THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 16, Issue of June 5. pp. 9570-9574,199O Printed in U.S. A.

Purification and Characterization of Recombinant Plasminogen Activator Inhibitor- 1 from Escherichia coZi*

(Received for publication, August 4, 1989)

Thomas M. Reilly*, Ramnath SeetharamQ, Jodie L. Dukei, Gary L. Davis, Sandra K. Pierce, Harry L. Walton, David Kingsley, and William P. Sisk From the E. I. du Pont de Nemours and Company, Medical Prodllcts Department, Experimental Station, Wilmington, Delaware 19880-0400 and the §Medical Products Department, Glasgow Site, Newark, Delaware 19719-6101

A recombinant form of plasminogen activator inhib- itor-l (rPAI-1) has been purified from lysates of pCE1200, a bacterial expression vector containing the full length PAI- gene, by utilizing sequential anion exchange and cation exchange chromatography on Q- Sepharose and S-Sepharose columns. Approximately 140 mg of rPAI-1, estimated at 98% purity on the basis of analytical high performance liquid chromatogra- phy, could be obtained from 200 g wet weight of cells. The purified protein exhibited a single Coomassie Blue-stainable band at the region of M, = 42,000 by sodium dodecyl sulfate-polyacrylamide gel electropho- resis and an NHz-terminal amino acid sequence con- sistent with the expected translation product of the pCE1200 PAI- insert. The rPAI-1 rapidly inhibited single- and two-chain tissue plasminogen activators, as well as urokinase, with apparent second order rate constants in the range of 2-5 x 10’ M-’ se’. A specific activity measurement of 250,000 units/mg was caleu- lated for the rPAI-1 based on its ability to inhibit the enzymatic activity of a single-chain tissue plasminogen activator. Stability studies showed that the activity of the rPAI-1 was very stable when stored at tempera- tures of 25 “C or lower, but decayed within hours when stored at 37 ‘C. Sodium dodecyl sulfate treatment, which partially activates the latent form of natural PAI-1, inactivated rPAI- 1. These results show that the purified rPAI-1 produced from pCE1200 displays many of the properties associated with the biologically active form of natural PAI- 1.

Tissue-type plasminogen activator (tPA)l is a serine pro- tease which converts the proenzyme plasminogen to the fi- brinolytic enzyme plasmin (1,2). The activity of tPA is highly stimulated by fibrin (3) and specifically inhibited by plasmin- ogen activator inhibitors (PAIs) (4). At present, four different types of PA1 have been identified including: PAI-1, originally identified in plasma and in endothelial cell culture fluids (5, 6); PAI-2, first demonstrated in human placenta and in preg- nancy plasma (7, 8); PAI-3, first demonstrated in urine and

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence and reprint requests should be ad- dressed.

1 The abbreviations used are: tPA, tissue-type plasminogen acti- vator; uPA, urokinase-type plasminogen activator; PA, plasminogen activator(s); PAI, plasminogen activator inhibitor(s); rPAI-1, recom- binant plasminogen activator inhibitor-l; SDS, sodium dodecyl sul- fate; PAGE, polyacrylamide gel electrophoresis; S2251, D-Val-Leu- Lys-p-nitroanilide; HPLC, high performance liquid chromatography.

subsequently shown to be identical with protein C inhibitor (9); and the protease nexin, identified in the conditioned medium of fibroblasts and other cells (10). However, because of the second order rate constants for its interaction with single-chain tPA (3.7 X lo7 M-' s-l) and two-chain tPA (1.6 x 108~-’ SK’), which are among the highest reported for enzyme- inhibitor interactions, it is generally accepted that PAI- is the principal physiological inhibitor of tPA (11, 12). Most tPA in plasma circulates complexed to PAl-1 (13, 14).

Elevated plasma PAl-1 levels are associated with an in- creased risk of thromboembolic disease (15) suggesting a critical role for the protein in the regulation of in uiuo fibrin- olysis. Increased PAI- levels have also been found in several other clinical situations including septicemia, a variety of malignancies and liver disease (16-18). However, critical bi- ological and pharmacological studies with purified protein will be required to define precise roles for PAI- in various path- ophysiological conditions.

