Production of vampire bat plasminogen activator DSPA α1 in CHO and insect cells

journal of

wotcchnology ELSEVIER

Journal of Biotechnology 39 (1995) 75-83

Production of vampire bat plasminogen activator DSPA al in CHO and insect cells T. Petri a~*,G. Langer a, P. Bringmann a, L. Cashion b, S. Shallow b, W.-D. Schleuning a, P. Donner a a Research Laboratories of Schering AG, Berlin, Germany b Be&x Biosciences, Richmond, CA, USA

Received 31 May 1994; accepted 9 November 1994

Abstract Salivary plasminogen activator from the vampire bat Desmodus rotundus (DSPA cull is a promising new thrombolytic agent. Continuous growth of a stably transfected, methotrexate amplified, dhfr- CHO cell line yields up to 60 mg 1-l of DSPA (~1. Utilizing an engineered baculovirus 10 mg 1-l were produced in batches of Sf 9 insect cells. Recombinant DSPA al is purified from both sources using a one-step purification protocol. Although

differences in glycosylation were detected, enzymatic activity and fibrin cofactor dependency are unaffected when DSPA al derived from the two expression systems is compared. Keywords:

Bat plasminogen activator; DSPA (~1; Production; CHO cell; Sf 9 insect cell

1. Introduction

Natural and recombinant plasminogen activators such as streptococcal streptokinase and human tissue-type plasminogen activator (t-PA) are used in the therapy of vascular occlusive diseases (Collen and Lijnen, 1991). However, the high doses of streptokinase and t-PA required for rapid thrombolysis lead to the systemic generation of plasmin, the degradation of clotting factors and potentially to bleeding complications.

* Corresponding author: Schering AG, Institute of Cell and Molecular Biology, S109, Miillerstr. 178, D-13342 Berlin, Germany. 0168-1656/95/$09.50

Recently, the cloning of a novel family of plasminogen activators with exceptional fibrin specificity from the salivary gland of the vampire bat Desmodus rotundus was reported (Garde11 et al., 1989; Kraetzschmar et al., 1991). Four different D. rotundus salivary plasminogen activators (DSPA’s) cDNAs were cloned (DSPA (~1, a2, p and y), which differed in their domain structures, amino acid sequences and potential N-glycosylation sites (Kraetzschmar et al., 1991). Transient expression of the four DSPA isozymes in COS cells as well as the construction of stable BHK cell lines expressing each form was recently described (Kraetzschmar et al., 1991; Kraetzschmar et al., 1992). Biochemical and pharmacological studies have shown that DSPA (~1 exhibits a

0 1995 Elsevier Science B.V. All rights reserved

SSDI 0168-1656(94)00146-4

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2. Materials and methods

EcoRl(-) has successfully been used for transient DSPA ul expression in COS-cell transfection experiments (Kraetzschmar et al., 1991). pUDHFR I contains a 2627 bp C&z1 fragment cloned into the AccI cleaved and dephosphorylated vector pUC 19. The CluI fragment contains an entire SV40 late promoter based dihydrofolate reductase expression cassette isolated from the plasmid pPA207 (Berlex Biosciences, Richmond, CA, USA, personal communication). For the construction of the plasmid pBTLl1 (Fig. 1) the baculovirus transfer vector pVL1392 (Invitrogen) was digested with EcoRI and ligated with the EcoRI fragment derived from pSVPAl1 carrying the complete DSPA crl cDNA sequence. The structures of all plasmids are summarized in Fig. 1.

