PROTEIN EXPRESSION AND PURIFICATION
ARTICLE NO.
11, 179–184 (1997)
PT970775
Expression in Escherichia coli of the Thermostable DNA
Polymerase from Pyrococcus furiosus
Chunlin Lu and Harold P. Erickson1
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710
Received March 24, 1997, and in revised form June 6, 1997
Pfu, the DNA polymerase from Pyrococcus furiosus,
has the lowest error rate of any known polymerase in
polymerase chain reaction (PCR) amplification. Previously the protein has been purified from P. furiosus
bacterial cultures, and a recombinant form has been
produced in a baculovirus system. We have produced
a pET plasmid for expression of Pfu in Escherichia
coli (the expression plasmid pETpfu is available from
ATCC, Accession No. 87496) and found that this plasmid is toxic or unstable in the expressing strain
BL21(DE3), even in the absence of induction. However,
the plasmid was stable in BL21(DE3) containing the
pLysS plasmid, which suppresses expression prior to
induction, and a 90-kDa protein was expressed upon
addition of isopropyl b-D-thiogalactopyranoside. The
protein was purified by heating (to denature E. coli
proteins), followed by chromatography on P11 phosphocellulose and mono Q columns. The purified protein had the same activity as the commercially obtained baculovirus-expressed Pfu in both DNA polymerase and PCR reactions. This bacterial expression
system appears to be the method of choice for production of Pfu. q 1997 Academic Press
of nonproofreading Taq DNA polymerase, and 2- to 30fold lower than other proofreading enzymes (4–6). In
addition to PCR2 amplification of DNA with minimal
error, mixtures of Pfu with a nonproofreading enzyme
(e.g., Taq, KTLA) are useful for amplifying long DNA
segments with higher fidelity (4,5).
Pfu was initially characterized from protein isolated
directly from Pyrococcus furiosus (7), but this thermophilic, anerobic bacterium is difficult to grow to obtain
large quantities of protein. A major advance was expression of recombinant Pfu in a baculovirus-mediated
system (8). In the system of Mroczkowski et al. (8), the
Pfu was not produced as a cytoplasmic protein but was
fused with the signal sequence from either human placental alkaline phosphatase or the Apis melifica melittin, so it was secreted into the medium. The baculovirus
system makes possible production of commercial quantities, but is more difficult and expensive than bacterial
expression. In the present study we explored the problems encountered in expressing Pfu in Escherichia coli
and describe an expression system that produces milligram quantities of Pfu with minimal expenditure.
METHODS
Construction of Expression Plasmid pETpfu
The DNA polymerase from Pyrococcus furiosus, referred to here as Pfu, has gained considerable attention
in the field of DNA amplification. It is a thermophilic
DNA polymerase with an integrated 3*–5* exonuclease
activity that corrects errors introduced during the polymerization. This error correction is thought to be associated with its intrinsic exonuclease activity. Several
thermophilic DNA polymerases, including Pfu, Vent,
deep Vent, Pwo, and UlTma have proofreading ability,
but they differ in their error rate (1–4). The error rate
for Pfu is reported to be 7- to 10-fold lower than that
1
To whom correspondence should be addressed. Fax: (919) 6843687. E-mail: H.Erickson@cellbio.duke.edu.
Based on the DNA sequence of Pfu DNA polymerase
gene (Accession No. D12983), two primers were synthesized: the amino-terminal sense primer, pfu-s, 5*AGACATATGATTTTAGATGTGGATTACA-3*, added
a unique NdeI site (underline), which includes an ATG
starting site of translation, followed by the sequence
coding the first seven amino acids; and the antisense
primer, pfu-as, 5*-CTAGGATTTTTTAATGTTAAGC2
Abbreviations used: PCR, polymerase chain reaction; dNTP, dinucleotide triphosphate; IPTG, isopropyl b-D-thiogalactopyranoside;
PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; BSA, bovine serum albumin; SSC, standard sodium citrate; DEAE, diethylaminoethyl.
