A Engineering a High-Affinity Scaffold for Non-Chromatographic Protein Purification Via Intein-Mediated Cleavage

ARTICLE
Engineering a High-Affinity Scaffold for
Non-Chromatographic Protein Purification
Via Intein-Mediated Cleavage
Fang Liu, Shen-Long Tsai, Bhawna Madan, Wilfred Chen
Department of Chemical and Biomolecular Engineering, University of Delaware, Newark,
Delaware 1971; telephone: 302-831-6327; fax: 302-831-1048; e-mail: [email protected]
ABSTRACT: While protein purification has long been dominated by standard chromatography, the relatively high cost
and complex scale-up have promoted the development of
alternative non-chromatographic separation methods. Here
we developed a new non-chromatographic affinity method
for the purification of proteins expressed in Escherichia coli.
The approach is to genetically fuse the target proteins with
an affinity tag. Direct purification and recovery can be
achieved using a thermo-responsive elastin-like protein
(ELP) scaffold containing the capturing domain. Naturally
occurring cohesin–dockerin pairs, which are high-affinity
protein complex responsible for the formation of cellulosome in anaerobic bacteria, were used as the model. By
exploiting the highly specific interaction between the dockerin and cohesin domain from Clostridium thermocellum
and the reversible aggregation property of ELP, highly
purified and active dockerin-tagged proteins, such as the
endoglucanase CelA, chloramphenicol acetyl transferase
(CAT), and enhanced green fluorescence protein (EGFP),
were recovered directly from crude cell extracts in a single
thermal precipitation step with yields achieving over 90%.
Incorporation of a self-cleaving intein domain enabled rapid
removal of the affinity tag from the target proteins, which
was subsequently removed by another cycle of thermal
precipitation. This method offers great flexibility as a
wide range of affinity tags and ligands can be used.
Biotechnol. Bioeng. 2012;109: 2829–2835.
ß 2012 Wiley Periodicals, Inc.
KEYWORDS: elastin; ELP; bioseparation
Introduction
Extensive efforts have been invested in the development of
simple and efficient methods for protein purification to
Correspondence to: W. Chen
Contract grant sponsor: NSF
Contract grant number: CBET1116090, CBET0965953
Received 19 December 2011; Revision received 2 April 2012; Accepted 23 April 2012
Accepted manuscript online 7 May 2012;
Article first published online 17 May 2012 in Wiley Online Library
(http://onlinelibrary.wiley.com/doi/10.1002/bit.24545/abstract)
DOI 10.1002/bit.24545
ß 2012 Wiley Periodicals, Inc.
meet the substantially growing global market for protein
drugs. Purification based on affinity tags such as His-tag,
FLAG, maltose binding protein (MBP), and glutathione
S-transferase (GST) (Terpe, 2003) have been developed as a
complementary approach to chromatography, and many
affinity resins are commercially available. Despite their ease
of operation, the widespread usage of affinity tags for largescale purification has been hindered by the need of expensive
resins and the further removal of affinity tags by protease
treatments (Fong et al., 2010). To overcome these problems,
one approach was recently reported based on the use of the
self-cleaving intein domain for tag separation and the
elastin-like polypeptide (ELP) as a reversible aggregating tag
for purification (Banki et al., 2005). However, it has shown
that fusions with a large ELP partner (30–50 kDa) can
significantly reduce cell growth and the overall expression of
ELP fusion proteins (Banki et al., 2005; Kostal et al., 2001;
Shimazu et al., 2003). Although shorter ELP domains of
4 kDa have been used for purification (Lim et al., 2007),
the requirement of very high salt concentrations and the
relatively low recovery (50%) make this strategy unattractive for practical applications.
Here we report a new non-chromatographic affinity
purification scheme in which the ELP-affinity-capturing
proteins and target proteins are separately expressed (Fig. 1).
The target protein is fused to an intein domain in the Cterminus, followed by a small affinity tag, which allows
affinity purification and the subsequent tag removal to yield
the protein of interest. Separately, an ELP fusion protein
containing a capturing domain for the affinity tag is
expressed and used to capture the target fusion protein.
