©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression and Folding of Recombinant Bovine Prethrombin-2 and Its Activation to Thrombin (*)

(Received for publication, August 31, 1994; and in revised form, November 2, 1994)

Elsie E. DiBella Muriel C. Maurer (§) Harold A. Scheraga (¶)

From the Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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/Kwas 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.


INTRODUCTION

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 Aalpha and Bbeta chains, releasing two FpAs^1 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^1 of the A chain to Cys of the B chain. (^2)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^1 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^1. 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 Aalpha 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^8, Val, and Leu^9 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^8, 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(4)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.


EXPERIMENTAL PROCEDURES

Materials

Bovine prothrombin was a generous gift from Francis J. Castellino (University of Notre Dame), and S2238 was a generous gift from Ty Fanella (Kabi Pharmacia). Ultrapure guanidine hydrochloride was obtained from ICN, and Factor Xa was purchased from Boehringer Mannheim. E. carinatus snake venom, p-APMSF, and bovine fibrinogen (96% clottable) were purchased from Sigma. Restriction enzymes and other enzymes for DNA sequencing and cloning were from U. S. Biochemical Corp., Promega Biotech, and New England Biolabs. All other materials were reagent-grade.

Plasmids and Bacterial Strains

Plasmid pBII III, containing an almost full-length bovine prothrombin cDNA insert, was kindly provided by Ross T. A. MacGillivray (University of British Columbia) (40) . Cloning vector M13mp18 was purchased from U. S. Biochemical Corp.. The expression vector used was pGEMEX-2 (Promega Corp). Host E. coli strains for cloning and mutagenesis were JM101 and Sure (Stratagene). The E. coli strain used for expression was HMS174(DE3)-pLys(S) (Novagen).

Plasmid Constructs

The prothrombin cDNA was removed from pBII III as a PstI fragment and subcloned into PstI-digested M13mp18, generating mPTh-1. Results from dideoxy sequencing indicated that the prothrombin cDNA was missing the codons for the 10 C-terminal amino acids. The bovine prethrombin-2 gene was amplified by polymerase chain reaction using mPTh-1 as the template. The oligonucleotides used in the polymerase chain reaction reaction, 5`-TTTTGAATTCTATGGCAATCGTCGAGGGACGCACG-3` and 5`-TTTTTTAAGCTTAAGAACCCAGACGGTCGATAACTTTCTGAATCCACTTCTTCAGGCGGAAGACGTG-3`, were designed to incorporate: (i) codons for the last 10 amino acids, (ii) two restriction sites: a 5`-EcoRI site and a 3`-HindIII site, and (iii) the factor Xa site before the start of prethrombin-2. The prethrombin-2 gene as an EcoRI/HindIII fragment was subcloned into EcoRI/HindIII-digested pGEMEX-2 to produce pGPTh-36.

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.

Expression of Recombinant Prethrombin-2

E. coli HMS174(DE3)-pLys(S) harboring pThr-1 were grown at 37 °C in 5 ml of 2 times YT (4% tryptone, 1% yeast extract, 10 mM NaCl, 1.4 mM KCl) containing 200 µg/ml ampicillin and 34 µg/ml chloramphenicol. The overnight cultures were diluted 1/200 into 1 liter of 2 times YT containing 200 µg/ml ampicillin and 34 µg/ml chloramphenicol and grown at 37 °C with shaking. When the cells reached an OD = 0.4-1.0, expression from the T7 promoter was induced by adding isopropyl-1-thio-beta-D-galactopyranoside to 0.4 mM, and growing was continued for another 4 to 12 h. The cells were then harvested by centrifugation at 7,800 rpm in a Beckman JA-10 rotor for 25 min at 4 °C.

Purification of Inclusion Bodies

The cell pellet was resuspended in 50 ml of 25 mM Tris, 2 mM EDTA, pH 8.0. The cell suspension was incubated at 37 °C for 30 min in the presence of lysozyme (0.2 mg/ml). The cells were then sonicated on ice for 5 min with 5-s pulses. The inclusion bodies were recovered by centrifugation at 7,800 rpm in a Beckman JA-10 rotor for 25 min at 4 °C. The inclusion body pellet was washed twice by resuspension in 50 ml of 0.5% Triton X-100, 2 mM EDTA, pH 8.0, followed by centrifugation at 7,800 rpm in a Beckman JA-10 rotor for 25 min at 4 °C.

