EP1123400A1 - Gene for a thrombin inhibitor from the triatoma genus and protein with a thrombin-inhibiting effect - Google Patents

Gene for a thrombin inhibitor from the triatoma genus and protein with a thrombin-inhibiting effect

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Publication number
EP1123400A1
EP1123400A1 EP99953879A EP99953879A EP1123400A1 EP 1123400 A1 EP1123400 A1 EP 1123400A1 EP 99953879 A EP99953879 A EP 99953879A EP 99953879 A EP99953879 A EP 99953879A EP 1123400 A1 EP1123400 A1 EP 1123400A1
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EP
European Patent Office
Prior art keywords
thrombin
sequence
protein
amino acids
domain
Prior art date
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EP99953879A
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German (de)
French (fr)
Inventor
Katrin Mende
Ute Lange
Götz NOWAK
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Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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Publication of EP1123400A1 publication Critical patent/EP1123400A1/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects

Definitions

  • the invention relates to a recombinant deoxyribonucleic acid (DNA) which can be isolated from Dipetalogaster maximus, encodes a protein with thrombin-inhibiting activity and a nucleotide sequence corresponding to that in SEQ ID NO. 1 specified nucleotide sequence or a nucleotide sequence derived therefrom, which under stringent conditions with the in SEQ ID NO. 1 specified nucleotide sequence hybridized.
  • the invention further relates to a protein with thrombin-inhibiting activity, which has a length of 344 amino acids and at positions 13 to 117, 125 to 229 and 234 to 342 has the domains DI, DU and DIII with the following conserved sequences:
  • Blood-sucking animals such as leeches, predatory bugs, bats etc. form proteins with an anticoagulant effect in order to prevent coagulation of the host blood.
  • the best studied is the anticoagulant hirudin from the leech Hirudo medicinalis, which has been characterized as a specific thrombin inhibitor and is used as a genetically accessible r-hirudin for the treatment of thromboembolic disorders.
  • a previously described thrombin inhibitor from the predatory bug Rhodnius prolixus has recently been isolated, its amino acid sequence elucidated, cloned and recombinantly expressed in Escherichia coli (DE-OS 41 36 513).
  • DE-OS 195 04 776 describes a protein with a thrombin-inhibiting effect from the predatory bug Dipetalogaster maxi mus. However, only the isolation process as such and an incomplete N-terminal amino acid sequence are described.
  • Thrombin inhibitors are suitable for the prophylaxis and treatment of thromboembolic disorders and for use in the development and manufacture of biocompatible implant materials.
  • the present invention is therefore based on the object of providing a DNA which encodes a protein with a thrombin-inhibiting effect.
  • the DNA is said to be easily replicated in host microorganisms.
  • specific sequence regions of the thrombin inhibitor are to be characterized from the DNA and put into a direct correlation to the activity of the inhibitor.
  • the encoded protein is said to be easy to isolate and to be similarly potent in the clotting activity of thrombin or inhibit more than hirudin.
  • thrombin should no longer be able to cleave low molecular weight chromogenic substrates by the addition of the inhibitor according to the invention.
  • the recombinant thrombin inhibitor should contain at least one of the three domains derived from the nucleotide sequence.
  • this object is achieved by a recombinant deoxyribonucleic acid which can be isolated from Dipetalogaster maximus and which is characterized in that it encodes a protein with thrombin-inhibiting activity and a nucleotide sequence corresponding to that in SEQ ID NO. 1 specified nucleotide sequence, or a nucleotide sequence derived therefrom, which under stringent conditions with the in SEQ ID NO. 1 indicated nucleotide sequence hybridized, and by a protein with thrombin-inhibiting activity, which has a length of 344 amino acids and has the following domains DI, DU and DIII with the conserved sequences at positions 13 to 117, 125 to 229 and 234 to 342 :
  • the invention further relates to a protein consisting only of the sequence of domain II, i.e. has the following sequence:
  • the invention further relates to a DNA sequence which, after expression in a prokaryotic or eukaryotic cell, encodes a protein with thrombin-inhibiting activity, the DNA sequence being selected from:
  • the DNA sequence according to (c) has a nucleotide sequence corresponding to that in SEQ ID NO. 1 specified nucleotide sequence or a sequence derived therefrom, which under stringent conditions with the in SEQ ID NO. 1 indicated nucleotide sequence hybridized, and transformed with this vector microorganisms.
  • Any vectors and microorganisms can be used according to the invention. Examples of the vector include pBluescript KS II
  • transformable microorganisms according to the invention include E. coli TOP10F ', E. coli BL21 or E. coli JM105.
  • the SEQ ID NO. 1 derived nucleotide sequence hybridizes to this sequence under stringent conditions. Any stringent hybridization conditions can be used as long as a DNA sequence with the above-mentioned properties is obtained.
  • the hybridization preferably takes place 15 to 40 ° C. below the melting temperature of the DNA. Examples of stringent hybridization conditions are:
  • Probe labeling is carried out using random prime labeling, hybridization solution according to Sambrbok et al. , Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY, 1982, mixed with 5% dextran sulfate,
  • the DNA sequence encoding the thrombin inhibitor can be isolated from the stomach (homogenized stomach tissue) of Dipetalogaster maxi mus.
  • a corresponding DNA can also be obtained from body fluids from other predatory bugs, e.g. Salivary glands from Dipetalogaster maximus, salivary glands from Triatoma pallidipennis, gastrointestinal tract from Triatoma pallidipennis, and Rhodnius prolixus (whole animal) can be isolated.
  • primers are produced with the aid of the known thrombin inhibitor obtained from the saliva-intestinal punctate from Dipetalogaster maximus, with which a cDNA library for the proteins of the stomach was produced. Using this cDNA library, further primers were synthesized, with which the complete sequence was then found.
  • Finding the DNA sequence encoding a thrombin inhibitor in the stomach of Dipetalogaster maximus depends crucially on the development of a suitable primer.
