Stable Expression and Purification of a Secreted Human Recombinant Prethrombin2 and Its Activation...

PROTEIN EXPRESSION AND PURIFICATION 10, 214–225 (1997)ARTICLE NO. PT970733

Stable Expression and Purification of a Secreted HumanRecombinant Prethrombin-2 and Its Activation to Thrombin

Giulia Russo,* Alain Gast,† Ernst-Jurgen Schlaeger,†Antonietta Angiolillo,* and Concetta Pietropaolo**Dipartimento di Biochimica e Biotecnologie Mediche, Universita di Napoli ‘‘Federico II,’’ Via Sergio Pansini 5,Naples I-80131, Italy; and †Pharmaceutical Division, Preclinical Research Department,F. Hoffmann–La Roche, Ltd., Grenzacherstrasse 124, CH-4070 Basel, Switzerland

Received December 18, 1996, and in revised form February 10, 1997

tion of hemostasis, thrombin can interact with a wideA human prothrombin cDNA has been engineered to variety of cell types leading to a broad range of effects

obtain a cDNA coding for a secreted form of human such as platelet aggregation, chemotactic response,prethrombin-2. The secreted prethrombin-2 has been growth promotion, and neurite retraction (1). A throm-produced in a mammalian expression system using bin receptor has been identified in human (2,3), ham-DXB11 cells, a mutant strain of CHO cells in which the ster (4), and rat (5) tissues. It belongs to a seven-trans-dihydrofolate reductase gene has been deleted, and an membrane-domain G-protein-coupled receptor classexpression vector carrying the dihydrofolate reduc- that is activated by limited proteolysis by thrombintase cDNA. Methotrexate-induced gene amplification through a unique mechanism, i.e., the proteolysis un-favored an efficient production of the recombinant masks a tethered ligand.protein which accumulated in the culture medium of The multiplicity of functions exerted by thrombin inthe DXB11 cells. Growth in suspension of the stable physiologically and pathologically relevant processestransformants in an airlift fermenter resulted in the has boosted research aimed at defining the fine struc-production of 25 mg/L recombinant prethrombin-2. ture of the thrombin molecule and at dissecting it. TheThe recombinant protein was purified using single-

most widely used approach has been to produce recom-step affinity chromatography on a recombinant-hiru-binant prothrombin mutants that can be activated indin column and activated by agarose gel-immobilizedvitro (6). These studies help both to clarify the mecha-ecarin. All purified recombinant prethrombin-2 wasnisms by which thrombin interacts with different mole-activated and the generated recombinant thrombincules on different cell types and to devise molecularshowed catalytic properties identical to those oftools to control each of the functions exerted by throm-plasma-derived a-thrombin. This expression systembin (7). We have cloned a human prothrombin cDNAcan be used to prepare mutants of prethrombin-2 forand we have engineered it so as to obtain a cDNA cod-structure–function studies investigating thrombin in-ing for prethrombin-2, a physiological precursor of a-teractions with substrate proteins, inhibitors, and cell

membranes. q 1997 Academic Press thrombin, which is efficiently secreted by eukaryoticcells thus facilitating the purification process. A mam-malian expression system, in a dihydrofolate reduc-tase-deficient (dhfr0) CHO strain (DXB11), is potenti-ated by methotrexate (MTX)-induced gene amplifica-Thrombin (EC 3.4.21.5) is a serine protease thattion. This results in a higher yield of the activeplays a central role in hemostasis and regulates bothrecombinant protein compared to the expression sys-the procoagulant and the anti-coagulant pathway. Intems reported so far (8). Furthermore, we show thatfact, thrombin catalyzes the formation of insoluble fi-recombinant human prethrombin-2 can bind hirudinbrin, initiating further reactions that are of decisiveand that this interaction can be used in a one-stepimportance in hemostasis as well as in the inductionaffinity chromatography procedure to purify to homoge-of thrombosis. On the other hand, thrombin modifiedneity the secreted, recombinant prethrombin-2. Fi-by the interaction with thrombomodulin on the surfacenally, the recombinant prethrombin-2 is quantitativelyof endothelial cells can activate an anti-coagulant path-

way via protein C. In addition to its role in the regula- activated by ecarin, and the generated recombinant

214 1046-5928/97 $25.00Copyright q 1997 by Academic Press

All rights of reproduction in any form reserved.

AID PEP 0733 / 6q10$$$121 06-05-97 00:09:27 pepa AP: PEP

HUMAN RECOMBINANT PRETHROMBIN-2 PRODUCTION 215

thrombin is structurally correct and functionally ac- with HindIII to allow the insertion of the s-prethrom-bin-2 cDNA. This cloning strategy restored an XbaI sitetive.at the 3*-terminus of the s-prethrombin-2 cDNA. Theresulting vector, pDEX-s-prethrombin-2 (Fig. 1), wasMATERIALS AND METHODSused in all transfection experiments to generate tran-

Materials. Human a-thrombin was purified follow- sient as well as stable transformants.ing the procedure described by Fenton et al. (9). The

Cell culture, DNA transfection, and cell line selection.thrombin preparation contained 90% a-thrombin as de-HeLa (ATCC-CCL 2.2) and HepG2 (16) cells weretermined by active-site titration with p-nitrophenyl-p*-grown in Dulbecco’s modified MEM medium, supple-guanidinobenzoate (NPGB) (10,11) and SDS–PAGE.mented with 10% fetal calf serum, 5 mM glutamine,The chromogenic substrate for thrombin was H-D-Phe-and penicillin–streptomycin (50 U/ml and 50 mg/ml,pipecolyl-L-Arg-paranitroanilide (S-2238) from Chro-respectively). For the transient expression assay, DNAmogenix AB (Endotell, Allschwil, Switzerland). Ecarintransfections were performed using the calcium phos-purified from Echis carinatus venom and ecarin aga-phate precipitation technique (17). Cells (1 1 106 cells/rose gel were kindly provided by Dr. K. Stocker (Penta-10-cm dish or 5 1 105 cells/6-cm dish) were transfectedpharm, Basel, Switzerland). Recombinant desulfatohi-with 20 and 10 mg of plasmid DNA, respectively; therudin (CGP 39393) was obtained from Dr. R. B. Walliscalcium phosphate precipitate remained on the cells(Ciba Pharmaceuticals, Horsham, Sussex, UK). Allfor 16–18 h. For RNA and protein analysis, transfectedother reagents were reagent grade.cells and culture medium were harvested 24 h later.