Unlike the commercially available resins that are based on
heterogeneous interaction with the affinity tag, this
approach is a homogeneous method that offers a high
degree of freedom for the ligands, and thereby facilitates
affinity interactions in the solution phase.
This method offers a great deal of flexibility in the
selection of affinity tags and the respective capturing
domains. A naturally occurring cohesin–dockerin (Coh–
Doc) pair from Clostridium thermocellum (CT), which is a
high-affinity protein complex responsible for the position-
Biotechnology and Bioengineering, Vol. 109, No. 11, November, 2012
2829
Figure 1.
A Schematic of the proposed non-chromatographic affinity purification scheme showing the steps involved. Insert: A schematic of the fusion proteins.
specific self-assembly of cellulosome in anaerobic bacteria to
degrade cellulose (Bayer et al., 2004), was used as the model.
This particular protein pair was chosen based on their
reported sub-nanomolar affinity, relative small size, and
thermo-stability. In addition, the Ca2þ-mediated interaction between CohCT and DocCT has been recently exploited
for affinity purification of proteins using CohCT-immobilized cellulose beads (Craig et al., 2006). In this report, we
successfully implemented the new affinity-purification
scheme based on CohCT–DocCT interaction. By exploiting
the reversible temperature precipitation property of ELP
and the pH induced self-cleaving property of intein,
separation of both the DocCT fusion proteins from cell
lysates and the cleaved intein tags from the target proteins
was easily achieved.
Materials and Methods
Strains and Plasmids
All procedures for DNA manipulation were performed
according to standard methods (Sambrook and Russel,
2003). The high fidelity Phusion DNA polymerase (Thermo
Scientific, Odessa, TX) and Taq DNA polymerase (Promega,
Madison, WI) were used for PCR amplification with
a S1000TM Thermal Cycler (Bio-Rad, Hercules, CA).
Escherichia coli strain DH5a (F F80lacZDM15
D(lacZYA-argF) U169 recA1 endA1 hsdR17 (rK, mKþ)
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Biotechnology and Bioengineering, Vol. 109, No. 11, November, 2012
phoA supE44 l thi-1 gyrA96 relA1) was used as the host
for genetic manipulations.
The ELP[KV8F-40] polypeptide was constructed as
previously reported (Lim et al., 2007) by overlapping
oligonuceotides (Integrated DNA Technologies, Coralville,
IA). The gene fragment was inserted into pET24(a) to
generate pET24(a)-ELP[KV8F-40] which contained several
restriction sites for inserting gene of interest at C- or
N-terminus of ELP. A gene fragment coding for the cohesin
domain from C. thermocellum was amplified from pScaf-ctf
(Tsai et al., 2009) with the forward primer 50 -CGCGGATCCAAACCACCTGCAACAACAAAAC-30 and the
reverse primer 50 -CCGCTCGAGTTATATATCTCCAACATTTACTCCACCG-30 . The PCR product was then
digested and ligated into BamHI and XhoI digested
pET24(a)-ELP[KV8F-40] to form pET24(a)-ELP[KV8
F-40]-CohCT.
To construct the expression vector for DocCT-inteinCAT, the gene encoding for the dockerin domain from
C. thermocellum was obtained by PCR from plasmid pETAt
(Tsai et al., 2009) with the forward primer 50 -GGAATTCCATATGGGAAACTTCCCGAATCCTTTG-30 and the
reverse primer 50 -GCGGAGCTCACATAAGGTAGGTGGGGTATGC-30 . The amplified fragment was cloned into
NdeI and SacI linearized plasmid pET21(þ)-EICAT (Banki
et al., 2005), which was a gracious gift from Dr. David W.
Wood from Ohio State University, to generate the plasmid
pET21(þ)-DICAT. A second gene fragment encoding for
the enhanced green fluorescent protein was PCR amplified
from pEGFP-n1 (Clontech Laboratories, Inc., Mountain
View, CA) with primers 50 -GCGCTGTACACAACATGGTGAGCAAGGGCGAGGAG-30 and 50 -CCAAGCTTTTAGTGATGGTGATGGTGATGTTTATAGAGCTCGTCCATGCCGAG-30 and ligated with pET21(þ)-DICAT using
BsrGI and HindIII sites. The resulting plasmid pET21(þ)DIEGFP coded for DocCT-intein-EGFP.