  • primers were synthesized based on the well-known thrombin inhibitor rhodniin. With this primer, however, none Amplification of a DNA encoding a thrombin inhibitor from the stomach of Dipetalogaster maximus can be achieved.
  • a primer sequence that was synthesized based on the thrombin-inhibiting protein from the saliva-intestinal punctate from Dipetalogaster maximus could be used to amplify the thrombin inhibitor from the stomach of Dipetalogaster maximus.
  • Codon Usage determines the probability with which an amino acid is encoded by different codons. So far, no DNA sequence from a thrombin inhibitor in Dipetalogaster maximus and thus no codon usage was known.
  • all possible base combinations had to be taken into account, which reduces the specificity of the primers (strongly) if several base ambiguities are installed.
  • an amplification of the cDNA was possible Thrombin inhibitors from the stomach of D. maximus can be achieved within 29 cycles.
  • the DNA sequence isolated from the stomach of Dipetalogaster maximus has that in the attached sequence listing under SEQ ID NO. 1 specified sequence or a sequence derived therefrom which hybridizes with this sequence under stringent conditions. It encodes a new thrombin inhibitor which differs from the previously known thrombin inhibitors both in sequence and in its properties.
  • the Dipetalogaster maximus inhibitor consists of three repetitive sections, as indicated above, and each of these domains contains the conserved sequences in bold:
  • each domain also contains two structural regions (printed in bold in the above sequences) which are somewhat similar to one another and have the sequence printed in bold as a consensus sequence. Due to the high homology of the domains DI and DU to the amino acid sequence of a natural thrombin inhibitor isolated from the saliva-intestinal punctate of D. maximus, these domains are directly linked to the activity of the thrombin inhibitor, as was also confirmed experimentally.
  • domains can be cloned as separate sequence components into other thrombin inhibitors or replace corresponding segments in other thrombin inhibitors, as a result of which even more potent thrombin inhibitors can be obtained.
  • the domain is linked to a sequence or a sequence section of another thrombin inhibitor, e.g. of an exoside inhibitor, ligated, cloned into a suitable vector and expressed.
  • the serine protease thrombin is an important, multifunctional component of the hemostatic system. Thrombin not only catalyzes the formation of fibrin, but also activates various coagulation factors and platelets, has a direct influence on the vascular endothelium and averages nonhematic static cellular effects. Furthermore, neuronal functions of thrombin in brain development are also known. An influence of thrombin on the formation and development of cancer and secondary tumors has also been described. These functions of thrombin were and are the starting point for the isolation and modification of effective thrombin inhibitors for antithrombin therapy.
  • thrombin inhibitor sequences with specific areas of other inhibitors of coagulation factors, a multifunctional inhibitor can be created which can simultaneously inhibit several enzymes of the coagulation apparatus.
  • Specific mutations in the thrombin inhibitor sequence can also increase the specificity of the inhibitor for thrombin.
  • the arginine in the reactive side loop of the front domain areas of the inhibitor according to the invention can be replaced by a histidine.
  • thrombin While arginine is also a typical cleavage site for the serine protease trypsin, histidine is considered to be a specific thrombin cleavage site for the specific thrombin inhibitor rhodniin.
  • the specificity of the inhibitor according to the invention for thrombin can thus be increased by the amino acid exchange.
  • the isolation and characterization of such new anticoagulants is essential for the use of anticoagulant proteins for the diagnosis, therapy and prophylaxis of thrombin-mediated diseases. For example, the entire front portion of domain I or II, which is responsible for binding to the ' active center, can be coupled with an exoside inhibitor (such as triabin). Double binding to thrombin is thus possible.
  • sequences can be connected to one another by ligations, cloned into the expression vector pGEX-5X-1, then expressed and isolated. It is also possible to couple the C-terminal region of hirudin, which is also responsible for binding to the anion exoside of the thrombin inhibitor, with the front regions of domains I and II. Domains I and II of the thrombin inhibitor according to the invention D. maximus are very identical to each other, but are very different from domain III. By coupling the front regions of domains I and II with the rear region of domain III, an altered activity of the protein expressed from domain III could be obtained.
  • the complete cDNA for the protein according to the invention with thrombin-inhibiting activity is contained in the plasmid pV / 6.
  • This plasmid was deposited on February 24, 1998 with the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ), Mascheroder Weg lb, D-38124 Braunschweig under the deposit number DSM 12033 in accordance with the terms of the Budapest Treaty.
  • the cDNA can be used in expression systems known per se, such as, for example, prokaryotic expression systems (the glutathione-S-transferase (GST) gene fusion system is, for example, a system for the expression, purification and detection of fusion proteins in E. coli using pGEX plasmid vectors) or eukaryotic expression systems (e.g. ' transfection of hamster kidney cells with expression vectors which contain the DNA for the thrombin inhibitor, and subsequent detection and purification of the proteins formed by means of CAT -Elisa (Boehringer Mannheim, Cat.No. 1363727)) can be expressed.
  • the protein can be isolated and purified in a manner known per se.
  • the protein obtained in this way is suitable as such or together with other active ingredients for packaging in preparations for diagnosis, prophylaxis and therapy of thrombin-mediated diseases. These preparations can be made by methods known in the pharmaceutical formulation art.
  • Figure 1 shows a 219 bp cDNA fragment from pPCRDip34 (+ deduced amino acid sequence) which was used to screen the ⁇ gtlO cDNA library from the stomach of D. maximus.
  • FIG. 2 shows the results of the expression of the thrombin inhibitor gene in different tissues from D. maximus: lane 1 + 3: mRNA from salivary glands lane 2 + 4: mRNA from stomach
  • FIG. 7 shows the restriction map of the 1.439 kb EcoRI fragment from pV / 6, which was cloned into the EcoRI site of the polylinker from pBluescript KS. This fragment carries a 1.035 kb cDNA which codes for a thrombin inhibitor from the stomach of D. maximus and is shown hatched in the figure.