Site-directed mutagenesis and construction of a DXB11, a dihydrofolate reductase-defective (dhfr0)pDEX-recombinant prethrombin-2 vector. A cDNA mutant CHO cell line (18), was obtained from Dr. L.coding for the prethrombin-2 fragment of the human Chasin (Columbia University, New York). The cellsprothrombin molecule was constructed by site-directed were maintained in F12 medium in the presence of 10%mutagenesis (12) starting from a 5*-truncated pro- fetal calf serum and antibiotics (penicillin–streptomy-thrombin cDNA clone in M13mp18 (13). A 28-mer oligo- cin 50 U/ml and 50 mg/ml, respectively). For DNA trans-nucleotide (5*-GGGCCATCGAATTCCACCGCCACA- fection experiments 20 mg of pDEX-s-prethrombin-2AG-3*) was used to insert an EcoRI recognition se- DNA was used to transfect exponentially growing cellsquence in the region where Factor Xa mediates the (5 1 105 cells/6-cm dish) by the calcium phosphate pre-proteolytic cut generating prethrombin-2. cipitation technique (17). The calcium phosphate pre-

The cDNA sequence coding for the leader peptide cipitate remained on the cells for 9 h. Seventy-twoof the human tissue-type plasminogen activator was hours later the cells were split 1:3 in 10-cm dishes con-derived from the Tndhbb vector, kindly provided by M. taining nucleoside-free F12 medium supplementedReff (14), as a HindIII–BglII fragment and cloned into with 10% dialyzed fetal bovine serum (selection me-the HindIII–BamHI sites of M13mp19. The resulting dium). Transfected cells were fed every 4–5 days withvector is designated ‘‘mp19-Lp.’’ selection medium until colonies appeared, which wasThe prethrombin-2 cDNA sequence delimited by about 12–15 days after subculturing. These trans-EcoRI sites in M13mp18 was excised and cloned into

formants were pooled in a T75 flask and grown in selec-the EcoRI site of the mp19-Lp vector. The clone con-tive medium until confluent. At confluence, the cellstaining the correct orientation of the prethrombin-2were distributed as follows: 1 1 106 cells were platedcDNA was used in a second round of mutagenesis di-in a 10-cm dish and grown in mass culture. Cell lysatesrected by a 38-mer synthetic oligonucleotide (5*-GTA-and medium were then analyzed for the expression ofCTCACTTGTGGCGGTTCTGGCTCCTCTTCTGA-recombinant s-prethrombin-2 by Western blot (19); theATC-3*), which enabled us to construct a correct cDNAremaining cells were plated in a 24-well plate: 6 wellssequence. The cDNA, namely recombinant secretedat 5 1 104 cells/well and 18 wells at 5 1 103 cells/prethrombin-2 (s-prethrombin-2), coded for a chimericwell. Two days later the medium was replaced with theprotein containing a leader peptide derived from theselection medium containing 20 nM MTX. Individualhuman tissue-type plasminogen activator followed byresistant colonies were isolated, expanded, and thenthe prethrombin-2 amino acid sequence. The resultinganalyzed for the expression of recombinant s-prethrom-M13 vector was designated ‘‘mp19-s-prethrombin-2.’’bin-2 by Western blot. Transformants expressingThe mp19-s-prethrombin-2 DNA was linearized withhigher amounts of recombinant prethrombin-2 wereEcoRI, the ends were filled with the Klenow fragmenttreated with several rounds of increasing concentra-of DNA polymerase I, then the s-prethrombin-2 cDNAtions of MTX.was released by cutting with HindIII endonuclease and

RNA analysis. The expression of the recombinantwas cloned into the pDEX vector, a derivative of thes-prethrombin-2 cDNA was revealed by Northern blotsT4DHFR vector (15). This vector had been linearized

with XbaI and the ends were made blunt and then cut analysis on total RNA prepared from either transient


RUSSO ET AL.216

FIG. 1. Map of pDEX-s-prethrombin-2 vector for the expression of a secreted prethrombin-2. For details see Materials and Methods andDeen et al. (15).

or stable transformants. The RNA was prepared ac- Antibodies. Polyclonal anti-human prothrombinantibodies from rabbit were from Nordic (Tilburg, Thecording to Chomczynski and Sacchi (20), fractionated

on 1.5% agarose, blotted onto a nitrocellulose filter, and Netherlands). Antibodies against D-Phe-Pro-Arg-chlo-romethylketone (PPACK)-inactivated human a-throm-hybridized with a specific probe, 32P-labeled by nick

translation or random priming (21). bin were raised in rabbits, and the IgG fraction of theantiserum was purified on a protein A–Sepharose col-Protein analysis. For protein analysis cells wereumn (Pharmacia, Uppsala, Sweden). An aliquot of thisplated on a 6-cm dish. When cells reached confluence,IgG fraction (0.84 mg/ml) was biotinylated by incuba-medium (3 ml) was collected and centrifuged at 3000tion with 50 mg/ml Biotin-X-NHS (Calbiochem, Sanrpm to remove cell debris, then 3 ml of a 50% acetone–Diego, CA) for 30 min at room temperature and exten-20% trichloroacetic acid (TCA) solution was added andsive dialysis at 47C against 10 mM Hepes, 150 mMthe sample was kept for 1 h on ice. The precipitate wasNaCl, pH 7.4. These antibodies were used in the ELISAthen collected by centrifugation and resuspended in 10(see below).ml of 0.5 M Tris–base containing 3% SDS. The cells

were collected in 1 ml phosphate-buffered saline (PBS), Quantification of recombinant prethrombin-2 andthrombin by ELISA. The amount of recombinant pro-centrifuged, and resuspended in 100 ml of lysis buffer

containing 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 tein present in the cell culture supernatants and inthe pooled chromatographic fractions (see below) wasmM MgCl2, 1% Triton X-100, and phenylmethylsulfo-

nyl fluoride, freshly added to a final concentration of estimated by a sandwich ELISA. Between each step,the wells were washed three times with 200 ml of PBS–0.25 mM. The lysate was kept on ice for 5 min, then

centrifuged at 13,000 rpm for 10 min in an Eppendorf 0.05% Tween 20. The wells of microtiter plates werecoated overnight at 47C with 100 ml (2 mg/ml) of rabbitcentrifuge. Fifty microliters of the supernatant was

TCA precipitated and resuspended in 25 ml of 0.5 M IgG against PPACK–a-thrombin in 0.1 M carbonatebuffer, pH 9.6. After saturation with 2% BSA in PBSTris–base–3% SDS. Protein samples were analyzed on

a 12% acrylamide SDS–PAGE (22). The production of for 2 h at 377C, 100 ml of sample (recombinant pre-thrombin-2 or thrombin) diluted in PBS–0.05% Tweenthe recombinant prethrombin-2 was revealed by West-

ern blot (19). 20 containing 1% BSA (PBS–BSA) was added to the



TABLE 1wells and incubated for 2 h at 377C. After washing, 100ml of rabbit biotinylated IgG against PPACK–throm- Purification of Recombinant Prethrombin-2 by

Hirudin–Sepharose Affinity Chromatographybin, 2.5 mg/ml in PBS–BSA, was added to the wellsand kept for 2 h at 377C. The bound biotinylated anti-

Volume Protein r-Thrombin Purity Yieldbody was detected using preformed streptavidin–bio-Sample (ml) (mg) (mg) (%) (%)tinylated horseradish peroxidase complexes (Amer-

sham, UK) and ABTS (2,2*-azino-di[3-ethylbenzothia- Starting medium 108 338 27.1 8.0 100.0zolin sulfonate (6)] (Boehringer Mannheim, FRG) as Flowthrough 157 314 0.4 0.1 1.5

Wash 57 3 0.1 3.3 0.4peroxidase substrate. Absorbance was measured at 4051 M NaCl eluate 116 29 26.3 90.7 97.1nm using the microtiter plate reader. The linear rangesGly, pH 2.8 eluate 84 1 0.1 10.0 0.4of standard curves determined with a-thrombin and

recombinant prethrombin-2 were about 0–11 and 0– Note. Recombinant prethrombin-2 was purified from a 10-fold-con-15 ng/ml, respectively. centrated DXB11 supernatant using a recombinant-hirudin–Sepha-

rose column, as described under Materials and Methods. ProteinLarge-scale production of recombinant prethrombin-concentration was determined by dye-binding using the BCA protein

