EP0403598A1

EP0403598A1 - tPA-LIKE POLYPEPTIDES, THEIR MANUFACTURE AND USE

Info

Publication number: EP0403598A1
Application number: EP89909751A
Authority: EP
Inventors: Alfred Bach; Martin Schmidt; Karl-Hermann Strube; Verena Baldinger; Margarete Schwarz
Original assignee: BASF SE
Current assignee: BASF SE
Priority date: 1988-09-17
Filing date: 1989-09-07
Publication date: 1990-12-27
Also published as: WO1990003436A1; DE3831714A1; JPH04500306A

Abstract

On décrit des polypeptides analogues au tPA, ainsi que leur fabrication. Les nouveaux polypeptides sont appropriés pour combattre des maladies.TPA-like polypeptides and their manufacture are described. The new polypeptides are suitable for fighting diseases.

Description

tPA-like polypeptides, their production and use

description

The present invention relates to new tPA-like polypeptides with plasma activator activity, processes for their preparation and their use in combating diseases.

The human, mature tissue plasmin activator (tPA) is a polypeptide of 527 amino acids and a molecular weight of about 68 kD (Pennica et al. 1983, Nature 301, 214-221 and Ny et al. 1984, Proc. Natl. Acad. Sei USA 81, 5355-5359) (see Fig. La-lc). The molecule contains several distinct regions, so-called domains, to which various functions are assigned. The N-terminus is formed by a finger region that is homologous to the fibronectin. This is followed by a region that is similar to several growth factors. This is followed by two Kringel domains. The protease domain follows a cleavage site at which the single-chain molecule can be cleaved into two chains; this contains the active center and has homology to other serine proteases.

Several properties make the tissue plasminogen activator an interesting molecule: tPA has a strong affinity for fibrin, the plasminogen activation is dependent on fibrin; as a protease, tPA cleaves plasminogen to plasmin, which in turn breaks down fibrin into cleavage products. tPA can be inhibited by plasminogen activator inhibitors. Furthermore, tPA has a relatively short half-life; the molecule breaks down rapidly in the liver.

These factors have the effect that tPA specifically activates plasminogen to plasmin in blood clots and causes the vascular current to be restored by breaking down the fibrin. tPA can therefore be used in fibrinolytic therapy, e.g. B. after a heart attack.

It has now been found that tPA-like polypeptides which have the amino acid sequence of tPA or sequences derived therefrom, but in which 1-6 tripeptides are replaced by the tripeptide RGD, have improved properties.

sequences derived from the tPA are to be understood as naturally occurring allele variants and synthetic variants which, in their primary structure, agree more than 90% with the natural tPÄ and have a plasminogen-activating effect. The new polypeptides preferably contain one or more RGD

(= Arg-Gly-Asp) tripeptides instead of one tripeptide each in the tPA with unchanged total length of the molecule.

Those polypeptides which contain the tripeptide RGD in position 130-132 are particularly preferred. Also preferred are polypeptides which contain one to two additional RGD tripeptides in addition to the RGD sequence in position 130-132.

The invention further relates to DNA sequences for the mutated

Encode polypeptides, as well as vectors containing these DNA sequences.

The new polypeptides can be produced genetically using known methods. The starting point for the constructions described here is a cDNA clone, hereinafter called pUCtpa, which contains the coding sequence of tPA and 5'- and 3'- untranslated regions *. After a "leader" and pro sequence, the mature protein begins with Ser (1) and ends after Pro (527).

PUCtPA is isolated as follows: RNA is isolated from human uterine tissue and transcribed into double-stranded cDNA. After inserting this cDNA into the commercially available cloning vector pUC9, a cDNA library is created. The methods used can be found, for example, in Maniatis et al., Molecular Cloning, CSH-press. 5 “Screening” of such gene banks with radioactively labeled oligonucleotide probes has also become a widely used and described method. A cDNA clone containing the coding region and adjacent regions can be isolated by this method.