2.1. Plasmid construction

2.2. CHO cell culture

Plasmid pSVPAl1 has been described by Kraetzschmar et al. (1991). In brief, pSVPAl1 is a Klenow fill-in, blunt-end fusion of an EcoRI fragment containing the DSPA al cDNA and the XbaI digested vector pSVL-EcoRI( - >, a derivative of the mammalian expression vector pSVL (Pharmacia) in which the single EcoRI site had been removed by filling in and religation. pSVL-

dhfr- CHO cells (Urlaub and Chasin, 1980) were obtained through Berlex Biosciences (Richmond, CA) and cultivated in aMEM with nucleosides (Gibco), containing 2.5% fetal calf serum (Gibco) and 0.01% Serextent (Hana Biological& The cells were transfected applying the calcium phosphate method (Chen and Okayama, 19881 and dhfr’ positive cells were selected in

superior pharmacological profile if compared to t-PA or streptokinase (Witt et al., 1992; Muschick et al., 1993). To obtain the large amounts of DSPA (~1 necessary for detailed pharmacological and clinical studies recombinant Chinese hamster ovarian (CHO) cells stably secreting high levels of DSPA al were established. In parallel a recombinant Autogruphica California Nuclear Polyhedrosis Virus (AcNPV) for the batchwise expression in infected insect cells was constructed. The purified recombinant proteins from the two expression systems are compared in terms of electrophoretic mobility, glycosylation and fibrin specificity.

VPI-InIron

XPA

Fig. 1. The structure of the plasmids pSVPAl1 and pUDHFR 1 used for the transfection of the dhfr- CHO cells and of the plasmid pBTL used for the construction of the recombinant baculovirus. Recombination sequences denote the homologous sequences in the plasmid pBTL which allow the integration into the baculovirus genome via homologous recombination.

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aMEM without nucleosides, supplemented with 2.5% dialysed fetal calf serum (FCS) and 0.01% Serextend. 2.3. Insect cell culture, baculovirus propagation and plaque assay Sf 9 cells (obtained as part of the “MaxBac Baculovirus expression system”, Invitrogen, San Diego, CA) were cultivated as described by the supplier. Baculoviruses were propagated in about 70% confluent monolayers of Sf 9 cells. Following removal of the growth medium (TNM-FH medium, from Invitrogen, supplemented with 10% FCS) the cells were infected with the virus inoculum (about 1 ml per 9-cm petri dish or an equivalent inoculum volume in case of other culture vessels). Unadsorbed virus was removed after 1 h at room temperature and the cells were incubated with fresh growth medium for up to 7 d. The virus containing cell supernatants were cleared from cellular debris by centrifugation at 1000 xg for 10 min and either stored at 4°C for up to 4 weeks or at -70°C for longer periods. For the baculovirus plaque assay 50% confluent monolayers of Sf 9 cells in 35mm petri dishes were infected with serial lo-fold dilutions of the virus suspension. After an adsorption period of 1 h cells were overlaid with growth medium prewarmed to 33°C containing 1% agarose (“low melting point agarose for plaque techniques”, Serva, Germany) and incubated for 5 to 7 d. Plaques could be detected macroscopically by staining with a 0.03% dilution of neutral red in phosphate-buffered saline (PBS) deficient of Ca*+ and Mg*+ (5 ml per 9-cm petri dish).

subsequently 0.75 ml of fresh medium were added followed by the transfection buffer containing the DNA. The cells were incubated for 4 h at room temperature, washed once with growth medium and incubated with fresh medium for 5 to 7 d at 27°C. The appearance of the wild-type virus specific polyhedra structures could be observed under the microscope during this time. When the majority of cells was lysed the supernatant was harvested for plating the virus on Sf 9 cell monolayers as described above. Recombinant viruses were identified by the inclusion body negative plaque morphology. 2.5. Preparation of high molecular weight DNA High molecular weight (hmw) genomic DNA was prepared using standard techniques. Briefly, cells obtained by trypsinization were resuspended in 0.1 ml phosphate-buffered saline (PBS) and 9.3 ml lysisbuffer (100 mM Tris, pH 8, 100 mM NaC1, 10 mM EDTA), subsequently 0.5 ml of 10% SDS and 0.1 ml of Proteinase K (10 mg ml-‘) were added. Cells were lysed by gently inverting the solution several times and incubation at 55°C for 30 min and another hour at 37°C. Following two extractions using an equal volume phenol/chloroform (70:30) saturated with 10 mM Tris, pH 7.5, 1 mM EDTA, ammonium acetate was added to a final concentration of 300 mM. The hmw DNA was recovered after the addition of 2 ~01s. of ethanol with a drawn out Pasteur pipette. The DNA was washed in 70% of ethanol for 5 min, air dried for another 10 min, redissolved overnight at 4°C in 2 ml double distilled H,O and aliquots used directly for PCR amplification.