Expression and Purification of ELP[KV8F-40]-CohCT
Protein
The ELP[KV8F-40]-CohCT fusion protein was expressed in
E. coli BLR [F ompT hsdSB (rB m
B ] gal dcm(DE3)
D(srl-recA)306::Tn10(TetR);
Novagen,
Madison,WI].
Overnight cultures were inoculated into 200 mL Terrific
broth (TB) medium supplemented with 100 mg/mL
ampicillin and incubated at 378C until OD600 reached
0.5. Isopropyl-b-D-thiogalactopyranoside (IPTG) was
added to a final concentration of 1 mM and cells were
grown at 258C overnight. Cells were harvested by
centrifugation, resuspended in binding buffer (50 mM
Tris–HCl, pH 7.5, 100 mM NaCl, 10 mM CaCl2), and lysed
by ultrasonic disruption using a sonicator. The cell lysate
was centrifuged to remove insoluble cell debris.
Purification of the ELP fusion protein was achieved by
several cycles of inverse phase transition (Gao et al., 2006).
NaCl was added to the cell lysates to a final concentration of
2 M and the mixture was incubated at 378C for 10 min
before centrifuging for 15 min at 15,000 rpm at the same
temperature. The pellet was resuspended in ice-cold binding
buffer and centrifuged for 15 min at 15,000 rpm at 48C to
remove the insoluble cellular proteins. This precipitation
and resolubilization process was repeated a second time and
the purity of the protein was determined by 10% SDS–PAGE
electrophoresis followed by Coomassie blue staining. ELP
concentrations were determined by spectrophotometric
measurements (UV-1800, Shimadzu, Columbia, MD) at
215 nm (e215 ¼ 65.7 (mg/mL)1 cm1).
Binding, Cleavage and Recovery of Dockerin-Tagged
Proteins
Cell lysates containing dockerin-tagged proteins were
incubated with purified ELP[KV8F-40]-DocCT for 1 h at
room temperature in the binding buffer. After incubation,
NaCl was added to a final concentration of 2 M and the
mixture was heated at 378C for 10 min and centrifuged for
15 min at 15,000 rpm at the same temperature. The pellet
was resuspended in ice-cold cleaving buffer (1 PBS, 40 mM
Bis–Tris, pH 6.2, supplemented with 10 mM CaCl2) and the
sample was incubated at 378C overnight for intein cleavage
(Fong et al., 2010). Once the cleavage reaction was
completed, another thermal cycle was used to precipitate
the ELP–DI complex and the final purified product in the
supernatant was transferred into a fresh tube. The remaining
salt in the purified product was removed by dialysis using
the 6,000–8,000 Da MWCO Dialysis Membrane (Spectrum,
Rancho Dominguez, CA).
Protein Quantification and Activity Assays
The concentrations of protein samples collected during
purification and the final purified samples were measured
using the Bradford method (Bradford, 1976) (Bio-Rad,
Hercules, CA). The activity of CelA was determined as
previously reported (Tsai et al., 2010). Chloramphenicol
acetyl transferase (CAT) activity was measured in reaction
with chloramphenicol in the presence of acetyl coenzyme A
followed by the reaction with 5,50 -dithio-bis (2-nitrobenzoic
acid; DNTB; Sigma, St. Louise, MO). The increase in A412nm
was monitored at 258C using a temperature-controlled
spectrophotometer (Shimadzu). The fluorescence intensity
of EGFP was measured by a Synergy H4 Hybrid Multi-Mode
Microplate Reader (BioTek, Winooski, Vermont). The
excitation wavelength was 488 nm and the emission
wavelength was 520 nm.
Results and Discussion
Expression of Dockerin-Tagged Proteins
Expression and Functionality of the ELP-CohCT Fusion
E. coli strain BL21(DE3) (F ompT gal dcm lon hsdSB(rB
m
B ) lDE3) expressing either the endoglucanase CelA from
C. thermocellum containing its native dockerin (CelADocCT) from plasmid pETAt (Tsai et al., 2009), DocCTintein-CAT or DocCT-intein-EGFP were grown in Luria–
Bertani (LB) medium supplemented with 1.5% glycerol,
20 mM CaCl2 and appropriate antibiotics (50 mg/mL
kanamycin or 100 mg/mL ampicillin) at 378C until OD600
reached 1.2–1.5. Protein expression was induced by 400 mM
IPTG. Cells expressing CelA-DocCT were grown at 258C for
1 h and cells expressing DocCT-intein-CAT and DocCTintein-EGFP were grown at 208C for 3 h. Cells were
harvested and resuspended in the binding buffer followed
by sonication.