0 parts of the DNA sequence of PUC9tPA are easily accessible with the help of restriction enzymes. These fragments, optionally in conjunction with chemically synthesized oligonucleotides, adapters or gene fragments, can be used to clone the DNA sequences which code for the new polypeptides. The incorporation of the gene fragments or synthetic 5 DNA sequences into cloning vectors, e.g. B. the commercial plasmids pBR322, pUCS or 9, pUCl8 or 19, Ml3mpl8 or Ml3mpl9 is carried out in a known manner. Also. the genes or gene fragments can be provided with suitable chemically synthesized control regions or isolated from bacteria, phages, eukaryotic cells or their viruses, which control the of the proteins. The transformation or transfection of the hybrid plasma obtained in this way into suitable host organisms is also known and has been described in detail. The hybrid plasmids can also be provided with corresponding signal sequences which allow the polypeptides to be secreted into the medium. For expression in mammalian cells, vectors can be used which place the gene to be expressed, in this case the mutated tPA cDNA, under the control of the mouse metallothionein or the SV40 viral promoter. The presence of the methionine start codon and the leader / prosequence of the tPA gene is necessary for the expression. Clones are then isolated which have copies of these vectors as episomes or integrated into the genome. For example, the integration and expression of the foreign gene based on the bovine papillo virus are particularly advantageous. In connection with prokaryotic sequences that code for replication in bacterial cells and antibiotic resistance, the construction of so-called “shuttle” vectors is possible. The plasmid is first constructed and propagated in bacterial cells; the conversion into the eukaryotic cells then takes place, for example into the mouse fibroblast cell line C127. It goes without saying that other cell systems, for example yeast, insect cells and other mammalian cells, such as CHO, L and 293 cells, can also be used for the expression.

These eukaryotic expression systems have the advantage that they are able to secrete their products effectively and mostly in their native form. They also have the ability to modify their ^" products post-translationally.

For example, the tissue plasma activator receives glycoside side chains at amino acids 117, 184 and 448 (Asn) when expressed in eukaryotic cells. Bacteria are unable to synthesize glycoside side chains. Most eukaryotic proteins expressed in bacteria, as well as tPA and the polypeptides derived from it according to the invention, occur in the cell as denatured inclusion bodies and must be renatured by protein chemistry. Furthermore, bacteria are often unable to split off the initiator amino acid methionine from the finished protein. These difficulties can be avoided by using secretion systems.

Due to the degeneration of the genetic code, it is also possible to use other DNA sequences, e.g. to use chemically synthesized genes with different DNA sequences for the expression of the new polypeptides.

The new polypeptides are purified by separating them from the culture medium by affinity and ion exchange chromatography using known methods.

The polypeptides obtained according to the invention have improved properties in at least one of the points mentioned below: clot specificity, half-life, inhibitor binding, proteolytic activity and can therefore be used for thrombolysis. They show improved properties compared to human tissue plasminogen activator. The invention therefore also relates to medicaments which contain at least one of the new polypeptides, optionally in a pharmaceutically acceptable carrier or binder. The drugs can also combinations of the new proteins with other fibrinolytics, such as prourokinase, urokinase or streptokinase or their derivatives, as well as with other pharmaceutical proteins, e.g. B. Superoxide disase included. Further embodiments of the invention are described in more detail in the following examples.

^" For genetic engineering methods, see, for example, the manual by Maniatis et al., Molecular Cloπing, Cold Spring Harbor Laboratory, 1982.

example 1

Isolation of a cDNA K-ion for human tissue plasminogen activator

a) RNA preparation

30 g of human uterine tissue producing tPA was in

6 M guanidinium thiocyanate, 5 mM sodium citrate (pH 7.0), 0.1 M 2-

Mercaptoethanol, 0.5% Sarcosyl digested in the Ultra-Turrax®. Coarse cell debris was centrifuged off at 3000 rp. The RNA was sedimented by centrifugation through a 5.7 M CsCl cushion overnight at 45,000 rpm. The polyA ⁺ -containing RNA fraction was then separated by affinity chromatography on oligo (dT) cellulose.