  • the stomach of two predatory bugs was frozen at -70 ° C, cut and placed in 1 ml of stock buffer preheated to 45 ° C (200 mM NaCl, 200 mM Tris-HCl pH 7.5, 1.5 mM MgCl 2 , 2% SDS) transferred containing 25 ul RNase / protein degradation solution (INVITROGENE Corporation). To shred the tissue, this suspension was passed several times through a syringe with a size 21 needle and then incubated in a 45 ° C. water bath for 30 minutes. Insoluble material was separated by centrifugation at 5,000 rpm for 5 minutes.
  • the lysate was adjusted to a salt concentration of 0.5 M by adding 63 ⁇ l of 5M NaCl stock solution per ml of lysate. After shearing the DNA using a sterile syringe with a size 18 to 20 needle, an oligo (dT) cellulose tablet was added, which had dissolved after 2 min. The mixture was shaken for 20 min at room temperature, then the oligo (dT) cellulose was pelleted for 8 min at 5000 rpm, carefully in 1.3 ml binding buffer (500 mM NaCl, 10 mM Tris-HCl pH 7.5, in water added with DEPC ) dissolved and then pelleted again by centrifugation. Repeat this washing step five times.
  • oligo (dT) cellulose tablet was added, which had dissolved after 2 min. The mixture was shaken for 20 min at room temperature, then the oligo (dT) cellulose was pelleted for 8 min at 5000 rpm, carefully in 1.3 ml binding
  • the mixture was placed in a spin tube / centrifuge tube set (INVITROGENE Corporation) and centrifuged for 10 seconds at room temperature and 6000 rpm. The cellulose in the spin tube was washed with binding buffer and then centrifuged for 10 seconds at 6000 rpm. This washing step was repeated five times.
  • the non-PolyA + RNA was rinsed by adding twice 200 ⁇ l wash buffer (250 mM NaCl, 10 mM Tris-HCl pH 7.5, in DEPC water) and centrifugation for 10 seconds at 6000 rpm.
  • the spin tube was then placed in a new centrifuge tube and the PolyA + RNA was detached from the cellulose by adding 100 ⁇ l of elution buffer (10 mM Tris-HCl pH 7.5 in DEPC water) twice and then centrifuging.
  • the PolyA + RNA was precipitated by adding 10 ⁇ l glycogen (2 mg / ml DEPC water), 30 ⁇ l 2 M sodium acetate and 600 ⁇ l 100% ethanol, centrifuged in a cooled centrifuge at 16000 rpm for 15 min and in 10 ⁇ l Elution buffer resuspended. PolyA + RNA isolation is also possible with other commercially available kits.
  • the isolated mRNA was used for the synthesis of single-stranded cDNA, using AMV reverse transcriptase, in polymerase chain reaction (PCR) amplifications (cDNA Cycle ® Kit). 10 ng mRNA in a total volume of 11.5 ⁇ l water were mixed with 1 ⁇ l oligo dT primer (0.2 ⁇ g / ⁇ l), incubated for 10 min at 65 ° C. and then left at room temperature for 2 min.
  • PCR polymerase chain reaction
  • RNA-cDNA hybrids were denatured by incubating at 95 ° C. for 2 minutes and then placing the sample on ice. The cDNA synthesis was repeated by adding 0.5 .mu.l AMV reverse transcriptase to the denatured mixture and incubating again at 42 ° C. for 60 min with subsequent denaturation.
  • each of the Action mixes containing single-stranded cDNA were used directly with lxPCR buffer, 2 mM dNTPs, 1 U Taq polymerase and 1 ⁇ M of the oligonucleotides DIPETALOLG1 and DIPOU3 as well as DIPETALOLG1 and DIPOU6 for the amplification of the cDNA by means of PCR.
  • the total volume of the PCR mixture was 50 ⁇ l.
  • N A, G, T, C
  • DIPETALOLG1 The specific oligonucleotides DIPETALOLG1, DIPOU3 and DIPOU6 were derived from the protein sequence of a thrombin inhibitor isolated from Dipetalogaster's saliva intestinal punctate. DIPETALOLG1 was derived from the sequence FQGNPCEC, DIPOU3 from VCGSD and DIPOU6 from QSFSDPHT. The corresponding sequence information was communicated to EUROGENTEC, which took over the synthesis of the primers.
  • 2 ⁇ l of the PCR products obtained in this way were cloned in a conventional manner using 2 U T4 DNA ligase, 50 ng vector pCR2.1 in lx ligation buffer in pCR2.1. The ligation is carried out at 14 ° C for 12 h.
  • the plasmids obtained in this way were propagated by their transformation into competent TOP10F'-E. coli cells. 2 ⁇ l each of the ligation mixtures were added to 50 ⁇ l competent cells, mixed carefully, incubated on ice for 30 min, then incubated at 42 ° C. for 30 seconds and then leave on ice for 2 min. After adding 450 ⁇ l SOC medium and culturing the transformation batches at 37 ° C. and 225 rpm for 1 h, the bacteria were plated on LB agar containing 50 ⁇ g / ml ampicillin and X-Gal and for 15 h at 37 ° C. cultured.
  • the cloned PCR products were sequenced using commercially available T7 sequencing kits according to the Sanger dideoxy chain termination method (Proc. Natl. Acad. Sei., USA 74: 5463-5467).
  • the PCR amplifications were carried out with PCR core reagents - Gene Amp; the conditions were 1 min., 94.5 ° C. denaturation, 1 min. 55 ° C annealing, 3 min., 72 ° C, extension, duration 29 cycles.
  • the starting point for the preparation of a cDNA library was the mRNA isolated from the stomach of D. maximus.
  • RNA 2 to 5 ⁇ g mRNA were first used for the synthesis of double-stranded cDNA (using a copy kit cDNA synthesis system) according to the Gubler-Hoffmann method (Gene 25: 263, 1987).
  • the mRNA was mixed with 5 ⁇ l oligo dT primer and distilled water to a final volume of 32.5 ⁇ l.