It has been demonstrated that the expression level of
thioredoxin (trx) fused to an ELP[KV7F] domain, where the
ionizable lysine residues enhance the salt sensitivity and the
hydrophobic pheylalanine residues lower the transition
temperature, is twofold higher than that fused to an
ELP[V5A2G5] domain (Lim et al., 2007). This increased
expression in combination with the lower level of salt
required for purification make the ELP[KV7F] domain an
attractive alternative to generate ELP fusions. In this work, a
modified ELP domain containing 40 VPGXG repeats, with
the guest residue (X) composed of Lys (K), Val (V), and Phe
(F) at a ratio of 1:8:1, was used. This design slightly reduces
the hydrophobicity, allowing better resolubilization. A 17.6kDa cohesin (CohCT) domain from C. thermocelum was
Liu et al.: Protein Purification Via Intein-Mediated Cleavage
Biotechnology and Bioengineering
2831
Figure 3. SDS–PAGE analysis of samples taken over the course of the
ELP[KV8F-40]-CohCT and CelA-DocCT binding experiment. Lane 1: E. coli BL21
harboring pET24(a) as a control; (Lane 2) CelA-DocCT total cell lysates; (Lane 3)
purified ELP[KV8F-40]-CohCT; (Lane 4) supernatant (soluble fraction) after binding and
precipitation of the ELP complex; (Lane 5) resolubilized ELP-CohCT/CelA-DocCT
complex.
Figure 2. Purification of ELP[KV8F-40]-CohCT fusion by two cycles of inverse
phase transition. Lane 1: Total cell lysate of E. coli BLR expressing the ELP[KV8F-40]CohCT fusion protein; (Lane 2) purified ELP[KV8F-40]-CohCT fusion protein (35.3 kDa).
then fused to the ELP domain to generate ELP-CohCT.
Purification of the ELP fusion protein was achieved by two
cycles of inverse phase transition (Stiborova et al., 2003).
From the SDS–PAGE gel, highly purified ELP-CohCT
fusion proteins were obtained from cell lysates with over
97% recovery (Fig. 2). The yield of the fusion protein was
typically 250 mg from 1 L of culture.
Purification of CelA-DocCT by Affinity Precipitation
The ability of ELP-CohCT to act as a capturing scaffold to
recover dockerin-tagged proteins directly from cell lysates
was first demonstrated by purifying the endoglucanase
CelA fused with a 9.8-kDa dockerin tag (DocCT) from
C. thermocelum (Tsai et al., 2009). Separation of CelADocCT from the cell lysate was achieved by the high-affinity
interaction between DocCT and CohCT and by coprecipitation with the bound ELP tag during the inverse
phase transition (Fig. 3). The recovered complex was then
dissolved in cold buffer and only purified CelA-DocCT
remained with ELP-CohCT (Fig. 3, lane 5), a result
consistent with the highly specific nature of the CohCT/
DocCT pair employed. More importantly, almost 100%
of all ELP-CohCT and CelA-DocCT was detected in the
resolubilized complex, indicating that even the larger ELP
complex can be precipitated and recovered efficiently. The
purification result was further quantified by measuring the
CelA activity in different fractions during purification
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Biotechnology and Bioengineering, Vol. 109, No. 11, November, 2012
(Table I). Around 90% of the CelA enzyme activity was
detected from the resolubilized fraction, consistent with the
SDS–PAGE observation. Combined together, the successful
purification of CelA-DocCT by the capturing scaffold ELPCohCT demonstrated the feasibility of our new affinityprecipitation scheme.
Purification of Target Proteins by Affinity Precipitation
and Intein Cleavage
To remove the DocCT tag from the target protein, a pHsensitive, self-cleaving mini-intein (18 kDa) was incorporated between the target protein and the DocCT tag. Two
model proteins, enhanced green fluorescence protein
(EGFP) and CAT, were used for the initial demonstration.