b) Preparation of the cDNA bank

Using the enzyme reverse transcriptase (AMV) and oligo (dT) 12-18 as starters, the polyA ⁺ RNA was rewritten into single-stranded cDNA. The second strand was synthesized using E. coli DNA polymerase I. Sall linkers were set up on the double-stranded cDNA with the aid of the enzyme T4-DNA ligase. The commercially available plasmid pUC9 was linearized with the restriction enzyme Sall. Both DNAs were ligated together and transformed with the hybrid CaCl2-treated, competent cells of the E. coli strain HB101 thus obtained. The cells were plated on LB plates with 100 g / ml A picillin - and incubated overnight at 37 ° C.

c) Screening the cDNA library

About 500,000 colonies were transferred to nitroceϊlulose filters, replica-plated, lysed with 0.5 N NaOH / 1.5 M NaCl and the denatured DNA was firmly bound to the feed by baking at 80 ° C. for 2 hours. The filters were placed in 6 x SET buffer (1 x SET = 0.15 M NaCl, 15mM Tris / HCL pH 7.4, 1mM EDTA), 0.1% SDS and 5 x Denhardt's solution (100 x Denhardt = 1 g Ficoll, 1 g polyvinylpyrrolidone, 1 g BSA per 50 ml) for 4 h, prehybridized at 68 ° C (saturation of non-specific binding sites for nucleic acids).

Using a DNA synthesizer, three oligonucleotide probes, each comprising 17 bases of the tPA-DNA, were produced. They consist of the following sequences:

5 'TGCAGATCACTTGGTAA 3' 5 'CCAGGCCCAGTGCCTGG 3' 5 'TCCAGTCCGGCAGCTGC 3'

These probes were labeled with j - 32p-ATP at the 5 'end. They were then incubated with the prehybridized filters in a solution containing 6 x SET, 0.1% SDS, 5 x Denhardt's and 10% dextran sulfate overnight at 42 ° C with gentle shaking. The filters are then washed several times in 6 x SET / 0.1% SDS at 42 ° C., dried and exposed to an X-ray film. Clones that gave a radioactive response during screening were isolated and grown up. A clone, hereinafter called pUCtPA, contained an approximately 2.1 kb insert which contained the coding region and 5 'and 3' non-coding regions (FIG. 2). Plasmid DNA from pUCtPA was prepared by lysozyme digestion and SDS-alkali treatment of the bacterial culture and subsequent CsCl gradient centrifugation.

Example 2

Production of single-stranded tPA-encoding DNA

The starting point was the plasmid pUCtPA described in Example 1. It was cut preparatively with the restriction enzyme Sall. The fragments obtained were separated by gel electrophoresis. The Sall fragment, which contained the DNA sequence encoding tPA, was eluted electrophoretically from the gel. 30 ng of this fragment were ligated at 4 ° C. with 100 ng of the Sall cut, commercially available cloning vector mpl9. The volume of the ligation mixture was 10 μl. The ligation was terminated by heating at 80 ° C. for δ minutes.

1/10 volume of this ligation mixture was used to transform 100 μl competent JM 101 cells. After the transformation had ended, 60 μl of 0.2 MIPTG solution and 120 μl of XGal (20 mg / ml) were added to the transformation mixture. This approach was carried out in NZYDT top agar on NZYDT agar plates plated. The medium NZYDT is commercially available. Clones containing the tPA cDNA could be identified due to the lack of blue staining of the plaques. The orientation of the 5 tPA cDNA within the plasmid was determined by means of DNA sequence analysis (Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74, 5463-67).

The mPl9 plasmid, which contained the cDNA of tPA in the orientation that the + strand is the tPA cDNA component of the phage genome when single-stranded M13 phages are formed, was designated as mptPA. 0

If bacteria of the Staπmies JM 101 or cells derived therefrom are transformed with the plasmid mptPA, single-stranded bacteriophages which contain the + strand of the tPA cDNA are released into the medium when these cells multiply. The single-stranded DNA can then be isolated using 15 conventional and widely described methods.