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All rights of reproduction in any form reserved.
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LU AND ERICKSON
3*, which matches the carboxyl-terminal sequence, including the stop codon. DNA amplification was performed using 2.5 units of Pfu DNA polymerase (Stratagene) in a 100-ml reaction mixture of PCR reaction
buffer (supplied by vendor), 1 mM each primer (pfu-s
and pfu-as), 0.2 mM each dNTP, 0.2 mg P. furiosus genomic DNA (provided by Drs. Frank Jenney and Michael
Adams, University of Georgia, Athens, GA). Prior to
cycling, the reaction mixture was heated to 957C for 2
min, followed by 30 cycles of 947C for 40 s, 557C for 1
min, 727C for 2.5 min, and a final extension at 727C
for 7 min. After final extension, 2.5 units of Taq DNA
polymerase (Stratagene) was added and incubation at
727C continued for another 5 min (this was to add overhanging A’s for subsequent A/T cloning). A predicted
2.4-kb fragment was agarose gel-purified using QIAEX
II gel extraction kit (Qiagen) and subcloned into
pT7Blue T-Vector (Novagen). This vector has an additional NdeI site very close to the A/T cloning site. A
2.4-kb fragment with one NdeI site at each end was
recovered from the pT7Blue plasmid digested by NdeI.
One of the NdeI sites was from the primer pfu-s, the
other was from the pT7 Blue T-vector. The expression
vector pET11 (Novagen) was cut with NdeI, and the
2.4-kb fragment was ligated into it. The plasmids were
initially propagated in E. coli DH5a, and the orientation of the ligated segment was checked by restriction
enzyme digestion. Clones with the correct orientation
were selected and designated pETpfu. To test for
protein expression, pETpfu was transformed into
BL21(DE3) or BL21(DE3) containing plasmids pLysS
or pLysE (Novagen) using a standard protocol (9, and
Novagen literature).
Purification of Pfu DNA Polymerase
Pfu was expressed in strain BL21(DE3) pLysS carrying
plasmid pETpfu. Fifty milliliters of overnight culture
was collected by centrifugation and transferred to 1 liter
(500 ml 1 2) LB medium (Gibco) plus 100 mg/ml ampicillin and 34 mg/ml chloramphenicol (Sigma). This culture
was grown at 377C to an A600 of 0.5, then induced with
0.5 mM isopropyl b-D-thiogalactopyranoside (IPTG), and
allowed to grow for another 3 h. Cells were collected at
6000 rpm, 47C for 10 min (Gsa rotor, Sorvall), and resuspended in 20 ml of cold resuspension buffer (50 mM Tris–
HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA) plus 1 mM
PMSF and 0.2 mg/ml lysozyme (Sigma). The mix was
incubated on ice for 2 h and frozen overnight at 0207C.
The bacteria were thawed at room temperature, and
MgCl2 was added to 10 mM, DNase I to 0.2 mg/ml, and
incubated at room temperature for 30 min. To shear remaining DNA (genomic DNA and plasmids), sonication
was performed three times on ice for 30 s with a 30-s
interval. Cell walls and insoluble debris were removed
by centrifugation at 40,000 rpm, 47C for 30 min (rotor
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Type 45, Beckman). For purification, 2- to 3-ml samples
of this supernatant were aliquoted into 10 mm diameter
plastic tubes, and these were immersed in a 727C water
bath for 10 min, cooled on ice for 20 min, and then centrifuged (25,000 rpm, 47C for 15 min, Type 45 rotor, Beckman) to remove denatured E. coli proteins. The clarified
supernatant was collected and applied directly onto a
1.0 1 16-cm cellulose phosphate column (P11, Whatman)
preequilibrated with 50 mM Tris–HCl, pH 8.0, 1 mM
EDTA at room temperature overnight. After loading, the
column was washed intensively until the UV absorption
returned to the baseline. Protein was eluted with a 60ml linear gradient of 0–1.0 M KCl prepared in 50 mM
Tris–HCl, pH 8.0, 1 mM EDTA. Four-milliliter fractions
were collected at 0.5 ml/min and assayed using 12%
SDS–PAGE. Major fractions containing a prominent 90kDa protein were combined and dialyzed against 50 mM
Tris–HCl, pH 8.0, overnight.
