(Received for publication, August 31, 1994; and in revised form, November 2, 1994)
From the
Bovine prethrombin-2 has been produced in Escherichia coli using a T7 expression system. The expressed prethrombin-2 formed
intracellular inclusion bodies which were solubilized by reversible
sulfonation of the cysteines in the presence of 7 M guanidine
hydrochloride. Sulfonated prethrombin-2 was refolded in the presence of
4 M guanidine hydrochloride, using oxidized and reduced
glutathione as the redox couple. The folded protein was purified by
heparin affinity chromatography and activated to thrombin with Echis carinatus snake venom. The resulting thrombin was also
purified by heparin affinity chromatography. Kinetic constants were
determined for the hydrolysis of
H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline
by recombinant thrombin (k = 123 ±
10 s
and K
=
2.91 ± 0.3 µM). These values are in good agreement
with those determined for wild-type thrombin (k
= 97 ± 8 s
and K
= 2.71 ± 0.25
µM). From the thrombin-mediated release of fibrinopeptide
A from fibrinogen, k
/K
was found to be the same for recombinant (17.3 ± 1.2
µM
s
) and wild-type
(16.7 ± 2.0 µM
s
) thrombin. These results, taken together
with circular dichroism spectra and the elution position of
prethrombin-2 from a heparin affinity resin, indicate that
prethrombin-2 was folded into a conformation similar to that of the
wild-type protein. In addition, since E. coli produces
deglycosylated enzymes, these findings suggest that the carbohydrate on
the B chain of wild-type thrombin does not affect the amidolytic and
fibrinolytic activities of thrombin. Finally, this expression system
can be used to prepare mutants of prethrombin-2 for future
structure-function studies involving thrombin and its substrates; some
preliminary results of this type are presented here.
Thrombin (EC 3.4.21.5) is a multifunctional protein playing a
key role in the blood clotting cascade(1) . In one of its
reactions, this enzyme acts as a catalyst in the first of three
reversible steps in the conversion of fibrinogen to fibrin(2) .
Thrombin cleaves fibrinogen at four Arg-Gly bonds, one in each of the
two A and B
chains, releasing two FpAs
and two
FpBs(3) . The resulting fibrin monomer polymerizes, ultimately
to fibrin, which is stabilized by factor XIIIa-catalyzed
cross-links(4) . In addition to cleaving fibrinogen, thrombin
also activates platelets (5) and the blood coagulation factors
V, VIII(6, 7) , XI(8) , and XIII(9) .
In the presence of thrombomodulin, thrombin triggers the major
anticoagulant pathway by activating protein C(10) .
Bovine
thrombin is a glycoprotein consisting of two polypeptides, a
259-residue B chain and a smaller 49-residue A chain, connected by a
disulfide bond, Cys of the A chain to Cys
of
the B chain. (
)The B chain contains all the residues
required for catalysis (12, 13) and is homologous to
the catalytic domains of other trypsin-like serine proteases including
trypsin, chymotrypsin, and elastase(14) . The A chain lies
along the side of thrombin opposite the active site(15) .
Chymotrypsin is the only serine protease containing a region that is
clearly homologous to the A chain of thrombin(16) . In addition
to the interchain disulfide bond, there are three disulfides in the
serine protease (B chain) domain of thrombin
[Cys
-Cys
],
[Cys
-Cys
], and
[Cys
-Cys
].
Thrombin circulates
in the blood as the inactive zymogen prothrombin which is converted to
thrombin by two sequential factor Xa cleavages at
Arg-Thr
and Arg
-Ile
(Fig. 1). Factor Xa activation of prothrombin, in the
absence of the rest of the prothrombinase complex (factor Va, calcium,
and phospholipid), has been shown to cleave initially at
Arg
-Thr
. This cleavage produces
prethrombin-2, the smallest single-chain precursor to thrombin, and
prothrombin fragment 1.2(17) .
Figure 1: Prothrombin activation. Prothrombin contains two factor Xa cleavage sites and one thrombin cleavage site. Cleavage at the second factor Xa site results in the formation of the two-chain meizothrombin, which is the same size as prothrombin. Cleavage at the thrombin site produces prethrombin-1 releasing prothrombin fragment 1. Cleavage of prothrombin at the first factor Xa site leads to prethrombin-2, which is the smallest single-chain precursor to thrombin and the same size as thrombin. Cleavage at both factor Xa sites leads to the formation of thrombin.