Otherwise, if not specifically described, the recommendations of the manufacturers of the enzymes and plasmids for the enzymatic and isolation of the plasmids were followed. 0

Example 3

Introduction of substitutions in the tPA cDNA

25 The following describes substitutions which all have the effect that, after expression of the substituted cDNA, the amino acid motif RGD is contained one or more times at different sites in the polypeptide tPA. All of these polypeptides are 527 amino acids in length. The starting point for the substitutions was the mptPA described in Example 2.

30

0.04 pmol of the single-stranded mptPA was reacted with 0.5 pmol of an oligonucleotide (21-27mer), the sequence of which was complementary to the region of the mptPA DNA to be changed apart from 1-3 base changes. The oligonucleotide was previously phosphorylated using the T polynucleotide kinase

35 lated. A typical mutagenization approach was as follows:

single-stranded mptPA DNA 1 μl (0.04 pmol) kinased oligonucleotide

(including ATP from kinization reaction) 1 μl (0.5 pmol)

40 10 x ligase buffer 5 μl

1 mM dNTPs 2.5 μl (50 μM final concentration)

1 mM ATP 4 μl (100 μM final concentration)

H ₂ 0 36.5 ul The mutagenization mixture was incubated at 37 ° C. for 15 min, then cooled to room temperature and finally 1 unit of Klenow fragment (Boehringer Mannheim) was added. After incubation at room temperature for one hour, 200 units of T ligase (Biolabs) were added. The mixture was then incubated at 4 ° C. for 16 h. Then 5 μl of this approach were used to transform components of JM 101 cells. The resulting phage plaques were transferred to nitrocellulose. The filters were then washed in 5XSSC for 5 min, air-dried and "baked" at 80 ° C. for 2 h. Prehybridization and hybridization were carried out as described in Example 1. The filters were washed at 6 × SET / 0.1% SDS at a temperature of 55-65 ° C. depending on the base composition and length of the oligonucleotide hybridization sample used. Hybridization was carried out in each case with the oligonucleotide which was used in the corresponding mutagenization batch. Positive clones were made autoradiographically visible. These substitutions had the effect that the tPA cDNA contained in mpl8 now codes for a polypeptide which contains the amino acid motif RGD and three further amino acid substitutions at one or more locations, the total number of amino acids of these polypeptides in each case being 527.

The mptPADNA described in Example 2 was generated using the oligonucleotides

C _B 5 'AGCAGTCACT GGATCCCTCA GAGC 3' C _Xm 5 'ACGTGGCCCG GGTATCTATT TC 3'

C _A 5 'AACTTTTGAC GGGCCCTGAG TGGCA 3' ^c xh 5 'CAGGCCGCAG CTCGAGCAGG AGGG 3'

mutated in such a way that additional recognition parts for the restriction enzyme were generated between the individual protein domain-coding DNA segments. These recognition sites allow the individual protein domain-coding DNA sections to be cut out in a targeted manner. This mutated mptPADNA was the starting point for the following examples 3.1 to 3.13.

Example 3.1

An oligonucleotide with the following sequence was used for the mutagenization as described in Example 3:

5 'TCTGCGTTTTATCACCTCTGCAGAT 3'

The washing temperature was 60 ° C in 6xSET / 0.1% SDS.

The resulting positive clones were named mptPA-RGDl. Example 3.2

For the mutagenization, as described in Example 3, an oligonucleotide with the following sequence was used: 5

5 'CCACCCGGCTCGCCTCTGAGCACA 3'

The washing temperature was 62 ° C in 6xSET / 0.1% SDS. The resulting positive clones were named mptPA-RGD2. 10

Example 3.3

For the mutagenization, as described in Example 3, an oligonucleotide with the following sequence was used: 15

5 'ACCAGCAATAATCCCCCCGGTTGCT 3'

The washing temperature was 60 ° C in 6xSET / 0.1% SDS. The resulting positive clones were named mptPA-RGD3. 20th

Example 3.4

For the mutagenization, as described in Example 3, an oligonucleotide with the following sequence was used: 25