recombinant viruses

2.6. Determination of the DSPA al by quantitative PCR

Approx. 2 x lo6 Sf 9 cells were seeded into a 25-cm* tissue culture flask and allowed to attach for 4 to 16 h resulting in a 60% confluent monolayer. 1 pg of AcNPV DNA (Invitrogen) and 2 pg of vector DNA (pBTL11) containing the DSPA cDNA were mixed thoroughly with 0.75 ml of the transfection buffer (Invitrogen). The growth medium was then removed from the monolayer,

PCR products of 651 bp and 481 bp specific for DSPA crl and exon 7 of Chinese hamster tk gene (Lewis, 19861, respectively, were obtained using 1 pg of hmw genomic DNA. (PCR conditions: 25 cycles each consisting of 30 s denaturation at 94°C 45 s annealing at 58°C and 120 s elongation at 72°C.) The amount of template DNA was reduced to l-10 ng and the conditions

2.4. Transfection of Sf 9 cells and isolation of

copy number

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changed to 22 cycles of 94°C for 60 s, then 50°C for 60 s, followed by 72°C for 90 s. The latter conditions were empirically determined and resulted in exponential amplification of the products. In both cases the reaction mix was preheated to 94°C for 5 min prior to cycling. Aliquots of the specific, full-length amplification products were also sequenced directly with the help of both, amplification and internal primers using dye terminators and an automated DNA sequencer (model 373A, Applied Biosystems, USA) The sequence data obtained were in accordance with the original cDNA (data not shown). 2.7. Fibrin plate assay Fibrin plates containing fibrin and plasminogen and casein plates containing dry milk powder and plasminogen were prepared as described (Fischer et al., 1985). 2.8. Zymography For the zymographic analysis (Levin and Loskutoff, 1982) samples were separated without prior heat treatment by 12.5% SDS polyacrylamide gel electrophoresis under non reducing conditions. Gels were then rinsed twice in PBS containing 2.5% Triton X 100 (Serva) and after two additional washes with PBS placed on a fibrin or casein layer.

3. Results 3.1. Construction and amplification of recombinant CHO cells expressing DSPA al

dhfr CHO cells were transfected as described in Materials and methods with 5 pg Sal1 linearized pSVPA 11 and 0.5 pg EcoRI linearized pUDHFR I (Fig. 1). dhfr-positive cell clones were selected, picked with sterile cotton swabs and treated individually with increasing doses of methotrexate (MTX) from 0.005 to 1 PM. The DSPA ~yl production of these cell clones before and during treatment with MTX was monitored by fibrin plate assays. One clone termed 8/6 responded especially well to this MTX treatment with an increasing DSPA al production. Clone

DSPA alpha 1 (amplified) Genomic DNA (ng) 1

2

5

10

Thymidine Kinase (single COPY) Plasmid DNA (pg) 0

1

2

Genomic DNA (ng x 100)

5

124

7

3 613 (x 104)

2.9. DSPA (YI purification Recombinant DSPA cul from the supernatants of either CHO cells or baculovirus infected Sf 9 cells was purified by affinity chromatography using Erythrina Zatissima trypsin inhibitor (ET0 immobilized to sepharose (Heussen et al., 1984). Conditioned media were concentrated hundredfold using ultra filtration (YM 30 membranes, Amicon, USA) and applied to an ET1 affinity column. After washing with PBS/400 mM NaCl followed by PBS ETI-bound DSPA al was eluted with 50 mM Na acetate pH 4 containing 100 mM NaCl.

