DE3710364A1 - Vascular anticoagulant proteins, DNA which encode these, process for their preparation and their use - Google Patents

Abstract

The present invention relates to biologically active proteins, to the DNA molecules coding for the latter, to processes for their preparation and to the use thereof.

Description

The present invention relates to biological active proteins coding for this DNA molecules, processes for their manufacture and their use.

Proteins exist in most mammals have anticoagulant properties. These Proteins can be divided into three groups, the division on the different Mechanisms of action based.

• 1. Proteins that form a complex with the coagulation factor and thereby render the coagulation factor ineffective. These include the proteins:
  • a) Antithrombin-III (Tromb. Res. 5, 439-452 (1974))
  • b) α 1 protease inhibitor (Ann. Rev. Biochem. 52, 655-709 (1983))
  • c) α ₂ macroglobulin (Ann. Rev. Biochem. 52, 655-709 (1983))
  • d) C₁ inhibitor (Biochemistry 20, 2738-2743 (1981))
  • e) Protease Nexin (J. Biol. Chem. 258, 10439-10444, (1983)).
• 2. Proteins that have a clotting factor proteolytically cut and thereby the coagulation factor deactivate. It is the only protein of this type previously described protein C (J. Biol. Chem. 251, 355-363, (1976)).
• 3. Proteins that contain the negatively charged phospholipids shield and / or hydrolize so that the Phospholipid-dependent reactions of the Blood clotting mechanism can be inhibited. So far are just phospholipases that come from different Snake venom varieties have been isolated (Eu. J. Biochem. 112, 25-32 (1980)).

The gradual coagulation system has been intensively examined in recent years. It is understood as an amplifying multi-stage system of various interconnected proteolytic reactions in which an enzyme converts a zymogen into the active form (see Jackson CM, Nemerson Y., Ann. Rev. Biochem. 49, 765-811, (1980 )). The speed of this reaction is significantly increased by the presence of phospholipids and other cofactors such as factor V _a and factor VIII _a . In vivo, the procoagulation reactions are regulated by various inhibition mechanisms that prevent explosive thrombotic trauma after a slight activation of the coagulation cascade.

The anti-clotting mechanisms can be as follows can be classified (Rosenbert, R.D., Rosenbert, J.S., J. Clin. Invest. 74, 1-6 (1984)):

• 1. The serine protease factor X _a and thrombin are inactivated due to their binding to antithrombin III or to the antithrombin / heparin complex. Prothrombin activation as well as the formation of fibrin can be inhibited in this way. In addition to anti-thrombin III, there are various other plasma protease inhibitors such as α ₂ macroglobulin and antitrypsin, the effect of which is time-dependent.
• 2. The discovery of protein C led to the discovery of another anti-clotting mechanism. Once protein C has been activated, it acts as an anticoagulant through the selective proteolysis of the protein cofactors factor V _a and VIII _a , by which the prothrombinase and the enzyme that converts factor X are inactivated. Exposure of thrombin to fibrinogen, thereby preventing the formation of insoluble fibrin (Nossel, HL, Nature, 291, 165-167 (1981)).

Of the above, in the clotting process engaging body's proteins is such. Currently only that Anthribombin III in clinical use. As a significant one However, the disadvantage of using this Protein increase in bleeding tendency exposed.

All agents previously used as anticoagulants, whether the body's own or synthetic nature somehow the clotting factors are ineffective and This leads to side effects that are disadvantageous can affect the coagulation process.

Surprisingly, in addition to these proteins other endogenous substances are isolated, although the desired anticoagulant properties show under special conditions, but here the Do not increase the risk of bleeding. With major bleeding these proteins lose their anticoagulant properties Properties and thereby interfere with their use not those necessary for survival in such cases Coagulation processes. Since they were first isolated from highly vascularized tissue, they became "vascular anticoagulating proteins ", called VAC.

Made from highly vascularized tissues like Umbilical vessels and placenta isolated proteins have molecular weights of approx. 70 × 10³, approx. 60 × 10³, approx. 34 × 10³ and approx. 32 × 10³, of which the substances with the molecular weights 34 respectively 32 × 10³ consist of a single polypeptide chain. The exact biochemical characterization of this Proteins, as well as their isolation and purification takes place in EP-A-01 81 465 of May 21, 1986.

Proteins with VAC activity are natural Anticoagulants that are used in the Intervene in two coagulation cascades.

The first time they inhibit by the factors IXa and VIIIa catalyzed activation of factor X to Xa, the second time they prevent the split from Prothrombin to thrombin by factors Xa and Va is conveyed. Both activation steps are common that they are calcium ions and phospholipids need. Apparently VAC proteins can also use Phospholipids interact, and through this binding the activation steps of the Block coagulation factors.

The proteins close their properties interesting, pharmacologically extremely valuable Active ingredients. However, in order to supply them in sufficient quantities to have highly pure form available Manufactured in a way other than protein purification natural tissue required. The genetic engineering method.

The object of the present invention was therefore proteins genetically engineered with VAC activity.

This task was solved by the fact that Amino acid sequence from parts of the strong vascularized tissue isolated and highly purified Proteins with VAC activity (EPA 01 81 465) resolved was based on these sequences synthetic DNA probes were produced and hereby suitable cDNA libraries were searched. After isolation of cDNA hybridizing with the probes, the Sequence determination and corresponding manipulation these cDNA were in appropriate host systems for example in bacteria, yeasts or mammalian cells brought to expression.

One of the purified proteins became enzymatic split with trypsin. The resulting peptides were separated and selected fragments sequenced. The However, the sequence of the N-terminus resisted one direct analysis since the first amino acid is obviously blocked. The resulting Subfragments were sequenced. Based on these Information can be the beginning of information in a cDNA are found, especially since Molecular weight of the N-terminal fragment is known (FAB-MS).

The sequence information is listed below.

To prepare a suitable DNA probe basically three ways possible. Is a longer one Section of the protein, about 30 or more amino acids long, known, there is the possibility of considering one of the preferred codons used in mammals most likely sequence for the corresponding one create mRNA section. Such a probe is in the worst case about 66% homologous to actual Sequence. This is also the reason why the probe would have to be relatively long to include non-stringent ones To be able to hybridize conditions.

The second option is for a short Peptide segment, about six to seven amino acids long, all conceivable variations of oligonucleotides synthesize. Are such complex blends at Browse cDNA libraries can be used relatively many "wrong" positive clones be isolated. In addition, the hybridization the signal is very weak because it fits perfectly Single oligonucleotide only a small part of the Total mix makes up.

The third way does not avoid the variability of one Oligonucleotide probe, but ensures by choosing one special nucleotide triphosphate (ITP) that the searched (or "wrong") cDNA all molecules can bind the probe. So became the tryptic Peptide [?] Matching 23 base oligonucleotides synthesized using inosine triphosphate.

As mentioned earlier, VAC proteins can be made from strong vascularized tissue can be isolated. Tissue of The choice is umbilical cord vessels or placenta. Therefore and there it is known that in contrast to the placenta other special tissues expressed almost all genes the DNA probes mentioned above were used a placental cDNA library from placental Fabric was made in a conventional manner was looking for cDNA molecules encoding VAC proteins to search.

To search the cDNA library for cDNA coding for VAC protein, an oligonucleotide was synthesized in accordance with the sequence of the tryptic peptide P16 / II two and the Staph A peptide P20 / I / 6 ( FIG. 1). These oligonucleotides each represent a mixture of all variants that take into account every coding possibility of the corresponding mRNA. EBI-386 has 512 variations with a chain length of 20 nucleotides and matches the Staph-A peptide P20 / I / 6. In order to keep the variation in the oligonucleotide for the tryptic peptide P16 / II lower, two oligonucleotides (20-mers) were synthesized: EBI-387: 128 variations, EBI-388: 64 variations.

Furthermore, two oligonucleotides matching the tryptic P30 / I VAC peptide were synthesized using deoxynosin as base at "wobble" positions ( FIG. 2): EBI-118 and EBI-119. This substitution was described by E. Ohtsuka et al., J. Biol. Chem. 260/5 (1985), pp. 2605-2608, and Y. Takahashi et al., Proc. Nat. Acad. Sci. (1985) pp. 1931-1935. Inosine bases well with cytosine, but hardly interferes with the formation of the double helix if other nucleotides are offered as partners.

After each of these oligonucleotides in a known manner prints had been radiolabelled Hereby phage plates by known methods hybridizes.

From hybridization with EBI-386, -387 and -388 resulted in the phages Lamda-P11 / 3, Lambda-P6 / 5, Lambda P6 / 6. Hybridization with EBI-118 and -119 resulted in phages Lambda-Nr15, Lambda-Nr19 and Lambda No. 22.

The DNAs isolated from the phages were analyzed with EcoRI cut and isolated fragments. The cDNA inserts of the clones Lambda-P11 / 3, Lambda-P6 / 5 and Lambda-P6 / 6 had sizes from about 1300 to 1400 bp. Sequence analysis showed that all three clones of one and the same mRNA were derived. The 5′-end of the However, mRNA was missing in the cDNAs. The phage inserts Lambda-Nr15, Lambda-Nr19 and Lambda-Nr22 had lengths of approx. 1600, 1100 and 1000 bp. The sequence analysis left assume an almost complete cDNA.

The cDNAs of the two phage groups Lambda-P11 / 3, Lambda-P6 / 5 and Lambda-P6 / 6 and Lambda-Nr15, Lambda-Nr19 and Lambda-Nr22 are of two different ones mRNAs derived, as the sequence analysis showed. The EcoRI inserts of the three clones Lambda-P11 / 3, Lambda-P6 / 5 and Lambda-P6 / 6 were isolated and in the Ligated EcoRI-cut Bluescribe M13⁺ vector (Vector Cloning Systems, 3770 Tansy Street, San Diego, CA 92121, USA). The resulting clones became pP6 / 5, called pP6 / 6 and pP11 / 3.

The EcoRI inserts of the three clones Lambda-Nr15, Lambda-Nr19 and Lambda-Nr22 were isolated and in the Ligated EcoRI-cut Bluescribe M13⁺ vector. The resulting clones were pRH201, pRH202 and called pRH203.

In order to obtain further cDNA clones, the human, searched placental lambda-gt10 library again, this time with the radio-labeled EcoRI insert of the pP11 / 3 as a probe.

A total of 69 positive clones were obtained (Lambda-VAC1 to Lambda-VAC69).

12 of these clones were made on a small scale as above prepares the cDNA inserts with EcoRI released and isolated. It turned out that the Insert the clone Lambda-VAC10 the whole for VAC- Contains protein coding reading frames. To characterize the for VAC-alfa and VAC-beta coding cDNAs were a Northern blot experiment, Sequence analysis and a genomic Southern blot Analysis carried out.

The result is shown in Fig. 3. The cDNA of clone pP11 / 3 hydrides to a mRNA of the size of approximately 1700 bases ("VAC-alpha"), the cDNA of the clone pRH 203 to a mRNA of the size of approximately 2200 bases ("VAC-beta").

Firstly, the amount of radioactivity used as well the amount of mRNA applied per lane was approximately the same and second, the hybridization of a genome blot in same solution bands of equal intensity with both cDNAs revealed (see below), it can be concluded that the shorter mRNA ("VAC-alfa") in larger amounts than that longer ("VAC-beta") mRNA and placenta is represented.

For the sequence analysis of the VAC-alpha cDNA, the cDNA clones pP6 / 5, pP6 / 6 and pP11 / 3 total and those of the clones Lambda-VAC1 to -12 were partially sequenced. The result is shown in Fig. 4. A total of 1465 bases were sequenced. The cDNA has a long, open reading frame that can code for 320 amino acids. If the DNA sequence is translated into an amino acid sequence, all sequenced peptides of the tryptic fragments can be accommodated in this sequence ( FIG. 5). This cDNA is therefore the one whose corresponding mRNA codes for VAC protein. Since the sequences of the second isolated cDNA code for a similar but different protein from VAC, the name VAC-alfa is introduced here.

The first ATG codon (bases 35-37) goes in the same Reading frames advance a stop codon. The bases 30 to 38 meet the Kozak rule quite well (M. Kozak, Nature 1984, 308, pp. 241-246), which the consensus sequence in close to the translation start codon with CC (A / G) CCAUGG indicates the corresponding sequence here TCGCTATGG. The 3 ′ untranslated region is 471 Bases long. 15 bases before the start of the poly-A section is the polyadenylation sequence AATAAA (N.J. Proudfoot et al., Nature 263, (1976), pp. 211-214). Will be a chain length of 150 to 200 bases calculated for the poly-A section of the mRNA the total length of the mRNA based on the cDNA sequence 1600-1650 bases. Since in the Northern blot experiment higher value has been determined is the 5 ′ untranslated region in no cDNA complete contain.

In contrast to all, the cDNA of clone pP6 / 5 points other cDNA clones at position 100 C instead of A. This would make the triplet 98-100 (22nd codon) of Change GAA to GAC and for Asp instead of Glu encode. This deviation can have several causes have: a) the reverse transcriptase built a wrong one Nucleotide one, b) it is the transcripts two allers, different genes at this point or c) there are two non-allelic genes that are involved distinguish this position.

The long, open reading frame codes for a protein with 320 amino acids, of which the Met-1 is likely split off, and the following alanine on the Amino group, possibly through acylation, is blocked. The calculated molecular weight is 35,896 D and is higher than the value according to SDS-PAGE. However, the proportion is more loaded Amino acids (Asp, Glu, Lys, Arg, His) with 30.6% (98/320) above average compared to the average value of 25.1%.

This would change the migration behavior of the Explain proteins at SDS-PAGE. Within the strong charged amino acids outweigh the acidic amino acids (Asp and Glu) with 54 compared to the basic (Lys and Arg) with 41. This explains the acidic, isolelectric Point of the VAC-alfa protein (pI = 4.4 to 4.8) VAC-alfa contains only one triplet coding for cysteine (Amino acid position 361); a typical one N-glycosylation site (Asn-XXX-Ser / Thr) is not available.

A structural analysis of the amino acid sequence (modified according to Pustell, J et al., Nucleic Acids Res. 10 (1982) pp 4765-4782) shows a four-fold repetition of a 67 amino acid sequence ( FIG. 6), hereinafter referred to as "repeats". Within this sequence, 7 amino acids (10.4%) are conserved in all four repeats, 15 amino acids (22.4%) occur in three of four repeats, at 28 positions (41.8%) two repeats each contain the same amino acid.

A comparison with published literature data (JJ Gisow, FEBS Letters 203 (1986), pp. 99-103, MJ Geisow et al., TIBS 11 (1986, pp 420-423) surprisingly showed that VAC-alfa thus became a larger group Ca ⁺⁺ dependent phospholipid binding proteins are heard and a consensus sequence is described (Lys-Gly-fob-Gly-Thr-Asp-Glu-var-var-Leu-Ile-fil-Ile-Leu-Ala-fob-Arg; fob = hydrophobic , fil = hydrophilic, was = variable) that could be involved in Ca ⁺⁺ binding (MJ Geisow et al., Nature 320 (1986), pp. 636-638) This sequence can be found in each of the four repeated 67 Amino acid long sub-sequences of the proteins according to the invention ( FIG. 6) Also striking is the 6 amino acid section at the end of each repeat, which consists almost exclusively of hydrophobic amino acids ("oooooo" in FIG. 6).

