 50 mM Tris-HCl, pH 7.5, 15 mM
imidazole, 10% glycerol] and flash frozen in liquid nitrogen.
Lysates were thawed, then lysed by sonication and cleared by
centrifugation at 15,0006g for 30 minutes. His6-tagged CPD
fusion proteins were affinity purified by incubating the lysates in
batch with 0.5–1.0 mL Ni-NTA Agarose beads (Qiagen) with
shaking for 2–4 hrs at 4uC. The binding reaction was pelleted at
1,5006g, the supernatant was set aside, and the pelleted Ni2+-
NTA agarose beads were washed three times with lysis buffer. In
some cases, 10% of the Ni2+-NTA beads containing immobilized
CPD-His6fusion proteins were removed, pelleted and then His6-
tagged fusion protein eluted using high imidazole buffer [500 mM
NaCl, 50 mM Tris-HCl, pH 7.5, 175 mM imidazole, 10%
To liberate untagged target proteins into the supernatant
fractions, 300–500 mL lysis buffer was added to the Ni2+-NTA
beads containing CPD-His6 fusion proteins and the indicated
amount of inositol hexakisphosphate (InsP6, Calbiochem) was
added. In general, on-bead cleavage was allowed to proceed by
Figure 8. The CPD improves the solubility of mouse macrophage metalloelastin (MMP12) relative to a His6-affinity tag. (A) SDS-PAGE
analysis of His6-tagged MMP12 using Coomassie stain. MMP12 was fused to His6-tagged CPD, and the expression of the fusion protein relative to
His6-tagged MMP12 was compared. Asterisk indicates a putative chaperone protein that co-purifies with MMP12VDAL. The diagonal arrows indicate a
His6-tagged truncated MMP12 product that is also observed during MMP12 purification from inclusion bodies . His6-tagged proteins bound to the
Ni2+-NTA resin were incubated with 50 mM InsP6for 1 hr at room temperature, and the resin was washed three times, followed by elution of Ni2+-
bound proteins by 200 mM imidazole. CL, cleared lysate, +, IPTG induced culture, FT, flowthrough, IP6, elution from InsP6incubation, E, imidazole
elution prior to InsP6addition. (B) Additional purification of MMP12VDALby gel filtration chromatography. Inset, Coomassie stain of SDS-PAGE analysis
of gel filtration fractions of MMP12VDAL. (C) MMP12 activity assay. The activity of MMP12 purified under denaturing conditions and refolded (MMP12
(Refolded)) and MMP12VDALpurified using the CPD system (CPD method) against a standard fluorogenic substrate were compared. Comparable rates
of fluorogenic substrate cleavage are observed for MMP12 purified by the CPD method relative to the refolding method.
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nutating the beads in the presence of 50–100 mM InsP6for 1–2 hr
at either room temperature or 4uC. The beads were pelleted at
1,5006g, and the supernatant fraction was removed. The beads
were then washed 3–4 times with 300–500 mL lysis buffer, and
supernatant fractions retained. His6-tagged proteins remaining on
the beads (i.e. cleaved CPD-His6) were eluted using high imidazole
buffer [500 mM NaCl, 50 mM Tris-HCl, pH 7.5, 175 mM
imidazole, 10% glycerol] in 300–500 mL volumes. The elution
was repeated 3–4 times, and eluate fractions were collected.
Purification of His6-tagged proteins lacking the CPD was
performed in parallel.
This general procedure was followed with the following
exceptions: for purification of MMP12 constructs, the cultures
were grown at 16uC for 8 hr after IPTG induction, and 1 mM
tris(2-carboxyethyl)phosphine (TCEP) was added to the lysis buffer
to prevent misfolding of the protein. PfSENP1 and BirA protein
purifications were performed exclusively at room temperature,
since at 4uC, protein aggregation was observed. For removal of the
His6-tag from His6-PfSENP1, thrombin beads (Calbiochem) that
had been washed in PBS were added to the eluted His6-PfSENP1,
which had been buffer exchanged into PBS according to the
manufacturer’s instructions. Thrombin cleavage was allowed to
proceed with shaking overnight for 12 hr at room temperature.
Aliquots were taken before and after thrombin addition to monitor
cleavage efficiency. Thrombin cleaved, untagged PfSENP1 was
enriched by performing a subtractive Ni2+-NTA pull-down.
Untagged PfSENP1 from both methods was then buffer-
exchanged into gel filtration buffer (50 mM NaCl, 20 mM Tris
pH 8.0). Protein purifications were analyzed by SDS-PAGE and
Coomassie staining using GelCode Blue (Pierce). Purified protein
concentrations of purified were determined by Bradford assay
Purification of MMP12-His6
MMP12-His6was purified as previously described  with the
following modifications. The cell pellet was resuspended in
100 mM NaCl, 100 mM Tris pH 8.0, 5.0 mM EDTA, 0.5 mM
DTT, 100 mg/mL lysozyme and stirred for 2 hr. The cells were
sonicated then centrifuged at 10,000 rpm for 10 min. The
resulting inclusion bodies were washed two times and then
resuspended in 50 mL 6M guanidine hydrochloride, 10 mM Tris
pH 8.0 by stirring at 4uC overnight. The mixture was centrifuged
at 15,000 rpm for 30 min, and 2 mL aliquots of supernatant were
prepared. The supernatant was diluted 1:100 into denaturing
buffer [6M Urea, 50 mM Tris pH 8.0, 10 mM CaCl2, 30 mM
NaCl, 5 mM DTT] to a final concentration of 0.1–0.2 mg/mL.