0.3 0.6 (x 103)

2

3

0 13 (x 105)

Genomic equivalents Fig. 2. Determination of the DSPA (~1 gene copy number. A 651 bp DSPA (~1 specific PCR product was amplified from stable transfected hmw CHO cell DNA (l-10 ng, 300-3000 genomic equivalents) and compared to the results obtained from the amplification of plasmid DNA (l-5 pg, 14-70000 copies) mixed with unspecific murine DNA. The amplification of a 485 bp product of the single copy CHO cell thymidine kinase locus (l-400 ng, 32500-130000 genomic equivalents) was used as a control.

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8/6 was therefore recloned by limited dilution in 96-well plates and chosen for further studies.

3.2. DSPA al gene copy number in recombinant CHO cells

The expression levels obtained following stable transfection in CHO cells of heterologous DNAs were roughly proportional to the gene copy number estimated by the amplification processes. Therefore, the determination of the copy number of the transfected gene is very informative, since overexpression depends on gene copy number in addition to the site of chromosomal integration and the expression vector’s design. DSPA al-specific sequences were PCRamplified using serial dilutions of high molecular weight genomic DNA prepared from the cell clone 8/6. pSVPA 11 plasmid DNA mixed with unspecific murine genomic DNA was used as internal standard (genomic reconstitution; Saiki et al., 1988; Lee et al., 1987). Analysis of the relative signal intensities obtained from PCR

products separated by standard agarose gel electrophoresis (Fig. 2) revealed that some 320-350 copies of DSPA (~1 genes are present per haploid CHO cell genome. The data were independently confirmed in dot blotting experiments where aliquots of the PCR reactions were probed with 32P-labelled internal oligonucleotides (data not shown). 3.3. Continuous cultivation of DSPA CYIproducing CHO cells

A continuous cultivation using a subclone of clone 8/6 was set up in a Braun Biostat M bioreactor using cytodex 3 microcarriers (3 g l- ‘; Pharmacia). The bioreactor was inoculated initially at a density of 2 X 10’ cells cultured in a spinnerflask using the same type of microcarriers. The cells were grown up to a density of 5 x 10’ ml-’ in aMEM containing 5% FCS omitting nucleosides and methotrexate. Subsequently, perfusion was started using medium containing 0.5% FCS. The perfusion rate was adjusted to maintain

Level Control

Hollow Fiber Fermenter 1 mm I.D. : : ____

Harvest Flask

Fig. 3. A schematic representation

79

i-1 Medium ---Flask

of the set up used for the continuous cultivation of the DSPA al producing CHO cells.

x0

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Specific Production vs. Doublings

-92 kDa -66 kDa -45 kDa

-31 kDa

-22 kDa Fig. 4. Stability of the recombinant CHO cell clone X/6 expressing DSPA ~1. Cells were propagated in 75 cm2 tissue culture flasks and the DSPA crl productivity (pg DSPA (~1 per cell per d) was determined on fibrin plates using DSPA al as a standard. A DSPA cut standard preparation was identified using highly purified recombinant material characterized by amino acid analysis, A280 nm quantification and activity measurement using a chromogenic substrate S 2288 (Chromogenics AB, Sweden).

the glucose level at about 0.3 g 1-l. Utilizing a hollow fiber module (AG technology, Needham, MA, USA) cells were retained in the reactor during perfusion and a maximal cell density of 5 X lo6 ml ~ ’ was maintained. A schematic picture of the continuous cultivation system is given in Fig. 3. DSPA al production was monitored by a continuous passage without methotrexate in culture flasks. No significant decrease in the productivity per cell was observed during up to 40 population doublings (Fig. 4).

6 Fig. 5. Analysis of purified DSPA al produced by CHO or Sf 9 cells using 12.5% SDS polyacrylamide gel electrophoresis, Coomassie staining. Left gel: lanes 1 and 4, 5 pg DSPA; lanes 2 and 5, 5 pg DSPA, all from CHO cells; lanes 3 and 6, 3 pg DSPA from Sf 9 cells; lanes l-3 reduced samples, lanes 4-6 non reduced samples. Right gel: zymographic analysis of 0.5 pg DSPA (~1 from CHO cells (lane 1) and 0.5 wg DSPA al from Sf 9 cells (lane 2). After electrophoresis gels were incubated for 2 h at 37°C. Molecular mass markers (M) are expressed in kDa.