Sequence analysis of clones Nr15, Nr19 and Nr22 showed 1940 bp for the VAC-beta cDNA, which merges into a poly-A section ( FIG. 7). The polyadenylation signal AATAAA is found 16 bases in front of the poly-A section. However, this consensus sequence already occurs at nucleotide position 1704-1709. Why this sequence is not used as a polyadenylation signal is unknown. The additionally required sequence on the 3'-side of the AATAAA sequence YGTGTTYY (Gill A. et al., Nature 312 (1984), pp. 473-474) occurs only relatively far away (TGTGTTAT, position 1735-1742); possible that this explains the non-acceptance of this first polyadenylation sequence.

The cDNA contains a long, open reading frame that extends from the beginning of the cDNA to position 1087. He would encoding potential for 362 amino acids. For reasons of analogy to VAC-alfa and due to The fact that a 34,000 D protein is also available VAC cleaning occurs (see E.P.A. 1 81 465) the first nethionine codon (ATG, position 107-109) as Translation start accepted. The Kozak rule is here not as well fulfilled as with VAC-alfa (AAGAGATGG on Item 102-110).

The resulting protein (VAC-beta) is 327 amino acids long. It has cysteine residues (amino acid position 161, 206, 250 and 293) and a potential N-glycosylation site (Asn-Lys-Ser, amino acid position 225-227). The calculated molecular weight is 36,837 ( Fig. 9). VAC-beta also has an above-average number of charged residues: 97/327 (29.6%), the acidic amino acids (Asp + Glu: 49) predominating over the basic ones (Lys + Arg 42); an explanation for the lower molecular weight determined according to SDS-PAGE.

VAC-beta also shows an internal repetition of a 67 amino acid sequence ( FIG. 8). Within this sequence, 7 amino acids (10.4%) are conserved in all four repeats, 17 amino acids (25.4%) occur in three of four repeats, at 25 positions (37.7%) two repeats each contain the same amino acid. VAC-beta also shows good agreement with the 17 amino acid consensus sequence. The explanations for the VAC-alfa apply analogously to the VAC-beta.

The analysis of chromosomal DNA from human placenta according to Southern shows a complex picture. The DNA was cut with EcoRI, HindIII, BamHI and PstI. The DNA transferred to nitrocellulose was hybridized both with a VAC-alpha DNA (pP11 / 3) and with a VAC-beta DNA (pRH203). Although the filters were washed under stringent conditions, a relatively large number of bands resulted with each digestion ( FIG. 10). The comparison of the two blots shows that the cross-reaction of VAC-alpha and beta DNA can be excluded under these conditions. The large number of bands can either be explained by the existence of genes that are similar to the VAC-alpha or VAC-beta gene, or else it is a gene that is interrupted by many and / or long introns.

FIG. 11 shows the comparison of the amino acid sequences of VAC-alpha with VAC-beta. The repeated structures can be arranged in the same way in both proteins. The connecting peptides are also of the same length, with the exception of those between the 2nd and 3rd repeat. In VAC-alpha, a gap must be introduced into this connecting peptide in order to allow an optimal adaptation of the two sequences. The N-terminal peptide of the two proteins is of different lengths, 19 amino acids for VAC-alfa, 25 amino acids for VAC-beta. This peptide also has the least homology. The two proteins are identical at 176 out of 320 amino acid positions, which corresponds to a degree of homology of 55.0%.

At this point, the comparison of the Nucleotide sequences of the VAC alfa and beta cDNAs inserted. Become two genes and their products compared to each other, you can see on the DNA (= RNA) Greater homology than at the level Amino acid level, which is explained by the fact that already a change in one with the nucleic acid Base triplet is sufficient to add a new amino acid encode.

FIG. 12 shows the comparison of the coding regions of VAC-alpha and VAC-beta cDNA. Surprisingly, the DNAs only show a degree of homology of 54.2%, which is slightly less than the two proteins.

In Fig. 13, the Hydrophilizitätsprofile of the two proteins are shown. The algorithm of Hopp and Wood was used (TP Hopp et al., Proc.Natl.Acad.Sci. 78 (1981) pp. 3824-3828). The four repeat areas are indicated by the bars, the connecting peptides are framed in the sequence below. Surprisingly, the connecting peptide between the second and third repeat is particularly hydrophilic. In both VAC-alpha and VAC-beta there is an arginine in this peptide in an identical position. It would therefore be possible for this arginine to be a preferred target for a protease with trypsin-like specificity. The molecule would have to break down into two halves of approximately the same size. It would be conceivable that such a “half-molecule” could have a biological, for example an anticoagulant, effect. In addition to the targeted digestion of the protein, for example with a protease of trypsinar specificity, these half-molecules or half-molecules can be prepared in a variety of ways with minor modifications. It is thus possible to insert the DNA molecules coding for these half-molecules, which can be prepared by processes known per se, into suitable expression vectors in such a way that this DNA is regulated by the expression control sequences and to express the half-molecules by cultivating a host organism transformed with these vectors. to isolate and clean.

Half molecules with minor modifications are obtained for example by inserting cleavage sites that the complete protein for certain proteases give required specificity. So you can for example, a protein with a cleavage site Establish factor Xa-like specification by in the Section responsible for the linker sequence between the coded second and third repeat structure, a Oligonucleotide is inserted that for the Detection tetrapeptide encoding factor Xa protease. This gives you a complete protein that possibly changed properties, for example, changed stability towards proteolytic attack, but on the other hand one highly specific cleavage site for the factor Xa protease contains, which specifically produces two half-molecules and can be used therapeutically.

By specifically mutating the for proteins with DNA encoding VAC activity can also be Produce proteins that cause a proteolytic attack resist better. So possibly the Exchange of the VAC-alfa and VAC-beta common arginine between the second and third repeat structure e.g. B. histidine for better stability of the proteins. Also the targeted replacement of the other arginines and lysines with histidine, possible by replacing the corresponding one Codone leads to proteins that are complicated by trypsin can be hydrolyzed. These targeted mutations let the biological, for example anticoagulant properties of the Proteins / polypeptides essentially unaffected, on the other hand, however, lead to change, for example to improve the stability of the Proteins, d. H. to a change in half-life of the proteins.

In some cases it is, for example for the Expression of proteins beneficial, the mature, desired proteins / polypeptide a signal sequence or also a protein sequence specific to the host organism upstream, so that as an expression product Fusion protein arises for a signal peptide coding signal sequence can be of microbial origin or derived from mammalian cells; preferably that is Signal peptide homologous to the host organism. Around Convert products into the desired mature protein a specific gap point is required between the signal / fusion portion and the mature Protein. You can do this, for example, as above described an oligonucleotide at this point insert that for the recognition tetrapeptide of Factor Xa protease encoded. This makes it possible expressed fusion protein specifically with the Cleave factor Xa protease. The mature protein is obtained.

If you specifically replace those responsible for methionine Codons, other than that at amino acid position 1, by codons, which code for leucine, isoleucine or valine, so lets a possibly received immature or Fusion protein by BrCN cleavage into the mature protein convict.

Should prove that such modified proteins same or, if necessary, improved biological Have properties, this would be a possible way to increase the yield and, if necessary, a process for the production of proteins with better properties than the natural VAC proteins.

Targeted exchange is another targeted mutation of one, possibly several cysteines by serine or to call valine by the corresponding codons be replaced. Does this exchange affect the biological activity not detrimental, so this could Way to improve insulation and / or yield of Cause proteins. It cannot be ruled out here either an improvement in the properties of the proteins.

From Fig. 13 it is also easy to see that neither VAC-alfa nor VAC-beta have a longer, hydrophobic region which would either allow the secretion by, or the incorporation of the proteins into a membrane. It can therefore be assumed that VAC-alfa and VAC-beta are intracellular proteins. Wallner et al (Nature 320 (1986), pp. 77-81) report the structure of human lipocortin I, Saris et al. (Cell 46 (1986), pp. 201-212) and Huang et al (Cell 46 (1986), pp. 191-199) the structure of the murine or human calpactin I, which is also called lipocortin II. These proteins also belong to the class of Ca ⁺⁺ dependent phospholipid binding proteins. Their structure can be written in analogy to VAC-alpha and beta ( FIG. 14). The structural differences are essentially limited to the N-terminal non-repeat regions, which have 46 amino acids in Lipocortin I and 37 amino acids in Lipocortin II. However, the homologies between VAC-alfa and VAC-beta are more pronounced than those between VAC and lipocortin:

VAC-alfa-VAC-beta 55.0% VAC-alfa-Lipocortin I41.9% VAC-alfa-Lipocortin II43.8% VAC-beta-lipocortin I41.7% VAC-beta-lipocortin II 44.6%

It can therefore be assumed that the lipocortins also have an anticoagulant effect due to their analogous structure, and are therefore to be used as anticoagulants, and furthermore that this is a general property of this class of Ca ⁺⁺ -dependent phospholipid binding proteins. Furthermore, on the basis of the analogous structures, it can be assumed that the proteins according to the invention not only have the anticoagulant activity but also the properties of the class of Ca ⁺⁺ -dependent phospholipid binding proteins which are already known. The anti-inflammatory properties of lipocortins may be mentioned here by way of example. The present invention therefore also relates to the use of the proteins according to the invention as anti-inflammatory agents and the use of the proteins according to the invention in the treatment of all indications applicable to the lipocortins, such as rheumatic diseases. For this purpose, too, the products according to the invention corresponding to the natural proteins can be modified in the manner already mentioned and their properties may be improved. When comparing the proteins VAC-alpha, VAC-beta, Lipocortin I and Lipocortin II, it is found that the most striking difference between the proteins is in the N-terminal peptides, in particular between the VAC proteins on one and the lipocortins the other side. A modification according to the invention therefore provides for the mutual exchange of these N-terminal peptides. These modified proteins can be produced in that the DNA molecules coding for these N-terminal peptides are produced in a manner known per se, for example by oligonucleotide synthesis, and with the “residual DNA”, that for the remaining repeats and the linker sections encoded, ligated. Expression vectors provided with this DNA can be expressed in a suitable manner in suitable host organisms; the expressed protein is isolated and purified.

Many members of the Ca ⁺⁺ dependent phospholipid binding proteins bind to purified secretory vesicles or other components of the cytoskeleton (see RH Krestinger et al., Nature 320 (1986), p. 573 for a summary). During secretion, the vesicles isolate from the cell's Golgi apparatus, migrate to the cell membrane and fuse with it. The content of the vesicles is released from the cell. In analogy to the coupling of excitation with muscle contraction, it has been suggested (WW Douglas, Br. J. Pharmac. 34 (1968), 451) that the stimulus and the secretion are also coupled via Ca ⁺⁺ . The Ca ⁺⁺ dependent phospholipid binding proteins could play an important role here. In the conserved, 17 amino acid region of each repeat, VAC-alfa has 5 to 6 hydroxyl group-containing amino acids (Asp, Glu, Thr, Ser), three of which are each in an identical position. VAC-beta contains four of these amino acids in each repeat. Although none of the proteins has the EF-hand structure observed in calmodulin, troponin C, S-100 and parvalbumin (RH Kretsinger et al., CRC Crit. Rev. Biochem. 8 (1980), p119), which is found in these molecules for the Ca ⁺⁺ binding is responsible, the conservation of this subsection in each repeat leads to the conclusion that this region is responsible for the Ca ⁺⁺ binding. Targeted mutations in this area could attempt to change the biological activity of the proteins. Mutations according to the invention provide, for example, the complete or partial replacement of the regions coding for the 17 amino acids of the conserved region:

• a. from the VAC proteins through areas that the encode the corresponding amino acids of the lipocortins,
• b. from VAC-alfa through areas that have the appropriate Encode VAC-beta amino acids and vice versa,
• c. from Lipocortin I through areas that match the corresponding Encode amino acids of lipocortin II and vice versa,
• d. from the lipocortins through areas that the encode corresponding amino acids of the VAC proteins or
• e. for the remaining polypeptides / proteins by areas which are the corresponding amino acids of each other Encode polypeptides / proteins, by coding the corresponding ones for these amino acids Areas to be exchanged.

The analog structure mentioned above also suggests that proteins that have these analogies are both anti-inflammatory as well as anticoagulant effect show and accordingly therapeutic and / or can be used prophylactically.

The present invention also relates to DNA molecules that are used for a polypeptide / protein Repeat areas encode DNA molecules that are responsible for one of the Repeat areas encode DNA molecules, thereby characterized in that for complete repeats coding areas are rearranged by these encoded proteins, their production and use.

All available through this or similar mutations Proteins that are responsible for these coding DNAs Production and use are also the subject of present invention, the known in part in Proteins described for the present application, which for these coding DNAs, their known production and Use are expressly excluded.

The present invention also relates to DNA molecules of non-human origin made by these encoded proteins, their production and use, the in a manner analogous to the present invention are producible.

The invention relates not only to gene sequences that encode specifically for the proteins of the invention, but also modifications that are easy and routinely through mutation, degradation, transposition or Addition can be obtained. Any sequence for that encoded proteins of the invention (i.e. the biological activity spectrum that here ) and in comparison with those shown degenerate is also included; Professionals in this field are able to sequence DNA to degenerate coding regions. Everyone is the same Sequence for a polypeptide with the activity spectrum encoded the proteins of the invention, and with the sequences shown (or parts thereof) under stringent Conditions hybridized (e.g. conditions that for more than 85%, preferably more than 90% homology select) includes.

The hybridizations are in 6 × SSC / 5 × Denhardt's Solution / 0.1% SDS performed at 65 ° C. The degree of Stringency is in the washing step on DNA sequences with about 85% or more homology the conditions 0.2x SSC / 0.01% SDS / 65 ° C and for selection on DNA sequences with approx. 90% or more homology the conditions 0.1x SSC / 0.1% SDS 65 ° C suitable.

The invention further relates to expression vectors which contain a DNA sequence coding for VAC, which of an expression control sequence is regulated in such a way that in one with this expression vectors transformed host cell polypeptides with VAC activity be expressed.

The expression vectors of the present invention will e.g. B. prepared by inserting into a vector DNA, which contains an expression control sequence, a VAC inserts coding DNA sequence such that the Expression control sequence regulates said DNA sequence.

The choice of a suitable vector results from the Transformation provided host cell. Suitable hosts are, for example, microorganisms such as yeasts, e.g. B. Saccharomyces cerevisiae, and in particular Strains of bacteria that have no restriction or Modification enzyme feature, especially strains of Escherichia coli, e.g. B. E. coli X1776, E. coli HB101, E. coli W3110, E. coli HB101 / LM1035, E. coli JA221 (30) or E. coli K12 strain 294, Bacillus subtilis, Bacillus stearothermophilus, Pseudomonas, Haemophilus, Streptococcus and others, further cells higher Organisms, especially established human or animal Cell lines. Whole strains are preferred host cells of E. coli, in particular E. coli HB101, E. coli JM101 and E. coli W3110.