The concentration of PAX-l in normal plasma is very low (approximately 20 ng/mI), making purification of large quan- tities of PAI- from whole blood a formidable task (19). Alternatively, PAI- has been isolated from a number of different cell lines including bovine aortic endothelial cells (6), HT 1080 human fibrosarcoma cells (20), hepatoma tissue culture rat hepatoma cells (21), and human endothelial cells (22). However, the isolated protein from these sources exists chiefly as a latent form with a very low specific activity, as determined in PA inhibition assays (6, 23). This latent form can be partially activated by treatment with denaturants such as sodium dodecyl sulfate (SDS), guanidinium hydrochloride, and urea (24), or by treatment with phospholipids (25). Recent studies suggest that PAI- is synthesized as an active form which is rapidly converted to the latent form by some un- known mechanism (26).

Expression of the PAI- cDNA in either prokaryotic or eukaryotic cells has been described by a number of groups (27-31). However, expression levels of recombinant PAI- (rPAI-l), where reported, have been extremely low. Further- more, rPAI-1 from Escherichia coli has been characterized as being present almost exclusively in the latent form (32).

In the present study, we have purified and characterized a recombinant form of PAI- expressed in E. coli from the expression vector pCE1200.” Our results indicate that sub- stantial quantities of functionally active, stable protein may be readily purified from this vector, and that this rPAI-1 shares many properties in common with the active form of natural PAI-1.

’ Sisk, W. P., Davis, G., Kingsley, D., Seetharam, R., Chiu, A. T., and Reilly, T. M. (1990) Gene (Am&.) in press.

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EXPERIMENTAL PROCEDURES

Materials-Natural PAI-1, purified from human fibrosarcoma cells, was purchased from American Diagnostica and was activated with SDS treatment as described (24). One-chain tPA, two-chain tPA, and the low molecular weight form of urokinase were also purchased from American Diagnostica.

1 IU of tPA. For stability studies, rPAI-1 in 0.05 M Tris buffer, pH 8.3, containing 0.01% Tween 80, was preincubated for various times at either 4.25, or 37 “C prior to activity determinations. Second order

Bacterial Expression of Reconbinant PAZ-l-The expression of rPAI-1 encoded bv the nlasmid ~CE1200 in E. coli host TAP106 will be described in a separate report.’ Briefly, a Xgtll phage library from human endothelial cells was screened using oligonucleotide probes directed to the 5’ end of the PAI- gene. A cDNA clone was isolated which contained a 2.1-kilobase pair segment, and a fragment of this segment containing the full PAI- coding sequence and flanking noncoding sequences was inserted into a Pi. expression vector. The resulting construct, designated pCE1200, was used to transform E. coli TAPlOG. This bacterial lysogen contains a defective X prophage including a temperature-sensitive mutant repressor gene. At low temperature (i.e. 32 “C), the mutant repressor is active, while at a higher temperature (i.e. 42 “C), the repressor is inactivated initiating transcription at the PL promoter. Preinduction conditions require growth at 32 “C in LB-derived media supplemented with ampicillin until midlog growth phase. The culture is then induced by quickly raising the temperature to 42 “C (33). A standard induction time of 4 h at 42 “C was utilized in this study unless otherwise indicated.

Purification of Recombinant PAI-l-Lysates from temperature- induced E. coli pCE1200 containing the PAI- cDNA were prepared by suspending cells (200 g, wet weight) in 10 volumes of 50 mM sodium phosphate buffer, pH 6.0, over an ice bath and disrupting the cells either by sonication for 5 min using a medium tip probe (Heat Systems Ultrasonics) or by pressure disruption using a Gaulin model 15M-8TA homogenizer (APV Gaulin, Everett, MA) at 5,000 p.s.i. The suspension containing lysed cells was centrifuged at 16,000 X g for 20 min at 4 “C, the supernatant was collected, and the pellet was resuspended in 5 volumes of buffer and resonicated as above. Super- natant collected from a 16,000 x g centrifugation of this resonicated material was pooled with the first supernatant, filtered through a 5- micron filter, and pumped onto a Q-Sepharose fast flow (Pharmacia LKB Biotechnology Inc.) column (4.4 X 30 cm), equilibrated with 50 mM sodium phosphate, pH 6.0, at a flow rate of 10 ml/min. The column was washed with the same buffer, and the effluent was collected until absorbance at 280 nm was no longer measurable (approximately 0.5 column volumes). The effluent was then loaded onto a S-Sepharose (Pharmacia) column (4.4 x 30 cm) equilibrated with 50 mM sodium phosphate, pH 6.0, the column was washed with 1 column volume of buffer, and the protein eluted using a O-l M sodium chloride gradient in buffer. PAI-1, which eluted at approxi- mately 0.4 M sodium chloride, was estimated to be 85-90% pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) and bv analvtical reversed-chase HPLC (see below). The fractions containing PAI- were pooied, diluted 2.5fold with’water, and loaded onto another S-Sepharose column equilibrated with 50 mM sodium phosphate, pH 6.0. The column was washed with 2 column volumes each of starting buffer and 50 mM sodium phosphate buffer, pH 8.0, and the PAI- eluted using 150 mM sodium phosphate buffer, pH 8.6.