V

Absorbance

at 405 nm

1.6-

0

CHO

W SF9 1.41.21 o0.80.6-

3.4. Construction of recombinant baculouiruses

-

0.4-

Generation of recombinant viruses was achieved by transfection of Sf 9 cells with wildtype AcNPV DNA and pBTLl1. Seven days after the transfection serial lo-fold dilutions of the supernatant were prepared and used to infect lo4 Sf 9 cells contained in a well of a 96-well plate. The plate was incubated at 27°C for 5 days and the supernatant of each well was analysed by a fibrin plate assay. Since the supernatant taken from the well infected with the highest dilution gave a positive result in the fibrin plate assay it contained an enriched population of recombinant viruses. The virus population was then serially

0.2n ”

II I

GNA

L

’

SNA

’

MAA

’

DSA

’

PNA

Lectm

Fig. 6. Analysis of carbohydrate structures of DSPA cul produced by CHO or Sf 9 cells using digoxigenin labelled lectins in a 96-well plate type of assay (Application sheet, Boehringer Mannheim, Germany). Lectins with the following specificity were used: GNA, Galanthus niualis, N-glycan chains of the high mannose type; SNA, Sambucus nigra, sialic acid bound to galactose; MAA, Maackia amurensis, complex carbohydrates containing sialic acid; PNA, Peanut agglutinin, O-linked GalP(l-3) N-acetyl galactosamine; DSA, Datura stramonium, glycan structures of the complex and hybrid type.

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diluted and the recombinant virus plaque purified twice. Recombinant viruses showed an inclusion body negative plaque morphology. Baculovirus derived DSPA al was produced by infecting 5 X lo5 Sf 9 cells per ml in 250 ml medium with a multiplicity of about 1. Cell supernatants with a productivity of about 10 pg DSPA al per ml were harvested after 3 d.

from CHO or Sf 9 cells were investigated by lectin binding analysis (Fig. 6). As expected, Sf 9 cell derived DSPA (~1 contained predominantly carbohydrates of the high mannose type as shown by binding to the lectin GNA. Strong binding of DSPA cul from CHO cells to the lectins MAA and DSA indicated the presence of sialylated galactose residues and carbohydrate structures like Gala (l-4) GlcNac occurring in complex and hybrid N glycans. These data are in good agreement with the literature on different glycosylation patterns of mammalian and insect cells (Butters and Hughes, 1981). Since almost identical activities of DSPA al isolated from either Sf 9 or CHO cells were observed by fibrin plate assay (Fig. 7A), the observed differences in the glycosylation pattern did not lead to significant differences in specific activities of DSPA al. t-PA produced by recombinant CHO cells (Actilyse, Genentech, USA) showed a slightly higher enzymatic activity. However, on casein plates containing plasminogen roughly 500-fold higher amounts of DSPA (~1 from either CHO or Sf 9 cells were necessary to produce the same degree of fibrin independent lysis as tPA underlining the much higher fibrin cofactor dependency of DSPA al (Fig. 7B).

3.5. Recombinant DSPA al isolated from CHO and

Sf 9

cells

Recombinant DSPA al was isolated from the cell culture supernatants as described in Materials and methods. The purified material derived from CHO as well as from Sf 9 cells exhibited an apparent molecular mass of 52000 Da under reducing conditions after 12.5% SDS polyacrylamide gel electrophoresis (Fig. 5). Under non reducing conditions DSPA (~1 derived from Sf 9 cells migrated sightly faster. This was also observed for the zymographic analysis (Fig. 5) where equimolar amounts of both preparations were applied. The similar intensity of the lysis zones indicates a comparable specific activity of DSPA (~1 isolated from CHO and Sf 9 cells. The carbohydrate core structures of DSPA al

Fibrin

81

I

Casein

I 6 DSPACY 1 CHO [pmol]

DSPACi 1 Sf9

[pmol]

Actilyse [fmol]

Fig. 7. Fibrin cofactor requirement of DSPA al purified from CHO or Sf 9 cells: lysis zones of DSPA al purified from CHO or Sf 9 cells on a fibrin plate (A) or a casein plate containing plasminogen (B). fmol amounts of DSPA (~1 were applied to the fibrin plate, whereas pmol amounts were applied on the casein plate.