Basically, all vectors are suitable which the heterologous invention coding for the VAC Replicate DNA sequences in the chosen host and express.

Examples of vectors which are suitable for expressing the VAC gene in an E. coli strain are bacteriophages, e.g. B. derivatives of bacteriophage γ , or plasmids, such as in particular the plasmid colE1 and its derivatives, e.g. B. pMB9, pSF2124, pBR317 or pBR322. The preferred vectors of the present invention are derived from plasmid pBR322. Suitable vectors contain a complete replicon and a labeling gene, which enables the selection and identification of the microorganisms transformed with the expression plasmids on the basis of a phenotypic characteristic. Suitable markings give the microorganism, for example, resistance to heavy metals, antibiotics and the like. Furthermore, preferred vectors of the present invention contain recognition sequences for restriction endonucleases outside the replicon and marker gene regions, so that the DNA sequence coding for the amino acid sequence of VAC and optionally the expression control sequence can be inserted into these sites. A preferred vector, plasmid pBR322, contains an intact replicon, marker genes conferring resistance to tetracycline and ampicillin (tet ^R and amp ^R ) and a series of unique recognition sequences for restriction endonucleases, e.g. B. PstI (cuts in the amp ^R gene, the tet ^R gene remains intact), BamHI, HindXIII, SalI (cuts all in the tet ^R gene, the amp ^R gene remains intact), NruI and EcoRI.

Several expression control sequences can be used to regulate VAC expression. In particular, expression control sequences of strongly expressed genes of the host cell to be transformed are used. In the case of pBR322 as a hybrid vector and E. coli as the host organism, the expression control sequences (which include the promoter and the ribosomal binding site, among others) of the lactose operon, tryptophan operon, arabinose operon and the like, the β- lactamase gene, are the corresponding sequences of the phage γ N- Gene or the phage fd layer protein gene and others. While the promoter of the β- lactamase gene ( b -lac gene) is already contained in plasmid pBR322, the other expression control sequences have to be introduced into the plasmid. The preferred expression control sequence in the present invention is that of tryptophan operon (trp po) both from Scaratia marcescens and from E. coli and the alkaline phosphatase promoter or its hybrid.

In addition to these particularly common promoters, other microbial promoters have also been developed and used. The gene sequence for the proteins according to the invention can be used, for example, under the control of the leftwart promoter of the bacteriophage lambda (P _L ). This promoter is a particularly effective controllable promoter. Control is made possible by the lambda repressor, from which neighboring restriction interfaces are known. A temperature sensitive allele of this repressor gene can be inserted into a vector containing a protein sequence. If the temperature is increased to 42 ° C, the repressor is deactivated and the promoter activated. Using this system, it is possible to establish a clone bank in which a functional protein sequence is placed in the vicinity of a ribosome binding site at varying distances from the lambda P _L promoter. These clones can then be checked and selected with the highest yield.

Expression and translation of a sequence coding for the proteins of the invention can also be under control other regulatory systems that are "homologous" to the Organism can apply in its untransformed form, expire. So contains z. B. chromosomal DNA from one Lactose-dependent E. coli is a lactose or lac operon that by release of the enzyme beta-galactosidase Lactose breakdown enables.

The Lac control elements can be from the bacteriophage Lambda-plac5, which is infectious to E. coli will. The phage's lac operon can be by transduction come from the same bacterial species. Regulation systems in the inventive Processes can also be used plasmid DNA originate from the organism. The Lac promoter operator system can be induced by IPTG will.

Other promoter-operator systems or parts thereof can can also be used: for example Colicine E 1 operator, galactose operator, xylose A operator, tac Promoter u. Ä.

In addition to prokaryotes, eukaryotic micro can also be used organisms such as yeast cultures are used. Saccharomyces cerevisiae is the most widely used of the eukaryptic microorganisms, although a number other species is generally available.

Vectors suitable for replication and expression in yeast contain a yeast replication start and a selective one genetic marker for yeast. Hybrid vectors that one Yeast replication start included, e.g. B. the chromosomal autonomously replicating segment (ars), remain after the Transformation within the yeast cell exta-chromosomal are maintained and replicated autonomously in mitosis. To Expression in Saccharomyces is, for example, the plasmid YRp7 (Stinchcomb et al. Natur 282, 39 (1979); Kingsman et al., Gene 7, 141 (1979); Tschumper et al., Gene 10, 157 (1980)) and the plasmid YEp13 (Bwach et al., Gene 8, 121-133 (1979)) was used. The plasmid YRp7 contains the TRP1 gene, which is a selection marker for a mutant yeast, unable to grow in tryphthophane free medium, provides; for example ATCC No. 44076.

The existence of the TRP1 damage as a characteristic of the Yeast host genome is then an effective tool to demonstrate transformation when without tryptophan is cultivated. The situation is similar Plasmid YE13, which is the yeast gene LEU 2, used as a supplement a LEU-2 minus mutant can be used.

Other suitable labeling genes for yeast are in the case of auxotrophic yeast mutants, generally genes that Complement host defects. Appropriate genes provide for Prototrophy in an auxotopic mutant yeast, e.g. B. the URA3- and HIS3 gen. Preferably contain yeast hybrid vectors continue to start replication and a marker gene for a bacterial host, especially E. coli, so that the Construction and cloning of the hybrid vectors and their Precursors can be done in a bacterial host. Further suitable for expression in yeast Expression control sequences are, for example, those of the PHO3 or PHO5 gene, furthermore in glycolytic structure promoter involved, e.g. B. the PGK and GAPDH promoter.

Other suitable promoter sequences for yeast vectors contain the 5′-flanking region of the genes of ADH I (Ammerer G., Methods of Enzymology 101, 192-201 (1983)), 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255, 2073 (1980)) or other glycolytic enzymes (Kawaski and Fraenkel, BBRC 108, 1107-1112 (1982)) such as enolase, Glyceraldehyde-3-phosphate dehydrogenase, hexokinase, Pyruvate decarboxylase, phosphofructokinase, glucose-6 Phosphate isomerase, phosphoglucose isomerase and Glucokinase. When constructing suitable expressions plasmids can be those associated with these genes Termination sequences also in the expression vector on 3 ′ end of the sequence to be experimented to be used To enable polyadenylation and termination of the mRNA.

Other promoters that also have the advantage of transcription controlled by growth conditions are the promoter regions of the genes for alcohol dehydrogenase-2, isocytochrome C, acid phosphatase, degrading enzymes which are linked to the nitrogen metabolism mentioned above Glyceraldehyde-3-phosphate dehydrogenase and enzymes that are responsible for processing maltose and galactose. Promoters that are regulated by the yeast mating type locus, for example promoters of the BARI, MF α 1, STE2, STE3, STE5 genes, can be used in temperature-regulated systems by using temperature-dependent sir mutations. (Rhine Ph. D. Thesis, University of Oregon, Eugene, Oregon (1979), Herskowitz and Oshima, The Molecular Biology of the Yeast Saccharomyces, part I, 181-209 (1981), Cold Spring Harbor Laboratory). These mutations influence the expression of the resting mating-type cassettes of yeasts and thereby indirectly the matin-type dependent promoters.

In general, however, every plasmid is a vector Yeast-compatible promoter, original replication and Contains termination sequences, suitable.

Hybrid vectors containing the yeast 2µ plasmid DNA contain homologous sequences can be used. Such Hybrid vectors are created by recombination within the Cell already existing 2µ plasmids incorporated or replicate autonomously. 2µ sequences are especially for Suitable for plasmids with high transformation frequency and allow a high number of copies

In addition to microorganisms, cultures are more multi-cellular Organisms also have suitable host organisms. Basically each of these cultures can be used, whether from vertebrate or invertebrate animal cultures. However, there is great interest on vertebrate cells so that the multiplication of vertebrate cells in culture (tissue culture) in recent years a routine method (Tissue Culture, Academic Press, Kruse and Patterson, Editors (1973)). Examples of such useful host cells are VERO and HeLa cells, CHO cells and W138, BHK, COS-7 and MDCK cell lines. Expression vectors for these cells usually contain (if necessary) a replication starting point, a promoter, located in front of the gene to be expressed with all necessary ribosome binding sites, RNA splicin site, polyadenylation site and transcriptional termination sequences.

The controls come from the use in acidic animal cells functions on the expression vectors often from viral Material. For example, they usually come from used promoters from polyoma adenovirus 2, and particularly often from Simian Virus 40 (SV 40). The early and late end promoters of the SV 40 are particularly useful because both are easily obtained from the virus as a fragment, which also contains the SV 40 viral replication site. (Fiers et al., Nature 273, 113 (1978)). Can too smaller or larger fragments of SV 40 are used, provided they contain approximately 250 bp Sequence extending from the HindIII interface to the BglI Interface in the viral origin of replication is sufficient. In addition, it is also possible and often recommended Promoter or control sequences to use normally are linked to the desired gene sequences, provided that these control sequences are compatible with the host cell systems.

A replication start point can either be by appropriate Vector construction can be provided to an exogenous Starting point, for example from SV 40 or others viral sources (e.g. Polyoma, Adeno. VSV, PBV, etc.) or can by the chromosomal replication mechanisms of the Host cell to be provided. If the vector is in that Host cell chromosome integrated, that's enough the latter measure mostly consists of).

In particular, the invention relates to replication and expression vectors capable of phenotypic selections, which is an expression control sequence and one for the Contain amino acid sequence of VAC coding DNA sequence, said DNA sequence including transcription start signal and termination signal and translation start signal and -stop signal in said expression plasmid under regulation said expression control sequence is arranged so that in a transformed with said expression plasmid Host cell VAC is expressed.

To achieve effective expression, the VAC gene must be properly (in "phase") with the expression control sequence. It is advantageous to place the expression control sequence in the region between the main mRNA start and the ATG of the gene coding sequence, which is of course linked to the expression control sequence (eg the β -lac coding sequence when using the β -lac promoter), with the VAC gene, which preferably brings its own translation start signal (ATG) and translation stop signal (e.g. TAG). This ensures effective transcription and translation.

For example, a vector, in particular pBR322, with a restriction endonuclease cut and, if necessary after modification of the so formed linearized vector, one with corresponding Expression control sequence provided with restriction ends introduced. The expression control sequence contains on 3′-end (in the direction of translation) the recognition sequence a restriction endonuclease so that the expression control sequence already contained vector with said Restriction enzyme digested and with matching ends provided VAC gene can be used. This creates a Mixture of two hybrid plasmids which contain the gene in right or wrong orientation included. It is advantageous that the expression control sequence already contained vector with a second one Cleave restriction endonuclease within the vector DNA and in the resulting vector fragment with the right one Insert end-end VAC gene. All operations on Vector are preferably done in such a way that the Function of the replicon and at least one marker gene is not affected.

In a preferred embodiment of the present Invention becomes a vector derived from pBR322, which an expression control sequence, especially that of Tryptophan operon (trp po), contains at the 3′-end (between the main mRNA start and the first ATG) Recognition sequence for one, preferably cohesive, end forming, restriction endonuclease, e.g. B. EcoRI, with the restriction endonuclease mentioned and in Vector DNA part with a second restriction endonuclease, which forms flat or preferably cohesive ends, e.g. B. BamHI, digested, after which the vector thus linearized with the corresponding ends of the VAC-DNA (e.g. with a EcoRI end before the ATG start and a BamHI end after the Translation stop codon) is linked. The link is done in a known manner by pairing the complementary (cohesive) ends and ligation, e.g. B. with T₄ DNA ligase.

Preferably, the DNA sequences according to the invention can also in the expression plasmid pER103 (E. Rastl-Dworkin et al., Gene 21, 237-248 (1983) and EP-A-01 15 613 - deposited with the DSM under number DSM 2773 on December 20, 1983), in the plasmid parpER 33 (EP-A-01 15 613) or the plasmid pRH100 can be expressed because these vectors are all Contain regulatory elements that lead to a high Expression rate of the cloned genes lead. According to the invention as an expression vector for the synthetic protein gene uses the plasmid pRH100, which the adjustable tryptophan promoter from Serratia marcescens and contains an artificial ribosome binding site. To The production of the expression plasmid pRH100 was the Plasmid pER103 (Eva Dworkin-Rastl et al., Gene 21 (1983) 237-248, EP-A-01 15 613) with the restriction endonuclease HindIII linearized and the 5'terminal phosphate residues were removed.

The via the mRNA route, from genomic DNA or synthetic obtained, with corresponding (in particular EcoRI and BamHI-) end-capped VAC-DNA can be inserted into an expression plasmid also in a vector, e.g. B. pBR322, be cloned to larger amounts of VAC DNA, for example for sequence analysis. The Isolation of the clones containing the hybrid plasmid is, for example, with a VAC-DNA specific radio-labeled oligonucleotide sample (see above) carried out. The VAC-DNA is characterized for example, according to the Maxam and Gilbert method (11).

In another embodiment of the invention Parts of the VAC-DNA synthesized. The invention also relates to a method for producing transformed host cells, characterized in that one a host cell with an expression vector that is one of an expression control sequence for which Contains amino acid sequence of VAC coding DNA sequence, transformed.

Suitable host cells are, for example, the above-mentioned microorganisms, such as strains of Saccharomyces cerevisiae, Bacillus subtilis and in particular Escherichia coli. The transformation with the expression plasmids according to the invention takes place, for example, as described in the literature, for S. cerevisiae (12), B. subtilis (13) and E. coli (14). The transformed host cells are advantageously isolated from a selective nutrient medium to which the ciocid is added, to which the marker gene contained in the expression plasmid confers resistance. If, as preferred, the expression plasmids contain the amp ^R gene, ampicillin is accordingly added to the nutrient medium. Cells which do not contain the expression plasmid are killed in such a medium.

The invention also relates to the above-mentioned route available transformed host cells.

The transformed host cells can be used to produce Compounds with VAC activity can be used. The This is a method of making this compound characterized in that the transformed host cells cultivated and the product released from the host cells and is isolated.

The invention therefore relates above all to a method for Preparation of compounds with VAC activity and of Salt of such compounds, characterized in that with an expression plasmid which is one of a Expression control sequence regulated for Contains amino acid sequence of VAC coding DNA sequence, transformed host cells in a liquid nutrient medium, which assimilable carbon and nitrogen sources contains, can be cultivated and the product from the Host cells is released and isolated, and, if required to have a product available according to the process one suitable for breaking the disulfide bonds Reductant added and the available reduced Polypeptide optionally with a for the new formation of Treated disulfide bonds suitable oxidizing agents and, if desired, an available VAC connection transferred to another VAC connection mixture of compounds obtainable according to the process VAC activity is separated into the individual components and / or, if desired, a salt obtained in the Polypeptide and a obtained polypeptide in the the corresponding salt is transferred.

The present invention relates in particular to a Process for the preparation of VAC compounds of the formula.