The structure-function
relationship between thrombin and fibrinogen has been studied by
active-site mapping (18) and by NMR spectroscopy. For example,
the interactions between bovine thrombin and FpA 7-16 (residues
7-16 of the A chain of human fibrinogen) have been examined
by transfer NOE experiments(19, 20, 21) . The
results indicated that the bound FpA 7-16 forms a multiple turn
structure containing residues Glu
through
Val
. As a result, Phe
, Val
, and
Leu
form a hydrophobic cluster that is located near the
peptide cleavage site Arg
-Gly
in a position
to interact with a nonpolar binding site on thrombin. The NMR structure
of the bound peptide, which has been confirmed by x-ray
crystallographic studies (22, 23) , provided a
structural basis for the observation that Phe
, which is
highly conserved among fibrinogen species (24) , is necessary
for optimal thrombin-specific cleavage of the
Arg
-Gly
peptide bond(25) .
In the
interest of pursuing further structural studies to understand the
interactions between thrombin and its substrates, we have developed an
expression system for the production of recombinant thrombin and
mutants thereof. Currently, there are several mammalian systems in use
for the production of prothrombin and its
mutants(26, 27, 28, 29) . These
systems all produce approximately 0.5 to 8 µg of thrombin per ml of
cell culture. There have also been reports of mammalian expression
systems for prethrombin-1 (30) and meizothrombin(31) ,
achieving yields of 15-20 µg/ml, as well as for prethrombin-2
with no reported yield (32, 33) . A system has also
been developed in which thrombin activity is reconstituted by combining
wild-type 1-thrombin, a proteolytically derived fragment of human
thrombin, with recombinant
2-thrombin from Escherichia coli (yields of 11 mg of purified unfolded
2-thrombin per liter of
cell culture)(34, 35) .
1-Thrombin consists of
the A chain of thrombin linked by a disulfide bond to residues
16-148 of the B chain, while
2-thrombin consists of residues
149 to 247 of the B chain. Human prethrombin-2 has been overproduced in E. coli(36, 37) at a level of 8 mg of
purified, unfolded protein per liter of cell culture, but little
physical characterization of the recombinant refolded prethrombin-2 or
thrombin was reported.
We have chosen to use E. coli for
overexpression of bovine thrombin because this prokaryotic system is
easy to work with and scale up. Consequently, it has the potential for
producing higher yields of thrombin than are currently obtained from
mammalian systems. In addition, protein expression in E. coli facilitates both specific isotopic labeling with N,
C-labeled amino acids and nonspecific
labeling with [
N]H
Cl and
[
C]glucose of proteins for multidimensional NMR
studies. An E. coli system also has the added advantage of
producing deglycosylated thrombin which allows for functional analysis
of the role of carbohydrate in thrombin action. In addition,
elimination of heterogeneity that would arise from glycosylation makes
it easier to produce the large amounts of homogeneous material required
for NMR studies.
However, since the environment of the E. coli cytosol is reducing, proteins are often isolated without correct disulfide pairing and must later be refolded into their native conformation(38) . In our laboratory, wild-type and mutant preparations of bovine pancreatic ribonuclease A have been refolded successfully after isolation of inclusion bodies from E. coli(39) , followed by reversible sulfonation of cysteines to break any incorrect disulfide pairings and subsequently to stabilize the unfolded protein. In developing an E. coli system for the production of thrombin, a decision had to be made whether to work with the two-chain species, thrombin, or with one of its earlier single-chain precursors (Fig. 1). With a single-chain species, it should be easier to form the native disulfide pairings since the entropy loss when the two chains are connected is eliminated. An additional consideration was the number of disulfide bonds which must be formed. Prethrombin-2 has only four disulfide bonds, whereas prethrombin-1 has seven and prothrombin has twelve. If disulfide pairing occurs randomly, a native four-disulfide conformation statistically should be much more likely to form than one containing either seven or twelve disulfide bonds. While current views in the protein-folding literature would suggest that disulfide pairing does not occur randomly, nevertheless, less demands on intramolecular interactions are made in the formation of a species with fewer disulfide bonds. Therefore, bovine prethrombin-2, the smallest single-chain precursor of thrombin, was chosen for expression in E. coli.
In this paper, we describe the overexpression of bovine prethrombin-2 in E. coli. Prethrombin-2 is isolated as intracellular inclusion bodies which are purified, reduced, and reversibly blocked by sulfonation. Sulfonated prethrombin-2 is then refolded in the presence of high concentrations of GdnHCl, using GSSG and GSH as a redox couple. From circular dichroism spectra, it is found that recombinant prethrombin-2 is refolded into a native-like structure. Upon activation of prethrombin-2 with Echis carinatus snake venom, the resulting recombinant thrombin has kinetic properties similar to those of wild-type thrombin. This recombinant system can be used to produce thrombin and mutants thereof that can be used in further functional and structural studies.
pThr-1 was made by removing gene-10 from pGPTh-36 (Fig. 2). pGPTh-36 was first digested with NheI. The sticky end produced by NheI was turned into a blunt end by digestion with mung bean nuclease. After further digestion with MscI, the plasmid was run on an agarose gel, purified by using the Gene Clean Kit (Bio 101), and recircularized with T4 DNA ligase.