Characterization of the Purified rPAZ-l-Analytical SDS-PAGE (34) was performed utilizing 8-25% gradient gels with the Pharmacia Phast system. Proteins were visualized by staining with Coomassie Blue. Analytical HPLC was performed with an HP 1090 M HPLC system. A C4 Vydac column (4.6 x 50 mm) was equilibrated with 0.1% trifluoroacetic acid in water, and the samples were applied to the column in the same solvent. Samples were eluted using a gradient of acetonitrile containing 0.1% trifluoroacetic acid.

NH?-terminal sequence analysis was obtained using automated Edman degradation chemistry. An ABI model 470 A (Applied Bio- systems) or a Porton model PI 2090 (Porton Instruments) gas-phase sequencer was employed for the degradations (35). The respective phenylthiohydantoin-derivatives were identified in an on-line fash- ion, using the HPLC standards supplied by the manufacturers. Ap- proximately 0.5-2.0 nmol of rPAI-1 was used for the sequencing.

Assay of PAI- Actiuity-PAI- activity of natural or recombinant protein was determined essentially as described (36). PAI- was incubated with single-chain tPA for 10 min at 37 “C, and the residual activity of the tPA was measured by means of a spectrophotometric assay with the chromogenic substrate Val-Leu-Lys-p-nitroanilide (S2251) according to the manufacturer’s instructions (Kabi Vitrum). One unit of PAI- is defined by the amount of protein that inhibits

rate constants for the inhibition of one-chain tPA, two-chain tPA, and uPA by rPAI-1 were calculated from the half-times of inhibition determined graphically, as previously described (37). Rate constants are expressed as equal to or more than a particular value to reflect the notion that the rPAI-1 may not be 100% functional.

RESULTS

Purification of rPAZ-PAI- antigen levels in lysates pre- pared from temperature-induced E. coli pCE1200 containing the mature PAI- gene were approximately 100 Fg/ml, as measured by an enzyme-linked immunosorbent assay specific for PAI-1. This value represented nearly 10% of the total protein content in the lysates. A procedure for purifying the rPAI-1 from bacterial lysates was developed which involved sequential passage over anion exchange (Q-Sepharose) and cation exchange (S-Sepharose) columns. Using a sodium chlo- ride gradient, PAI- eluted from the S column at approxi- mately 400 mM sodium chloride and was estimated to be approximately 8590% pure by SDS-PAGE (Fig. 1). There- fore, the PAI-l-containing fractions were further chromato- graphed on a second S-Sepharose column, and the PAI- eluted with 150 mM sodium phosphate buffer, pH 8.6. The purified rPAI-1 exhibited a single Coomassie Blue-stainable band at 42 kDa (Fig. 1). This is consistent for an unglycosyl- ated protein of 381 residues, the predicted translation product contained within the expression plasmid pCE1200. Purity was estimated at 98% on the basis of this SDS-PAGE and analyt- ical HPLC (Fig. 2). An NHz-terminal sequence of Ser-Ile-Val- His-His-Pro-Pro-Ser-Tyr-Val-Ala-His-Leu-Ala-Ser-Asp- Phe-Gly-Val was determined for the purified protein. This represents the predicted sequence for the pCE1200 rPAI-1 translation product, accounting for removal of the NH*-ter- minal Met residue by aminopeptidases in E. coli.