  • To denature the RNA secondary structure the mixture was heated at 65 ° C. for 10 minutes and left at room temperature for 2 minutes. There were 1 ul placental RNase inhibitor, 10 ul 5xRT buffer, 2 ul 100 mM dNTP, 2.5 ul 80 mM sodium pyrophosphate and 2.0 ul reverse Transcriptase added and everything mixed gently.
  • the reaction mixture was collected by brief centrifugation at the bottom of the reaction vessel, incubated at 42 ° C. for 60 min and then cooled on ice for 2 min. After adding 166.5 ⁇ l second-strand buffer (191 mM KCl, 4.5 mM MgCl 2 , 15 mM NH 4 S0 4 ), 12.5 ⁇ l BSA (1 mg / ml), 5 ⁇ l 10 mM ß- NAD, 2.5 ⁇ l 1 M DTT, 10 ⁇ l RNase H (0.3 U / ⁇ l), 1 ⁇ l E. coli DNA ligase and 2.5 ⁇ l DNA polymerase, the mixture was mixed gently and successively for 90 min at 15 ° C. and incubated for 30 min at room temperature.
  • the cDNA synthesis was stopped by heating the mixture for 10 min at 70 ° C., 2 min incubation at room temperature and 2 min cooling on ice.
  • the generation of blunt ends of the cDNA was achieved by adding 1 ⁇ l of T4 DNA polymerase (10 U / ⁇ l) and incubating for 10 min at room temperature. Then 2 ul 0.5 M EDTA was added to stop the reaction.
  • the mixture was purified using 250 ⁇ l phenol / - chloroform.
  • the upper aqueous phase, which contains the double-stranded cDNA, was carefully removed and the DNA was precipitated with 5 ⁇ l of Mussei glycogen (2 mg / ml).
  • the double-stranded, smooth-ended cDNA was used to produce a gene bank in ⁇ gtlO bacteriophages using commercially available kits, such as, for example, the cDNA rapid adapter ligation module, the cDNA rapid cloning module ⁇ gtlO and ⁇ DNA packaging module , ligated.
  • the Dipetalogaster maximus cDNA library was screened after plating approximately 25,000 recombinant phages on nitrocellulose membranes under highly stringent conditions, using a 220 bp subcloned PCR fragment containing the cDNA of the D. maximus trombin inhibitor.
  • the DNA inserts were cut out of positive ⁇ phages and cloned into pBluescript according to standard methods.
  • the hybridization was carried out as follows:
  • Hybridization solution was according to the instructions from Sambrook et al. (1982), mixed with 5% dextran sulfa.
  • probes were marked using random prime labeling.
  • a maximum of 50 ng probe DNA was denatured in a final volume of 11 ⁇ l distilled water for 5 min at 95 ° C. and shocked on ice for 10 min.
  • 15 ⁇ l Random Prime buffer (0.67 M HEPES, 0.17 M Tris-HCl, 10 mM MgCl 2 , 33 mM 2-mercaptoethanol, 1.33 mg / ml BSA, 18 OD 260 U / ml oligodeoxyribonucleotide primer, pH 6.8), 16 ⁇ l distilled water,
  • Nitrocellulose membranes were used.
  • Sequencing TM kits performed. It was the in SEQ ID NO. 1 given sequence received.
  • the amino acid sequences FQGNPCEC and VCGSD of the thrombin inhibitor from the saliva of D. maximus were used to prepare sense and antisense primers for PCR and for sequencing. PCR amplification of the cDNA from the stomach of D. maximus using these primers resulted in the isolation of five PCR fragments that were cloned into pCR2.1.
  • the results from the sequence analysis show that the plasmids pV / 6, pH / 2, pVIl / 2 and pIII / 3 contain the complete cDNA for only one thrombin inhibitor.
  • the plasmid pIV / 4 contains only a part of the cDNA.
  • the gastric thrombin inhibitor cDNA from D. maximus has an open reading frame of 1032 base pairs that encode 344 amino acids.
  • the deduced amino acid sequence contains 3 repetitive regions, indicating that the protein consists of three domains. Amino acid sequence comparisons of these domains showed a high degree of similarity with one another and with the peptide sequence of the protein from the saliva of D. maximus. The greatest similarities were found between domains 1 and 2 from the stomach (identity 86%), also between the native protein from saliva and domain 2 from the stomach.
  • the expression clones are Escherichia coli JM 105 or BL21, transformed with the vector pGEX-5X-1 (from PHARMACIA Biotech), in the "multi cloning site" of which the DNA sequences of domain II of the dipetaline according to the invention were ligated.
  • the expression clones are grown in liquid or on solid 2xYT medium, which is mixed with ampicillin in a final concentration of 100 ⁇ g / ml.
  • 2xYT medium 16 g / 1 trypton
  • the expression of the fusion proteins (r-dipetalin coupled to glutathione S-transferase) is initiated by adding 100 mM IPTG to a final concentration of 0.75 M. A further incubation of the batches follows at 30 ° C.
  • the bacteria are then pelleted by centrifugation at 4 ° C. and 5500 rpm for 15 minutes. The supernatant is discarded and the bacteria are resuspended in 50 ⁇ l lxPBS buffer (ice cold) per ml culture volume.
  • the bacterial cells are destroyed using ultrasound when using a Sonicater. For this purpose, the ultrasound rod is dipped directly into the bacterial suspension and ultrasound is switched on twice for 20 seconds. After adding Triton X-100 to a final concentration of 1%, the mixture is shaken gently for 30 minutes. After centrifugation at 10,000 rpm and 4 ° C. for 10 minutes, the supernatant is collected in a new centrifuge tube.