Pfu was further purified by a Mono Q anion exchange column (HR 10/10, Pharmacia). The column
was equilibrated with 50 mM Tris – HCl, pH 8.0. Before loading, the Pfu sample was filtered using a 0.22mm filter unit (Millipore). The column was developed
with a 60-ml linear gradient of 0 – 1.0 M KCl in 50
mM Tris – HCl, pH 8.0, at 2 min/ml, 4 ml per fraction.
Each fraction was assayed by 12% SDS – PAGE. The
90-kDa protein eluted at 0.17 M KCl and was dialyzed
overnight against 100 mM Tris – HCl, pH 8.2, 0.2 mM
EDTA at 47C. Protein could be concentrated by Centriprep-30 (Amicon) and recovery exceeded 95% of
starting material.
The purified protein was stored frozen in Pfu storage
buffer (50 mM Tris–HCl, pH 8.2, 0.1 mM EDTA, 1 mM
DTT, 0.1% NP-40, 0.1% Tween 20, 50% (w/v) glycerol.
Protein was stable at 020 and 0807C for at least several months.
Determination of Protein Concentration
The concentration of purified Pfu was determined
from ultraviolet absorption, using the extinction coefficient for A278 Å 0.74 for 1 mg/ml, calculated from the
number of trp and tyr in the sequence (using the Protean program of DNAstar, Madison, WS). We also compared this concentration with that estimated by the
PIERCE BCA assay, using BSA as a standard. When
we assayed a Pfu solution calibrated by ultraviolet absorption to be 1 mg/ml, the BCA assay indicated a concentration of 0.65 mg/ml. Indirect assays like the BCA,
Lowry or Bradford are known to produce different color
intensity for different proteins, and in this case the
BCA assay substantially underestimates the concentration of Pfu. The validity of calculated A280 as an
accurate measure of protein concentration has been
verified previously (10,11).
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Pfu DNA POLYMERASE EXPRESSED IN E. coli
DNA Polymerase Assay
Pfu DNA polymerase activity was measured as described by Mroczkowski et al. (8). The assays were performed at 727C in a 25-ml reaction mixture containing
20 mM Tris–HCl, pH 7.5, 8 mM MgCl2 , 40 mg/ml BSA
(New England Biologicals), 0.5 A260 units of activated
calf thymus DNA (Pharmacia), 0.4 mM each of dATP,
dGTP, dCTP, TTP (Amersham), and 1 mCi [3H]TTP (30
Ci/mmol, Amersham). Reactions were stopped on ice
and 5-ml aliquots were spotted onto ion-exchange paper
discs (2.3 cm diameter, DE81, Whatman). Discs were
air dried and washed three times in 21 SSC buffer for
5 min each, once in 100% cold ethanol, then air-dried.
Incorporated radioactivity was counted using a liquid
scintillation counter (LS1800, Beckman). One unit of
Pfu DNA polymerase is defined as the amount of polymerase that incorporates 10 nmol of labeled 41 dNTP
into a DE81-bound form at 727C in 30 min (7,8). Recombinant Pfu DNA polymerase from Stratagene was used
as a positive control.