Figure 2: Construction of an expression vector for prethrombin-2. pGPTh-36 encodes for prethrombin-2 as a fusion protein with T7 gene 10. The coding region for the T7 gene 10 was removed from pGPTh-36 to produce pThr-1 as described under ``Experimental Procedures.'' pThr-1 encodes for prethrombin-2 with a 6-residue N-terminal tail MAIEGR.
All cloning steps were confirmed by the dideoxy sequencing method using the U. S. Biochemical Corp. Sequenase Version 2.0 kit.
The extent of
prethrombin-2 refolding was assessed by a chromogenic assay. An aliquot
of the dialyzed prethrombin-2 refolding solution (10-100 µl)
in 975 µl of 25 mM sodium phosphate, pH 7.4, containing
0.15 M NaCl and 0.1% PEG, was activated to thrombin by adding
3 µl of E. carinatus snake venom (1 mg/ml) and incubating
at 37 °C for 30 min. The snake venom was first pretreated with p-APMSF to inactivate contaminating serine proteases ()that interfere with measurements of S2238
activity(42) , and then desalted on a PD-10 column (Pharmacia)
into 20 mM Tris, pH 8.0 buffer. The chromogenic substrate
peptide S2238 (Kabi) at a final concentration of 0.1 mM was
then added to the thrombin solution, and the absorbance at 405 nm was
monitored at room temperature. All absorbance measurements were made on
a modified Cary Model 14 spectrophotometer(43) .
Due to the possibility
that lyophilization may lead to the inactivation of thrombin, the
measured molar extinction coefficients were used only to compare
wild-type and recombinant thrombin. Actual protein concentrations were
estimated using E = 19.5 at 280
nm(45) .
Samples for N-terminal sequencing were either desalted by
reverse-phase HPLC or separated by 10% SDS-polyacrylamide gel
electrophoresis and electroblotted onto polyvinylidene difluoride
membranes (Bio-Rad). For sequencing of isolated A and B chains of
thrombin, recombinant thrombin (50 µg/ml) in 3.5 M GdnHCl,
10 mM Tris, 1 mM EDTA, pH 8.0, was treated with 0.1 M dithiothreitol at 37 °C for 3 h. The A and B chains were
separated on a Brownlee Aquapore RP-300 column (100 2.1 mm)
equilibrated in 10% acetonitrile/0.1% trifluoroacetic acid using a
linear gradient from 10% acetonitrile/0.1% trifluoroacetic acid to 95%
acetonitrile/0.1% trifluoroacetic acid. N-terminal sequencing was
carried out at the Cornell Biotechnology facility.
The amount of product formed was calculated by using an extinction
coefficient of 9920 M cm
for p-nitroaniline at 405 nm(47) , and the
concentration of S2238 was determined by using an extinction
coefficient of 8270 M
cm
at 342 nm(47) . Values and standard deviations for K
and k
were calculated
from triplicate assays by least-squares fit to a straight line of a
plot of the inverse of the rate of p-nitroaniline release
against the inverse of the concentration of S2238, using the program
LINFIT(48) .
An HPLC method for following the
thrombin-mediated release of FpA from fibrinogen was initially
developed by Martinelli and Scheraga(49) . For this work, a
modification of the procedure of Lewis and Shafer (50) was
followed. Approximately 0.01 NIH unit/ml (3 ng/ml) of thrombin and 3.5
µM fibrinogen were incubated at 37 °C in 50 mM HPO
, 100 mM NaCl, 0.1% PEG 8000,
pH 7.5. At designated times between 1 and 14 min, an aliquot of the
reaction was quenched by adding 0.3 M perchloric acid. After
an additional 10 min at 37 °C, the pH was raised to
2 by
adding 0.14 M NaOH. An infinity time point (30 min) for the
FpA release assay was determined using thrombin at a concentration of
0.05 NIH unit/ml.
An LKB HPLC system consisting of a 2152 LC
controller, a 2151 pump, a 2141 variable wavelength detector, a 2221
integrator, and a 2157 autosampler was used for the detection of FpA.