The cultivation of the transformed according to the invention Host cells are made in a manner known per se. So can for the cultivation of the transformed according to the invention Host organisms different carbon sources be used. Examples of preferred carbon sources are assimilable carbohydrates, such as glucose, maltose, Mannitol or lactose, or an acetate, either alone or can be used in suitable mixtures. Suitable nitrogen sources are, for example, amino acids, such as casamino acids, peptides and proteins and their Degradation products such as trypton, peptone or meat extracts; furthermore yeast extracts, malt extract, as well as ammonium salts, e.g. B. ammonium chloride, sulfate or nitrate, either used alone or in suitable mixtures can. Inorganic salts that are also used can, for. B. sulfates, chlorides, phosphates and carbonates of sodium, potassium, magnesium and calcium.

Furthermore, the medium contains e.g. B. growth-promoting substances such as trace elements, for. As iron, zinc, manganese and the like, and preferably substances that exert a selection pressure and prevent the growth of cells that have lost the expression plasmid. For example, ampicillin is added to the medium if the expression plasmid contains an amp ^R gene. Such an addition of antibiotic substances also has the effect that contaminating, antibiotic-sensitive microorganisms are killed.

The cultivation conditions, such as temperature, pH of the Medium and fermentation time are selected so that maximele VAC titer can be obtained. How an E. coli or a yeast strain preferred in aerobic conditions submerged culture with shaking or stirring at a Temperature of about 20 to 40 ° C, preferably about 30 ° C, and a pH of 4 to 9, preferably about 30 ° C, and one pH from 4 to 9, preferably at pH 7, during about 4 until 20h. preferably 8 to 12h, cultivated. Doing so collects the expression product intracellularly.

When the cell density has reached a sufficient level, the cultivation is stopped and the product if required from the cells of the microorganism released. For this purpose the cells are destroyed, e.g. B. by treatment with a detergent such as SDS or Triton, or with lysozyme or a similar enzyme lysed. Alternatively or additionally, mechanical Forces such as shear forces (e.g. X press, French press, Dyno-Mill) or shaking with glass beads or aluminum oxide, or alternating freezing, e.g. B. in liquid nitrogen, and thawing e.g. B. to 30 ° to 40 ° C, and ultrasound for Apply cell breakage. The resulting mixture that Contains proteins, nucleic acids and other cell components, is after centrifugation in a conventional manner Enriched proteins. So z. B. most of the Non-protein components through polyethyleneimine treatment separated and the proteins including the VAC connections e.g. B. by saturating the solution with Ammonium sulfate or precipitated with other salts. Further Purification steps include, for example, chromatographic Methods such as ion exchange chromatography. HPLC, Reverse phase HPLC and the like. This is how the Separation of the mixed components by dialysis, after loading by means of gel or carrier-free electrophoresis, after Molecular size using a suitable Sephadex column Affinity chromatography, e.g. B. with antibodies, in particular monoclonal antibodies, or with thrombin coupled to a suitable carrier for Affinity chromatography, or by further, in particular Methods known from the literature.

For example, isolation includes expression VAC connections the following stages. Separation of the cells from the culture solution using Centrifugation; Production of a raw extract by Destroy the cells, e.g. B. by treatment with a lysing enzyme and / or alternating freezing and Thawing; Spin off the insoluble ones Components; Precipitation of the DNA by adding Polyethylene imine; Precipitation of the proteins by ammonium sulfate; Affinity chromatography of the dissolved precipitate an anti-VAC antibody monoclonal column; Desalination of thus obtained solution by means of dialysis or chromatography Sephadex G25 or Sephadex G10.

Further cleaning steps are followed by gel filtration Sephadex G50 (or G75) and reverse phase HPLC. Desalination again on Sephadex G25.

The test can be used to demonstrate VAC activity Anti-VAC antibodies (e.g. rabbit / mouse or monoclonal antibodies available from hybridoma cells) or can use the tests described in EPA 01 81 465 be used.

VAC proteins obtainable according to the process can in themselves converted into other VAC proteins (peptides) in a known manner will.

Studies have shown that the disulfide bridges for the anticoagulant activity of natural VAC without Meaning is. Depending on the choice of host cell, can not be excluded that a link of the Cysteine residues in the primary translation product with formation of disulfide bridges in a manner that differs from naturally occurring process differs. It is possible, that the "wrong" tertiary structure of the Product a reduction or even loss, possibly but also an improvement to the valuable pharmacological properties, especially the anticoagulant activity, what happens to Can be determined using the VAC assays mentioned above. In In such a case, it may be appropriate to Disulfide bonds with a suitable reducing agent cleave and the reduced polypeptide to re-link the Disulfide bonds with a suitable oxidizing agent to treat. Based on the VAC activity of the product formed it can be determined whether the selected conditions (Reducing and / or oxidizing agent) to the desired Have increased biological activity or whether the conditions must be modified in a known manner.

Suitable for splitting disulfide bridges Reducing agents are, for example, thiol compounds, such as Thiophenol, 4-nitrothiophenol, 1,4-butanedithiol and especially 1,4-dithiothreitol. The reduction will advantageously in an aqueous alkaline medium, for example in the dilute aqueous solution Alkali metal hydroxide, e.g. B. sodium hydroxide, Alkali metal carbonate, e.g. As sodium carbonate, or one organic base, in particular a tri-lower alkylamine, e.g. B. triethylamine, carried out at room temperature.

Oxidizing agents used to relink Disulfide bonds in the reduced polypeptides are suitable are, for example, atmospheric oxygen, which by a aqueous solution of the polypeptide, optionally an catalytic amount of a transition metal salt, e.g. B. Iron (III) sulfate, iron [III) chloride or Copper (II) sulfate, added, is passed; Iodine, also in the form of the potassium iodide adduct KJ₃, which preferably in alcoholic, e.g. B. methanolic, or aqueous alcoholic, e.g. B. aqueous methanol solution is used; Potassium hexacyanoferrate (III) in aqueous Solution; 1,2-Dÿodäthan or dimethyl azodicarboxylate or diethyl ester, which is in water or in a mixture consisting of water and a water-miscible Alcohol, e.g. As methanol, are reacted. The Oxidation is carried out especially at room temperature.

The separation of the reagents, especially the salts and the oxidizing or reducing agents and their Follow-up products from the desired VAC connection by methods known per se, for example by Molecular weight filtration, e.g. B. on Sephadex or Biogel.

A mixture of compounds obtainable according to the process with VAC activity can in a known manner in the individual components can be separated. Suitable Separation processes are, for example, chromatographic Process, e.g. B. adsorptious chromatography, Ion exchange chromatography, HPLC or reverse phase HPLC, multiplicative distribution or electrophoretic Methods, e.g. B. electrophoresis on cellulose acetate or Gel electrophoresis, especially polyacrylamide Gel electrophoresis ("PAGE").

The connections that can be produced according to the invention cannot only in free form, but also in the form of their salts, especially their pharmaceutically acceptable salts, are available. Because they have multiple amino acid residues with free Containing amino groups, the invention Connections z. B. in the form of acid addition salts are available. In particular come as acid addition salts physiologically acceptable salts with conventional, therapeutic applicable acids into consideration; than are inorganic acids the hydrohalic acids, such as the hydrochloric acid, but also sulfuric acid and phosphoric or pyrophosphoric acid to call; as organic acids are primarily Sulphonic acids, such as the benzene or p-toluenesulphonic acid or Lower alkanesulfonic acids, such as methanesulfonic acid, as well Carboxylic acid such as acetic acid, lactic acid, palmitic and Stearic acid, malic acid, tartaric acid, ascorbic acid and Citric acid suitable. Because the VAC connections too May contain amino acid residues with free carboxyl groups it also as a metal salt, especially as an alkali metal or Alkaline earth metal salt, e.g. B. sodium, calcium or Magnesium salt, or also as ammonium salt, derived from Ammonia or a physiologically acceptable, organic nitrogenous base. But since at the same time contain free carboxyl groups in free amino groups, they can also be present as internal salts.

Depending on the way of working, the inventive ones are obtained Free form compounds in the form of Acid addition salts or salts with bases. From the Acid addition salts and the salts with bases can be added in known manner, for example by adjusting the pH to the isoelectric point, the free Connections are won. Let the latter again by reacting with acids or bases, e.g. B. with such which form the above salts, and evaporation or Lyophilize therapeutically acceptable acid addition salts or win salts with bases.

The property of antibodies to specific antigens bind, is practically used outside the body in the qualitative and quantitative determination (Immunoassay) and when cleaning the antigens (Immunoaffinity chromatography). Serum of immunized animals usually contains a variety of different ones Antibodies with the same antigen on different To do so, binding sites react with different affinity but also antibodies against other antigens that the previous ones Reflect experiences of the individual. The successful one Use of antibodies for the determination and purification of But antigens require high specificity and Reproducibility.

Homogeneous antibodies that meet these requirements are by the one described by Köhler and Milstein (17) Hybridoma technology has become accessible. In principle there is the technique in that antibodies secrete B lymphocytes, e.g. B. from the spleen, immunized animals with Tumor cells are fused ("fused"). The formed hybridoma cells combine the ability to unlimited propagation through division with the ability to form a uniform type of antibody and to be eliminated. By cultivating in a selective Medium in which unfused tumor cells die, Hybridoma cells multiply, however, and by suitable ones Manipulations can clones, i. H. Cell populations that are derived from a single hybridoma cell and genetically are identical, won and cultivated and that by the Cells produced monoclonal antibodies can be isolated.

The present invention relates to monoclonal antibodies against VAC, hybridoma cells that have such antibodies produce and process for their manufacture. Prefers are hybridoma cell lines and those secreted by them monoclonal antibodies that react specifically with VAC. The process of producing monoclonal anti-VAC and Anti-VAC antibodies are characterized in that Mice immunized with VAC, such as B-lymphocytes immunized animals fused with myeloma cells, the hybridoma cells formed, then cloned in vitro or cultivated by injection in mice and from cultures Antibody isolated.

The invention further relates to immunoaffinity chromatography columns and test kits for immunoassays that contain these antibodies.

According to the method of the invention, mice, e.g. B. Balb / c mice, immunized in a manner known per se. In In a preferred embodiment, VAC becomes approximately weekly or even at larger intervals for several weeks, for example 5 to 12 weeks, injected until one sufficient number of antibody-producing B lymphocytes has formed.

Organs containing B-lymphocytes, e.g. B. spleen cells, the immunized mice are removed and with such Myeloma cells fused due to a mutation in selective culture medium. Such Myeloma cells are known and are, for example, those with the designation X63-Ag8, X63-Ag8.6.5.3, MPC-11, NS1-Ag4 / 1, MOPC-21 NS / 1 or SP 2/0. In a preferred one Embodiment are spleen cells immunized with mice Myeloma cells of the cell line X63-Ag8.6.5.3 fused.

The merger is carried out according to procedures known per se Mix the B lymphocytes and the myeloma cells with addition a cell fusion agent, such as polyethylene glycol, Sendai virus, Calcium chloride or lysolecithin performed. Preferably is in the presence of polyethylene glycol, for example with a molecular weight between 1000 and 4000. After the merger, the resulting hybrids become one known methods in a selective Culture medium as hypoxanthine, aminopterin and thymidine (HAT medium) is complemented, cultivated. Not fused myeloma cells cannot in this medium grow and die just like normal lymphocytes.

The supernatants of the hybridoma cultures can in themselves known methods on their content of specific Antibodies are checked, for example with Radio immunoassay or agglutination. Doing so surprisingly found that with the described Hybridoma cells can be obtained by the process Eliminate antibodies specifically against VAC. The Hybridoma cells, the antibodies of the desired specificity produce, are from the from the merger resulting mixture of different hybridoma cells selected by cloning. This will be done after a known process, called "limit dilution" cultures from a single growing cell scheduled.

Hybridoma cell clones are used for mass production Produce antibodies of the desired specificity, either cultivated in vitro in known medium or for Propagation injected into mice. In a preferred one Embodiment are hybridoma cells in with Pristan pretreated mice injected, ascites fluid removed and from it by precipitation with ammonium sulfate solution Antibody isolated.

The VAC obtained with the help of these hybridoma cells specific antibodies can be produced in a manner known per se for the production of immunoaffinity chromatography Columns are used. In a preferred one Embodiment of the invention becomes a suitable one Carrier material (suspended in a buffer solution) with a Antibody solution added, unbound portions then washed out and vacant positions of the Carrier material blocked.

The VAC obtained with the help of the hybridoma cells specific antibodies can be produced in a manner known per se can be used for the production of test kits. These Test kits can be based on different methods, for example on radio-immunoassay, latex agglutination, Spot tests, competitive or sandwich radio immunoassay, Enzyme immunoassay, immunofluorescence or immunochemical Enzyme tests. Such kits can be used in addition to ordinary ones Antibodies of various origins using antibody conjugates Contain enzymes or fluorescent carriers, for this VAC marked with radioactive isotopes such as J¹²⁵, or conjugated with Enzymes, for example with horseradish peroxidase or alkaline phosphatase, also enzyme substrates, suitable Buffers, gels, latex, polystyrene or other filling materials and carrier.

The known ones obtainable according to the present invention Proteins (peptides) have valuable pharmacological Properties on and can be prophylactic or in particular be used therapeutically.

The new VAC connections according to the invention can therefore in Analogy to natural VAC for therapy and Prophylaxis of thrombosis and thromboembolism, including for the prophylaxis of postoperative Thrombosis, for acute shock therapy (e.g. in septic or polytraumatic shock), for therapy consumption coagulopathies, hemodialysis, Hemoseparations, preserved blood and in the extracorporeal Circuit can be used.

The invention also relates to pharmaceutical Compositions which contain at least one of the compounds of the invention or their pharmaceutically acceptable salts, if appropriate together with a pharmaceutically acceptable carrier and / or auxiliary substances. These Compositions can be especially in the above indicated indications are used if, for. B. parenterally, such as intravenously, intracutaneously, subcutaneously or administered intramuscularly, or topically.

The invention also relates to the use of new compounds according to the invention and these containing pharmaceutical compositions for the prophylactic and therapeutic treatment of the human and animal body, especially at the above-mentioned clinical pictures, primarily to inhibit blood clotting within and outside the human and animal body.