PCR Amplification with Purified Pfu DNA
Polymerase
Pfu amplification activity was also evaluated by PCR
titration and compared to commercial recombinant Pfu
DNA polymerase (Stratagene). We diluted the protein
to 2.5 units/ml in Pfu dilution buffer: 50 mM Tris–HCl,
pH 8.2, 0.1 mM EDTA, 1 mM DTT, 0.1% NP-40, 0.1%
Tween 20, 50% glycerol (w/v), based on the assumed
25,000 units per milligram of Pfu protein. We also prepared our own 10X PCR reaction buffer according to
the recipe in the Stratagene catalog: 100 mM KCl, 60
mM (NH4)2SO4 , 200 mM Tris–HCl, pH 8.8, 20 mM
MgSO4 , 1% Triton X-100, and 1 mg/ml BSA (New England Biologicals). This prepared buffer gave identical
results to the one obtained from Stratagene. We synthesized two primers corresponding to the N- and Ctermini of the Mycoplasma genitalium FtsZ (MgFtsZ)
gene, sense primer, 5*-TTAACATATGGATGAAAATGAAACTCAATTCAA-3*; antisense primer, 5*-TTA
AGGATCCTTAGTAGATTTGGTTTTGGTGCT-3*
(underlined sequences were added for convenience of
cloning). The PCR reaction was done in a 50-ml mixture
containing 11 PCR buffer, 0.2 mM each primer, 0.2 mM
each dNTP, 50 ng plasmid which carried the MgFtsZ
gene (clone MG224, The Institute for Genomic Research, MD) and purified Pfu (up to 100 units) as indicated in the text or 1.25 units of Pfu DNA polymerase
from Stratagene. After amplification for 25 cycles (30
s at 947C, 1 min at 557C, and 1.5 min at 727C) 10 ml of
PCR mixture was electrophoresed on a 1% agarose gel
in 1X TBE.
RESULTS AND DISCUSSION
Construction of Expression Plasmid, pETpfu
The pET system is one of most powerful systems
developed for cloning and expression of recombinant
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proteins in E. coli (9). The pET11 vector has a very
strong and stringent T7lac promoter and can be
grown in combination with pLysS or pLysE to provide
additional stringency (12, 13). The coding region is
conveniently cloned into pET11 using an NdeI site,
which provides an ATG start codon (12). We amplified
the pfu gene using the proofreading Pfu DNA polymerase and cloned it into the NdeI site of pET11 as
described under Methods to produce the expression
plasmid pETpfu. An important feature of the pET11
vector is that it adds no tag or extra amino acids at
either end, so the expressed product should be identical to the native protein.
The expression plasmid pETpfu is available from
ATCC, Accession No. 87496.
Pfu Is Successfully Expressed in BL21 (DE3) pLysS
The level of expression was examined in different
strains at 30 and 377C. When pETpfu was transformed
into BL21(DE3), many colonies were formed at 307C,
but at 377C no colonies were obtained. When the colonies obtained at 307C were tested for protein expression
by induction with IPTG, no 90-kDa protein was seen
in SDS–PAGE. As a control, pETpfu was digested with
BamHI to delete about four-fifths of the Pfu gene; the
resultant truncated plasmid was able to propagate in
BL21(DE3) at 377C. These observations suggested that
the complete pETpfu might be producing a small
amount of Pfu protein prior to induction by IPTG and
that this was toxic to the E. coli. The colonies obtained
by growth at 307C may have undergone some rearrangement of the plasmid, since they were unable to
produce protein when induced by IPTG. This result
was surprising since we expected the thermophilic
polymerase to be inactive at 377C, and it has been demonstrated that Taq DNA polymerase can be easily expressed in E. coli (14). Nevertheless, we decided to see
if more stringent control of expression could permit
growth of the plasmid and expression of Pfu.
BL21(DE3) carrying a plasmid pLysS or pLysE,
which express T7 lysozyme in the bacterial cytoplasm,
provide much more stringent repression of protein expression from the pET vector in the absence of induction and can tolerate some plasmids expressing very
toxic proteins. The mechanism of the additional stringency is that the T7 lysozyme binds to and inactivates
T7 RNA polymerase (which is expressed at low levels
in the absence of induction) and inhibits transcription
(12,13). Following induction the amount of T7 RNA
polymerase is sufficient to overcome the lysozyme inhibition.