Inhibition of tPA by #AI-l-The purified rPAI-1 was compared with natural PAI-1, purified from human librosar- coma cells, for its ability to neutralize the enzymatic activity of tPA. A fixed amount of tPA (10 IU) was incubated with increasing amounts of either natural or rPAI-1, and the plasminogen-activating activity was determined by employing an amidolytic assay with the chromogenic substrate S2251. The results showed that the tPA activity was inhibited by increasing amounts of rPAI-1 (Fig. 3). Half-inhibition of the added tPA was achieved with approximately 20 ng of rPAI-1, yielding a specific activity of 0.25 unit/rig or 250,000 units/ mg. Subsequent lots of purified rPAI-1 ranged in activity from 0.2 unit to 0.4 unit/rig. These values represent the highest

12 3 4 5 MW x10-3

-97 ” -66

-43

-31

-22 T& 3t i - -16

FIG. 1. SDS-PAGE analysis of rPAI-1 from E. coli. Electro- phoresis was carried out as described in the text, and the gel was stained with Coomassie Blue. Lane I, protein eluted from the first S column with 400 mM NaCl; lane 2, protein eluted from the Q column; lane 3, lysate prepared from pCEl200; lane 4, rPAI-1 eluted from a second S column with 50 mM sodium phosphate buffer; lane 5, molecular weight markers.

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10 15 20 25 50

Time (mln)

FIG. 2. Analytical HPLC analysis of purified rPAI-1. Ana- lvtical HPLC was uerformed as described under “Exnerimental Pro- cedures” using an acetonitrile gradient to elute the sample.

1 1 10 100 1000 10000 PAI-1 (ng)

FIG. 3. Effect of SDS treatment on the ability of natural and rPAI-1 to inhibit single-chain tPA. 500 pg of natural or rPAl-1 (specific activity of 0.2 unit/rig) in 1 ml of 0.01 M Tris buffer, pH 8.0, was mixed with 10 ~1 of 10% SDS and incubated at 25 “C for 1 h. Subsequently, the SDS was neutralized by the addition of Triton X-100 at a concentration of l%, and samples were dialyzed for 24 h against 0.01 M Tris, pH 8.0, at 4 “C. Untreated samples were incubated for 1 h at 25 “C without detergent and then dialyzed as above; such incubation and dialysis had negligible effects on the activity of PAI- 1. The ability of each PAl-1 preparation to inhibit the enzymatic activity of 10 IU of tPA at the indicated concentrations was then assessed in an amidolytic assay employing the chromogenic substrate S2251.0, SDS-treated natural PAI-1; 0, untreated natural PAI-1; X, SDS-treated rPAl-1; n , untreated rPAl-1. Data are expressed as the percent of tPA activity observed in the absence of PAI-1.

activity reported for any recombinant form of PAI-1; the theoretical specific activity for PAI- is 650,000 units/mg when using tPA as the target enzyme (12). The activity of rPAI-1 was not increased by pretreating with SDS, a proce- dure which has been reported to promote conversion of latent to active PAI- (24). In fact, SDS treatment actually de- creased the activity of the rPAI-1 (Fig. 3).

In contrast, natural PAI- purified from human fibrosar- coma cells was essentially inactive without SDS treatment (Fig. 3). A specific activity of approximately 25,000 IU/mg was calculated for the denaturant-treated PAI-1. This lower activity, in comparison with the rPAI-1, may reflect irrevers- ible inactivation of some protein during the preparation of conditioned media and the subsequent purification steps (38, 39).

Rate Constants-The half-times of the inhibition of one- chain tPA, two-chain tPA, and uPA by rPAI-1 were used to calculate apparent second order rate constants (Table I). These values, in the range of 2-5 X lo7 M-’ s-l, are consistent with those reported for active, natural PAI- (37, 40).

Stability of rPAZ-l-Incubation of functionally active, nat- ural PAI- at 37 “C inactivates the protein, perhaps by con- verting PAI- to a latent form (24, 38). The effect of incuba- tion at different temperatures on the activity of rPAI-I was therefore determined. As shown in Fig. 4, rPAI-1 activity decayed by 50% at 37 “C in approximately 2 h. Analysis of subsequent lots revealed half-life values for rPAI-1 at 37 “C ranging from 70 to 120 min. These values compare favorably with the 90-min incubation period at 37 “C reported to pro- mote a 50% reduction in the PA1 activity in conditioned

TABLE I Kinetics of inactivation of UPA and tPA by rPAI-1

Inhibition assays were performed as described under “Experimen- tal Procedures” except that the incubation period of PA with rPAI-1 varied from O-20 min. The tH of inhibition was determined from eranhs of the data: k’ = 0.693/t8,.

Concentration of rPAI-I t, k’/[PAI-I]

A.

l3.