2 in DXB11 cells. The recombinant CHO–DXB11 assay kit from Pierce with BSA as standard. Recombinant thrombincells secreting human prethrombin-2 were grown in (r-Thrombin) concentration was determined after activation by

ecarin for 1 h at 377C using a chromogenic substrate assay with a-suspension using DHI medium supplemented withthrombin as standard. Purity is expressed as the fraction of the0.75% fetal calf serum in the presence of 20 nM MTX.amount of recombinant thrombin to that of the total proteins.DHI medium is a mixture of DME, Ham F12, and

IMDM in the respective proportions of 1:1:2 (by weight)supplemented with human transferrin (6 mg/ml), insu- (108 ml, starting medium in Table 1) was loaded ontolin (5 mg/ml), and Primatone RL (0.3%), as described the recombinant hirudin–Sepharose column (1.6 1 10previously (23). Addition of 1.33 ml/L of a lipid mixture, cm, 20 ml resin) equilibrated with 0.11 PBS at a flowwhich is used in insect cell culture (24), supports cell rate of about 0.5 ml/min. The column was washed withgrowth and productivity of these recombinant DXB11 the same buffer to remove nonspecifically bound mate-cells. rial. Recombinant prethrombin-2 was then eluted with

The scale-up production was performed in a 23-liter PBS containing 1 M NaCl at a flow rate of about 1airlift fermenter (Chemap AG, Switzerland) without ml/min. Preliminary experiments showed that boundselection pressure. The fermenter was inoculated with recombinant prethrombin-2 was quantitatively and21 105 cells/ml and cultured under standard conditions specifically recovered with this elution buffer. The elu-(pH 7.1–7.3, t Å 377C, pO2 Å 30%) for a final cell den- ate containing recombinant prethrombin-2 was imme-sity of 1.5–2 1 106 cells/ml. After reaching a cell titer diately deep frozen in small aliquots and kept at 0807Cof 1 1 106 cells/ml, the fermenter culture was fed with until use.a mixture of glucose, glutamine, other amino acids, and The concentration of recombinant prethrombin-2vitamins to support further growth and protein produc- was determined in the pooled fractions of each purifi-tion. After 7 days, the bioreactor was harvested and cation step after ecarin activation. An aliquot dilutedthe cells were separated by continuous centrifugation in 50 mM Tris, 100 mM NaCl, 0.1% PEG 6000, pH 7.8,(Heraeus Varifuge 20RS, Germany) at 107C. The cell- was incubated for 1 h at 377C with ecarin (molar ratiofree supernatant was immediately concentrated 10-fold ecarin:recombinant prethrombin-2 of about 1:5). Pre-using an Amicon SP20 ultrafiltration unit with a mem- liminary experiments showed that the activation of re-brane cutoff of 10 kDa and was stored in three portions combinant prethrombin-2 was complete after 1 h underof about 700 ml at 0807C, to be used for protein purifi- these conditions (data not shown). The amount of gen-cation. erated thrombin was assessed by following kinetically

the cleavage of S-2238 (final concentration 100 mM) atPurification of recombinant prethrombin-2. Recom-405 nm with a microtiter plate reader (Thermomax;binant prethrombin-2 was purified by affinity chroma-Molecular Devices, Menlo Park, CA). The concentrationtography with recombinant desulfatohirudin immobi-of generated active recombinant thrombin was readlized on cyanogen bromide-activated Sepharose-CL4Bfrom a standard curve obtained using a-thrombin. The(5 mg desulfatohirudin/ml resin), using the procedureprotein concentration was determined by dye-bindingdescribed by the manufacturer (Pharmacia, Sweden).using the bicinchoninic acid (BCA) protein assay kitAll purification steps were performed at 47C. An aliquot(Pierce, The Netherlands) with a-thrombin as stan-of 103 ml of concentrated supernatant was thawed atdard.377C and dialyzed (Spectrapor membrane, MW cutoff

12–14 kDa) extensively against 1:10 diluted PBS (0.11 Large-scale activation of recombinant prethrombin-2PBS) containing 0.01% thimerosal. After centrifuga- using ecarin-agarose. Purified recombinant pre-

thrombin-2 (3.125 ml at 0.2 mg/ml) was added to 3.75tion for 15 min at 10,000g, the recovered supernatant


RUSSO ET AL.218

ml of 50 mM Tris–HCl, 100 mM NaCl, 0.1% PEG 6000, a-thrombin 1 nM (final concentration 0.5 nM) wasadded to 125 ml of S-2238 (eight concentrations, 0.78–pH 7.8, and 3.2 ml ecarin (2.1 mg/ml resin, specific

activity 304 IU/mg ecarin) immobilized on agarose, pre- 100 mM final). The velocity of the reaction was moni-tored at 405 nm for 3 min with the kinetic microtiterequilibrated in the activation buffer. After incubation

at 377C for 1 h with mild shaking, the activation mix- autoreader. The amount of product formed was calcu-lated by using an extinction coefficient of 9920 M01 cm01ture was placed in a cellulose acetate centrifuge filter

unit (0.22 mm, Spin-X; Costar, Cambridge, MA) and for p-nitroaniline at 405 nm (27). Km values were de-rived from Eadee–Hoffstee plots (28) and kcat valuescentrifuged for 30 s at 10,000g. Preliminary experi-

ments showed that the activation of recombinant pre- were calculated using the formula kcat Å Vmax/concen-tration of thrombin.thrombin-2 was complete after 1 h under these condi-

tions (see Fig. 5). The activated recombinant prethrom- Inhibition of recombinant thrombin by napsagatran.bin-2 contained in the filtrate was immediately divided To evaluate further the functionality of the active siteinto 50-ml portions and deep frozen. All the functional of recombinant thrombin, we compared the inhibitorystudies described below were performed with this batch activity of napsagatran, a new potent and selectiveof activated recombinant prethrombin-2, namely re- thrombin inhibitor (29) against a-thrombin and recom-combinant thrombin. binant thrombin. Assay conditions were the same as

Amino-terminal sequence analysis and mass spec- for the determination of Km for S-2238. Recombinanttroscopy. Purified recombinant prethrombin-2 and re- thrombin or a-thrombin (final concentration 1 nM) wascombinant thrombin were subjected to automated Ed- incubated for 5 min with napsagatran (final concentra-man degradation over 10 to 20 cycles, and amino-termi- tions 0.15–10 nM). The reaction was started with S-nal sequence analysis was performed on an Applied 2238 (final concentration 100 mM) and monitored forBiosystems 494A pulsed liquid protein sequenator 5 min. IC50 values were derived from dose–response(Foster City, CA), with on-line phenylthiohydantoin- curves of the percentage of residual activity iterativelyamino-acid derivative detection. Before mass spectros- fitted to a sigmoid function [y Å 100/(1 / exp[a*(x 0copy analysis, the protein samples were desalted on a b)])] on a VAX computer under RS1 (BNN SoftwaremPoros R2/H column (0.8 1 5 mm). The analysis was Products Corporation, Cambridge, MA).performed in the positive ion mode, using a nanoelec- Plasma clotting activity. The clotting activity of re-trospray source (25) implemented on a triple quadru- combinant thrombin was measured in a thrombin timepole instrument (API III; SCIEX, Thornhill, Ontario, (TT) clotting assay using an automated coagulometerCanada). Scans between m/z 1300 and 1800 were re- (ACL 300R; Instrumentation Laboratory, Milan, Italy).corded with 0.2 average mass unit steps. Clotting was initiated by adding 75 ml of human ci-