Sulfonation of Recombinant Prethrombin-2

The washed inclusion body pellet was resuspended in 10 ml of 7 M GdnHCl, 10 mM Tris, 1 mM EDTA, pH 8.0. Reduction of disulfide bonds was accomplished by the addition of Na(2)SO(3) to 0.3 M and incubation at 37 °C for 30 min. Sulfonation was completed by the addition of 2/5 volume (4 ml) of 50 mM 2-nitro-5-thiosulfobenzoate and incubation of the solution for 15 min at room temperature in the dark (41) . The reaction was quenched by dialysis of the sulfonated recombinant prethrombin-2 (14 ml) against 3 changes of 4 liters of 0.7% acetic acid at 4 °C. These acidic conditions minimize unwanted disulfide shuffling. Sulfonated recombinant prethrombin-2 was found to be insoluble in the absence of denaturant, and the precipitate was recovered by centrifugation at 12,500 rpm in a Beckman JA-20 rotor for 20 min at 4 °C.

Purification of Recombinant Sulfonated Prethrombin-2

The insoluble sulfonated recombinant prethrombin-2 was resuspended in 10 ml of 7 M GdnHCl, 10 mM Tris, 1 mM EDTA, pH 8.0. The sulfonated protein was purified and desalted to remove GdnHCl using reverse phase HPLC on a Dynamax-300 Å C18 (10 mm times 25 cm) column (Rainin). The column was equilibrated in 23% acetonitrile/0.1% trifluoroacetic acid, and a linear gradient from 23% acetonitrile/0.1% trifluoroacetic acid to 50% acetonitrile/0.1% trifluoroacetic acid in 50 min was used to elute the protein. The purified enzyme was lyophilized and stored desiccated at -80 °C until needed.

Refolding of Recombinant Sulfonated Prethrombin-2

Lyophilized recombinant sulfonated prethrombin-2 was solubilized in 7 M GdnHCl, 10 mM Tris, 1 mM EDTA, pH 8.0, at a concentration of 4 mg/ml. The protein was diluted to a concentration of 25 µg/ml in folding buffer (0.1 M Na(2)HPO(4), 2 mM EDTA, 0.1% PEG, 0-4 M GdnHCl, 0.1 mM GSSG, 0.2 mM GSH, pH 7.4). The solution was allowed to incubate for 24 h at room temperature and was then dialyzed, to remove GdnHCl, GSSG, and GSH at 4 °C, against 3 changes of 4 liters of 25 mM sodium phosphate, pH 7.4, containing 2 mM EDTA and 0.1% PEG. The refolded prethrombin-2 remained soluble upon removal of GdnHCl. The typical scale of a refolding experiment was 5 mg of sulfonated prethrombin-2 in 200 ml of folding buffer.

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 (^3)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) .

Purification of Refolded Recombinant Prethrombin-2

Refolded prethrombin-2 in 25 mM sodium phosphate, 2 mM EDTA, 0.1% PEG, pH 7.4, was purified on a 1-ml HiTrap heparin affinity column (Pharmacia) equilibrated with 25 mM sodium phosphate, pH 7.4. The sample was loaded at 1.2 ml/min and was eluted with a linear gradient from 0 M to 1 M NaCl in 25 mM sodium phosphate, pH 7.4. Recombinant prethrombin-2 was either activated immediately to thrombin or stored at -80 °C until needed.