Since EGFP is a monomeric protein and CAT is a trimeric
protein, their successful purification will validate the
versatility of this method in purifying different types of
protein. To demonstrate that the use of the smaller DocCT
domain could improve the overall protein yield, expression
of DocCT-intein-CAT (DI-CAT) and ELP-intein-CAT (EICAT; a gift from Prof. David Wood) was first compared.
As shown in the SDS–PAGE (Fig. 4), a significantly higher
level of DI-CAT was produced. This was further verified by
detecting a 5.5-fold higher specific CAT activity in the cell
Table I.
Specific CelA activities during purification.
Step
Cell lysate
Soluble fraction
Purified product (CelA-DocCT)
Enzyme activity (U/L)
103.9
23.8
93.8
Figure 4. Comparison of DocCT-intein-CAT (D) and ELP-intein-CAT (E) expression by SDS–PAGE analysis.
lysates (data not shown), consistent with the SDS–PAGE
analysis. The yield of DI-CAT under this condition was
110 mg/L.
To further demonstrate the DI tag removal, an overall
purification scheme similar to that described above was
employed except that the DI tag was further removed by
lowering the pH to 6.2 (Wood et al., 1999) followed by a
subsequent cycle of inverse phase transition. Samples from
each purification step of EGFP were analyzed by SDS–PAGE
(Fig. 4A). After binding with the ELP-CohCT capturing
scaffold, only DocCT-intein-EGFP (DI-EGFP) was codetected in the resolubilized pellet (Fig. 5A, lane 5). After
changing to the cleaving buffer (pH 6.2) to promote intein
cleavage, the 58-kDa DI-EGFP precursor was cleaved
completely to the 31-kDa DocCT-intein (DI) portion and
the 27-kDa target EGFP (Fig. 5A, lane 6) after overnight
incubation. The cleaved DI tag, which was still bound to
ELP-CohCT, was removed by co-precipitation with ELPCohCT, resulting in only EGFP in the supernatant
(Fig. 5A, lane 8). The EGFP fluorescence intensity was
measured for each step of the purification to quantify the
recovery. Over 87% the original EGFP fluorescence in the
cell lysate was recovered after the first binding step. This
lower recovery can be attributed to a small amount of
partially degraded EGFP detected in the supernatant that
cannot interact with ELP-CohCT. In comparison, 100%
recovery of GFP fluorescence was obtained after the cleavage
and precipitation step, agreeable with the complete recovery
of EGFP as shown in the SDS–PAGE (Table II).
A similar purification scheme was used for a larger
trimeric protein, CAT (Fig. 5B and Table III). Again, only
DocCT-intein-CAT (DI-CAT) was separated from the cell
lysate after binding and co-precipitation with ELP-CohCT
Figure 5. SDS–PAGE analysis of samples taken over the course of (A) DocCTintein-EGFP purification and (B) DocCT-intein-CAT purification. Total cell lysates of
E. coli BL21 expressing DocCT-intein fusion proteins without (Lane 1) or with (Lane 2)
IPTG induction. Lane 3: purified ELP[KV8F-40]-CohCT; (Lane 4) supernatant after
binding and precipitation of ELP complex; (Lane 5) resolubilized ELP complex before
the intein cleavage reaction; (Lane 6) ELP complex after the overnight cleavage
reaction; (Lane 7) resolubilized DI bound ELP complex after cleavage; (Lane 8) purified
target protein in the supernatant.
Table II.
EGFP intensities during each purification step.
Specific
Fluorescence
activity of
Purification
unit (U/mL) EGFP (U/mg)
fold
Step
Cell lysate
Soluble fraction
Purified precursor (DI-EGFP)
Purified product (EGFP)
3,018
123
2,626
2,626
1193.8
49.3
21008.0
45124.4
1.0
17.6
37.8
Samples were diluted 20 times before measurements.
(Fig. 5B, lane 5). The intein cleavage was complete after
overnight reaction (Fig. 5B, lane 6) and the cleaved CAT
proteins were separated from the bound DocCT tag
(Fig. 5B, lane 8). However, a small amount of the cleaved
Table III.