5 'CTGTGCTCCAATCGCCCCTGTAGC 3'

The washing temperature was 60 ° C in 6xSET / 0.1% SDS. The resulting positive clones were named mptPA-RGD4. 30

Example 3.5

For the mutagenization, as described in Example 3, an oligonucleotide with the following sequence was used: 35

5 'GGTGCACTCGTCGCCTCTCTCCGCTG 3'

The washing temperature was 64 ° C in δxSET / 0.1% SDS. The resulting positive clones were named mptPA-RGD5. 40 Example 3.6

5 'GATGCCGTCTCCCCTCCGCCCGC 3'

The washing temperature was 60 ° C in 6xSET / 0.1% SDS. The resulting positive clones were named mptPA-RGD6.

Example 3.7

5 'CAGGAACCGATCTCCGCGCGACCTCC 3'

The washing temperature was 65 ° C in 6xSET / 0.1% SDS. The resulting positive clones were named mptPA-RGD7.

Example 3.8

5 'GCTCTCCTGGTCACCGCGGGACGAA 3'

The washing temperature was 61 ° C in 6xSET / 0.1% SDS. The resulting positive clones were named mptPA-RGD10.

Example 3.9

5 'GCTGCAGGTCCCCCCGGGGAAGGCA 3'

The washing temperature was 65 ° C in 6xSET / 0.1% SDS. The resulting positive clones were named mptPA-RGDll. 10

Example 3.10

5 'AGTGTCTCCATTACACAGCATG 3'

The washing temperature was 55 ° C in 6xSET / 0.1% SDS. The resulting positive clones were named mptPA-RGDl2. 10

Example 3.11

5 'TGGGGCCCGTCGCCCCGAGTGTC 3'

The washing temperature was 60 ° C in 6xSET / 0.1% SDS. The resulting positive clones were named mptPA-RGDl3. 20th

Example 3.12

5 'CGAATCGCCCCGGCAGGCGTC 3'

The washing temperature was 55 ° C in 6xSET / 0.1% SDS. The resulting positive clones were named mptPA-RGD14. 30

Example 3.13

5 'GGTCGCATGTCGCCACGAATCCAG 3'

The washing temperature was 58 ° C in 6xSET / 0.1% SDS. The resulting positive clones were named mptPA-RGDlδ. 40 Example 4

Construction of vectors for the expression of the modified tPA-DNA

DNA of the monkey virus SV40 was cut with the restriction enzymes Ba HI and Bell and the 0.24 kb fragment was prepared by gel electrophoresis (Fig. 5). The ends were filled with the Klenow fragment of the DNA polymerasel in the presence of the four deoxynucleotide triphosphates dATP, dCTP, dGTP and dTTP. Xhol linkers were then ligated.

In parallel, the commercially available vector pUClδ was linearized with the enzyme Smal 1. Then Xhol linkers were also used. DNA of this vector ("pUClβXho") was inerted with Xhol 1, treated with alkaline phosphatase and ligated with the 0.24 kb XhoI-SV40 fragment (see above). PSVpA was created. pSVpA DNA was preparatively cleaved with Xhol and incubated as ^* above with Klenow polymerase in the presence of the four dNTPs. The 0.24 kb fragment was gel isolated. At the same time, the eukaryotic expression vector CL28XhoBPV, created by ligation of CL28X and pB2-2 (according to Reddy et al. 1987, DNA 6, 461-72), was partially cut with the restriction enzyme Xbal, ie it was incubated for a limited time so that molecules arose that were split at only one of the two Xbal recognition sequences, i.e. linearized (Fig. 6). The approach was then implemented with Klenow polymerase and dNTPs as described. The linear molecules were then isolated by gel electrophoresis.

The linear pCL28XhoBPV fragments were then ligated with the pretreated 0.24 kb fragment from SV40. After transformation and screening of minilysates, a clone was isolated which carried the SV40 fragment in the former Xbal site approximately 0.15 kB 3 'to the Xhol site; this DNA ("pCL28XhoBPV-SVpolyA") carried the SV40 transcription stop signals of the "early" genes.