Large Scale Activation of Recombinant Folded Prethrombin-2 to Thrombin Using E. carinatus Snake Venom

Typically, 20 µl of snake venom (1 mg/ml), inactivated as described above, were added to 100 µg of recombinant prethrombin-2 in 25 mM sodium phosphate, pH 7.4, containing 0.4 M NaCl. After incubation at 37 °C for 3 h, the solution was diluted 1:8 in 50 mM phosphate buffer, pH 6.5 (H(3)PO(4) adjusted to pH 6.5 with NaOH) to lower the NaCl concentration. Recombinant thrombin was purified on a heparin affinity column equilibrated in 50 mM phosphate buffer, pH 6.5, and eluted with a linear gradient from 0 M to 1.0 M NaCl in the same buffer. The thrombin fractions were combined and stored immediately at -80 °C until needed.

Preparation of Wild-type Bovine Thrombin and Prethrombin-2

Bovine thrombin was prepared by the procedure of Ni et al.(19) . Bovine prethrombin-2 was prepared by a modification of the procedure of Mann(44) . Bovine prothrombin (8 mg/ml) was activated with 25 µg/ml factor Xa. The mixture was incubated at 37 °C until results from a fibrinogen clotting assay indicated that the sample contained 1% thrombin. The reaction was then immediately quenched by lowering the pH to 6.0 and adding p-APMSF to 0.25 mM. The solution was desalted on a PD-10 column into 50 mM phosphate buffer, pH 6.5, and then loaded onto a Mono S column (Pharmacia) equilibrated with the same buffer. Prethrombin-2 was eluted using a linear gradient from 0 to 1 M NaCl in the same buffer.

Determination of Extinction Coefficient

An extinction coefficient for wild-type and recombinant bovine thrombin was determined by quantitative amino acid analysis. Wild-type or recombinant thrombin (45 µg/ml) was desalted by reverse-phase HPLC on a Brownlee Aquapore RP-300 column (100 times 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 in 30 min. After lyophilization, the samples were resuspended in distilled and deionized water, and the absorbance at 280 nm was recorded. Aliquots of the thrombin solution in separate hydrolysis tubes were lyophilized. Quantitative amino acid analysis was carried out in triplicate at the Cornell Biotechnology facility using the Waters Pico-Tag chemistry.

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) .

Mass Spectroscopy and N-terminal Sequencing

Samples for mass spectral analysis were desalted by reverse-phase HPLC. Data were collected at the Cornell Biotechnology facility using a Finnegan Lasermat MALDTOF mass spectrometer.

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 times 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.

Circular Dichroism Measurements

Wild-type and recombinant prethrombin-2 samples were desalted on PD-10 columns (Pharmacia) into 10 mM phosphate buffer, pH 6.5 (H(3)PO(4) adjusted to pH 6.5 with NaOH). CD spectra were recorded on a Cary 14 spectrophotometer, modified for circular dichroism measurements and equipped with a Pockel cell(46) , kept at a constant temperature of 23 °C. The concentrations of prethrombin-2 used in these experiments were between 30 and 43 µg/ml. The resultant spectra were compared in terms of mean residue ellipticities.

Kinetic Analysis of Recombinant Thrombin

To assess the catalytic efficiency of recombinant thrombin, kinetic constants were determined for the cleavage of S2238 and the release of FpA from fibrinogen. The release of p-nitroaniline resulting from the hydrolysis of S2238 was followed by measuring the rate of increase in absorbance at 405 nm. The assays were carried out in polymethacrylate cuvettes at 37 °C in assay buffer (50 mM TrisbulletHCl, 0.15 M NaCl, 0.1% PEG, pH 7.4). Both the 10 nM thrombin stock solution and the 2-30 µM S2238 stock solutions were prepared in assay buffer. In addition, the thrombin solution contained 1 mg/ml bovine serum albumin. The absorbance at 342 nm of 990 µl of S2238 stock was recorded, then 10 µl of thrombin stock (10 nM) were added, and the absorbance at 405 nm was monitored for 5-10 min at 37 °C.

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(m) 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 H(3)PO(4), 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 times 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](t))/([FpA] - [FpA](0))} against time using the program LINFIT(48) , where [FpA](0) is a baseline correction.