The dosage depends primarily on the specific form of administration and the purpose of Therapy or prophylaxis. The size of the individual doses as well as the administration schedule can best be seen on an individual assessment of each To be determined: the required methods for determining relevant Blood factors are familiar to the person skilled in the art. Normally is the therapeutically effective injection Amount of the compounds according to the invention in Dose range from about 0.005 to about 0.1 mg / kg Body weight. The range of about 0.01 is preferred up to about 0.05 mg / kg body weight. The administration is done by intravenous, intramuscular or subcutaneous injection. Accordingly included pharmaceutical preparations for parenteral use Administration in single dose form depending on the type of application, about 0.4 to about 7.5 mg per dose the compound of the invention. In addition to the active ingredient contain these pharmaceutical compositions usually a buffer, e.g. B. one Phosphate buffer that maintains the pH between about 3.5 and 7 should hold, and also sodium chloride, mannitol or Sorbitol to stop isotonicity. You can in freeze-dried or dissolved form, where Solutions an antibacterial Preservatives, e.g. B. 0.2 to 0.3% 4-hydroxybenzoic acid methyl ester or ethyl ester, can contain. A preparation for the topical Can be used as an aqueous solution, lotion or jelly, oily solution or suspension, or fatty or in particular emulsion ointment are present. A preparation in An aqueous solution is obtained, for example in that the active ingredients according to the invention or a therapeutically acceptable salt thereof in one dissolves aqueous buffer solution from pH 4 to 6.5 and desired case, another active ingredient, e.g. B. a Anti-inflammatory agent, and / or a polymeric auxiliary, e.g. B. polyvinyl pyrrolidone, and / or a Preservatives added. The concentration of the Active ingredient is about 0.1 to about 1.5 mg, preferably 0.25 to 1.0 mg, in 10 ml of a solution or 10 g of a jelly.

An oily form of application for the topical Administration is obtained, for example, by Suspending the active ingredients according to the invention or a therapeutically acceptable salt thereof in one Oil, optionally with the addition of swelling agents, such as Aluminum stearate, and / or surfactants (Surfactants) whose HLB value ("hydrophilic-lipophilic- balance ") is less than 10, like fatty acid monoesters polyhydric alcohols, e.g. B. glycerol monostearate, Sorbitan monolaurate, sorbitan monostearate or Sorbitan monooleate. A fatty ointment is obtained e.g. B. by suspending the active compounds according to the invention or their salts in a spreadable fat base, optionally with the addition of a surfactant with an HLB value under 10. An emulsion ointment is obtained from Trituration of an aqueous solution of the invention Active ingredients or their salts in a soft, spreadable fat pad with the addition of a surfactant, whose HLB value is below 10. All of these topical Application forms can also contain preservatives contain. The concentration of the active ingredient is 0.1 to 1.5 mg, preferably 0.25 to 0.1 mg, approximately 10 g of the basic mass.

In addition to the ones described above and their analog ones pharmaceutical compositions for a direct medical use on the human body or a mammal present invention also pharmaceutical Medical compositions and preparations Use outside of the human living body or the mammals. Such compositions and Preparations are primarily used as anticoagulant additive to blood that is outside the body of a circulation or treatment (e.g. extracorporeal circulation or dialysis in artificial Kidneys), preservation or modification (e.g. Hemoseparation) is subjected. In your Composition are such preparations as Stock solutions or preparations in Single dose form, those described above Similar to injectables; expediently but the amount or concentration of active ingredient on the Volume of the blood to be treated. Depending on The specific dose is about the appropriate dose 0.01 to about 1.0 mg of active ingredient / l of blood, the upper Boundary may still be crossed without danger.

In particular, the invention relates to the Supporting examples described for those for VAC proteins coding VAC molecules, such VAC molecules containing expression plasmids, with such Expression plasmids transformed microorganisms, monoclonal antibodies against VAC, hybridoma cells that produce such antibodies to test kits for Immunoassays containing those antibodies that are found in the methods described to their examples Preparation and that described in the examples Process for the production of proteins / polypeptides with VAC activity using the transformed Microorganisms, as well as those in the examples described new VAC connections.

Is used in the present application by vascular-anticoagulant proteins or in the Short form VAC protein spoken, that means if not otherwise stated that polypeptides / Proteins are meant that are essentially those Have properties that are inherent in the proteins, which were first described in EPA 18 14 465. These properties can be seen through those there specified test and characterization procedures determine and verify (VAC activity).

This definition also includes Polypeptides / proteins that like aggregations for example dimers, trimers or tetramers represent, even if in the aggregate form not per se or only limited biological Have activities provided that this in vivo or in vitro in at least one active Component to be transferred.

Are within the scope of the present invention Get polypeptides / proteins that are not per se or have limited biological activity, for example fusion proteins or so-called "pro-drugs", polypeptides / proteins, for the expert in a manner known per se into the active components are transferable, for example by in vitro or in vivo processing or polypeptides / proteins that first Develop their activity in vivo, so should this under the definition vascular-anticoagulant, in short VAC proteins and thus the subject of belong to the present invention.

Under "Connections with VAC activity" are also such, from the transformed host cells mentioned expressed polypeptides understood which one VAC effect and a positive reaction with anti-VAC antibodies show and which the Primary structure of VAC or a derivative thereof Have structure. Under VAC connections with a structure derived from the primary structure of VAC Modified VAC connections are understood, whereby the modification in a shortening of the Primary structure of the VAC, in a rearrangement of the Repeat structure or in a modification that too changes in stability or activity.

VAC-DNA / gene stands for in the present invention DNA molecules responsible for the VAC proteins defined above encode.

The following examples and drawings are for Illustration of the invention and are not intended in any Restrict way.

To simplify the examples below, are often recurring methods briefly described.

Plasmids are designated with a small "p" followed of capital letters and numbers. Starting plasmids are commercially available or without restriction. You can also publish such plasmids using Methods are constructed.

"Cutting" or "digesting" DNA refers to that catalytic cleavage of the DNA by means of Restriction endonucleases (restriction enzymes) on for these specific sites, called restriction sites. Restriction endonucleases are commercially available and are among those recommended by the manufacturers Conditions (buffer, bovine serum albumin (BSA) as Carrier protein (DTT) as oxidation protection) used. Restriction endonucleases are labeled with a Uppercase letters, mostly followed by lowercase letters and usually called a Roman numeral. The Letters depend on the microorganism from which the restriction endonuclease in question (e.g. Sma I: Serratia marcescens). Usually about 1µg DNA with one or more units of the enzyme approximately Cut 20 µl buffer solution. Usually one Incubation time 48522 00070 552 001000280000000200012000285914841100040 0002003710364 00004 48403 used from 1 hour at 37 ° C can but according to the manufacturer's instructions for use can be varied. After cutting, sometimes 5'phosphate group by incubation with alkaline Phosphatase removed as calf intestine (CIP). This is for Preventing an unwanted reaction of the specific Site in a subsequent ligase reaction (e.g. Circularization of a linearized plasmid without Insertion of a second DNA fragment). Unless otherwise specified, DNA fragments are made after cutting usually not with restriction endonucleases dephosphorylated. Reaction conditions for the Incubation with alkaline phosphatase are e.g. B. the M13 Cloning and Sequencing Guide (Cloning and Sequencing handbook, Amersham, PI / 129/83/12). After incubation, protein is extracted by using Removed phenol and chloroform, and DNA from the aqueous phase precipitated by the addition of ethanol.

"Isolation" of a particular DNA fragment means the separation of the cut DNA on a z. B. 1% Agarose gel. After electrophoresis and the Make the DNA visible in UV light by staining with Ethidium bromide (EtBr) becomes the desired fragment using the applied molecular weight marker localized and by further electrophoresis on DE 81 Paper (Schleicher and Schüll) bound. The DNA will by rinsing with low salt buffer (200 mM NaCl, 20 mM Tris pH = 7.5, 1 mM EDTA) and then with a high salt buffer (1 M NaCl, 20 mM Tris pH = 7.5, 1 mM EDTA) eluted. The DNA is made by adding ethanol precipitated. "Southern analysis" is the method through the presence of a particular DNA fragment in a DNA mixture by hybridization with a known, labeled oligonucleotide probe or a labeled DNA fragment is detected. Southern In the following analysis means nothing else specified the separation of the DNA mixture a 1% agorose gel, denaturation and transfer Nitrocellulose filter (Schleicher and Schüll, BA 85) using the method of E. Southern, J. Mol. Biol. 98 (1978), pp.503-517, and hybridization as described in R. Hauptmann et al., Nucleic Acids Res. 13 (1985), pp.4739-4749.

"Transformation" means introducing DNA into an organism so that the DNA can be replicated there, either extra-chromosomal or as chromosomal Integrant. Transformation of E. coli follows that in M13 Cloning and Sequencing Guide (Cloning and Sequencing Handbook, Amersham, PI / 129/83/12) Method.

"Sequencing" a DNA means analyzing the Nucleotide sequence in a DNA. To do this, the DNA is included different restriction enzymes cut, and the Fragments in appropriately cut M13 mp8, mp9, mp18 or mp19 double stranded DNA introduced, or the DNA is scanned using ultrasound, followed by repairs the ends and size selection cut in Sma I, dphosphorylated M13 mp8 DNA (Shotgun method) brought in. After the transformation of E. coli JM 101 becomes single-stranded DNA from recombinant M13 phages according to the M13 Cloning and Sequencing manual (Cloning and Sequencing Handbook, Fa Amersham, PI / 129/83/12) isolated and according to Sanger's Diedoxymethode (F. Sanger et al., Proc. Natl. Acad. Sci. 74 (1977), pp. 5463-5467). The The sequences are evaluated using the originally developed by R. Staden (R. Staden, Nucleic Aceds Res. 10 (1982), pp.4731-4751) and by Ch. Piegler modified (C. Pieler 1987, dissertation, University of Vienna) computer programs.

"Ligate" refers to the process of forming Phosphodiester bonds between two ends of Double stranded DNA fragments. Usually will between 0.02 and 0.2 µg DNA fragments in 10 µl with about 5 units of T4 DNA ligase ("ligase") in one suitable buffer solution (T. Maniatis et al., Molecular cloning, 1982, p.474).

"Preparation" of DNA from transformants means Isolation of the plasmid DNA from bacteria using the Alkenian SDS method modified according to Birnboim and Doly (T. Maniatis et al., Molecular cloning, 1982, pp. 368-369) omitting the lysozyme. In doing so the bacteria from 1.5 and 50 ml culture used.

"Oligonucleotides" are short polydeoxynucleotides that be chemically synthesized. The Applied Biosystems Model 381A synthesizer used. The Oligonucleotides are designed according to the 381A model User Manual (Applied Biosystems) revised and purified by polyacrylamide gel electrophores (PAGE).

"Phosphorylation" means enzymatic Transfer of the gamma-phosphate residue from ATP to a free 5′OH group of a nucleic acid, usually a Oligonucleotide. Up to 100 pMol of the oligonucleotide with 10 units T 4 -Poly-nucleotide kinase in the presence of 100 pmol ATP in suitable buffer solution (70 mM Tris, pH = 7.6, 10 mM MgCl₂, 5 mM DTT) phosphorylated at 37 ° C for 30 minutes. The reaction is usually caused by heating for 10 minutes Stopped at 100 ° C.

Some abbreviations used are briefly explained will:

bp base pairs BSA Binder Serum Albumin DTTDithiothreit EDTA ethylenedinitrilotetraacetic acid, sodium salt SDS sodium dodecyl sulfate TrisTris (hydroxymethyl) aminomethane Denhardt 0.01% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02 BSA LB10 g / l tryptone, 5 g / l yeast extract, 5 g / l NaCl 1 × SSC150 mM NaCl, 15 mM tri-sodium citrate, pH = 7 TE10 mM Tris pH = 8.0, 1 mM EDTA

List of pictures:

1Screening oligonucleotides EBI-386, -387 and -388 1A Cryptic Fragments 1BHPLC 1CHPLC 1D / 1E Permeation HPLC 1FReverse phase HPLC 1GSDS gel electrophoresis 2Screening oligonucleotides EBI-118 and EBI-119 3 Northern blot analysis with VAC-alfa and VAC-beta cDNA 4VAC-alfa cDNA sequence 5 Arrangement of the peptide sequences in the VAC-alpha cDNA-derived amino acid sequence 6 Repeats four times in the VAC-alpha protein 7VAC-beta cDNA sequence 8 Repeats four times in the VAC-beta protein 9 Amino acid composition of VAC-alfa and VAC-beta 10 Genomic Southern blot analysis with VAC-alfa and VAC-beta cDNA 11 Amino acid comparison of VAC-alfa with VAC-beta 12 Nucleotide comparison between VAC alfa and beta cDNA 13 Hydrophilicity plot of VAC-alfa and VAC-beta 14 Comparison of VAC-alfa, VAC-beta, Lipocortin I and Lipocortin II 15 Sequence of the promoter and terminator part in pRH284 16 Construction of pR291 17 Construction of pRH212 18 Construction of PRH292

Example 0

The one isolated from umbilical cord vessels and / or placenta and cleaned material was using a Reverse phase HPLC cleaned.

Stationary PhaseBakerbond WP-RP 18, 4.6 × 250 mm, 5 µm particles, 300 A pores Mobile phase A 0.1% trifluoroacetic acid in water, pH 2.2 Mobile phase B 0.1% trifluoroacetic acid in acetonitrile Gradient 20-68% B in 24 min Flow 1 ml / min Detection UV, 214 nm

Following this cleaning stage, both were Materials, each the substance that the Molecular weight was 32,000, digested with trypsin.

Reaction conditions

30 µg VAC from placenta in 135 µl 0.15 M NH₄HCO₃, pH 8.0

+ 2% w / w trypsin (Worthington) 6 hours at 37 ° C

+ 2% w / w trypsin (Worthingon), overnight at 37 ° C
30 µg VAC from umbilical cord in 100 µl 1% NH₄HCO₃, pH 8.0

+ 2% w / w trypsin (Worthington) 6 hours at 37 ° C

+ 2% w / w trypsin (Worthington), overnight at 37 ° C

The fragments obtained were analyzed using HPLC separated and sequenced with a Applied Biosystems type 470A gas phase sequencer, Program 02 RPTH fed.

HPLC separation conditions

Stationary PhaseµBondapak C 18, 3.8 × 200 mm, 10 µ particles Mobile phase A0.1% trifluoroacetic acid in water, pH 2.2 Mobile phase B0.1% trifluoroacetic acid in acetonitrile gradient0-55% B in 55 min flow 1 ml / min Detection UV, 214 nm (upper trace)
280 nm (lower trace)

In addition to tryptic digestion, that was about Reverse phase HPLC purified material also one more BrCN cleavage. These cleavage peptides were also sequenced and with the data of the peptides from the compared tryptic digestion.

BrCN cleavage

111 µg VAC purified in RP-HPLC in 111 µl 70% formic acid dissolved. This already contained the 250 fold molar excess of BrCN (90 µg). The incubation took place in the dark, 17 hours at room temperature. 100 ul was used for the HPLC separation.

HPLC column µBondapak C 18 Mobile phase A 0.1% trifluoroacetic acid in water Mobile phase B 0.1% trifluoroacetic acid in acetonitrile Gradient 0-70% B in 70 min Flow 1 ml / min Detection UV, 214 and 280 nm

A comparison of the results achieved with this as well as the Analysis by gel permeation HPLC and SDS gel electrophoresis proves the identity of VAC Placenta and VAC from umbilical cord.