We found that colonies could be obtained at 377C
when pETpfu was used to transform BL21(DE3) carrying either pLysS or pLys E. Moreover, expression of
Pfu protein was obtained after induction with IPTG,
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FIG. 1. SDS–PAGE analysis of Pfu overexpressed in BL21(DE3)
carrying pLysS or pLysE. Cells carrying pETpfu plasmid were grown
to A600 Å 0.5 and induced with 0.5 mM IPTG (/) for 2 h or grown
without IPTG (0) as a control. Cells from 100-ml culture were resuspended in 11 SDS sample buffer and boiled before loading. A 20-ml
sample was analyzed on 12% SDS–PAGE and stained with Coomassie blue. Heat stable proteins (lane HS) were those remaining in the
supernatant following 10 min at 727C and centrifugation. Lane M is
prestained protein markers, their size indicated on the left.
as shown in Fig. 1. After induction by IPTG, a 90-kDa
protein was produced clearly in strain pLysS cells and
was also made in pLysE at a low level. pLysE produces
more lysozyme than pLysS, and this frequently reduces
the expression level. A 118-kDa protein was also expressed following IPTG induction of pETpfu in pLysS,
not in pLysE. We also observed this 118-kDa protein
when expressing another protein in the pLysS system.
The Vent DNA polymerase from T. litoralis was also
successfully expressed from a pET plasmid in the
BL21(DE3), pLysS system (3). This previous study did
not compare expression with pLysE or without a lysozyme plasmid. In our study the pLysS system is clearly
superior for producing Pfu.
FIG. 2. SDS–PAGE analysis of Pfu purified by P11 chromatography. The chromatography was performed as described under Methods. Ten microliters of each fraction was applied to 12% SDS–PAGE.
Fraction numbers are shown on top. ST is starting material; FT is
the flow-through of the column loading.
P11 column, as described previously for purification of
the baculovirus-expressed protein (8). Pfu eluted at
0.48 M KCl and was significantly purified (Fig. 2, fractions 8–10). At this stage, Pfu was pure enough for
most applications, but for the analyses of activity reported below Pfu was purified further by chromatography on a Mono Q anion-exchange column (Fig. 3). The
Purification of Pfu DNA Polymerase
Pfu was expressed as a soluble form in the cytosol.
To reduce contamination by DNA the lysed bacteria
were extensively digested with DNase and sonicated,
as described under Methods. We then used the thermophilic property of Pfu and eliminated most E. coli proteins by heating to 727C for 10 min and centrifuging to
eliminate denatured proteins (Fig. 1, lane HS). Several
E. coli proteins still remained soluble after the heating
step. The soluble supernatant from the heating step
was then chromatographed on a cellulose phosphate
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FIG. 3. SDS–PAGE analysis of Pfu purified on the Mono Q column.
Fractions 8–10 from Fig. 2 were pooled, dialyzed, and applied to the
Mono Q. The SDS–PAGE was run under the same conditions as
described in the legend to Fig. 2. Fraction 4 was collected and used
for activity assays.
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Pfu DNA POLYMERASE EXPRESSED IN E. coli
TABLE 1
Purification of Pfu Expressed in E. coli
Purification step
1.
2.
3.
4.
Total protein
(mg)a
Specific activity
(units/mg)
Purity
(%)b
NDc
50
7
3.7
ND
ND
21000
22500
6
40
82
97
Total bacteria lysate
Heat-soluble proteins
Cellulose phosphate (P11) chromatography
Mono Q (anion-exchange) chromatography
a
The concentration of heat-soluble proteins was determined using the BCA assay and BSA as the standard. The concentration of protein
in Steps 3 and 4 was determined by ultraviolet spectroscopy, using the extinction coefficient A278 Å 0.74 for 1 mg/ml.
b
Purity of Pfu at each step was estimated by analyzing digitized images of the SDS–PAGE gel using NIH Image 1.60 software.
c
ND, not determined.
peak of Pfu eluted at 0.17 M KCl, followed by a trail of
dilute protein. The peak fraction still contained most
of the weak, lower molecular weight bands seen in the
starting material, suggesting that these may be proteolytic products of Pfu itself.