Plasmid DNA from pCL28XhoBPV-SVpolyA was linearized with the restriction enzyme Xhol and treated with alkaline phosphatase. At the same time, mptPARGDl-15 was cleaved with the restriction enzyme Sall and the 2.1 kb fragment was isolated. Both fragments were linked together using T4 ligase. After transformation and minilysates, a clone was isolated which contained the mutated tPA DNA simply and in the correct orientation: pCL28BPV-tPA-RGDl-15. Example 5

Transfektϊoπ and establishment of cell lines

5 C127I cells (J. Virol. 26 (1978) 292; ATCC catalog of cell lines and hybrido as 5th edition, 1985, pl42) were transfected with BPV expression plasmids using the calcium phosphate coprecipitation method (Virology 52 (1973) / 456, DNA cloning; volume II; ed. DMGlover IRL Press, pages 1 3ff and 213 (1985)).

10 5 x 105 cl27l cells were seeded in DMEM (Dulbecco's Modified Eagles Medium) + 10% FCS (fetal calf serum) in 60 mm petri dishes. The next day the medium was changed to MEM (Modified Eagles Medium) with 25mM Hepes + 10% FCS. A Ca phosphate coprecipitate was formed with 10 μg of CsCl-purified plasmid DNA, which was carefully applied to the Cl27l cells

15 was applied. The cells were kept at 37 ° C for 4 h; 7% C0 ₂ incubated. Subsequent glycerol shock treatment significantly increased the efficiency of the transfection. For this purpose, the medium was withdrawn from the cells 4 h after application of the precipitate. The cells were 3 min. with 2 ml each of 15% glycerol / HBS (DNA cloning Vol. II, page 152) each

20 60 mm petri dish incubated at room temperature. The glycerol / HBS solution was drawn off, the cell lawn was washed with 3 ml of DMEM + 10% FCS. The cells were washed with DMEM + 10% FCS at 37 ° C; 7% C0 ₂ incubated. The DMEM + 10% FCS was withdrawn three times a week and replaced with fresh one. After 2-3 weeks there were transfected cells that did

25 BPV genomes contain, to recognize as collections transformed cells, so-called foci. .

Foci expressing the tPA muteins described above were identified by a casein agar overlay (see below). After 2-3 h incubation at 37 ° C; 7% C0 ₂ were in the cloudy casein agar lysis yards on the

30 sites can be recognized where there are tPA-expressing cells. After removing the casein agar, the cells located at these sites were isolated by the "cloning-cylinder" method (DNA cloning Vol. II, p. 220). The cell lines obtained were then grown using standard methods in DMEM + 10% FCS.

35

For production, the cell lines were kept in serum-free DMEM after reaching confluence. The tPA mutein from the serum-free cell culture supernatant obtained in this way can now be characterized and purified.

40 The following solutions are required for the agar overlay test mentioned above:

Overlay agarose

1) 8% skim milk solution in H ₂ 0 are boiled at 100 ° C for 30 min and then cooled in a 37 ° C water bath.

2) A 2% solution of low-melting agarose in PBS is cooled after autoclaving in a 37 ° C water bath. Then double-concentrated DMEM is added in a 1: 1 ratio.

3) 0.64 mg of plasminogen is dissolved in 1 ml of H ₂ O. The overlay agarose is produced by pipetting together 16 ml of solution 2, 4 ml of solution 1 and 0.4 ml of plasminogen. After mixing, this is kept in a 37 ° C water bath.

To carry out the test, the cells are washed twice in 60 mm petri dishes with serum-free medium. Then 2 ml of "overlay agarose" are carefully pipetted into the petri dishes. The petri dishes are left to cool and solidify the agarose at room temperature. It is then incubated for 2-3 hours in an incubator at 37 ° C. with the addition of 7% CO ₂ and the size and number of the lysis yards are determined.