Gel permeation HPLC

Stationary PhaseWaters I-125, 7.8 × 600 mm, 10 µm particles Mobile phase B0.5M Na₂SO₄, 0.02M Na₂PO₄, pH 7.0, 25% propylene glycol, 0.04% Tween 20 Flow 0.5 ml / min Detection UV, 214 nm

SDS gel electrophoresis

SDS-Gel 15% gel strength 0.7 mm electrophoresis conditions 20 mA / plate, 2-3 hours running time staining Coomassie Blue samples: 8 µg VAC from umbilical cord or
: 7 µg VAC from placenta

Example 1 Generation of a human placental cDNA library a) Total RNA isolation from placenta

GT: 5 M guanidinium thiocyanate, 50 mM Tris pH = 7.4, 25 mM EDTA. Before use 8% (v / v) beta-mercaptoethanol clog. 20 ml GT are 5 to 10 minutes before Use chilled on ice, GT should not precipitate. GH: 6 M guanidium hydrochloride, 25 mM EDTA, 10 mM beta-mercaptoethanol, pH = 7.0. Ice cooling.

1 g frozen and mechanically pulverized placenta are in 20 ml GT (0 ° C) 20 seconds with maximum Speed mixed with a Polytron (Brinkmann). The Volume of the homogenate is determined in 0.3 vol ethanol (-20 ° C) poured, mixed and immediately at 12000 rpm 5 ′ -10 ° C (Beckmann JA 21 centrifuge, JS13.1 rotor) centrifuged. A possible protein film as well as the The supernatant is removed. 10 ml ice-cold GH are the Pellet added and 10 seconds with the Polytron homogenized. The suspension is 5 minutes at -10 ° C and Centrifuged at 12000 rpm. The supernatant turns into a transferred sterile Corex tube, discarded the pellet. 0.025 vol 1 M acetic acid and 0.75 vol cold ethanol (-20 ° C) added and mixed well. To 2 hours incubation at -20 ° C is 10 minutes at 6000 centrifuged at -20 ° C (JA20 rotor). The protein film and the supernatant is carefully removed. 2 ml GH (0 ° C) are added to the pellet, the pellet resuspended, and the suspension in a 15 ml Corex tubes transferred. With another 8 ml of GH that will rinsed old Corex tubes, the solution with the 2 ml united. It is important that the entire pellet is suspended, possibly by mild sonication post-treat. 0.025 vol l M acetic acid and 0.5 vol cold ethanol (-70 ° C) are added and about 2 hours incubated at -20 ° C. Centrifugation (6000 rpm, 10 min, JA20 rotor), dissolve and precipitate twice repeated, halving the total amount of GH to 5 ml becomes. After the last centrifugation, none should Protein film may be more visible above the solution, otherwise repeat this cleaning step. The pellet will water treated with 5 ml diethyl pyrocarbonate (0 ° C) 2 Vortexed minutes. The clear solution is decanted, too any remaining pellet remains Water and vortexed until the solution is clear. By adding 0.1 vol 3 M Na acetate (pH = 5.8), 2.5 vol Ethanol and 1 hour incubation at 0 ° C at -70 ° C RNA spun down at 6000 rpm for 10 minutes at 4 ° C. To solve and precipitate is repeated once. At long last the RNA is stored in ethanol at -20 ° C.

b) poly-A ^± RNA isolation

0.5 mg oligo dT cellulose (Collaborative Research, Typ 3, binding capacity: 5 mg poly-A⁺-RNA / g) in Binding buffer suspended (200 mM NaCl, 0.2% SDS, 10 mM Tris, pH = 7.4, 1 mM EDTA), 1 ml of this suspension is packed into a column and successively with 10 ml Water, 10 ml 0.1 N NaOH, 10 ml water and ultimately with Washed 10 ml binding buffer. The total RNA is made up the alkaline solution by centrifugation (10 min., 6000 rpm, JA20 rotor pelleted. Approx. 10 mg RNA are in 4.5 ml of water dissolved. After adding 50 µl 2% SDS the solution is heated to 70 ° C. for 3 minutes and immediately chilled on ice. After adding 50 µl 1M Tris (pH = 7.4), 10 ul 0.5 M EDTA and 200 ul 5 M NaCl the solution is immediately applied to the column. The one from the Column dripping solution is put back on the column applied. This procedure is done three times in total repeated. Then the column with 30 ml Binding buffer washed. The bound RNA is labeled 5 ml 0.2 SDS eluted. The column is washed with 10 ml of water, 10 ml Washed 0.1 N NaOH and 10 ml H₂O and with 10 ml Binding buffer equilibrated. The RNA solution will heated again to 70 ° C for 3 minutes, quickly on ice cooled, 50 µl 1 M Tris pH = 7.4, 10 µl 0.5 M EDTA and 200 ul 5 M NaCl added and onto the column applied. The solution going through is total applied three times to the oligo-dT column. After this Washing the RNA is eluted with 30 ml of 0.2% SDS.

By adding 0.1 vol 3M Na acetate pH = 5.6 and 2.5 vol Ethanol and incubation at -20 ° C (16 hours) the RNA failed. The RNA is centrifuged off (10,000 rpm, 15 min., JA-20 rotor) and in water with a Concentration of 1 µg / µl dissolved.

c) Construction of the Lambda gt10 cDNA library

The synthesis of the cDNA, the methylation of the internal EcoRI sites, the attachment of EcoRI linkers, the Regrooving with EcoRI, separating the short DNA fragments, ligating with the lambda gt10 arms and the ligated DNA was packaged in vitro with the Amersham cDNA synthesis system (RPN 1256) and the cDNA cloning system Lambda-gt10 (Amersham, RPN 1257). The attached work instructions were accurate adhered to. Starting from about 3 µg ultimately about 0.5 × 10⁶ recombinant lambda gt10 Preserve phages.

Example 2 cDNA isolation a) Search the Lambda-gt10 library with Oligonucleotides

To search the cDNA library for cDNA coding for VAC protein, an oligonucleotide was synthesized in accordance with the sequences of the tryptic peptide P16 / II two and the Staph A peptide P20 / I / 6 ( FIG. 1). These oligonucleotides each represent a mixture of all variants that take into account every coding possibility of the corresponding mRNA. EBI-386 has 512 variations with a chain length of 20 nucleotides and matches the Staph-A peptide P20 / I / 6. In order to keep the variation in the oligonucleotide for the tryptic peptide P16 / II lower, two oligonucleotides (20-mers) were synthesized: EBI-387: 128 variations, EBI-388: 64 variations.

Furthermore, two oligonucleotides were synthesized using the tryptic P30 / I VAC peptide using deoxinosin as base at "wobble" positions ( FIG. 2): EBI-119. This substitution was described by E. Ohtsuka et al., J. Biol. Chem. 260/5 (1985), pp. 2605-2608, and Y. Takahashi et al., Proc. Nat. Acad. Sci. (1985) pp. 1931-1935. Inosine bases well with cytosine, but hardly interferes with the formation of the double helix if other nucleotides are offered as partners.

Each 60 pmol gamma-32 P-ATP (Amersham, PB 218, 5000 Ci / mmol) in 30 µl solution using 20 units T₄ polynucleotide kinase phosphorylated. The solution contained 70 mM Tris pH 7.5, 10 mM MgCl₂ and 5 mM DTT, the incubation period was 30 minutes. By adding of 30 mM EDTA and heating to 70 ° C for 5 minutes the reaction stopped. The labeled oligonucleotide was determined by column chromatography on Biogel P6DG (Biorad) separated from non-built-in radioactivity. Between 70 and 110 × 10⁶ cpm per oligonucleotide Phosphor-32 built in.

15 pmoles of EBI-118 and EBI-119 were placed in 90 µl each Use of 45 pmol gamma-32 P-ATP (Amersham, PB 218, <5000 Ci / mmol) with 10 units T₄ polynucleotide kinase phosphorylated and then cleaned using Biogel P6DG. A total of 70 × 10⁶ cpm phosphorus-32 were installed.

About 1.2 × 10⁶ pfu (plaque forming units) recombinant phages of a human, placental cDNA Library in phage lambda-gt10 were used to Infect E. coli C600. About 50,000 phages were made per 13.5 cm petri dish plated (LB, 10 mM MgSO₄, 15 g / l Bacto agar). After the plaques were almost confluent, 2 prints from each plate Nitrocellulose filter manufactured (Schleicher and Schüll, BA 85) (T. Maniatis et al., Molecular Cloning, 1982, pp 320-321).

The filters were treated 3 x 20 minutes in TE at 100 ° C and then washed overnight at 65 ° C:

Wash solution: 50 mM Tris pH = 8
1 M NaCl
1mM EDTA
0.1% SDS

The filters were then at 49 ° C for 4 hours pre-hybridized:

Prehybridization solution: 6 × SSC
5 × Denhardt
0.1% SDS
1 mM Na pyrophosphate
50 mM NaH₂PO₄, pH = 6.5
100 µM ATP
80 µg / ml denatured herring
Sperm DNA

The hybridization took place in the same solution Use the entire amount of labeled oligonucleotides. The double prints of plates 1 to 6 were made with EBI-386, those plates 7-12 with EBI-387 and EBI-388 Hybridized at 49 ° C for 16 hours. The prints of the plates 13-24 were 16 hours with EBI-118 and EBI-119 at 37 ° C hybridizes.

After hybridization, the filters were changed twice rinsed with 6 × SSC / 0.01% SDS, 2 × 30 minutes at Room temperature with 6 × SSC / 0.01% SDS and 3 × 10 minutes washed in the same solution at 49 ° C or 37 ° C. After air drying, the filters were attached to Kodak X-Omat S film at -70 ° C using an intensifying screen exposed.

Lambda phages using hybridization on both filters the oligonucleotide were used the same hybridization protocol for homogeneity cleaned.

From hybridization with EBI-386, -387 and -388 resulted in the phages Lambda-P11 / 3, Lambda-P6 / 5, Lambda P6 / 6. Hybridization with EBI-118 and -119 resulted in phages Lambda-Nr15, Lambda-Nr19 and Lambda No. 22.

b) Isolation of the cDNA inserts

E. coli C 600 was 2.5 × 10⁶ pfu (plague forming units) phage infected and with 6 ml top agarose (0.7% Agarose, 10 mM MgSO₄, LB) on 13.5 cm agar plates (LB, 10 mM MgSO₄, 1.5% agarose, 0.2% glucose) plated. After 5½ hours incubation at 37 ° C the plagues were confluent. The plates were cooled at 4 ° C for 30 minutes and the agarose with 10 ml lambda diluent (10 mM Tris, pH = 8, 10 mM MgSO₄, 0.1 mM EDTA) layered. The Phages were grown overnight at 4 ° C under mild Shake eluted. The supernatant phage suspension (approx. 8 ml) were transferred into Corex tubes and 10 Minutes at 15,000 rpm (4 ° C, JA 21 centrifuge) centrifuged. The supernatant was in Polycarbonate tubes decanted and at 50,000 rpm (20 ° C, L70 Beckmann centrifuge, 20 ° C, 50 Ti rotor) to centrifuged omega²t = 3 × 10¹⁰ (approx. 23 minutes). The pelleted phages were removed after removal of the Resuspend the supernatant in 0.5 ml lambda diluent and transferred to Eppendorf tubes. The suspension was by briefly centrifuging non-suspended Particles freed, the supernatant in a new tube filled. After adding 10 µl / ml RNaseA and 1 µg / ml DNase I was incubated at 37 ° C for 30 minutes. After encore of 25 mM EDTA, 25 mM Tris, pH = 8, 0.2% SDS became 30 Incubated for minutes at 70 ° C. Next 1 vol Phenol / chloroform (1: 1) added, and proteins through Extracted tilting the tube. After 2 minutes The centrifugation in the Eppendorf centrifuge was the aqueous phase extracted with 1 vol chloroform (only Tilt, not vortex). After phase separation by Centrifuge the supernatant 1/20 vol 3 M Na acetate and 1 ml of ethanol were added. The fell Phage DNA filamentous. After 5 minutes of incubation the DNA was centrifuged at 0 ° C. (10 minutes). The Pellet was washed once with 70% ethanol, dried and dissolved in 50 µl TE overnight (4 ° C).

The DNAs were cut with EcoRI and the resulting fragments separated on a 1% agarose gel. The cDNA inserts of the clones Lambda-P11 / 3, Lambda-P6 / 5 and Lambda P6 / 6 had sizes from about 1300 to 1400 bp. The Sequence analysis showed that all three clones of one and same mRNA were derived. The 5'-end of the mRNA however, was missing from the cDNAs. The phage inserts Lambda-Nr15, Lambda-Nr19 and Lambda-Nr22 had lengths of approx. 1600, 1100 and 1000 bp. The sequence analysis left assume an almost complete cDNA. The cDNAs of the two phage groups Lambda-P11 / 3, Lambda-P6 / 5 and Lambda-P6 / 6 and Lambda-Nr15, Lambda-Nr19 and Lambda-Nr22 are of two different ones mRNAs derived, like the subsequent sequence analysis revealed.

The EcoRI inserts of the three clones Lambda-P11 / 3, Lambda-P6 / 5 and Lambda-P6 / 6 were isolated and used in the Ligated EcoRI-cut Bluescribe M13⁺ vector (Vector Cloning Systems, 3770 Tansy Street, San Diego, CA 92121, USA). The resulting clones became pP6 / 5, called pP6 / 6 and pP11 / 3.

The EcoRI inserts of the three clones Lambda-Nr15, Lambda-Nr19 and Lambda-Nr22 were isolated and in the Ligated EcoRI-cut Bluescribe M13⁺ vector. The resulting clones were pRH201, pRH202 and called pRH203.

c) Additional VAC cDNA clones

In order to obtain further cDNA clones, the human, searched placental lambda-gt10 library again, this time with the EcoRI insert of pP11 / 3 as a probe. This DNA fragment was translated by nick radioactively labeled (T. Maniatis, Molecular Cloning, 1982, pp. 109-112, no DNase I used). A total of 4 × 10⁵ phages were searched on 8 plates. The Treatment of the nitrocellulose filter was carried out as in T. Maniatis, Molecular Cloning, 1982, pp. 320-321 described. The hybridization solution contained 6 × SSC, 5 × Denhardt's SDS and 20 × 10⁶ cpm pP11 / 3 Insert. The hybridization time was 16 h at 65 ° C. The filters were then at 3 × 10 minutes Room temperature with 6 × SSC / 0.1% SDS and 3 × 45 minutes at 65 ° C and washed with 0.2 × SSC. A total of 69 Clones reacting positively (Lambda-VAC1 bis Lambda VAC69).

12 of these clones were made on a small scale as above prepares the cDNA inserts with EcoRI released and separated on a 1% agarose gel. It was found that the insert of the clone Lambda-VAC10 the entire coding for VAC protein Contains reading frames.