Following the P11 column, we obtained 6 mg of purified, active Pfu, as determined by the A280 , from a 1
liter bacterial culture. This culture was induced with
IPTG at A600 Å 0.5, but to check for optimal expression
we tested other cultures induced at A600 of 0.5, 1.0, and
1.2. All cultures were grown for total 5 h after seeding
cells from a 50-ml overnight culture. Cultures induced
at higher optical density produced more cell mass, but
the yield of Pfu after P11 chromatography was similar,
around 6 mg per liter. Vent DNA polymerase was produced in a similar system, however, only 0.5 mg purified protein per liter culture was obtained (3). We have
found that reproducible high yields of Pfu are obtained
from freshly transformed cells, whereas expression
from a freezer stock gave erratic and lower yields. Table
1 summarizes the steps of Pfu purification.
Activity of Bacterially Expressed Pfu
The purified Pfu from E. coli was fully functional as
a DNA polymerase when tested for incorporation of
deoxyribonucleotides into DEAE-paper bound form.
One unit of Pfu was defined as the amount of protein
that catalyzed the incorporation of 10 nmol total nucleotide into a DEAE-bound form in 30 min at 727C. The
specific activity of Mono Q-purified Pfu was 22,500
units per milligram of Pfu. This is similar to the activity
reported for Pfu directly purified from P. furiosus
(31,713 units/mg (7)) or from the baculovirus expression (26,000 units per mg (8)). Pfu purified by the P11
column without the Mono Q step had 93% the activity
of Mono Q purified protein, confirming that the Mono
Q column gave only a small additional purification (Table 1). Previous reports did not indicate how protein
concentration was determined. Our determination of
protein concentration was based on the A280 , using the
calculated extinction coefficient. If we had used the
BCA assay, referenced to BSA, to estimate protein concentration (Methods), we would have reported 35,700
units per milligram.
The activity of recombinant Pfu was also examined
in the PCR reaction, titrating the Pfu concentration
over a large range. Pfu (0.125–100 units) was used in
a 50-ml standard reaction, and the results were compared to Stratagene recombinant Pfu at 1.25 units (Fig.
4). The template was 50 ng of a plasmid containing the
1.2 kb ftsZ gene from M. genitalium and primers to
amplify the whole gene. When fairly large amounts of
FIG. 4. Activity assay of purified Pfu in the PCR titration. The amount of Pfu in units is shown on top. Reactions shown in a and b were
run separately. Lane M, 1-kb DNA ladder (Gibco); S, 1.25 units of Pfu from Stratagene as a positive control.
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Pfu (20–100 units) were used, no PCR product was
made (Fig. 4a). It is possible that high levels of Pfu
inhibited the reaction or that the exonuclease activity
digested primers. It has been reported that large
amounts of Taq DNA polymerase inhibited PCR as well
(14). Six units of Pfu amplified the expected 1.2 kb ftsZ
gene, but when Pfu was reduced to 2 units nonspecific
products were reduced and specific ftsZ was increased.
In a separate experiment, 2.5–0.125 units of Pfu was
tested. Pfu at 1.25–2.5 units showed a similar amplification efficiency, somewhat higher than the amplification with 1.25 units of Stratagene Pfu (Fig. 4b). Our
purified Pfu and Stratagen Pfu at 0.5 unit or less did
not amplify DNA under our conditions. We did not titrate the activity of Stratagen Pfu at high concentration (greater than 20 units), since concentrated Pfu was
not available commercially.
In summary, the Pfu expressed in E. coli can easily
be prepared in milligram quantities, and it appears to
have the same activity as that purified from P. furiosus
or that expressed in the baculovirus system. The key
to expression appears to be to suppress the basal level
of expression of the toxic protein prior to induction. The
combination of pET and pLysS has worked well in our
present study, but we expect other expression systems
that provide stringent control of expression should also
work.
ACKNOWLEDGMENTS
We thank Drs. Frank Jenney and Michael Adams, University of
Georgia, Athens, GA for providing genomic DNA from P. furiosus.
This work was supported by NIH Grant GM28553.
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