Active site titration with NPGB. The active site trated platelet-poor plasma to 75 ml of recombinantconcentration of recombinant thrombin was deter- thrombin or a-thrombin diluted in TT buffer containingmined in microtiter plates with the active site titrant 100 mM CaCl2 (Thrombin Time IL Test kit from Instru-NPGB (10), according to the method described by mentation Laboratory). Four concentrations of recom-Fasco and Fenton (11). binant thrombin or a-thrombin were tested in parallel:

3.75, 7.5, 15, and 30 nM (final concentrations). Concen-Titration with recombinant hirudin. All reactantstrations required for a clotting time of 100 s were deter-were diluted in 50 mM Tris–HCl, 100 mM NaCl, 0.1%mined from plots of concentration versus clotting time.PEG 6000, pH 7.8, and the assay was performed at

room temperature in microtiter plates. Recombinant Platelet aggregating activity. To determine the pro-thrombin (2 nM based on protein concentration) was aggregatory activity of recombinant thrombin in com-incubated for 5 min with known amounts of recombi- parison with a-thrombin, we measured platelet aggre-nant hirudin (0.3–2 nM based on amino acid analysis). gation in human platelet-rich plasma or with gel-fil-After the addition of 100 mM S-2238, the velocity of the tered platelets.reaction was followed at 405 nm for 5 min using a Platelet-rich plasma was isolated by centrifugationkinetic microtiter autoreader. Under these experimen- of citrated whole blood for 10 min at 160g. Gly-Pro-tal conditions, hirudin inhibits thrombin in a stoichio- Arg-Pro (Bachem, Bubendorf, Switzerland) was addedmetric manner. Thus, the plots of the velocities versus to a concentration of 2 mM to prevent fibrin polymeriza-the concentration of hirudin can be fitted by linear re- tion (30). Aggregation was initiated by the addition ofgression, the intercept on the x axis corresponding to various amounts of recombinant thrombin or a-throm-the concentration of active thrombin (26). bin and the change of light transmission was measured

in a dual-channel aggregometer (ELVI, Milan, Italy) atDetermination of the Michaelis constant (Km) for S-377C. One concentration of recombinant thrombin and2238. Assay conditions were the same as for titrationone concentration of a-thrombin were tested in parallelwith recombinant hirudin. One hundred twenty-five

microliters of activated recombinant prethrombin-2 or for each experiment.



Platelets were isolated from fresh human platelet-rich plasma by gel filtration on Sephadex G25 (Phar-macia) in Tyrode’s buffer containing 0.2% BSA. CaCl2

was added to a concentration of 1 mM and the plateletswere left to settle for 20 min at 377C before use. Thegel-filtered platelets were incubated in the presenceof 0.8 mM human fibrinogen (fibronectin free; IMCO,Stockholm, Sweden). Aggregation was initiated by theaddition of various amounts of recombinant thrombinor a-thrombin as described above.

Extent of aggregation was determined by measuringthe peak value of percentage of transmission. The con-centrations required for 20% of maximal stimulationof platelet aggregation (EC20) were determined fromplots of the concentration dependence of aggregation.

RESULTS AND DISCUSSION

Transient expression of recombinant prethrombin-2.We used a cDNA coding for human prothrombin (19)to generate, by oligonucleotide-directed mutagenesis, aprethrombin-2 cDNA. This cDNA was inserted into avaccinia virus-derived vector and expressed in HepG2 FIG. 2. Transient expression of recombinant prethrombin-2 in

HeLa and HepG2 cells. (A) Northern blot analysis. 10 mg of totalcells (G. Russo, unpublished). A protein of the expectedRNA prepared from HeLa and HepG2 cells 48 h after transfectionmolecular size, recognized by an anti-prothrombin an-was analyzed. (B) Western blot analysis. Western blotting was per-tiserum, was produced, but the efficiency was rather formed as described elsewhere (19). Lanes a, cell lysate or TCA-

low compared to the production of prothrombin in the precipitated culture medium from control cultures. Lanes b, cell ly-same expression system and, furthermore, the recombi- sate or TCA-precipitated culture medium from cultures transfected

with 10 mg of pDEX-s-prethrombin-2 DNA.nant protein was retained in the cells, thus making itspurification and functional characterization difficult.

To improve efficiency, we provided the recombinantthrombin mRNA (2.1 kb) (19). The amounts of RNAprotein with a leader peptide that could direct the se-analyzed for HepG2 and for HeLa cells were compara-cretion of the product into the culture medium. To thisble, as controlled by probing the filter with a b-actinaim we cloned a sequence coding for the leader peptideprobe (not shown); however, a better transfection effi-of the human tissue-type plasminogen activator (14)ciency could be responsible for the higher level of re-into M13mp19, and engineered an incomplete cDNAcombinant prethrombin-2 RNA in HeLa cells. Figurecoding for human prothrombin in order to obtain a2B shows the results of the immunodetection of recom-cDNA coding for a secreted form of prethrombin-2. Webinant prethrombin-2 in culture media and cell lysates,then cloned this s-prethrombin-2 cDNA in pDEX, a de-using an anti-prothrombin antiserum (19). A specificrivative of the sT4DHFR vector (15) to produce theband of about 36 kDa, as expected for the processedpDEX-s-prethrombin-2 vector (Fig. 1). The pDEX-s-recombinant prethrombin-2, was present in the cultureprethrombin-2 vector was tested for transient expres-media from both HepG2 and HeLa cells. Thus, in bothsion in HepG2, a cell line producing constitutively pro-transient expression systems the recombinant pre-thrombin in addition to several other plasma proteinsthrombin-2 RNA was expressed and the protein was(16), and in HeLa cells, in order to verify whether theefficiently secreted into the cell culture medium. Inrecombinant prethrombin-2 would be properly se-fact, in the Western blot shown in Fig. 2B, cell lysatescreted. Ten micrograms of pDEX-s-prethrombin-2 DNAare completely devoid of recombinant prethrombin-2,was transfected into both cell types; 48 h after transfec-indicating that an efficient processing of the recombi-tion, media and cells were harvested and RNA andnant protein by the leader peptidase had occurred. Inprotein were analyzed. Figure 2A shows a Northernaddition, the antiserum detected the prothrombin en-analysis on RNA from HepG2 and HeLa cells probeddogenously produced (72 kDa) and secreted in HepG2with a prothrombin cDNA labeled with 32P by nickculture medium.translation (21). An RNA species of the expected molec-

ular size (1.2 kb) was specifically recognized by the Constitutive, stable expression of recombinant pre-thrombin-2 in DXB11 cells. To produce large amountsprothrombin cDNA probe in both cell types; in HepG2

cells the probe also recognized the endogenous pro- of recombinant prethrombin-2, we chose to transfect


RUSSO ET AL.220

various clones resistant to MTX were fractionated onSDS–PAGE and probed with anti-prothrombin antise-rum. The recombinant prethrombin-2 is detected as adoublet in the medium of the dhfr/ cells. The presenceof two molecular species, in contrast with the sharpsingle band obtained by transient expression in HeLaand HepG2 cells (Fig. 2B), is due to a heterogeneousglycosylation pattern in DXB11 cells, since the treat-ment with 4 mg/ml tunicamycin, a well-characterizedinhibitor of N-glycosylation, resulted in a single proteinband with a reduced apparent molecular size (resultsnot shown).