C.

uPA (9.2 x lo-‘*) One-chain tPA (9.9 x lo-‘*) Two-chain tPA

M s hf-’ s-1

1.5 x 1o-‘o si?3 25.9 x 10’ 3.0 x lo-‘O 546 25.0 x 107 1.2 x lo-lo 5161 ~3.6 x 10’ 2.4 x IO-” 5133 22.2 x lo7 3.0 x lo-‘O 546 a.1 x lo7

(9.9 x lo-‘*) 6.0 x lo-“’ 526 24.4 x lo7

0 24 4s 72 9s IZO 144 168 192 Time (hr)

FIG. 4. Stability of rPAI-1 to incubation at different tem- peratures. 500 pg of rPAl-1 (specific activity of 0.2 unit/rig) in 1 ml of 0.01 M Tris buffer, pH 8.0, was incubated at either 4 “C (O), 25 “C (O), or 37 “C (x). At the indicated times, the ability of 50 ng of rPAI- 1 from each temperature group to inhibit 10 IU of tPA was determined by means of an amidolytic assay. Data are expressed as the percent of tPA activity observed in the absence of PAI-1.

TABLE II Effect of incubation at 42 “C on the expression and activity of rPAI-1

by the pCE12OO/TAP106 bacterial expression vector The bacterial culture was induced by raising the temperature to

42 “C, and, at the indicated time points, l-ml samples were collected. Lysates prepared from these samples were assayed for PAl-1 levels and activity as described under “Experimental Procedures.” The level of PAI- in the uninduced culture was negligible. All values are the mean of trinlicate determinations.

Time PAI- antieen PAI- activitv Smcifk activitv

dml IV/ml IUlng 19.7 4,875 0.24 67.8 21,250 0.31

109.1 25,500 0.23 145.2 27,000 0.19 212.5 25,800 0.12 283 10,500 0.037 310 9.380 0.030

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medium from human endothelial cells (39). At a lower tem- perature of 25 “C, the rPAI-1 lost 50% of its activity after incubation for 2 days. The rPAI-1 was very stable at 4 “C, with only a 15% loss in activity observed following a ‘I-day incubation period (Fig. 4). No decrease in specific activity has been observed following storage of rPAI-1 at -20 “C for a l- year period.

The temperature sensitivity of rPAI-1 prompted us to ex- amine protein expression by the pCE1200 bacterial expression vector since induction of this particular vector requires incu- bation at 42 “C. For this purpose, samples were collected from a bacterial culture at various time points after incubation at 42 “C, crude lysates were prepared, and PAI- antigen levels as well as activity levels were determined for each sample. The results of this analysis, shown in Table II, indicate that the antigen level of PAI- increases continuously during the course of the 7-h study. However, the specific activity of the rPAI-1, expressed as the ratio of activity to antigen, actually peaked at 2 h and declined thereafter. Apparently prolonged incubation of the pCE1200 expression system at 42 “C! results in a greater quantity of rPAI-1, but with a lower degree of activity.

DISCUSSION

We have developed a procedure for the purification of rPAI- 1 from the E. cd expression vector pCE1200 in sufficient amounts to allow biochemical characterization of the protein. Approximately 140 mg of protein could be purified from 200 g wet weight of cells over a period of 9 days, using a procedure that included sequential application of material over anion and cation exchange columns. Purity of the protein collected from a second cation column run was assessed at 98% on the basis of analytical SDS-PAGE and HPLC. The purified pro- tein exhibited a single Coomassie Blue-stainable band at 42 kDa and an NHa-terminal amino acid sequence consistent with the unglycosylated translation product of the pCEl200. From other studies (41,42), two forms of mature natural PAI- 1 have been identified: a 379-amino acid protein with an amino terminus of Val-His-His; and a 381-amino acid protein with an amino terminus of Ser-Ala-Val-His-His. The rPAI-1 described in this report corresponds to the 381-amino acid form with the exception of an IIe substitution for Ala at residue 2.

The recombinant protein was functionally active, as as- sessed in studies where enzymatic activity of tPA was meas- ured using an amidolytic assay. Specific activity calculations yielded a value for rPAI-1 of 250,000 units/mg, making it among the highest value reported for purified PAI- from any source. The theoretical specific activity of PAI- is 650,000 IU/mg when using tPA as the target enzyme (12). Our results confirm that glycosylation is not required for functional ac- tivity of PAI- (29). The rPAI-1 was remarkably stable at 4 “C, with little loss in activity observed following a 7-day incubation period. Activity of the rPAI-1 did decay upon storage at higher temperatures of 25 “C and 37 “C, as has been reported for PAI- purified from other sources (24). However, the 2-h half-life compares favorably with the 90-min half-life reported for PAI- activity in conditioned medium of human endothelial cells (39).