For each time point, 900 µl were loaded onto a Brownlee Aquapore
RP-300 column (100 2.1 mm) equilibrated in 10%
acetonitrile/0.1% trifluoroacetic acid, and FpA was eluted with a
linear gradient from 10% acetonitrile/0.1% trifluoroacetic acid to 35%
acetonitrile/0.1% trifluoroacetic acid in 35 min at a flow rate of 0.5
ml/min. Kinetic constants and standard deviations were determined by
least-squares fit to a straight line of a plot of
-ln{([FpA]
-
[FpA]
)/([FpA]
-
[FpA]
)} against time using the program
LINFIT(48) , where [FpA]
is a baseline
correction.
Figure 3:
Silver-stained gel of recombinant
prethrombin-2 and thrombin. The following samples were run on a 10% SDS
reducing gel: lane 1, molecular mass standards; lane
2, sulfonated prethrombin-2 (5 µg); lane 3,
folded recombinant prethrombin-2 off of a heparin affinity column; lane 4, 20 µg of snake venom; lane 5, folded
recombinant prethrombin-2 after incubation with snake venom; lane
6, purified recombinant thrombin. The samples for the gel were
prepared by activating 100 µg of folded and purified recombinant
prethrombin-2 with 20 µg of E. carinatus snake venom at 37
°C for 3 h and purifying the resulting thrombin by heparin affinity
chromatography as described under ``Experimental
Procedures.'' Aliquots (
5 µg each) were removed before
addition of snake venom, at the end of the 3-h incubation, and after
purification. The protein was precipitated by the addition of 1 ml of
acetone, incubation at -80 °C for 30 min, followed by
centrifugation in a microcentrifuge for 20 min. The protein pellet was
resuspended in 50 µl of SDS-reducing sample buffer, and 20 µl
(
2 µg) were loaded onto a 10% SDS
gel.
During dialysis to remove the denaturant and redox couple, 50%
of the prethrombin-2 precipitated out of solution. The resultant pellet
could be recycled through the folding procedure, and more active
recombinant prethrombin-2 could be isolated. The yields of isolated,
active prethrombin-2 from the first and second recycling steps
(1-4% of starting material) are similar to the original folding
trial (1.5-2.7%).
Figure 4: Heparin affinity chromatography of refolded recombinant prethrombin-2 and thrombin. a, 10 mg of recombinant sulfonated prethrombin-2 were refolded in 500 ml of buffer as described under ``Experimental Procedures.'' After dialysis to remove GdnHCl, the sample was centrifuged to remove the insoluble protein. The soluble refolded prethrombin-2 was loaded onto a heparin affinity column equilibrated in 25 mM sodium phosphate, pH 7.4. The prethrombin-2 was eluted with a linear gradient from 0-1 M NaCl in the same buffer. b, 60 µg of recombinant prethrombin-2 were activated with E. carinatus snake venom for 3 h at 37 °C. The activated material was loaded onto a heparin affinity resin equilibrated with 50 mM sodium phosphate, pH 6.5, and thrombin was eluted with a 30-ml gradient from 0-1 M NaCl in the same buffer.
We have developed a T7 expression system for the
overproduction of prethrombin-2 and its mutants in E. coli which produce high levels of recombinant protein
(80-100 mg/liter). Recombinant prethrombin-2 is produced as
inclusion bodies which are purified with the four disulfide bonds
reduced and sulfonated. Because of the insolubility of sulfonated
prethrombin-2, it is refolded in the presence of 4 M GdnHCl,
resulting in a recovery of 1.0-4.0% folded prethrombin-2 from a
single folding trial. Refolded prethrombin-2 is purified by specific
elution from a heparin affinity resin suggesting that the heparin
binding site is intact (Fig. 4a). Further evidence for
native-like structure is provided by the CD spectra which show that the
overall backbone structure of refolded prethrombin-2 is similar to that
of wild-type bovine prethrombin-2. After purification on the heparin
column, the recombinant prethrombin-2 is activated to thrombin using E. carinatus snake venom. Recombinant thrombin has been shown
to be equivalent to wild-type bovine thrombin in terms of S2238
activity and FpA cleavage of fibrinogen (Table 1). These results
demonstrate that the structure of the active site is equivalent to that
of wild-type bovine thrombin. The FpA assay results also show that the
fibrinogen anionic binding site is in a structure comparable with that
of the wild-type protein. Since recombinant prethrombin-2 is produced
in a deglycosylated form, the results presented here demonstrate that
carbohydrate does not play a role in the catalytic activity of thrombin
toward S2238 and fibrinogen, although it may have a function in some of
the other cellular roles of thrombin.