Table 2. Comparison of CPD-mediated and thrombin-mediated purification of PfSENP1.
Step 1Prepare soluble lysate (1 hr)Prepare soluble lysate (1 hr)
Step 2 IMAC purification (2 hr)IMAC purification (1 hr)
Step 3On-bead cleavage; collect supernatant (2 hr)Imidazole elution
Step 4 Concentrate protein (0.5 hr)Buffer exchange and concentrate protein (0.5 hr)
Step 5 Gel filtration chromatography (1 hr)Thrombin cleavage overnight (.12 hr)
Step 6 Concentrate protein (0.5 hr) Remove His6-tag and uncleaved fusion with IMAC (1 hr)
Step 7Concentrate protein and buffer exchange (0.5 hr)
Step 8 Gel filtration chromatography (1hr)
Concentrate protein (0.5 hr)
Total time5 hr
Table 1. Target proteins expressed and purified by CPD purification method.
Target protein Yielda(mg/L culture) Yield (nmol/L culture)Activity
GFPVDAL(CPD method) 3.3105 Fluorescence at 511 nm
gp130(ICD)VDAL(CPD method) 5.9 188n/a
3.7 115 n/a
BirAVDAL(CPD method) 10.9202 Biotinylates LHHILDAQKMVWNHR BirA biotinylation site
12.0 90Biotinylates LHHILDAQKMVWNHR BirA biotinylation site
PfSENP1VDAL(CPD method)2.0 67Cleaves PfSUMO
GSHMPfSENP1 1.446Cleaves PfSUMO
2.1 148Binds Orai1
MMP12VDAL(CPD method) 1.447 Cleaves fluorogenic peptide substrate Mca-PLGLDL(Dpa)AR
MMP12 (refolded)23767 Cleaves fluorogenic peptide substrate Mca-PLGLDL(Dpa)AR
aProtein yield per litre of culture.
bYield difficult to assess since GST-fusion protein degrades and falls out of solution over time.
One-Step Affinity Purification
PLoS ONE | www.plosone.org9 December 2009 | Volume 4 | Issue 12 | e8119
The protein was then dialyzed for 24 hr in 2 L refolding buffer 1
[3 M Urea, 50 mM Tris pH 8.0, 10 mM CaCl2, 30 mM NaCl,
5 mM DTT]. The partially refolded protein was then dialyzed in
4 L of refolding buffer 2 [1 M Urea, 50 mM HEPES pH 7.4,
10 mM CaCl2, 5 mM DTT). The buffer exchanged protein was
then purified using tandem 5 mL MonoQ and SP Sepharose (GE
Healthcare) at 4uC. After loading the protein on the column, the
column was washed with 50 mL of refolding buffer 2 without
DTT at 1 M, 0.5, and 0 M urea, respectively. The protein was
eluted from the SP column in 500 mM NaCl, 50 mM HEPES
pH 7.4, 10 mM CaCl2.
Gel Filtration Chromatography
Untagged PfSENP1 obtained from either thrombin or InsP6-
mediated cleavage was concentrated using a 10 kDa Centricon
concentrator (Millipore) and buffer exchanged into 50 mM NaCl,
20 mM Tris pH 8.0 and purified on a Superdex 200 10/30
column (GE Healthcare) equilibrated in the same buffer. For
MMP12, the gel filtration buffer contained 150 mM NaCl,
50 mM Tris pH 7.4, 10 mM TCEP. Gel filtrations were
performed at 4uC.
Fluorescence of purified GFP at 511 nm was verified using a
Molecular Devices fmax plate reader in black 96-well plates and
488 nm excitation. The activity of MMP12 was determined using
the fluorogenic substrate Mca-PLGLDL(Dpa)AR (Mca, (7-meth-
oxycoumarin-4-yl)acetyl, Dpa, N-3-(2,4-dinitrophenyl)-L-2,3-dia-
minopropionyl, Anaspec). Reactions were performed in the assay
buffer (50 mM Tris pH 7.4, 150 mM NaCl, 10 mM CaCl2,
0,02% NaN3, 5 mM TCEP) at 37uC. The substrate was used at
10 mM and the protein at 0.2 mM. The substrate hydrolysis was
monitored continuously in a fluorescent plate reader (Molecular
Devices) using an excitation wavelength of 325 nm and an
emission wavelength of 395 nm.
are underlined, and the HA tag is shown in italics.bRE - Restric-
Found at: doi:10.1371/journal.pone.0008119.s001 (0.06 MB
Primers used in Study.aRestriction enzyme sequences
et al. (2008) Mol Cell 31: 737–748. 2. Ponder EL, et al. (2009)
under review Nat Chem Biol. 3. Park CY, et al. (2009) Cell 136:
Found at: doi:10.1371/journal.pone.0008119.s002 (0.07 MB
Strains used in study. 1. Skiniotis G and Lupardus, PJ,
We thank Chris Overall for kindly providing the pET41a-mMMP12
expression construct, and Chan Young Park for providing the GST-CAD-
His6construct used to clone the CAD domain of Stim1.
Conceived and designed the experiments: AS PJL MM ELP AMS KCG
MB. Performed the experiments: AS PJL MM ELP AMS. Analyzed the
data: AS PJL MM ELP AMS. Contributed reagents/materials/analysis
tools: AS PJL MM ELP AMS KCG. Wrote the paper: AS MB.
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