Specific CAT activities during purification.
Step
Enzyme
activity
(U/mL)
Specific enzyme
activity
(U/mg)
Cell lysate
Soluble fraction
Purified precursor (DI-CAT)
Purified product (CAT)
4461.88
448.53
4152.74
3326.27
1440.8
362.5
21241.6
47564.8
Liu et al.: Protein Purification Via Intein-Mediated Cleavage
Biotechnology and Bioengineering
Purification
fold
1.0
14.7
33.0
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CAT proteins was found to remain in the precipitated
fraction. This might be due to some aggregation of the
trimeric CAT during thermal precipitation. This was further
confirmed by the CAT activity assay, which indicated over
93% recovery from the cell lysates but only 75% after the
intein cleavage. Although, premature intein cleavage has
been reported to be a problem for the lower protein recovery
(22% for GFP) in the earlier study (Banki et al., 2005), we
observed very limited premature cleavage for both DI-EGFP
and DI-CAT, likely the result of using a smaller fusion tag
and the shorter induction time.
Optimizing the Amount of Capturing Scaffolds for
Purification
The initial purification experiments were performed by
using an excess amount of ELP-CohCT scaffold to ensure
the complete precipitation and recovery of the scaffold–
protein complex. Because of the high binding affinity
between CohCT and DocCT (kD ¼ 109 M1) (Pages et al.,
1999), even a 1:1 stoichiometric ratio should result in close
to 100% binding. To test this feasibility, Na2SO4, which has
been shown to substantially improve the precipitation
efficiency even at lower ELP concentrations (Fong et al.,
2009) was used. From Figure 6 and Table IV, it is clear that
nearly all DI-EGFP was captured and removed from the cell
lysates even at the 1:1 ratio. The use of 1 M Na2SO4 has no
effect on both intein cleavage and the EGFP fluorescent
property. The final EGFP recovery of 84% is similar to that
obtained using an excess amount of ELP-CohCT. Since
even a stoichiometric amount of ELP capturing scaffold is
sufficient to directly remove all dockerin-tagged proteins,
this can substantially lower the cost, making this purification
method very attractive in practice.
Table IV. EGFP intensities during each purification step using an
ELP[KV8F-40]-CohCT/DocCT-intein-EGFP ratio of 1:1.
Step
Cell lysate
Soluble fraction
Purified precursor (DI-EGFP)
Purified product (EGFP)
Specific
Fluorescence
activity of
Purification
unit (U/mL) EGFP (U/mg)
fold
3,197
247
2,694
2,678
846.1
90.5
8026.1
29070.8
1
13.6
34.4
Samples were diluted 20 times before measurements.
Conclusions
In summary, by combining the Ca2þ-dependent, highaffinity interaction between CohCT/DocCT, the thermal
triggered reversible precipitation property of the ELP
domain, and the intein-mediated self-cleavage for tag
removal, we demonstrated the concept of a fast and costeffective purification method for recombinant protein
purification. The capability of modulating the individual
components of the system, such as the use of different
affinity pairs, ELP tags, and/or intein domains, makes
this method very flexible for the purification of a wide
range of proteins from different recombinant hosts.
Since the cohesin–dockerin interaction has been shown to
be reversible by the addition of EDTA to remove the
bound Ca2þ (Craig et al., 2006), we will explore the
possibility of reusing the capturing ELP scaffold using this
method of regeneration.
This work was supported by grants CBET1116090 and CBET0965953
from NSF. We thank Prof. David Wood for the mini-intein
constructs.
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Figure 6. SDS–PAGE analysis of samples taken over the course of EGFP
purification using an ELP[KV8F-40]-CohCT/DocCT-intein-EGFP ratio of 1:1. Total cell
lysates of E. coli BL21 expressing DocCT-intein-EGFP without (Lane 1) or with (Lane 2)
IPTG induction. Lane 3: purified ELP[KV8F-40]-CohCT; (Lane 4) supernatant after
binding and precipitation of ELP complex; (Lane 5) resolubilized ELP complex before
the intein cleavage reaction; (Lane 6) ELP complex after the overnight cleavage
reaction; (Lane 7) resolubilized DI bound ELP complex after cleavage; (Lane 8) purified
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