Example 3 Characterization of the cDNAs coding for VAC-alfa and VAC-beta a) Northern blot experiment

5 µl water, 16 µl were added to 2 µg poly-A⁺ RNA Formamide, 6 µl formaldehyde and 3 µl 0.1 M NaOH added, the solution incubated at 68 ° C for 10 minutes and then chilled on ice. After adding 5 µl Dye solution (0.4% bromophenol blue and xylene cyanol each in 50% glycerol, 1 mM EDTA) the RNA was on a Formamide agarose gel separated (1.5% agarose, 10 mM Na phosphate pH = 7.6, 1 mM EDTA, 5 mM Na acetate, 6% Formaldehyde, electrophoresis 100 V, 3 hours, Running buffer like gel buffer, without formaldehyde). As Total RNA was applied for reference. That trail was separated after electrophoresis and washed with EtBr stained to reveal the location of the 28 and 18 S rRNA determine. The rest of the gel was twice 10 minutes washed in 10 × SSC, and the RNA exposed to 20 × SSC Transfer nitro cellulose filter. The filter was with Washed 2 × SSC, dried and 2 hours in a vacuum Baked at 80 ° C. 1 µg pP11 / 3 and PRH203 were added with the Multiprime DNA labeling system (Amersham, RPM 1601) radioactively labeled. The nitro cellulose filter was 2 h at 65 ° C in 6 × SSC / 5 × Denhart's / 0.1% SDS pre-hybridized. One track at a time was with 180 × 10⁶ cpm pP11 / 3 or pRH203 hybridized (16h 65 ° C). The filters became 30 twice Minutes at room temperature with 6 × SSC / 0.01% SDS and twice 30 minutes at 65 ° C with 0.2 × SSC / 0.1% SDS washed, dried and attached to Kodak X-Omat S film Exposed reinforcement film.

The result is shown in Fig. 3. The cDNA of clone pP11 / 3 hybridizes to an mDNA of the size of approximately 1700 bases ("VAC-alpha"), the cDNA of the clone pRH 203 to an mRNA of the size of approximately 2200 bases ("VAC-beta").

Firstly, the amount of radioactivity used as well the amount of mRNA applied per lane was approximately the same and second, the hybridization of a genome blot in same solution bands of equal intensity with both cDNAs (see below), it can be concluded that the shorter mRNA ("VAC-alfa") in larger amounts than that longer ("VAC-beta") mRNA is represented in the placenta.

b) Sequence analysis of the VAC-alpha cDNA

The cDNA clones pP6 / 5, pP6 / 6 and pP11 / 3 were totally sequenced, and those of the clones Lambda-VAC1 to -12 were partially sequenced. The result is shown in Fig. 4. A total of 1465 bases were sequenced. The cDNA has a long, open reading frame that can code for 320 amino acids. If the DNA sequence is translated into an amino acid sequence, all sequenced peptides can be accommodated in this sequence ( FIG. 5). This cDNA is therefore the one whose corresponding mRNA codes for VAC protein. Since the sequences of the second isolated cDNA (see below) encode a similar but different protein from VAC, the name VAC-alfa is introduced here.

The first ATG codon (bases 35-37) goes in the same Reading frames advance a stop codon. The bases 30 to 38 meet the Kozak rule quite well (M. Kozak, Nature 1984, 308, pp. 241-246), which the consensus sequence in close to the translation start codons with CC (A / G) CCAUGG indicates the corresponding sequence here TCGCTATGG. The 3 ′ untranslated region is 471 Bases long. 15 bases before the start of the poly-A section is the polyadenylation sequence AATAAA (N.J. Proudfoot et al., Nature 263 (1976), pp. 211-214). Will be a chain length of 150 to 200 bases calculated for the poly-A section of the mRNA the total length of the mRNA based on the cDNA sequence 1600-1650 bases. Since in the Northern blot experiment higher value has been determined is the 5 ′ untranslated region in no cDNA complete contain.

In contrast to all, the cDNA of clone pP6 / 5 points other cDNA clones at position 100 C instead of A. This would make the triplet 98-100 (22nd codon) of Change GAA to GAC and for Asp instead of Glu encode. This deviation can have several causes have: a) the reverse transcriptase built a wrong one Nucleotide one, b) it is the transcripts two alleles, different genes at this point or c) there are two non-allelic genes that are involved distinguish this position.

The long, open reading frame encodes a 320 amino acid protein from which the Met-1 is likely to be cleaved and the subsequent alanine at the amino group is blocked, possibly by acylation. The calculated molecular weight is 35 896 D and is higher than the value according to SDS-PAGE. However, the proportion of charged amino acids (Asp, Glu, Lys, Arg, His) is above average at 30.6% (98/320) compared to the average value of 25.1%. This would explain the changed migration behavior of the protein on SDS-PAGE. Within the highly charged amino acids, the acidic amino acids (Asp and Glu) predominate with 54 over the basic ones (Lys and Arg) with 41. This explains the acidic, isoelectric point of the VAC-alfa protein (pI = 4.4 to 4.8) VAC-alfa contains only a triplet coding for cysteine (amino acid position 316); a typical N-glycosylation site (Asn-XXX-Ser / Thr) is not available. A structural analysis of the amino acid sequence (modified according to Pustell, J et al., Nucleic Acids Res. 10 (1982) pp 4765-4782) shows a four-fold repetition of a 67 amino acid long sequence ( FIG. 6), hereinafter referred to as "repeats". Within this sequence, 7 amino acids (10.4%) are conserved in all four repeats, 15 amino acids (22.4%) occur in three of four repeats, at 28 positions (41.8%) two repeats each contain the same amino acid.

A comparison with published literature data (MJ Geisow, FEBS Letters 203 (1986), pp. 99-103, MJ Geisow et al., TIBS 11 (1986), pp. 420-423) surprisingly showed that VAC-alfa became one belongs to a larger group of Ca ⁺⁺ dependent phospholipid binding proteins. A consensus sequence is described (Lys-Gly-fob-Gly-Thr-Asp-Glu-var-var-Leu-Ile-fil-Ile-Leu-Ala-fob-Arg; fob-hydrophobic, fil-hydro-phil, var = variable), which could be involved in Ca ⁺⁺ binding (MJ Geisow et al., Nature 320 (1986), pp. 636-638). This sequence is found in each of the four repeated 67-amino acid sub-sequences of the proteins according to the invention ( FIG. 6). Also striking is the 6 amino acid section at the end of each repeat, which consists almost exclusively of hydrophobic amino acids ("oooooo" in Fig. 6).

c) Sequence analysis of the VAC-beta cDNA

The VAC-beta cDNA sequence of clones No. 15, No. 19 and No. 22 resulted in 1940 bp and merges into a poly-A section ( FIG. 7). The polyadenylation signal AATAAA is found 16 bases in front of the poly-A section. However, this consensus sequence already occurs at nucleotide position 1704-1709. Why this sequence is not used as a polyadenylation signal is unknown. The additionally required sequence on the 3 ′ side of the AATAAA sequence, YGTGTTYY (Gill A. et al., Nature 312 (1984), pp. 473-474) occurs only relatively far away (TGTGTTAT, position 1735-1742); possible that this explains the non-acceptance of this first polyadenylation sequence.

The cDNA contains a long, open reading frame that extends from the beginning of the cDNA to position 1087. He would contain coding potential for 362 amino acids. For reasons of analogy to VAC-alfa and due to The fact that a 34,000 D protein in the VAC cleaning occurs (see E.P.A. 1 81 465) the first methionine codon (ATG, position 107-109) as Translation start accepted. The Kozak rule is here not as well fulfilled as with VAC-alfa (AAGAGATGG) Item 102-110).

The resulting protein (VAC-beta) would be 327 amino acids long. It has 4 cysteine residues (amino acid position 161, 206, 250 and 293) and a potential N-glycosylation site (Asn-Lys-Ser, amino acid position 225-227). The calculated molecular weight is 36 837 ( Fig. 9). VAC-beta also contains an above-average number of charged residues: 97/327 (29%), the acidic amino acids (Asp + Glu: 49) predominating over the basic ones (Lys + Arg 42); an explanation for the low molecular weight determined according to SDS-PAGE.

VAC-beta also shows an internal repetition of a 67 amino acid sequence ( FIG. 8). Within this sequence, 7 amino acids (10.4%) are conserved in all four repeats, 17 amino acids (25.4%) occur in three of four repeats, at 25 positions (37.7%) two repeats each contain the same amino acid. VAC-beta also shows good agreement with the 17 amino acid consensus sequence. The explanations for the VAC-alfa apply analogously to VAC-beta.

d) Genomic Southern blot analysis

The analysis of chromosomal DNA from human placenta according to Southern shows a complex picture. The DNA was cut with EcoRI, HindIII, BamHI and PstI. The DNA transferred to nitrocellulose was hybridized both with a VAC-alpha DNA (pP11 / 3) and with a VAC-beta DNA (pRH203). The filters were washed under stringent conditions (see point a)), ie with 0.2 × SSC and 65 ° C. Nevertheless, a relatively large number of bands resulted with each digestion ( FIG. 10). The comparison of the two blots shows that the cross-reaction of VAC-alpha and beta DNA can be excluded under these conditions. The large number of bands can either be explained by the existence of genes that are similar to the VAC-alpha or VAC-beta gene, or else it is a gene that is interrupted by many and / or long introns.

Example 4 Protein analysis

FIG. 11 shows the comparison of the amino acid sequences of VAC-alpha with VAC-beta. The repeated structures can be arranged in the same way in both proteins. The connecting peptides are also of the same length, with the exception of those between the 2nd and 3rd repeat. In VAC-alpha, a gap must be introduced into this connecting peptide in order to allow an optimal adaptation of the two sequences. The N-terminal peptide of the two proteins is of different lengths, 19 amino acids for VAC-alfa, 25 amino acids for VAC-beta. This peptide also has the least homology. The two proteins are identical at 176 out of 320 amino acid positions, which corresponds to a degree of homology of 55.0%.

At this point, the comparison of the Nucleotide sequences of the VAC alfa and beta cDNAs inserted. Become two genes and their products compared to each other, you can see on the DNA (= RNA) Greater homology than at the level Amino acid level, which is explained by the fact that already a change in one with the nucleic acid Base triplet is sufficient to add a new amino acid encode.

FIG. 12 shows the comparison of the coding regions of VAC-alpha and VAC-beta cDNA. Surprisingly, the DNAs only show a degree of homology of 54.2%, which is slightly less than the two proteins.

In Fig. 13, the Hydrophilizitätsprofile of the two proteins are shown. The algorithm of Hopp and Wood was used (TP Hopp et al., Proc.Natl.Acad.Sci. 78 (1981) pp.3824-3828). The four repeat areas are indicated by the bars, the connecting peptides are framed in the sequence below. It is striking that the connecting peptide between the second and third repeat is particularly hydrophilic. In both VAC-alpha and VAC-beta there is an arginine in this peptide in an identical position. It would therefore be possible for this arginine to be a preferred target for a protease with trypsin-like specificity. The molecule would have to break down into two halves of approximately the same size. It would be conceivable that such a “half-molecule” could have a biological, for example an anticoagulant, effect. On the other hand, the replacement of this arginine by e.g. B. histidine give the VAC proteins greater stability.

From Fig. 13 it can easily be seen that neither VAC-alfa nor VAC-beta have a longer, hydrophobic region which would allow either the secretion by or the incorporation of the proteins into a membrane. It can therefore be assumed that VAC-alfa and VAC-beta are intracellular proteins. Wallner et al (Nature 320 (1986), pp. 77-81) report the structure of human lipoxortin I, Saris et al. (Cell 46 (1986), pp. 201-212) and Huang et al. (Cell 46 (1986), pp. 191-199) The structure of the murine or human calpactin I, which is also called lipocortin II. These proteins also belong to the class of Ca ⁺⁺ dependent phospholipid binding proteins. Their structure can be written in analogy to VAC-alpha and beta ( FIG. 14). The structural differences are essentially limited to the N-terminal non-repeat regions, which have 46 amino acids in Lipocortin I and 37 amino acids in Lipocortin II. However, the homologies between VAC-alfa and VAC-beta are more pronounced than those between VAC and lipocortin:

VAC-alfa - VAC-beta55.0% VAC-alfa - Lipocortin I41.9% VAC-alfa - Lipocortin II 43.8% VAC-beta - Lipocortin I41.7% VAC-beta - Lipocortin II44.6%

It can therefore be assumed that the lipocortins also have an anticoagulant effect due to their analogous structure, and are therefore to be used as anticoagulants, and furthermore that this is a general property of this class of Ca ⁺⁺ -dependent phospholipid binding proteins.

Many members of the Ca ⁺⁺ dependent phospholipid binding proteins bind to purified secretory vesicles or other components of the cytoskeleton (see RH Kretsinger et al., Nature 320 (1986), p. 573 for a summary). During secretion, the vesicles isolate from the cell's Golgi apparatus, migrate to the cell membrane and fuse with it. The content of the vesicles is released from the cell. In analogy to the coupling of excitation with muscle contraction, it was proposed (WW Douglas, Br. J. Pharmac. 34 (1968), 451) that the stimulus and the secretion are also coupled via Ca ⁺⁺ . The Ca ⁺⁺ dependent phospholipid binding proteins could play an important role here. In the conserved, 17 amino acid region of each repeat, VAC-alfa has 5 to 6 hydroxyl group-containing amino acids (Asp, Glu, Thr, Ser), three of which are each in an identical position. VAC-beta contains four of these amino acids in each repeat. Although none of the proteins has the EF-hand structure observed in calmodullin, troponin C, S-100 and parvalbumin (RH Kretsinger et al., CRC Crit. Rev. Biochem. 8 (1980), p119), which is found in these molecules for the Ca ⁺⁺ binding is responsible, the conservation of this subsection in each repeat leads to the conclusion that this region is responsible for the Ca ⁺⁺ binding. Targeted mutations in this area could attempt to change the biological activity of VAC proteins.

Example 5 Expression of VAC-alfa in E. coli a) pRH284T: expression vector with the alkaline Phosphatase promoter and terminator

The gene for alkaline phosphatase (PhoA) from E. coli is subject to strict regulation. In In the presence of phosphate, the gene becomes complete switched off, in the absence of phosphate in the medium there is gene expression. H. Shuttleworth et al., Nucleic Acids Res. 14 (1986), p. 8689 and C.N. Chang et al., Gene 44 (1986), pp. 121-125 described the Nucleotide sequence of this gene.

The promoter region was formed from several oligonucleotides of the PhoA gene and in EcoRI-ClaI cut pAT153 inserted. Before the ribosomal Binding site, an XhoI site was introduced. The Orignial EcoRI site is inserted when the synthetic DNA fragment destroyed. After ribosomal binding site was a Translation start ATG provided, the G of which is the first Nucleotide of a SacI (= SstI) site. The Expression vector can be cut by cutting with SacI linearize this point and the 3 ′ overhang Treatment with DNA polymerase I - Klenow fragment in Convert the presence of dGTP to a straight end. This allows any gene at this point for correct expression it must be inserted with the first base of the coding region.