  • the fusion proteins are separated from the other proteins by means of a glutathione-Sepharose 4B matrix placed in appropriate columns (PHARMACIA Biotech). The fusion proteins bind to the matrix and all others are flushed through the column. After washing the matrix three times with ice-cold lxPBS buffer, the fusion proteins are separated from the matrix using 900 ⁇ l of a specific elution buffer (10 mM reduced glutathione in 50 mM Tris-HCl (pH 8.0)). The recombinant dipetalin is then split off from its fusion partner, the glutathione S-transferase, using 20 units of factor Xa and purified and concentrated by means of HPLC. A pure protein is obtained which corresponds to the sequence of domain II.
  • the coagulation tests were carried out in a CL4 coagulometer (Behnk-Elektronik, Norderstedt) at 37 ° C. • The determination of the activated partial thromboplastin time (aPTT) was carried out according to the instructions of the manufacturer (Boehringer Mannheim) (FIG. 3).
  • thrombin clotting time TT
  • FCT fibrinogen clotting time
  • the D. maximus recombinant thrombin inhibitor has an anticoagulant activity similar to that of hirudin and the natural D. maximus inhibitor. 2. Methodology for determining the Ki value
  • the dissociation constant Ki was determined according to Stone S.R. and Hofsteenge J., 1986, Biochem. 25: 4622-4628. The test was carried out at 25 ° C in 0.05 M Tris-HCl buffer (pH 8.0), the 0.1 M NaCl, 0.1% PEG 6000, r-inhibitor and HD-Phe-Pip-Arg- p-Nitroaniline (S2238, final concentration 50 ⁇ M).
  • the r inhibitor was measured at various final concentrations (0 pM, 7.35 pM, 14.7 pM, 29.4 pM, 44.1 pM, 58.8 pM, 73.5 pM, 88.2 pM, 102.9 pM and 117.6 pM) added.
  • the reaction was started by adding thrombin titrated at the active site (final concentration 77.5 pM).
  • the p-nitroaniline formation was recorded over 20 min at 405 n.
  • the r-inhibitor of D. maximus acted as a slow and binding thrombin inhibitor under the experimental conditions according to the invention.
  • the Ki value was calculated using the nonlinear regression according to the theory of slow and binding inhibitors.
  • the Ki value of the r inhibitor is 49.3 x 10 "15 M ⁇ 22.28 x 10 " 15 M.
  • the molecular weight was determined by MALDI-TOF analysis (Matrix Assisted Laser Desorption Time-Of-Flight). This analysis was carried out on a linearly operated "HP G2024A" device (Hewlett Packard). A stock solution consisting of 20 mg dihydroxyacetophenone (DHAP, Fluka) and 5 mg ammonium citrate in 1 ml 80% isopropanol / water. 0.5 ul of the matrix and the ' r inhibitor were mixed directly on the MALDI target and dried under reduced pressure on the "HP G2024A" sample preparation device. The analysis was carried out using predefined settings for the laser energy and TOF parameters.
  • the molecular weight for the D. maximus r inhibitor is 13 kDa.
  • domain II is responsible for the thrombin inhibitory effect of the protein according to the invention.
  • Domain II isolated already has thrombin-inhibiting activity. This domain can now, if appropriate and for therapeutic reasons, e.g. immunological tolerance, retention in the organism is required, be cloned into other proteins, the additional protein structures must not adversely affect the thrombin inhibitor effect.

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Abstract

The invention relates to a recombinant deoxyribonucleic acid which can be isolated from Dipetalogaster maximus, encoding a protein with a thrombin-inhibiting effect and a nucleotide sequence corresponding to the nucleotide sequence cited in SEQ ID NO. 1 or a nucleotide sequence derived therefrom that can be hybridized in stringent conditions with the nucleotide sequence cited in SEQ ID NO. 1. The invention also relates to a protein that has a thrombin-inhibiting effect, characterized in that it has a length of 344 amino acids, exhibiting in positions 13-117, 125-229 and 234-342 domains DI, DII, DIII with conserved sequences in previous sections of the three domains: domain I:VCGSDGNGTYSNPCMLNC, amino acids 27-43; domain II: VCGSDGNTYSNPCMLTC, amino acids 139-155; domain III: VCGTDGRTYPNICVLKC, amino acids 248-264 in the rear sections of the three domains: domain I; VCGDDQITYLNLCHLEC, amino acids 80-96, domain II: VCGDDEITYRNLCHLEC, amino acids 192-208, domain III: VCGTDGKTYGNLCMLGC, amino acids 305-321 and is encoded by the above-mentioned DNA sequence.

Description

GEN FÜR EINEN THROMBININHIBITOR AUS RAUBWANZEN UND PROTEIN MIT THROMBINHEMMENDER WIRKUNG GEN FOR A THROMBIN INHIBITOR FROM RUBBER BUGS AND PROTEIN WITH THROMBIN INHIBITING EFFECT
BESCHREIBUNGDESCRIPTION
Die Erfindung betrifft eine rekombinante Desoxyribonukleinsäure (DNA) , die aus Dipetalogaster maximus isolierbar ist, ein Protein mit thrombinhemmender Wirkung codiert und eine Nukleotidsequenz, entsprechend der in SEQ ID NO. 1 angegebenen Nukleotidsequenz oder eine davon abgeleitete Nukleotidsequenz, die unter stringenten Bedingungen mit der in SEQ ID NO. 1 angegebenen Nukleotidsequenz hybridisiert, aufweist. Die Erfindung betrifft ferner ein Protein mit thrombinhemmender Wirkung, das eine Länge von 344 Aminosäuren besitzt und an den Positionen 13 bis 117, 125 bis 229 und 234 bis 342 die Domänen DI, DU und DIII mit den folgenden konservierten Sequenzen besitzt:The invention relates to a recombinant deoxyribonucleic acid (DNA) which can be isolated from Dipetalogaster maximus, encodes a protein with thrombin-inhibiting activity and a nucleotide sequence corresponding to that in SEQ ID NO. 1 specified nucleotide sequence or a nucleotide sequence derived therefrom, which under stringent conditions with the in SEQ ID NO. 1 specified nucleotide sequence hybridized. The invention further relates to a protein with thrombin-inhibiting activity, which has a length of 344 amino acids and at positions 13 to 117, 125 to 229 and 234 to 342 has the domains DI, DU and DIII with the following conserved sequences:
in den vorderen Abschnitten der drei Domänen: Domäne I VCGSDGNTYSNPCMLNC Aminosäuren 27 bis 43 Domäne II VCGSDGNTYSNPCMLTC Aminosäuren 139 bis 155 Domäne III VCGTDGRTYPNICVLKC Aminosäuren 248 bis 264 in den hinteren Abschnitten der drei Domänen: Domäne I VCGDDQITYLNLCHLEC Aminosäuren 80 bis 96 Domäne II VCGDDEITYRNLCHLEC Aminosäuren 192 bis 208 Domäne III VCGTDGKTYGNLCMLGC Aminosäuren 305 bis 321.in the front sections of the three domains: Domain I VCGSDGNTYSNPCMLNC amino acids 27 to 43 Domain II VCGSDGNTYSNPCMLTC amino acids 139 to 155 Domain III VCGTDGRTYPNICVLKC amino acids 248 to 264 in the back sections of the three domains: Domain I VCGDDQITYLNLCHLEC amino acids 80 to 96 Domain II VCGDDEITYRNLCHLEC amino acids 192 to 208 Domain III VCGTDGKTYGNLCMLGC amino acids 305 to 321.