The amounts of recombinant protein obtained fromthe various MTX-resistant clones correlate with thespecific RNA amplification pattern. With the best pro-ducer clones (clone 6 and clone 8) we obtained 10–12 mg of recombinant protein per milliliter of culturemedium/107 cells in 12 h.

FIG. 3. Expression of the recombinant prethrombin-2 in DXB11 Genomic analysis of the stable recombinant pre-cells. (A) Northern blot analysis. 10 mg of total RNA from the untrans-thrombin-2 transformants. The integration pattern offected cells (DXB11), the transfected cells exposed to selection me-

dium (dhfr/ pool), and three different clones of MTX-resistant cells the pDEX-s-prethrombin-2 DNA in the genome of thewas fractionated on 1.5% agarose gel, transferred onto a nitrocellu- stable transformants was determined by a Southernlose filter, and probed with a recombinant prethrombin-2 cDNA analysis (21) carried out on the DNA of three trans-probe 32P-labeled by nick translation. (B) The same nitrocellulose

formant cell lines and on the dhfr/ pool of transformantfilter as in A was probed with a 32P-labeled synthetic oligonucleotideclones, using a recombinant prethrombin-2 and a dhfrcorresponding to a 28S rRNA sequence representing a single-

stranded domain which is conserved among species (40), as an inter- cDNA probe (Fig. 4). In clone 6 and clone 8, both resis-nal standard to quantitate the RNA on the filter. (C) Immunodetec- tant to 40 nM MTX, both probes detected two majortion by Western blotting of the recombinant prethrombin-2 in un- fragments of 5.4 and 3.5 bp, respectively, showing thattransfected DXB11 cells, transfected cells exposed to selection me-

the recombinant prethrombin-2 and the dhfr cDNA un-dium (dhfr/ pool), and MTX-resistant clones. Human a-thrombinpurified from plasma was used as a control. derwent the same fate and that there were two major

integration sites in the DXB11 genome. The integrationsites were different in clone 80 where a major rear-rangement seems to have taken place. Indeed, a DNApDEX-s-prethrombin-2 DNA into DXB11, a mutant fragment is recognized by the recombinant prethrom-strain of CHO cells in which the dhfr locus has been bin-2 probe but not by the dhfr probe. However, indeleted (18). In these cells the dhfr gene carried by the all the transformant lines analyzed the observed genepDEX-s-prethrombin-2 vector can be used as a selec-amplification can account for the enhanced productiontion and amplification marker. After transfection,of the recombinant prethrombin-2.dhfr/ clones were selected using a nucleoside-free me-

Production, purification, and activation of recombi-dium.nant prethrombin-2. Recombinant prethrombin-2 byThe dhfr/ cells were then exposed to increasing con-DXB11 transformants (clone 6) was produced in a 23-centrations of MTX to amplify the gene and so increaseliter airlift fermenter, as described under Materialsthe production of the heterologous recombinant pre-and Methods. The concentration of recombinant pre-thrombin-2. Several rounds of MTX selection were car-thrombin-2 in the concentrated cell-free supernatantried out, starting with 20 nM MTX. Figure 3 shows thewas about 250 mg/L as measured with a sandwichRNA (Fig. 3A) and protein (Fig. 3C) analysis of someELISA. Thus, the total amount of recombinant pre-dhfr/ clones resistant to MTX. A different amount ofthrombin-2 produced in the fermenter was 575 mg.specific mRNA was detected in different clones after

Prethrombin-2 has been shown to bind several mac-treatment with the same concentration of MTX. Fur-romolecular ligands of thrombin, such as fibrinogenthermore, the recombinant prethrombin-2 RNA(31), heparin, and hirugen, a synthetic dodecapeptideamount in a clone resistant to 80 nM MTX is lower thancorresponding to the 53–64 carboxyl-terminal aminothe RNA level in two other clones resistant to 40 nMacids of hirudin (32,33). We hypothesized that pre-MTX. The MTX selection was stopped for each clonethrombin-2 could also bind hirudin, and evaluatedwhen no further amplification occurred.whether this interaction could be exploited to purifyFigure 3C shows the results obtained when culture

media from a pool of dhfr/ clones (dhfr/ pool) or from the recombinant prethrombin-2. Indeed, preliminary



showed that recombinant prethrombin-2 was quantita-tively converted to thrombin after 1-h incubation at 377C,as analyzed by SDS–PAGE and amidolytic activity (Fig.5). The electrophoretic mobility of reduced recombinantprethrombin-2 (Fig. 5, 0-time lane) was, as expected,slightly lower than that of human a-thrombin (Fig. 5,a-thr lane). Unreduced recombinant prethrombin-2 andplasma-derived prethrombin-2 had the same electropho-retic mobility as the reduced proteins (not shown). Thisactivation procedure was used to activate about 0.6 mgof purified recombinant prethrombin-2 and all functionalstudies were performed with the same batch of recombi-nant thrombin.

FIG. 4. Southern blot analysis of DXB11 cell lines resistant to MTXand secreting recombinant prethrombin-2. Genomic DNA was pre-pared from DXB11 and from the clones indicated. 15 mg DNA wasdigested to completion with HindIII restriction endonuclease andfractionated on a 0.8% agarose gel. pDEX-s-prethr indicates the vec-tor DNA analyzed under the same conditions. Following transfer ofthe gel onto a nitrocellulose filter, the filter was cut and the twofilters obtained were hybridized to a 32P-labeled recombinant pre-thrombin-2 cDNA probe (prethr-probe) or a DHFR cDNA probe(DHFR-probe). The numbers between the two analyses are molecularsize markers, in kilobase pairs.

experiments demonstrated that prethrombin-2 bindson a hirudin–Sepharose column and that the boundmaterial can be quantitatively eluted with a phosphatebuffer containing 1 M NaCl (data not shown), thus sug-gesting that the anion-binding exosite of prethrombin-2 is, at least partially, accessible for hirudin as it is forfibrinogen and hirugen. With this one-step method (seeMaterials and Methods), we obtained a good yield ofpurified recombinant prethrombin-2 from DXB11 su-pernatant (Table 1). The apparent purity of recombi-