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4. Discussion

Recombinant CHO cells as well as Sf 9 cells infected with a recombinant baculovirus can both be used for the efficient production of DSPA al. Stable expression of DSPA (~1 by CHO cells even in the absence of methotrexate for at least 40 cell doublings compares favourably with other work, where a rapid loss of productivity of t-PA producing CHO cell lines after omission of methotrexate was observed (Weidle et al., 1988). Meanwhile methotrexate independency and stability of DSPA al production has been confirmed by continuous growth in 10-l bioreactors for several weeks. Compared with other expression systems the productivity of CHO cell clone S/6 is exceptionally high. From a continuous l-1 bioreactor system (Fig. 3) with a cell density maintained at 5 X lo6 ml-’ and a daily harvest of 1 1 conditioned medium 60 mg of DSPA al are produced. This corresponds to a cell productivity of 12 pg per cell per day. We did not achieve a similar productivity with our insect cell system. 10 pg DSPA (~1 were isolated per ml conditioned medium after a 3 d culture. No attempts were made so far for the optimization of the insect cell production system with respect to time of harvest, cell density at the time of infection, etc. The DSPA al gene copy number was 320-350 copies per haploid CHO genome. In comparison with other published data (Weidle et al., 1988) where up to 1000 gene copies per cell after MTX amplification have been reported, our estimated copy number is rather moderate and may not alone explain the high productivity of the CHO clone 8/6. A favourable integration site of the DSPA al gene in proximity to a strong enhancer could be the reason for this outstanding productivity. Recombinant DSPA (~1 is conveniently purified from conditioned media using immobilized Erythrina latissima inhibitor. There is no loss of enzymatic activity after elution with an acidic buffer. The purity (Fig. 5) of the isolated material was sufficient for a biochemical comparison of both DSPA al produced by recombinant CHO or insect cells. As expected, the glycosylation patterns of DSPA cul produced in the two recom-

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binant cell lines were different. Like natural occurring DSPA (~1, material produced by CHO clone 8/6 exhibited carbohydrate structures of the complex type containing sialic acid and of the mannose rich or hybrid type. The occurrence of high mannose carbohydrate structures was enhanced in DSPA al produced by insect cells. The unique fibrin specificity of natural DSPA c-u1(Schleuning et al., 1992) was previously found to be unaltered in recombinant DSPA al transiently expressed in COS cells (Kraetzschmar et al., 1991). This property was likewise maintained in DSPA (~1 obtained from the two expression systems described here. In the absence of the fibrin cofactor DSPA al was about .500- to lOOOfold less active than t-PA. In spite of differences in glycosylation, the enzymatic activity of DSPA al produced by recombinant CHO or insect cells was similar. This corroborates the previous finding that enzymatic activity is fully retained after glycopeptidase F treatment and that glycosylation of DSPA is not a prerequisite for plasminogen activation (Schleuning et al., 1992). The CHO expression system (especially the CHO cell clone S/6) provides the basis for production of DSPA al under GMP conditions. Pharmacological studies (Witt et al., 1992; Muschick et al., 1993) have demonstrated a superior profile of DSPA al if compared to other plasminogen activators currently in use. Given the high yields of the described expression system, DSPA al may in addition be produced at lower cost. Acknowledgements

The authors thank J. Dieckmann, 0. Lange, A. Liese, D. Roben, D. Schmidt and A. Toben for skilful technical assistance, and I. Kuhn for advice on the bacuIovirus/Sf 9 insect cell expression system. References T.D. and Hughes, R.C. (1981) Isolation and characterization of mosquito cell membrane glycoproteins. Biochim. Biophys. Acta 640, 655-671.