Blutsaugende Tiere, wie Blutegel, Raubwanzen, Fledermäuse etc. bilden Proteine mit blutgerinnungshemmender Wirkung, um die Koagulation des Wirtsbluts zu verhindern. Am besten untersucht ist der Blutgerinnungshemmstoff Hirudin aus dem Blutegel Hirudo medicinalis, der als spezifischer Thrombinin- hibitor charakterisiert wurde und als gentechnisch zugängliches r-Hirudin zur Therapie thromboembolischer Erkrankungen eingesetzt wird. Ein früher beschriebener Thrombinhemmstoff aus der Raubwanze Rhodnius prolixus wurde in neuerer Zeit isoliert, in seiner Aminosäuresequenz aufgeklärt, cloniert und in Escherichia coli rekombinant exprimiert (DE-OS 41 36 513) . Die DE-OS 195 04 776 beschreibt ein Protein mit thrombinhemmender Wirkung aus der Raubwanze Dipetalogaster maxi mus . Beschrieben sind jedoch nur das Isolierungsverfahren als solches und eine unvollständige N-terminale Aminosäuresequenz.Blood-sucking animals such as leeches, predatory bugs, bats etc. form proteins with an anticoagulant effect in order to prevent coagulation of the host blood. The best studied is the anticoagulant hirudin from the leech Hirudo medicinalis, which has been characterized as a specific thrombin inhibitor and is used as a genetically accessible r-hirudin for the treatment of thromboembolic disorders. A previously described thrombin inhibitor from the predatory bug Rhodnius prolixus has recently been isolated, its amino acid sequence elucidated, cloned and recombinantly expressed in Escherichia coli (DE-OS 41 36 513). DE-OS 195 04 776 describes a protein with a thrombin-inhibiting effect from the predatory bug Dipetalogaster maxi mus. However, only the isolation process as such and an incomplete N-terminal amino acid sequence are described.
Thrombininhibitoren eignen sich zur Prophylaxe und Behandlung von thromboembolisehen Erkrankungen und zum Einsatz bei der Entwicklung und Herstellung biokompatibler Implantatwerkstoffe.Thrombin inhibitors are suitable for the prophylaxis and treatment of thromboembolic disorders and for use in the development and manufacture of biocompatible implant materials.
Die meisten natürlichen Thrombininhibitoren aus blutsaugenden Tieren wurden entweder aus eingefrorenen, homogenisierten Tieren, wie Rhodniin (J. Biol . Chem. (1993), 268: 16216- 16222) , oder als Punktate aus den entsprechenden Organen (Speicheldrüsen, Darm) wie Triabin (J". Biol . Chem. (1995), 270: 28629-28643) gewonnen. Auch in der DE-OS 195 04 776 wird die Isolation eines Thrombininhibitors aus D. maximus als Punktat des Darminhaltes beschrieben. Diese Methoden sind jedoch mit einem enormen Aufwand verbunden:Most natural thrombin inhibitors from blood-sucking animals were either from frozen, homogenized animals, such as Rhodniin (J. Biol. Chem. (1993), 268: 16216- 16222), or as punctures from the corresponding organs (salivary glands, intestines) such as triabin (J. " Biol. Chem. (1995), 270: 28629-28643). Also in DE-OS 195 04 776 the isolation of a Thrombin inhibitors from D. maximus are described as punctures of the intestinal contents, but these methods are associated with an enormous effort:
1. Es ist eine Tierhaltung der entsprechenden Raubwanzen notwendig.1. It is necessary to keep the predatory bugs in stock.
2. Es müssen stets genügend Tiere zur Verfügung stehen.2. There must always be enough animals available.
3. Aus den Raubwanzen können nur geringe Mengen der Inhibi- toren isoliert und gereinigt werden, die für eine Anwendung in der Diagnostik, Prophylaxe und Therapie bei weitem nicht ausreichen.3. Only small amounts of the inhibitors can be isolated and cleaned from the predatory bugs, which are far from sufficient for use in diagnostics, prophylaxis and therapy.