Example 6

Isolation of a new polypeptide

After sterile filtration and the addition of 0.01% © Tween 80, the tPA mutein was isolated from the serum-free cell culture supernatant obtained according to Example 5 by affinity chromatography on Erythrina trypsin inhibitor Sepharose (ETI-Sepharose, 1 cm x 3 cm). To produce the affinity matrix, 5 mg of ETI per 1 ml of CNBr-activated Sepharose 4B were coupled. The gel material was equilibrated with 20 mM Na phosphate, 0.15 M NaCl, 0.01% © Tween 80, pH 7.0 before applying the cell supernatant. After application, the gel material was treated with the same buffer in order to remove non-specifically bound material. The specifically bound mutein was then desorbed by elution with 0.1 M glycine, 0.1 M arginine / HCl, 0.01% © Tween 80, pH 3.0. The eluate was combined and adjusted to pH 5.0 with 0.1 M sodium hydroxide solution. The following polypeptides were obtained, which differ as follows from the tPA shown in Fig. La-d:

6.01 8G, 9D, 46G, 177S, 263S

56.02 28G, 29D, 46G, 177S, 263S

6.03 31G, 32D, 46G, 177S, 263S

6.04 103D, 46G, 177S, 263S

6.05 109R, 111D, 46G, 177S, 263S

6.06 131G, 46G, 177S, 263S 106.07 301R, 303D, 46G, 177S, 263S

6.08 384G, 385D, 46G, 177S, 263S

6.09 398R, 399G, 46G, 177S, 263S

6.10 458R, 46G, 177S, 263S

6.11 463G, 464D, 46G, 177S, 263S 156.12 475R, 46G, 177S, 263S

6.13 523G, 524D, 46G, 177S, 263S

The numerals denote the amino acid positions in which the polypeptide differs from the tPA shown in Fig. La-d, while the letter stands for the 20 amino acid, which is in the mutein at the position indicated by the digit.

Example 7

25 Characterization of the oligosaccharide content

5-10 μg of the mutein obtained according to Example 6 were separated by SDS gel electrophoresis and then transferred to nitrocellulose membranes. After saturation with PBS buffer (2 mM phosphate,

30 150 mM NaCl, pH 7.4), which contained 0.1% © Tween 20, pH 7.4, the membrane was incubated for 2 hours with lectin from Griffonia Si plicifolia coupled to peroxidase. After removing unbound material with the aid of TBS buffer (10 M Tris, 150 mM NaCl, pH 7.4), the nitrocellulose was kept in 50 mM Tris, 150 mM NaCl, 0.02% H ₂ O for 5-10 min ₂ and 0.5 mg / ml

Incubated 354-chloro-1-naphthol. Positive reactions became visible within 5 min by blue coloring of the protein band. The reaction was then stopped by transferring the nitrocellulose membrane into water.

The oligosaccharide residues contain α-linked galactose residues which, according to 40 Fig. 8, are linked to β-linked subterminal galactose residues. The α-galactose residues are preferably localized on the galactose ⁽ T_ (numbering of the sugar residues see Fig. 8). The other subterminal galactose residues are either substituted by sialic acid or other α-linked galactose.

Claims

1. tPA-like polypeptides, characterized in that they have the amino acid sequence of tPA or sequences derived therefrom, but in which 1-6 tripeptides are replaced by the tripeptide RGD.

2. DNA sequences which code for the polypeptides according to claim 1.

3. Vectors containing gene sequences which code for the polypeptides according to claim 1.

4. Genetic engineering process for the production of peptide sequences according to claim 1, characterized in that a gene is expressed in a host organism which codes for the peptide sequences according to claim 1.

5. Medicament containing at least one polypeptide according to claim 1, optionally in a pharmaceutically acceptable carrier or binder.

6. Medicament according to claim 5, comprising the combination of at least one polypeptide according to claim 1 and another fibrinolytic.

7. Process for the preparation of tPA-like polypeptides which

Having amino acid sequence of the tPA or sequences derived therefrom, but in which 1-6 tripeptides are replaced by the tripeptide RGD, characterized in that a gene is expressed in a host organism which codes for the peptide sequences.