While many procedures have been described for purification of PAI-1, in most instances the resulting protein has been functionally inactive or at best a few percent active (6, 20,21, 22, 24). These latent PAI- preparations may be activated by treatment with denaturants such as SDS; however, even fol- lowing such treatment, most isolated PAI- preparations con- tain less than 5% active molecules. This low degree of acti-

vation may be due to irreversible inactivation of some protein during the purification process (38, 39). Interestingly, treat- ment with SDS, which increased the activity of native PAI- purified from human fibrosarcoma cells, actually decreased the activity of the purified rPAI-1. These results are consist- ent with findings from a recent study (43) which suggested that SDS exerts dual effects on PAI-1: enhancing the inhibi- tory activity of essentially inactive or latent PAI- prepara- tions, while partially inactivating active PAI- preparations.

More recently, it has been suggested that activity of PAI- is dependent upon its association with vitronectin, an adhe- sive protein found in plasma (44), platelets (45), urine (46), and the extracellular matrix (47). Functionally active PAI- purified from HT 1080 cells (48) and from HepG2 hepatoma cells (43) was associated with vitronectin and/or its NH2- terminal fragments. In contrast, purified HT 1080 PAI- which did not contain detectable amounts of vitronectin ex- pressed only limited inhibitory activity (43, 48). We have observed that vitronectin inhibits the decay of rPAI-1 activity following incubation at 37 “C (data not shown). These results suggest that the activity of rPAI-1, like that of the natural protein (43, 48, 49), is stabilized by vitronectin. The physio- logical relevance of the association between PAI- and vitro- nectin remains to be established.

An alternative approach to purifying native PAI- from cultured cells or plasma is to explore the properties of recom- binant PAI-1. A number of groups have expressed the PAI- cDNA in either prokaryotic or eukaroytic cells (27-31, 41). However, biological activity of rPAI-1 from E. coli was re- ported only in the reverse fibrin autography assay (28, 29), a procedure which is known to promote conversion of latent to active protein. A subsequent report has indicated that rPAI- 1 from E. coli is expressed almost predominantly in the inactive form (32). PAI- has recently been purified from Chinese hamster ovary cells which had been transfected with cDNA of PAI- (31). As with PAI- purified from plasma, only the high &f, fraction, most likely representing PAI- complexed with vitronectin from the fetal bovine serum uti- lized in the growth medium, was functionally active. Further- more, the expression level of rPAI-1 in transfected Chinese hamster ovary cells was low and variable (0.1 mg/Iiter).

In view of these previous reports on both natural and rPAI- Is, it was surprising that the M, = 42,000 rPAI-1 in this study displayed significant functional activity in the absence of any discrete binding protein. The unique activity of this particular protein in comparison with natural and recombinant PAI-1s from other sources may relate to temperature sensitivity of PAI-1. Since incubation of PAI- at 37 “C results in a loss of activity (39), prolonged incubation of PAI-l-producing cells at 37 “C or higher may actually promote inactivation of the natural or recombinant protein in the conditioned medium prior to its collection for harvesting and eventual purification. This hypothesis is supported by the results in Table II which show that, beyond 2 h, the activity of rPAI-1 decreases with increased incubation time of the pCEl200 expression vector at 42 “C. We are currently investigating whether the bulk of this inactive rPAI-1 collected at the later time points repre- sents latent or irreversibly inactivated protein. Another im- portant consideration for maintaining activity of purified PAI- may relate to the ionic strength of the buffer used for storage. We have observed a considerable amount of aggre- gation of purified rPAI-1 when the ionic strength of the phosphate buffer was reduced below 150 mM, and this aggre- gated material was functionally inactive. The possible de- pendence of PAI- activity on high ionic strength also requires investigation.

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9574 Recombinant Plasminogen Activator Inhibitor-l

Further experiments are needed to precisely define biolog- ical roles for PAI- in various physiological and pathophysi- ological states. The ability to produce and purify this recom- binant form of PAI-1, which shares many properties in com- mon with natural active PAI-1, is an important step in defining such roles for PAI-1.

Acknowledgments-We would like to thank Walt Manger and Gene Corman for the NH*-terminal sequencing, Shubada Kaemekar and Alex Boon for technical assistance, Dr. Richard Yates for fermenta- tion analysis, and Claire Stecher for preparation of the manuscript.

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