4. Die Präparation des Tiermaterials ist sehr aufwendig.4. The preparation of the animal material is very complex.
Der vorliegenden Erfindung liegt somit die Aufgabe zugrunde, eine DNA bereitzustellen, die ein Protein mit thrombinhemmender Wirkung codiert . Die DNA soll sich leicht in Wirtsmikroorganismen vermehren lassen. Ferner sollen aus der DNA spezifische Sequenzbereiche des Thrombininhibitors charakterisiert werden und in eine direkte Korrelation zu der Aktivität des Inhibitors gesetzt werden. So sollen durch Kopplung dieser Sequenzbereiche mit spezifischen Thrombininhibitor-Sequenzen anderer Organismen noch potentere Thrombininhibitoren gewonnen werden. Das codierte Protein soll leicht isolierbar sein und soll die Gerinnungsaktivität des Thrombins ähnlich stark oder stärker als Hirudin hemmen. Ferner soll es eine amidoly- tische Thrombinhemmaktivität, vergleichbar mit der von r-Hirudin besitzen, d.h. Thrombin soll durch die Anlagerung des erfindungsgemäßen Inhibitors nicht mehr in der Lage sein, niedermolekulare chromogene Substrate zu spalten. Der rekom- binante Thrombininhibitor sollte wenigstens eine der drei Domänen, die von der Nukleotidsequenz abgeleitet sind, beinhalten.The present invention is therefore based on the object of providing a DNA which encodes a protein with a thrombin-inhibiting effect. The DNA is said to be easily replicated in host microorganisms. Furthermore, specific sequence regions of the thrombin inhibitor are to be characterized from the DNA and put into a direct correlation to the activity of the inhibitor. Thus, by coupling these sequence regions with specific thrombin inhibitor sequences from other organisms, even more potent thrombin inhibitors are to be obtained. The encoded protein is said to be easy to isolate and to be similarly potent in the clotting activity of thrombin or inhibit more than hirudin. Furthermore, it should have an amidolytic thrombin inhibitory activity comparable to that of r-hirudin, ie thrombin should no longer be able to cleave low molecular weight chromogenic substrates by the addition of the inhibitor according to the invention. The recombinant thrombin inhibitor should contain at least one of the three domains derived from the nucleotide sequence.
Erfindungsgemäß wird diese Aufgabe durch eine rekombinante Desoxyribonukleinsäure gelöst, die aus Dipetalogaster maximus isolierbar ist und dadurch gekennzeichnet ist, daß sie ein Protein mit thrombinhemmender Wirkung codiert und eine Nukleotidsequenz entsprechend der in SEQ ID NO. 1 angegebenen Nukleotidsequenz, oder eine davon abgeleitete Nukleotidsequenz, die unter stringenten Bedingungen mit der in SEQ ID NO. 1 angegebenen Nukleotidsequenz hybridisiert, aufweist, sowie durch ein Protein mit thrombinhemmender Wirkung, das eine Länge von 344 Aminosäuren besitzt und an den Positionen 13 bis 117, 125 bis 229 und 234 bis 342 die folgenden Domänen DI, DU und DIII mit den konservierten Sequenzen besitzt:According to the invention, this object is achieved by a recombinant deoxyribonucleic acid which can be isolated from Dipetalogaster maximus and which is characterized in that it encodes a protein with thrombin-inhibiting activity and a nucleotide sequence corresponding to that in SEQ ID NO. 1 specified nucleotide sequence, or a nucleotide sequence derived therefrom, which under stringent conditions with the in SEQ ID NO. 1 indicated nucleotide sequence hybridized, and by a protein with thrombin-inhibiting activity, which has a length of 344 amino acids and has the following domains DI, DU and DIII with the conserved sequences at positions 13 to 117, 125 to 229 and 234 to 342 :
in den vorderen Abschnitten der drei Domänen: Domäne I VCGSDGNTYSNPCMLNC Aminosäuren 27 bis 43 Domäne II VCGSDGNTYSNPCMLTC Aminosäuren 139 bis 155 Domäne III VCGTDGRTYPNICVLKC Aminosäuren 248 bis 264 in den hinteren Abschnitten der drei Domänen: Domäne I VCGDDQITYLNLCHLEC Aminosäuren 80 bis 96 Domäne II VCGDDEITYRNLCHLEC Aminosäuren 192 bis 208 Domäne III VCGTDGKTYGNLCMLGC Aminosäuren 305 bis 321.in the front sections of the three domains: Domain I VCGSDGNTYSNPCMLNC amino acids 27 to 43 Domain II VCGSDGNTYSNPCMLTC amino acids 139 to 155 Domain III VCGTDGRTYPNICVLKC amino acids 248 to 264 in the back sections of the three domains: Domain I VCGDDQITYLNLCHLEC amino acids 80 to 96 Domain II VCGDDEITYRNLCHLEC amino acids 192 to 208 Domain III VCGTDGKTYGNLCMLGC amino acids 305 to 321.