FIG. 5. Time course of recombinant prethrombin-2 (r-prethrombin-nant prethrombin-2 was about 90% in agreement with2) activation by ecarin-agarose, analyzed by SDS–PAGE (A) and

the value estimated by SDS–PAGE (see Fig. 5). The thrombin amidolytic activity (B). 125 ml of purified recombinant pre-amount of immunoreactive protein determined by thrombin-2, 0.2 mg/ml, was added to 150 ml of 50 mM Tris–HCl, 100

mM NaCl, 0.1% PEG 6000, pH 7.8, mixed with 125 ml of ecarinELISA was slightly lower but in the same range as thatimmobilized on agarose, and incubated at 377C with mild shaking.estimated after activation with ecarin in a chromogenicAt the indicated time points, the activation mixture was placed in asubstrate assay (data not shown). Based on protein and cellulose acetate centrifuge filter unit (0.22 mm, Spin-X; Costar) and

thrombin concentration, about 25 mg of purified recom- centrifuged for 30 s at 10,000g. (A) An aliquot of 50 ml, containingbinant prethrombin-2 can be recovered from 1 liter of 1.9 mg protein, was withdrawn and subjected to SDS–PAGE (10%

polyacrylamide) under reducing conditions (4% 2-mercaptoethanol).culture medium (Table 1).The gel was stained with Coomassie blue R-250 (Sigma). a-thr, a-Ecarin immobilized on agarose was chosen to activatethrombin standard (3 mg). (B) At the same time points, an aliquot ofpurified recombinant prethrombin-2 to thrombin so as 25 ml was withdrawn to measure the concentration of generated

to avoid further purification steps to eliminate ecarin recombinant thrombin, using a chromogenic substrate assay with a-thrombin as a standard, as described under Materials and Methods.from the generated thrombin. Preliminary experiments


RUSSO ET AL.222

Thus, this stable DXB11 transformant allows theproduction of recombinant prethrombin-2 with yieldshigher than those obtained with other expression sys-tems. Furthermore, the use of immobilized ecarin toconvert prethrombin-2 into thrombin avoids furtherpurification steps which could reduce the overall yieldof recombinant thrombin. Bovine prethrombin-2 hasbeen expressed in Escherichia coli (34), extracted frominclusion bodies, and refolded in vitro. The yield of re-combinant protein based on the absorbancy at 280 nmis 30–40 mg/L of bacterial culture, but, after activation,only 600–800 mg of purified protein could be recoveredfrom 30–40 mg of starting material. Human prethrom-bin-2 also has been produced in E. coli (8); the proteinresulting from the activation of purified recombinantprethrombin-2 is 100-fold less active than thrombingenerated from the activation of human prothrombin.Recombinant human prethrombin-2 has been producedin CV-1 cells for studies on the exposure of thrombinanion-binding exosite in various prothrombin deriva-tives; however, there is no information about the yieldof this expression system (35).

Amino-terminal sequence and mass spectroscopy ofrecombinant prethrombin-2 and recombinant throm-bin. The N-terminus amino acid analysis of purifiedrecombinant prethrombin-2 showed that 75% of the re-combinant protein had a correct N-terminus (TATSEY-QTFFNPRT), whereas 25% of the recombinant proteinwas two amino acids shorter at its N-terminus (TSE-YQTFFNPRT). However, the N-termini of the A and Bchains of recombinant thrombin were identical to thoseof a-thrombin, indicating that ecarin cleaved betweenthe disulfide-linked A and B chains of recombinant pre-thrombin-2 at Arg320-Ile321 (human prothrombin num-bering), as expected, and that both forms of prethrom-bin-2 could be quantitatively activated.

FIG. 6. Mass spectrometry analysis of purified recombinant pre-The purified recombinant prethrombin-2 and recom-thrombin-2 (A) and recombinant thrombin (B). After desalting, thebinant thrombin were also analyzed by ISP-mass spec- protein samples were analyzed in the positive ion mode, using a

trometry (Fig. 6). For recombinant prethrombin-2, five nanoelectrospray source implemented on a triple quadrupole instru-major peaks corresponding to an average molecular ment (API III; SCIEX). Scans between m/z 1300 and m/z 1800 were

recorded with 0.2 average mass unit steps.mass of 36,964, 37,138, 37,312, 37,489, and 37,850 Daare present. Based on the results of the N-terminusamino acid sequence analysis, we expected two proteinswith predicted (nonglycosylated) average molecular sis, we expected a protein with a predicted (nonglyco-masses of 35,182 and 35,354 Da. The mass shifts of sylated) average molecular mass of 33,810 Da. The1782 and 1784 Da, respectively, are most probably due mass shift of 1787 Da and the mass differences betweento N-glycosylation of the Asn373, the potential glycosyla- the determined masses (about 324 Da) were in goodtion site of thrombin. The additional masses of 37,312, agreement with the values found for recombinant pre-37,489, and 37,850 Da (average difference with the de- thrombin-2. For comparison, we also analyzed a-termined masses of 350 Da) might be due to glycosyla- thrombin. Two major peaks with average moleculartion microheterogeneity. masses of 35,771 and 36,063 Da were detected (data

For recombinant thrombin, we detected four major not shown). The mass shifts of 2253 and 1961 Da, whichpeaks corresponding to an average molecular mass of are higher than the values found for recombinant35,597, 35,921, 36,243, and 36,563 Da. Based on the thrombin, suggest that the recombinant and plasma-

derived thrombin have different glycosylation patterns.results of the N-terminus amino acid sequence analy-



TABLE 2 thrombin against macromolecular substrates, we com-pared the procoagulant and proaggregatory activitiesConcentration of Recombinant Thrombin,

as Measured by Different Assays of recombinant thrombin with those of a-thrombin inhuman plasma. The concentration of recombinant

Total concentration Active site concentration thrombin needed to induce a clotting time of 100 s was(mM) (mM) 5.3 { 0.1 nM, versus 5.8 { 0.1 nM for a-thrombin. This

indicates that recombinant thrombin has the same pro-Protein ELISA NPGB Rec hirudincoagulant activity as a-thrombin (Table 3). To assess

r-Thrombin 1.28 { 0.08 1.31 { 0.07 1.35 { 0.12 1.12 { 0.13 the proaggregatory activity of recombinant thrombin,(n Å 2) (n Å 8) (n Å 3) (n Å 2) we determined the concentration of recombinant and

a-thrombin inducing a similar platelet aggregatory ef-Note. Purified recombinant prethrombin-2 was activated to throm- fect, using platelet-rich plasma or gel-filtered platelets.bin by ecarin immobilized on agarose, as described under Materials

In platelet-rich plasma, recombinant thrombin and a-and Methods. The protein concentration of recombinant thrombin (r-thrombin) was determined by dye-binding using the BCA protein thrombin at a concentration of 3 nM induced a maximalassay kit from Pierce with a-thrombin as a standard. The immunore- aggregation of platelets (data not shown). Recombinantactive concentration was determined by ELISA, using a polyclonal thrombin at a concentration of 2.6 nM induced an ag-antiserum against PPACK-thrombin, as described under Materials

gregation response similar to that of 2.5 nM a-thrombinand Methods, with a-thrombin as a standard. Active site concentra-(Table 3). On gel-filtered platelets, recombinant throm-tions were determined with the active site titrant NPGB, and by

back-titration with recombinant hirudin (see Materials and Meth- bin and a-thrombin at a concentration of 2 nM inducedods). Values are the means { SEM of n experiments. a maximal aggregation of platelets (data not shown).

Recombinant thrombin at a concentration of 1 nM in-duced an aggregation response similar to that of a-

This observation is in line with the different glycosyla- thrombin at a concentration of 1.1 nM (Table 3). Thesetion patterns recently reported for human plasma-de- results demonstrate that recombinant thrombin hasrived and recombinant prothrombin produced in CHO the same platelet aggregatory activity as a-thrombin.cells (36). Taken together, these data indicate that recombinant

prethrombin-2 is structurally correct and that its acti-Functional characterization of recombinant throm-bin. The concentration of recombinant thrombin esti- vation by ecarin generates a fully active thrombin with

catalytic properties identical to those of a-thrombinmated by four different methods is shown in Table 2.It is worth noting that the concentration of the immu- from human plasma.