Butters,

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Chen, C.A. and Okayama, H. (1988) Calcium phosphatemediated gene transfer: a highly efficient transfection system for stably transforming cells with a plasmid. Biotechniques 6, 632-634. Cohen, D. and Lijnen, H.R. (1991) Basic and clinical aspects of fibrinolysis and thrombolysis. Blood 78, 3114-3124. Fischer, R., Wailer, E.K., Grossi, G., Thompson, D., Tizard, R. and Schleuning, W.-D. (1985) Isolation and characterization of the human tissue-type plasminogen activator structural gene including its 5’ flanking region. J. Biol. Chem. 260, 11223-11230. Gardell, S.J., Duong, L.T., Diehl, R.E., York, J.D., Hare, T.R., Register, R.B., Jacobs, J.W., Dixon, R.A.F. and Friedman, P.A. (1989) Isolation, characterization and cDNA cloning of a vampire bat salivary plasminogen activator. J. Biol. Chem. 64, 17947-17952. Heussen, C., Joubert, F. and Dowdle, E. (1984) Purification of human tissue type plasminogen activator. J. Biol. Chem. 259, 11635-11638. Kraetzschmar, J., Haendler, B., Langer, G., Boidol, W., Bringmann, P., Alagon, A., Donner, P. and Schleuning, W.-D. (1991) The plasminogen activator family from the salivary gland of the vampire bat Desmodus rotundus: cloning and expression. Gene 105, 229-237. Kraetzschmar, J., Haendler, B., Bringmann, P., Dinter, H., Hess, H., Donner, P. and Schleuning, W.-D. (1992) Highlevel secretion of the four salivary plasminogen activators from the vampire bat Desmodus rotundus by stably transfected baby hamster kidney cells. Gene 116, 281-284. Lee, M.-S., Chang, K.-S., Cabanillas, F., Freireich, E.J., Trujillo, J.M. and Stass, S.A. (1987) Detection of minimal residual cells carrying the t(14;18) by DNA sequence amplification. Science 237, 175-178.

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Levin, E.G. and Loskutoff, D.J. (1982) Cultured endothelial cells produce both urokinase and tissue-type plasminogen activators. J. Cell Biol. 94, 631-636. Lewis, J.A. (1986) Structure and expression of the Chinese hamster thimidine kinase gene. Mol. Cell. Biol. 6, 19982010. Muschick, P., Zeggert, D., Donner, P. and Witt, W. (1993) Thrombolytic properties of Desmodus (vampire bat) salivary plasminogen activator DSPA cul, Alteplase and streptokinase following intravenous bolus injektion in a rabbit model of carotid artery thrombosis. Fibrinolysis 7,284-290. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Ehrlich, H.A. (1988) Primer directed enzymatic amplification of DNA with thermostable DNA polymerase. Science 239, 487-491. Schleuning, W.-D., Alagon, A., Boidol, W., Bringmann, P., Petri, T., Kraetzschmar, J., Haendler, B., Langer, G., Baldus, B., Witt, W. and Donner, P. (1992) Plasminogen activators from the saliva of Desmodus rotundus (Common vampire bat): Unique fibrin specificity. NY Acad. Sci. 667, 395403. Urlaub, G. and Chasin, L.A. (1980) Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc. Nat]. Acad. Sci. USA 77, 4216-4220. Weidle, U.H., Buckel, P. and Wienberg, J. (1988) Amplified expression constructs for tissue-type plasminogen activator in Chinese hamster ovary cells: instabilitiy in the absence of selective pressure. Gene 66, 193-203. Witt, W., Baldus, B., Bringmann, P., Cashion, L., Donner, P. and Schleuning, W.-D. (1992) Thrombolytic properties of Desmodus rotundus (vampire bat) salivary plasminogen activator in experimental pulmonary embolism in rats. Blood 79 (51, 1213-1217.