RESULTS

Expression of Bovine Prethrombin-2

After polymerase chain reaction amplification, the prethrombin-2 gene was cloned into pGEMEX-2, a fusion protein expression vector based on the T7 expression system developed by Studier and Moffatt(51) . Previous work in our laboratory demonstrated that the pGEMEX-2 system produces high yields of bovine ribonuclease A(39) . Prethrombin-2 was expressed in pGPTh-36 as a fusion protein with the 260-residue T7 gene 10 protein. To avoid the need for enzymatic or chemical cleavage of a fusion protein, the coding region for T7 gene 10 was removed from pGPTh-36. The resulting plasmid pThr-1 expressed the prethrombin-2 gene with a 6-amino acid N-terminal tail MAIEGR which would be removed after folding (Fig. 2). pThr-1 produced unpurified recombinant prethrombin-2 at levels of 80-100 mg/liter of cells.

Purification of Recombinant Prethrombin-2

Recombinant prethrombin-2 produced in E. coli was purified as described under ``Experimental Procedures.'' After reduction, the protein cysteines were reversibly sulfonated to prevent disulfide shuffling due to air oxidation and to create a homogeneous starting material for refolding. The reverse-phase HPLC purified protein migrated on an SDS gel with an apparent molecular mass of 37,000 Da, which is close to the calculated molecular mass of 36,600 Da, and was 85% pure based on silver staining (Fig. 3, lane 2). After sulfonation and reverse-phase HPLC purification, the yields of purified, recombinant sulfonated prethrombin-2 were 30-40 mg/liter of cells based on absorbance at 280 nm.


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.



Characterization of Sulfonated Recombinant Prethrombin-2

N-terminal sequencing of recombinant sulfonated prethrombin-2 indicated that the Met at position 1 was missing; however, the subsequent 12 N-terminal amino acids (AIEGRTSEDHFQ) were present as expected. The molecular mass of recombinant sulfonated prethrombin-2 was 36,648 ± 73 Da by MALDTOF mass spectrometry. This experimentally determined value was equivalent to the calculated molecular mass of 36,600 Da.

Refolding of Recombinant Prethrombin-2

A variety of experimental conditions was explored to convert sulfonated prethrombin-2 into its native four-disulfide conformation. Any prethrombin-2 which, after activation to thrombin, was capable of hydrolyzing S2238, was defined as containing folded protein and is henceforth described as refolded recombinant prethrombin-2. Strong denaturant was needed to keep the sulfonated prethrombin-2 soluble during the folding process. Studies were conducted to optimize the amount of active prethrombin-2 recovered while minimizing the concentration of denaturant required. Refolding in 4 M GdnHCl led to approximately twice the amount of active prethrombin-2 as compared to refolding in 2 M GdnHCl. After refolding in 1 M GdnHCl, less than 1% of active prethrombin-2 could be recovered compared to refolding in 4 M GdnHCl. In addition, refolding in 4 M GdnHCl was 9-fold more effective than refolding in 2 M GdnSCN and 45-fold more than in 4 M urea. Based on these studies, it was concluded that the optimal refolding buffer for prethrombin-2 should contain 4 M GdnHCl.

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%).

Purification of Refolded Recombinant Prethrombin-2

Purification of refolded recombinant prethrombin-2 on a heparin affinity column was designed to take advantage of the heparin binding site on prethrombin-2. As shown in Fig. 4a, elution with NaCl led to three main peaks. The protein from peak C elutes from the heparin affinity column in the same position in the NaCl gradient as wild-type prethrombin-2 (data not shown). In addition, following activation with E. carinatus snake venom, protein from peak C was the only one that exhibited S2238 activity. Based on these results, protein from peak C is proposed to be correctly folded recombinant prethrombin-2. Folded recombinant prethrombin-2 runs on an SDS reducing gel as a single band with an apparent molecular mass consistent with that expected (Fig. 3, lane 3). The other two peaks contained proteins that run similar to refolded prethrombin-2 or as lower molecular mass contaminants (data not shown). Correctly folded prethrombin-2 eluted from the heparin column at the highest NaCl concentration and probably has the most specific interaction with heparin. Based on absorbance at 280 nm, 600-800 µg of purified refolded prethrombin-2 can be recovered from a single folding experiment starting with 30-40 mg of sulfonated recombinant prethrombin-2 isolated from 1 liter of cells. Recycling of the precipitated protein through the folding procedure twice increases the yield to 0.9-1.3 mg/liter of cells.