In conclusion, we have established an efficient ex-noreactive species is nearly identical to the protein con-centration. These results confirm the high purity ofrecombinant prethrombin-2 and suggest that the con-

TABLE 3formations of the epitopes recognized by the polyclonalComparison of Recombinant Thrombinantibodies are similar in a-thrombin and recombinant

and a-Thrombin Activitiesthrombin. The active site concentration determined bytitration with NPGB and recombinant hirudin was

Assay r-Thrombin a-Thrombinvery similar and close to the total protein concentra-tion, indicating that nearly all recombinant thrombin

S-2238was in an active form. kcat (s01) 53.8 { 1.2 63.3 { 2.8To assess the catalytic efficiency of recombinant Km (mM) 1.5 { 0.1 1.6 { 0.2

kcat/Km (s01 mM01) 36.4 { 1.2 41.3 { 3.8thrombin, we compared the activities of recombinantn 6 4thrombin with those of a-thrombin in several assays.

Inhibition by napsagatranAs shown in Table 3, the kcat (53.8 { 1.2 s01) and Km IC50 (nM) 1.1 { 0.2 1.0 { 0.1(1.5 { 0.1 mM) of recombinant thrombin, determined n 3 3on the basis of the hydrolysis of the chromogenic sub- Clotting activity (human plasma)

Concentration (nM) for TT Å 100 s 5.3 { 0.1 5.8 { 0.1strate S-2238, are in good agreement with the kcat (63.3n 3 3{ 2.8 s01) and Km (1.6 { 0.2 mM) values of human a-

Platelet aggregation (human plasma)thrombin for the same substrate. These values of kcat EC20 (nM) (platelet-rich plasma) 2.6 2.5and Km of a-thrombin and recombinant thrombin lie EC20 (nM) (gel-filtered platelets) 1.0 1.1within the range of published values (6,37–39). In ad-

Note. Identical concentrations, based on the active site titrationdition, the IC50 values for the inhibition of recombi-with NPBG, of recombinant thrombin (r-thrombin) and a-thrombinnant thrombin and a-thrombin by napsagatran, a po-were used in the various assays. EC20 is the concentration requiredtent and selective thrombin inhibitor (29), were very for 20% of maximal stimulation of platelet aggregation. For experi-

similar (Table 3). mental details, see Materials and Methods. The results representthe means { SEM of n experiments except for platelet aggregation.To assess the proteolytic activity of recombinant


RUSSO ET AL.224

7. Gibbs, C. S., Coutre, S. E., Tsiang, M., Li, W-X., Jain, A. K.,pression system to produce a secreted form of humanDunn, K. E., Law, V. S., Mao, C. T., Matsumur, S. J., Mejza, S. J.,prethrombin-2, a single-chain immediate precursor ofPaborsky, S. L., and Leung, L. L. K. (1995) Conversion of throm-

a-thrombin. The advantages of this expression system bin into an anticoagulant by protein engineering. Nature 378,are: (i) The secreted protein efficiently accumulates in 413–416.the culture medium of the stable recombinant cell line. 8. So, I. S., Lee, S., Kim, S. W., Hahm, K., and Kim, J. (1992) Puri-(ii) The MTX-induced gene amplification has been ex- fication and activation of recombinant human prethrombin 2

produced in E. coli. Korean Biochem. J. 25, 60–65.ploited to select for high-producer cells resulting in a9. Fenton, J. W., II, Fasco, M. J., Stackrow, A. B., Aronson, D. L.,recombinant protein yield higher than in any other re-

Young, A. M., and Finlayson, J. S. (1977) Human thrombins.ported expression system. (iii) A fast single-step purifi-Production, evaluation, and properties of a-thrombin. J. Biol.cation procedure results in the quantitative recovery Chem. 252, 3587–3598.

of the recombinant protein from the cell culture me- 10. Chase, T., and Shaw, E. (1967) p-Nitrophenyl-p*-guanidinoben-dium. (iv) The purified recombinant prethrombin-2 is zoate HCl: A new active site titrant for trypsin. Biochem. Bio-fully activated by ecarin and the generated recombi- phys. Res. Commun. 29, 508–514.nant thrombin shows catalytic properties identical to 11. Fasco, M. J., and Fenton, J. W., II (1973) Specificity of thrombin.

I. Esterolytic properties of thrombin, plasmin, trypsin and chy-those of a-thrombin from human plasma. This recombi-motrypsin with substituted guanidino derivatives of p-nitrophe-nant secreted prethrombin-2 represents a very conve-nyl-p*-guanidinobenzoate. Arch. Biochem. Biophys. 152, 802–nient starting molecule for constructing thrombin mu-812.

tants to be used as tools to investigate the multiplicity12. Carter, P., Bedouelle, H., and Winter, G. (1985) Improved oligo-of thrombin interactions with substrate proteins, inhib- nucleotide site-directed mutagenesis using M13 vectors. Nucleic

itors, and cell membranes. Acids Res. 13, 4431–4443.

13. Costanzo, F., Castagnoli, L., Dente, L., Arcari, P., Smith, M.,Costanzo, P., Raugei, G., Izzo, P., Pietropaolo, T. C., Bougueleret,ACKNOWLEDGMENTSL., Cimino, F., Salvatore, F., and Cortese, R. (1983) Cloning ofseveral cDNA segments coding for human liver proteins. EMBOThe skillful assistance of Regina Visiello during the isolation ofJ. 2, 57–61.clones of recombinant prethrombin-2 is gratefully acknowledged. We

thank Dr. Christiane Chene for establishing the purification proce- 14. Connors, R. W., Sweet, R. W., Noveral, J. P., Pfarr, D. A., Frank-dure of recombinant thrombin, Urs Rothlisberger for the determina- lin, S., and Reff, M. E. (1988) DNA co-amplification of t-PA intion of the amino-terminus sequence, and Arno Friedlein for mass DHFR/ endothelial cells: In vitro characterization of the purifiedspectroscopy analysis. We also thank Klaus Christensen, Robert serine protease. DNA 7, 651–661.Ecabert, and Martine Singer for excellent technical assistance. This 15. Deen, K. C., McDougal, J. S., Inacker, R., Folena-Wasserman,work was supported by F. Hoffmann–La Roche, Ltd., Basel, Switzer- G., Arthos, J., Rosenberg, J., Maddon, P. J., Axel, R., and Sweetland, and by grants from the CNR (Progetto Finalizzato Biotecnologie R. W. (1988) A soluble form of CD4 (T4) protein inhibits AIDSe Biostrumentazioni, Progetto Finalizzato Ingegneria Genetica), virus infection. Nature 331, 82–84.from the Ministero della Universita e della Ricerca Scientifica e Tec-

16. Knowles, B. B., Howe, C. C., and Aden, D. P. (1980) Human he-nologica (MURST, Rome), and from EC (Biomed-2, BMH 4-CT96-patocellular carcinoma cell lines secrete the major plasma pro-0937).teins and hepatitis B surface antigen. Science 209, 497–499.