Recombinant sulfonated prethrombin-2 is insoluble in aqueous buffers, and refolding had to be carried out in the presence of high concentrations of denaturant. It was determined that folding in 4 M GdnHCl was necessary to obtain maximal formation of an active species. Bauer et al. (52) have shown that thrombin is completely unfolded in 1.5 M GdnHCl at pH 6.5. Therefore, it is reasonable to assume that sulfonated recombinant prethrombin-2 is completely unfolded in 4 M GdnHCl at pH 7.4. Even though aggregation is not observed, more folded, active prethrombin-2 is recovered in 4 M GdnHCl than in 2 M GdnHCl.
During refolding, sulfonated recombinant prethrombin-2 is incubated in 4 M GdnHCl with a redox couple, and reshuffling of disulfide bonds results. Under these conditions, it is likely that random disulfide bond formation occurs, a percentage of which contains the native disulfide pairing. On removal of the denaturant during dialysis, prethrombin-2 continues to fold with a fraction attaining the native conformation. For recombinant prethrombin-2, the native four-disulfide pairing is not favored over any of the other 104 possible combinations of four disulfides. Assuming that disulfide pairing occurs randomly, the amount of native pairing expected to form under denaturing conditions is 0.95%. Therefore, our folding yields of 1-4% suggest that most of the correctly refolded prethrombin-2 is being recovered and that there are no large losses due to co-precipitation with non-native folding species.
Other serine protease precursors, including trypsinogen and chymotrypsinogen, which are similar to thrombin in structure and sequence have been successfully refolded from both the reduced and the glutathione-blocked forms(53, 54, 55, 56) . Recovery yields from 10-70% were found, but minimal denaturant was necessary. We have found that, in the absence of denaturant, the reduced and the glutathione-blocked forms of prethrombin-2 are as insoluble as the sulfonated form. Folding from either the glutathione blocked or reduced form produced the same or less active prethrombin-2 than folding from the sulfonated form (data not shown).
Despite the low yield of refolding, this recombinant system does produce large amounts of denatured prethrombin-2 which, after folding, can be purified by heparin affinity chromatography. The specificity of folded prethrombin-2 for heparin provides a simple method for isolating correctly folded prethrombin-2 (Fig. 4a). Folded prethrombin-2 is purified away from other folding species and contaminants which elute earlier in the NaCl gradient and are not active after activation with E. carinatus snake venom. These species presumably contain non-native intermediates that may bind to the heparin matrix through a partially formed heparin binding site or in a less specific manner than folded prethrombin-2.
The heparin binding site can be exploited in the purification of active-site or other mutants of prethrombin-2 and thus thrombin. Any mutant of thrombin that maintains the heparin binding site should be able to be refolded and easily purified away from the misfolded protein on the heparin affinity column. This system can readily produce the quantities of mutant thrombin necessary for carrying out activity assays to assess the relationship between mutation and thrombin function. Currently, 0.9 to 1.3 mg of recombinant active prethrombin-2 and 0.5-0.7 mg of recombinant thrombin can be isolated from 1 liter of cells, and scale-up to the quantities of protein necessary for functional and structural studies appears to be obtainable.
Using site-directed mutagenesis, a mutant of prethrombin-2 has been made in which the cysteines of the disulfide bond linking the A and B chains covalently were replaced with alanines. This mutant, prethrombin-2 (C1A/C122A), will be used for further understanding of the role of the A chain in protein stability and substrate binding. Prethrombin-2 (C1A/C122A) is expressed in E. coli at levels comparable with that of wild-type prethrombin-2, and the correctly folded enzyme is isolated by heparin affinity chromatography. Activation of prethrombin-2 (C1A/C122A) to thrombin (C1A/C122A) is thought to yield the two chains held together, presumably by electrostatic interactions. Preliminary studies indicate that thrombin (C1A/C122A), as a mixture of A and B chains, is able to hydrolyze the chromogenic substrate S2238 and to clot fibrinogen (data not shown).
We have developed a system for the production of recombinant thrombin and mutants of thrombin and have demonstrated that recombinant prethrombin-2 can be refolded from the sulfonated form into an active enzyme. No major differences were found in the amidolytic and fibrinolytic activities of recombinant thrombin and wild-type thrombin which is important for further functional and structural studies. These findings, taken together with the specific elution position of recombinant thrombin from a heparin affinity matrix and the circular dichroism spectra, indicate that recombinant prethrombin-2 has been refolded to its native conformation. Also, these findings indicate that the carbohydrate chain on the B-chain of thrombin does not affect the amidolytic or fibrinolytic activities of thrombin. This recombinant prethrombin-2 system provides a method for producing mutants of thrombin for further functional and structural studies of the interactions of thrombin with fibrinogen.