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.



Circular Dichroism of Recombinant Prethrombin-2

Analysis of the CD spectra of wild-type and recombinant prethrombin-2 provided an additional method for characterizing these proteins physically, independent of activity measurements. The mean residue ellipticities of the two species are comparable, suggesting that they both contain the same overall folding pattern (data not shown). The proteins exhibit predominantly beta-sheet character consistent with x-ray crystallographic studies(15) .

Large Scale Activation and Purification of Recombinant Thrombin

E. carinatus snake venom at 5 µg/ml was used to activate prethrombin-2 (20 µg/ml) to thrombin. Thrombin was purified away from the snake venom by heparin affinity chromatography (Fig. 3, lanes 4-6 and Fig. 4b). E. carinatus snake venom cleaves between the disulfide-linked A and B chains of thrombin at the factor Xa cleavage site without loss of any amino acids. It was uncertain whether or not the venom would also cleave the additional factor Xa recognition sequence (IEGR) at the N terminus of the folded recombinant prethrombin-2. N-terminal sequencing of the isolated A and B chains from recombinant thrombin gave the expected sequences, showing that the additional factor Xa site had been cleaved and that the N terminus of the A chain is the same as that in wild-type thrombin. Based on absorbance at 280 nm, 0.5-0.7 mg of purified recombinant thrombin is recovered from 1 liter of cells.

Characterization of Recombinant Thrombin

The experimentally determined molar extinction coefficients at 280 nm were found to be approximately the same for both recombinant (10.5 ± 0.47 times 10^4M cm) and wild-type (9.41 ± 0.56 times 10^4Mcm) thrombin. The steady-state parameters for hydrolysis of the chromogenic substrate S2238 and for the release of FpA from fibrinogen were compared for recombinant and wild-type bovine thrombin. As shown in Table 1, k (123 ± 10 s) and K(m) (2.91 ± 0.3 µM) determined for the hydrolysis of S2238 by recombinant thrombin are in reasonable agreement with k (97 ± 8 s) and K(m) (2.71 ± 0.25 µM) determined for wild-type thrombin. From the thrombin-mediated release of FpA from fibrinogen, a value for k/K(m) of 17.3 ± 1.2 µM s was determined for recombinant thrombin and 16.7 ± 2.0 µM s for wild-type thrombin.




DISCUSSION

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).


CONCLUSIONS

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.


FOOTNOTES

*
This research was supported in part by National Institutes of Health Grant HL-30616 from the NHLBI of the National Institutes of Health. Preliminary results of this work were presented at the ASBMB/Biophysical Society Joint Meeting, Houston, TX, February 1992, Abstr. 1092. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a National Institutes of Health postdoctoral fellowship from the NHLBI(1992-1995).

To whom correspondence and reprint requests should be addressed. Tel.: 607-255-4034; Fax: 607-254-4700.

(^1)
The abbreviations used are: FpA, fibrinopeptide A; FpB, fibrinopeptide B; GdnHCl, guanidine hydrochloride; p-APMSF, p-aminophenylmethylsulfonyl fluoride; S2238, H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline; PEG, polyethylene glycol 8000; MALDTOF, matrix-assisted laser desorption time of flight; HPLC, high performance liquid chromatography.

(^2)
Thrombin residues are numbered according to the chymotrypsinogen numbering system(11) .

(^3)
The protease responsible for cleavage of prethrombin-2 to thrombin is not a serine protease; therefore, inactivation with p-APMSF does not affect the desired proteolytic activity.


ACKNOWLEDGEMENTS

We thank R. T. A. MacGillivray for providing the plasmid pBII III containing the bovine prothrombin cDNA, F. J. Castellino for providing bovine prothrombin, T. Fanella for the generous gift of S2238, and D. M. Rothwarf for helpful discussions throughout the course of this research.


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