The HinIII-SalI fragment of the pAT portion was removed and replaced by the alkaline phosphatase transcription terminator. The original SalI site was destroyed. For this purpose it was reintroduced in front of the terminator together with the BamHI site also deleted from pAT153. The sequence of the synthetically produced DNA is shown in FIG. 15.

b) Expression clone pRH291

The cDNA clone pP6 / 5 was treated with BglII and PstI cut and isolated the 980 bp fragment that the main part of the coding and about 200 bp 3'untranslated region contains. Means Oligonucleotides became the missing 5'end of coding region replaced. It was done by two Mutations (GGC → GGT, Gly-7 and ACT → ACC, Thr-8) at the same time a KpnI interface in the VAC cDNA introduced. The Oligonucleotides looked like this:

The oligonucleotides EBI-680, -677, -678 and -682 were phosphorylated at the 5 'end. EBI-678 and -680, EBI-677 and -679 as well as EBI-682 and -681 were heated in pairs to 100 ° C and slowly cooled (100 pmol in 20 µl each). The double-stranded oligonucleotides were combined and ligated. pRH284T was linearized with SacI, the 3 ′ overhanging end was reduced to a straight end by treatment with DNA polymerase I / Klenow fragment in the presence of dGTP, and trimmed with BamHI. Approx. 30 ng vector, 200 ng cDNA and 250 nMol oligonucleotides were ligated in 15 ul solution. Competent E. coli HB101 were transformed with the ligase solution. The resulting clone was named pRH291 ( Fig. 16).

Example 6 Vac-beta expression in E. coli a) Expression clone pRH212

The plasmit pER103 was used as the expression vector (E. Rastl-Dworkin et al., Gene 21 (1983), 237-248). The vector was linearized with HindIII. The 5 'overhanging end was partially filled with dATP and DNA polymerase I / Klenow fragment, and the remaining single-strand residue was digested with S1 nuclease. The vector was cut with BanHI and the large fragment isolated ( Fig. 17). The 440 bp MäIII-BamHI fragment containing the codons 13 to 157 was isolated from the clone pRH203. The missing 5 'end was supplemented by oligonucleotides:

Optimal codons were used for E. coli (e.g. Grantham, R. et al., Nucleic Acids Res. 8 (1980), 1893-1912). This codon exchange results in one new HindIII site at codon 5 to 7. Das Oligonucleotide EBI-306 was phosphorylated with EBI-306 hybridized and ligated to the VAC-beta fragment. To the re-cut with BamHI was the 5'terminal VAC fragment ligated into the prepared pER103 vector. The resulting clone was named pRH211. To the for VAC-beta coding region was added from the Clone pRH201 the 1230 bp BamHI-SpHI fragment isolated. The approximately 200 bp long pBR322 section of BamHI to SphI was removed from plasmid pRH211 and replaced by the corresponding VAC cDNA part. It clone pRH212 resulted. The EcoRI-BamHI fragment, that the Trp promoter (S. marcescens), the ribosomal Binding site and the synthetically produced beginning of the VAC-beta gene was obtained by sequencing checked.

b) Expression of VAC-beta (pRH212)

The plasmid-encoded VAC-beta was detected in the Maxizell system (A. Sancar et al., J. Bacteriol. 137 (1979), pp. 692-693.). Ecoli CSR603 (F, thr-1, leuB6, proA2, phr-1, reca1, argE3, thi-1, uvrA6, ara-14, lacY1, galK2, xyl-5, mtl-1, gyrA98 (nalA98), rpsL31, tsx-33, Lambda, supE44) was transformed with pRH212 and grown to an OD₆₀₀ at 37 ° C. 20 ml of culture were irradiated in an open petri dish with a UV-Germicid lamp (15W) from a distance of 50 cm for 5 seconds and then further incubated for 1 hour. 100 µg of D-cycloserine were added to the culture. After 16 hours, the bacteria were centrifuged off, washed twice with Hershey's saline solution (5.4 g / l NaCl, 3.0 g KCl, 1.1 g / l NH₄Cl, 15 mg / l CaCl₂ × 2 H₂O, 0.2 g / l MgCl₂ × 6 H₂O, 0.2 mg / l FeCl₃ × 6 H₂O, 87 mg / l KH₂POI₄, 12.1 g / l Tris pH = 7.4), resuspended in 5 ml Hershey medium (per 100 ml Hershey salts: 2 , 0 ml 20% glucose, 0.5 ml 2% threonine, 1.0 ml 1% leucine, 1.0 ml proline, 1.0 ml 2% arginine, 0.1 ml 0.1% thiamine HCl) and incubated with 20 µg / ml indole acrylic acid for 2 hours. After adding 5 µCi / ml ³⁵S-methionine and further incubation at 37 ° C (1 hour), the plasmid-encoded proteins were radiolabelled. The bacteria were centrifuged off and taken up in 200 μl SDS sample buffer (Lämmli-Gel System, e.g. BLG Davies et al., Methods in Molecular Biology (1986) pp. 306-310). The marked products were separated on a 15% acrylamide gel. The gel was dried and exposed to Kodak X-Omat S film. Purified VAC-alpha protein was run as a reference and visualized by staining. VAC-alfa runs somewhat faster than the PRH212-encoded VAC-beta, as was expected from the molecular weight. The expression of VAC-beta can also be stimulated by adding the inducer indolacrylic acid ( FIG. 18).

c) Expression clone pRH292

The expression enhancer pRH284T was with SacI linearized and the 3 'overhanging end with DNA polymerase I / Klenow fragment and dGTP in straight Ends transferred. The vector was created with SalI trimmed and the large fragment isolated. The HindIII-SalI insert from clone pRH212 were isolated. 0.2 pmol of the oligonucleotide pair

was ligated with 0.2 pmol VAC-beta insert and 0.015 pmol prepared pRH284T. E. coli HB101 was transformed with the ligase solution. The resulting clone was named pRH292 ( Fig. 19).

Claims (64)

1. DNA coding for a polypeptide / protein with essentially the biological properties of vascular-anticoagulant Proteins. 2. DNA according to claim 1, characterized in that it has the formula corresponds, where XXX stands for GAA or GAC. 3. DNA according to claim 1, characterized in that it has the formula corresponds. 4. DNA according to claim 1, characterized in that it has the formula corresponds, where XXX possibly stands for GAA or GAC and / or stands for a stop codon after the last AGA. 5. DNA according to claim 1, characterized in that it has the formula corresponds, an initiation codon possibly being in front of the first GAC. 6. DNA according to claim 1, characterized in that it has the formula corresponds, with a stop codon possibly after the last AGG. 7. DNA according to claim 1, characterized in that it has the formula corresponds, an initiation codon possibly being in front of the first GAT. 8. DNA according to any one of claims 1 to 3, characterized in that in the section for the linker sequence between the second and third repeat structure, an oligonucleotide is inserted, which codes for a recognition sequence of a specific protease. 9. DNA according to any one of claims 2 or 3, characterized in that the triplet coding for arginine +161 (VAC-alfa) or +167 (VAC-beta) is replaced by a codon that codes for an amino acid that is not of Trypsin is recognized as the preferred cleavage site, for example for Histidine. 10. DNA according to any one of claims 1 to 8, characterized in that the specifically replaced for lysine and / or arginine-encoding triplets are by triplets that code for histidine. 11. DNA according to any one of claims 1 to 3 and 5 to 10, characterized characterized in that or, if applicable, that for cysteine coding triplets is / are replaced by for serine or valine coding triplets. 12. DNA according to claim 1, characterized in that it is for a Polypeptide / protein encoded with repeat regions. 13. DNA according to claim 12, characterized in that it is for one of the Repeat areas coded. 14. DNA according to any one of claims 1 to 13, characterized in that rearranged the areas coding for the full repeats are. 15. DNA according to any one of claims 1-14, characterized in that the regions coding for the 17 amino acids of the converged region are modified. 16. DNA according to any one of claims 1 to 15, characterized in that the regions coding for the 17 amino acids of the conserved region are completely or partially replaced

• a. in the case of the VAC proteins by regions which code the corresponding amino acids in the pipocortins,
• b. in VAC-alfa by areas which encode the corresponding amino acids of the VAC-bata and vice versa,
• c. in the case of lipocortin I by regions which encode the corresponding amino acids of lipocortin II and vice versa,
• d. in the case of the lipocortins by regions which code the corresponding amino acids in the VAC proteins or
• e. for the remaining polypeptides / proteins by regions which code the corresponding amino acids of the other polypeptides / proteins.

17. DNA according to any one of claims 1 to 17, characterized in that the regions coding for the N-terminal peptide are exchanged are replaced by oligonucleotides which are essential for the N-terminal peptide the other, having a repeat structure. 18. DNA according to any one of the preceding claims, characterized in that that the N-terminal 5'-end homologous for each host Signal sequence is prepended. 19. DNA according to the preceding claim, characterized in that the Signal sequence at the 3'-end for a specific recognition cleavage site encodes a protease. 20. DNA according to any one of claims 1 to 11, characterized in that the N-terminal 5'-end for a fusion protein portion coding sequence is prepended. 21. DNA according to the preceding claim, characterized in that the DNA coding for the fusion protein portion at the 3'-end one specific recognition cleavage site encoding a protease. 22. DNA, characterized in that it is an allele to one of those in the DNA described above claims. 23. DNA, characterized in that it is a degenerative form of one of the DNA described in the preceding claims. 24. DNA, characterized in that it follows one of the DNA molecules hybridize one of claims 1 to 23 under conditions which recognize a homology greater than 85%, preferably greater than 90% leave, the DNA from natural, synthetic or can be of semi-synthetic origin and with the DNA molecules one of claims 1 to 23 by mutation, Nucleotide substitutions, nucleotide deletions, Nucleotide insertions and nucleotide inversions may be related and for a polypeptide / protein with essentially the biological Properties of the vascular-anticoagulant proteins are encoded. 25. DNA according to one of the preceding claims, characterized in that that it is inserted into a vector. 26. DNA according to claim 25, characterized in that the DNA according to a of the preceding claims in a vector in functional Is linked to an expression control sequence in Microorganisms and mammalian cells replicable. 27. DNA according to any one of claims 25 or 26, characterized in that the vector is a plasmid, replicable in prokaryotes. 28. DNA according to any one of claims 25 or 26, characterized in that the vector is a plasmid, replicable in eukaryotes. 29. DNA according to one of claims 25 or 26, characterized in that it is replicable in acidic animal cells. 30. host organism, characterized in that it with a DNA one of claims 25 to 29 is transformed. 31. Host organism according to claim 30, characterized in that it is a Prokaryote, preferably E. coli. 32. Host organism according to claim 30, characterized characterized as being a eukaryote. 33. Host organism according to claim 30, characterized characterized as being a mammalian cell line. 34. Process for the preparation of DNA molecules for Polypeptides / proteins essentially with the Properties of the vascular anticoagulant proteins encode, characterized in that from a genomic DNA library for this polypeptide coding gene isolated or from that to this gene belonging mRNA a complementary for this DNA encoding polypeptide using the enzyme Revese Transcriptase or that for that DNA encoding polypeptide using chemically and / or enzymatic process is produced. 35. The method according to the preceding claim, characterized characterized in that the DNA is one of the in the Claims 1 to 23 described corresponds. 36. Method for producing a DNA according to one of the Claims 25 to 29, characterized in that one into one with the help of restriction endonucleases cut vector DNA one with corresponding ends inserts provided DNA for a polypeptide in the essential with the properties of the vascular encodes anticoagulant proteins. 37. Method for producing a DNA according to one of the Claims 26 to 29, characterized in that one into one cut with restriction endonucleases Vector DNA containing expression control sequences a DNA with corresponding ends, which for a polypeptide with essentially the properties encoded by vascular anticoagulant proteins, so inserts that the expression control sequences the inserted DNA regulates. 38. Process for making transformed Host organisms according to any one of claims 30 to 33, characterized in that the host organism with one of the vectors according to any one of claims 25 to 29 transformed. 39. Polypeptide, essentially with the properties of vascular-anticoagulant proteins. 40. Polypeptide according to claim 39, characterized in that that it is from a DNA according to any one of claims 1 to 23 is encoded. 41. Polypeptide according to one of the preceding claims, completely free of homologous polypeptides. 42. Polypeptide according to one of the preceding claims, characterized in that it is a leader peptide contains. 43. Polypeptide according to one of the preceding claims, characterized in that there is a Contains fusion protein. 44. A process for the preparation of polypeptides essentially with the properties of the vascular-anticoagulant proteins, characterized in that

• a. a suitable host organism with genetic information coding for a VAC protein transformed,
• b. expresses the information on the production of the VAC protein in the host organism and
• c. the VAC protein is isolated.

45. The method according to claim 44, characterized in that the host organism according to claims 30 to 33 is defined. 46. The method according to claim 44 or 45, characterized characterized that the genetic information in the DNA molecules according to any one of claims 1 to 23 are included. 47. The method according to any one of claims 44 to 46, characterized characterized in that the VAC protein according to one of the claims 39 to 43 is defined. 48. Polypeptide can be produced according to one of claims 44 to 47. 49. Pharmaceutically tolerable salts of the polypeptides after a of claims 39 to 43 or 48 50. Use of the polypeptides according to one of claims 39 to 43 or 48 for therapeutic or prophylactic treatment of the human or animal body. 51. Use of the polypeptides according to one of claims 39 to 43 or 48 for the manufacture of pharmaceutical preparations. 52. Use of the polypeptides according to one of claims 39 to 43 or 48 as an anticoagulant. 53. Use of the polypeptides according to one of claims 39 to 43 or 48 as an anti-inflammatory agent. 54. Use of the polypeptides according to one of claims 39 to 43 or 48 as thrombin inhibiting agents. 55. Agent for therapeutic treatment, characterized in that that in addition to pharmaceutically inert carriers effective amount of a polypeptide according to any one of claims 39 contains up to 43 or 48. 56. Hybrid cell line, characterized in that it is monoclonal Antibodies against one of the polypeptides according to one of the Claims 39 to 43 or 48 secreted. 57. Monoclonal antibodies characterized in that they specifically the effect of the polypeptides according to one of the Neutralize claims 39 to 43 or 48 in whole or in part or specifically bind to one of said polypeptides. 58. Use of the monoclonal antibodies according to claim 57 for qualitative and / or quantitative determination of one of the Polypeptides according to one of claims 39 to 43 or 48. 59. Use of the monoclonal antibodies according to claim 57 for Purification of one of the polypeptides according to one of claims 39 to 43 or 48. 60. Test kit for the determination of polypeptides according to one of the Claims 39 to 43 or 48 characterized in that it contains monoclonal antibodies according to claim 57. 61. Method for the production of monoclonal antibodies according to Claim 57, characterized in that host animals with one of the Polypeptides according to one of claims 39 to 43 or 48 immunized B lymphocytes of these host animals with myeloma cells fused, which secrete the monoclonal antibodies Hybrid cell lines subcloned and cultivated in vitro or in vivo will. 62. Polypeptide according to one of claims 39 to 43 or 48, characterized characterized that it as Dimeres, Trimeres, Tetrameres or Multimer is present. 63. Pharmaceutically tolerable adducts or covalent compounds between the polypeptides according to any one of claims 39 to 43 or 48 with an inert carrier, for therapeutic or prophylactic treatment of human or animal Body. 64. Pharmaceutically tolerable adducts or covalent compounds between the polypeptides according to any one of claims 39 to 43 or 48 with, for example, polyethylene glycol, for therapeutic or prophylactic treatment of human or animal Body.