17. Wigler, M., Silverstein, S., Lee, L., Pellicer, A., Cheng, T., andREFERENCES Axel, R. (1977) Transfer of purified herpes virus thymidine ki-

nase gene to cultured mouse cells. Cell 11, 223–232.1. Stubbs, M. T., and Bode, W. (1993) A player of many parts: The

18. Urlaub, G., Kas, E., Carothers, A. M., and Chasin, L. (1983)spotlight falls on thrombin structure. Thromb. Res. 69, 1–58.Deletion of the diploid dihydrofolate reductase locus from cul-

2. Vu, T-K. H., Hung, D. T., Wheaton, V. I., and Coughlin, S. R. tured mammalian cells. Cell 33, 405–412.(1991) Molecular cloning of a functional thrombin receptor re-

19. Russo, G., Mertens, K., De Magistris, L., Lange, H., Cortese,veals a novel proteolytic mechanism of receptor activation. CellR., and Pietropaolo, C. (1991) Biologically active recombinant64, 1057–1068.prothrombin and antithrombin III expressed in a human hepa-

3. Vu, T-K. H., Wheaton, V. I., Hung, D. T., Charo, I., and Coughlin, toma/vaccinia system. Biotech. Appl. Biochem. 14, 222–233.S. R. (1991) Domains specifying thrombin–receptor interaction.

20. Chomczynski, P., and Sacchi, N. (1987) Single-step method ofNature 353, 674–677.RNA isolation by acid guanidinium thiocyanate–phenol–chloro-

4. Rasmussen, U. B., Vouret-Craviari, V., Jallat , S., Schlesinger, form extraction. Anal. Biochem. 162, 156–159.Y., Pages, G., Pavirani, A., Lecocq, J. P., Pouyssegur, J., and Van

21. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) ‘‘MolecularObberghen-Schilling, E. (1991) cDNA cloning and expression ofCloning: A Laboratory Manual,’’ 2nd ed., Cold Spring Harbora hamster alpha-thrombin receptor coupled to Ca2/ mobilization.Laboratory Press, Cold Spring Harbor, NY.FEBS Lett. 288, 123–128.

22. Laemmli, U. K. (1970) Cleavage of structural proteins during5. Zhong, C., Hayzer, D. J., Corson, M. A., and Runge, M. S. (1992)the assembly of the head of bacteriophage T4. Nature 227, 2922–Molecular cloning of the rat vascular smooth muscle thrombin2928.receptor: Evidence for in vivo regulation by basic fibroblast

23. Schlaeger, E-J., and Schumpp, B. (1992) Propagation of mousegrowth factor. J. Biol. Chem. 267, 16975–16979.myeloma cell line J558L producing human CD4 immunoglobulin6. Tsiang, M., Jain, A. K., Dunn, K. E., Rojas, M. E., Leung,G1. J. Immunol. Methods 146, 111–120.L. L. K., and Gibbs, C. S. (1995) Functional mapping of the sur-

face residues of human thrombin. J. Biol. Chem. 270, 16854– 24. Schlaeger, E-J., Foggetta, M., Vonach, J. M., and Christensen,K. (1993) SF-1, a low cost culture medium for the production of16863.



recombinant proteins in baculovirus infected insect cells. Bio- ski, A. (1994) The isomorphous structures of prethrombin-2, hir-ugen-, and PPACK-thrombin: Changes accompanying activationtechnol. Tech. 7, 183–188.and exosite binding to thrombin. Protein Sci. 3, 2254–2271.25. Wilm, M., and Mann, M. (1996) Analytical properties of the

nanoelectrospray ion source. Anal. Chem. 68, 1–8. 34. DiBella, E. E., Maurer, M. C., and Scheraga, H. A. (1994) Ex-pression and folding of recombinant bovine prethrombin-2 and26. Degryse, E. (1990) Determination of the specific activity of re-its activation to thrombin. J. Biol. Chem. 270, 163–169.combinant hirudin. Thromb. Res. 60, 433–443.

35. Wu, Q., Picard, V., Aiach, M., and Sadler, J. E. (1994) Activation-27. Lottenberg, R., and Jackson, C. M. (1983) Solution compositioninduced exposure of the thrombin anion-binding exosite. J. Biol.-dependent variation in extinction coefficients for p-nitroaniline.Chem. 269, 3725–3730.Biochim. Biophys. Acta 742, 558–564.

36. Fischer, B., Mitterer, A., Dorner, F., and Eibl, J. (1996) Compari-28. Hoffstee, B. H. (1952) On the evaluation of the constants Vm andson of N-glycan pattern of recombinant coagulation factors XIIKm in enzyme reactions. Science 116, 329–331.and IX expressed in Chinese hamster ovary (CHO) and African29. Hilpert, K., Ackermann, J., Banner, D. W., Gast, A., Gubernator,green monkey (Vero) cells. J. Thromb. Thrombol. 3, 57–62.K., Hadvary, P., Labler, L., Muller, K., Schmid, G., Tschopp,

T. B., and van de Waterbeemd, H. (1994) Design and synthesis 37. Wu, Q., Sheehan, J. P., Tsiang, M., Lentz, S. R., Birktoft, J. J.,of potent and highly selective thrombin inhibitors. J. Med. Chem. and Sadler, J. E. (1991) Single amino acid substitutions dissoci-37, 3889–3901. ate fibrinogen-clotting and thrombomodulin-binding activities of

human thrombin. Proc. Natl. Acad. Sci. USA 88, 6775–6779.30. Plow, E. F., and Marguerie, G. (1982) Inhibition of fibrinogenbinding to human platelets by the tetrapeptide glycyl-L-prolyl- 38. Sheehan, J. P., and Sadler, J. E. (1994) Molecular mapping ofL-arginyl-L-proline. Proc. Natl. Acad. Sci. USA 79, 3711–3715. the heparin binding exosite of thrombin. Proc. Natl. Acad. Sci.

USA 91, 5518–5522.31. Kaczmarek, E., Kaminski, M., and McDonagh, J. (1987) Fibrino-gen–Sepharose interaction with prothrombin, prethrombin 1, 39. Le Bonniec, B. F., Guinto, E. R., MacGillivray, R. T. A., Stone,prethrombin 2 and thrombin. Biochim. Biophys. Acta 914, 275– S. R., and Esmon, C. T. (1993) The role of thrombin’s Tyr-Pro-282. Pro-Trp motif in the interaction with fibrinogen, thrombomo-

dulin, protein C, antithrombin III, and the Kunitz inhibitors. J.32. Liu, L-W., Ye, J., Johnson, A. E., and Esmon, C. T. (1991) Proteo-Biol. Chem. 268, 19055–19061.lytic formation of either the two prothrombin activation interme-

diates results in formation of a hirugen-binding site. J. Biol. 40. Barbu, V., and Dautry, F. (1989) Northern blot normalizationChem. 266, 23632–23636. with a 28S rRNA oligonucleotide probe. Nucleic Acids Res. 17,

7115.33. Vijayalakshmi, J., Padmanabhan, K. P., Mann, K. G., and Tulin-


Stable Expression and Purification of a Secreted Human Recombinant Prethrombin2 and Its Activation...

Documents

Transcript of Stable Expression and Purification of a Secreted Human Recombinant Prethrombin2 and Its Activation...