Die Erfindung betrifft ferner ein Protein, das nur aus der Sequenz der Domäne II besteht, d.h. die folgende Sequenz besitzt :The invention further relates to a protein consisting only of the sequence of domain II, i.e. has the following sequence:
FQGNPCECPRALHRVCGSDGNTYSNPCMLTCAKHEGNPDLVQVHEGPCDEHDH DFEDTCQCDDTFQPVCGDDEITYRNLCHLECATFTTSPGVEVKHEGECHPETKFQGNPCECPRALHRVCGSDGNTYSNPCMLTCAKHEGNPDLVQVHEGPCDEHDH DFEDTCQCDDTFQPVCGDDEITYRNLCHLECATFTTSPGVEVKHEGECHPETK
Die Erfindung betrifft ferner eine DNA-Sequenz, die nach Expression in einer prokaryontisehen oder eukaryontisehen Zelle ein Protein mit thrombinhemmender Wirkung codiert, wobei die DNA-Sequenz ausgewählt ist aus:The invention further relates to a DNA sequence which, after expression in a prokaryotic or eukaryotic cell, encodes a protein with thrombin-inhibiting activity, the DNA sequence being selected from:
(a) der Sequenz gemäß SEQ ID NO. 1 oder dem komplementären Strang dazu,(a) the sequence according to SEQ ID NO. 1 or the complementary strand to it,
(b) der DNA-Sequenz, die mit der Sequenz von (a) oder Fragmenten davon hybridisiert,(b) the DNA sequence that hybridizes with the sequence of (a) or fragments thereof,
(c) der DNA, die wegen der Degeneriertheit des genetischen Codes mit der Sequenz von (a) oder (b) hybridisiert, und ein Protein mit der gleichen Aminosäuresequenz codiert,(c) the DNA which hybridizes to the sequence of (a) or (b) due to the degeneracy of the genetic code and which codes for a protein with the same amino acid sequence,
sowie einen Vektor, enthaltendas well as containing a vector
(a) DNA-Sequenzen zur Replikation des Vektors in E. coli ,(a) DNA sequences for replication of the vector in E. coli,
(b) DNA-Sequenzen zur Expression und Sekretion eines Proteins mit thrombinhemmender Wirkung in einem E. coli- Stamm, die einen Promotor einer Signalpeptid-Sequenz und gegebenenfalls einen Terminator codieren, (c) eine ein Protein mit thrombinhemmender Wirkung codierende DNA-Sequenz, die funktioneil mit den DNA-Sequenzen nach (b) verknüpft ist,(b) DNA sequences for the expression and secretion of a protein with thrombin-inhibiting activity in an E. coli Strain which encode a promoter of a signal peptide sequence and optionally a terminator, (c) a DNA sequence coding for a protein with thrombin-inhibiting activity and which is functionally linked to the DNA sequences according to (b),
der dadurch gekennzeichnet ist, daß die DNA-Sequenz nach (c) eine Nukleotidsequenz entsprechend der in SEQ ID NO. 1 angegebenen Nukleotidsequenz oder eine davon abgeleitete Sequenz, die unter stringenten Bedingungen mit der in SEQ ID NO. 1 angegebenen Nukleotidsequenz hybridisiert, aufweist, und mit diesem Vektor transformierte Mikroorganismen. Erfindungsgemäß können beliebige Vektoren und Mikroorganismen verwendet werden. Beispiele für den Vektor umfassen pBluescript KS IIwhich is characterized in that the DNA sequence according to (c) has a nucleotide sequence corresponding to that in SEQ ID NO. 1 specified nucleotide sequence or a sequence derived therefrom, which under stringent conditions with the in SEQ ID NO. 1 indicated nucleotide sequence hybridized, and transformed with this vector microorganisms. Any vectors and microorganisms can be used according to the invention. Examples of the vector include pBluescript KS II
(Stratagene, USA), pCR TM2.1 (Invitrogene Corporation, USA) oder pGEX-5X-l (Pharmacia Biotech) , die erfindungsgemäß transformiert wurden. Beispiele für erfindungsgemäß transformierbare Mikroorganismen umfassen E. coli TOP10F', E. coli BL21 oder E. coli JM105.(Stratagene, USA), pCR TM2.1 (Invitrogene Corporation, USA) or pGEX-5X-1 (Pharmacia Biotech), which were transformed according to the invention. Examples of transformable microorganisms according to the invention include E. coli TOP10F ', E. coli BL21 or E. coli JM105.
Die von SEQ ID NO. 1 abgeleitete Nukleotidsequenz hybridisiert unter stringenten Bedingungen mit dieser Sequenz. Es können beliebige stringente Hybridisierungsbedingungen verwendet werden solange eine DNA-Sequenz mit den oben genannten Eigenschaften erhalten wird. Bevorzugt erfolgt die Hybridisierung 15 bis 40°C unterhalb der Schmelztemperatur der DNA. Beispiele für stringente Hybridisierungsbedingungen sind:The SEQ ID NO. 1 derived nucleotide sequence hybridizes to this sequence under stringent conditions. Any stringent hybridization conditions can be used as long as a DNA sequence with the above-mentioned properties is obtained. The hybridization preferably takes place 15 to 40 ° C. below the melting temperature of the DNA. Examples of stringent hybridization conditions are:
die zu hybridisierende DNA wird an Nylon-N+-Membranen gebunden, Sonden-Markierung erfolgt mittels Random Prime Labeling, Hybridisierungslösung nach Sambrbok et al . , Molecular cloning: a laboratory manual, Cold Spring Harbor Labora- tory Press, Cold Spring Harbor NY, 1982, versetzt mit 5% Dextransulfat ,the DNA to be hybridized is bound to nylon N + membranes, Probe labeling is carried out using random prime labeling, hybridization solution according to Sambrbok et al. , Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY, 1982, mixed with 5% dextran sulfate,
Hybridisierungstemperatur von 54 bis 68°C, Hybridisierung über Nacht in einem Hybridisierungsofen.Hybridization temperature of 54 to 68 ° C, hybridization overnight in a hybridization oven.
Die den Thrombininhibitor codierende DNA-Sequenz ist aus dem Magen (homogenisiertes Magengewebe) von Dipetalogaster maxi mus isolierbar. Eine entsprechende DNA kann auch aus Körperflüssigkeiten anderer Raubwanzen, wie z.B. Speicheldrüsengewebe von Dipetalogaster maximus, Speicheldrüsen (Salivary glands) von Triatoma pallidipennis, Magen-Darm-Trakt von Triatoma pallidipennis , und Rhodnius prolixus (gesamtes Tier) isoliert werden.The DNA sequence encoding the thrombin inhibitor can be isolated from the stomach (homogenized stomach tissue) of Dipetalogaster maxi mus. A corresponding DNA can also be obtained from body fluids from other predatory bugs, e.g. Salivary glands from Dipetalogaster maximus, salivary glands from Triatoma pallidipennis, gastrointestinal tract from Triatoma pallidipennis, and Rhodnius prolixus (whole animal) can be isolated.
Zur Isolierung der erfidnungsgemäßen DNA-Sequenz werden mit Hilfe des aus dem Speichel-Darm-Punktat von Dipetalogaster maximus erhaltenen bekannten Thrombininhibitors Primer hergestellt, mit denen eine cDNA-Bank für die Proteine des Magens hergestellt wurde. Anhand dieser cDNA-Bank wurden weitere Primer synthetisiert, mit denen dann die vollständige Sequenz aufgefunden wurde.To isolate the DNA sequence according to the invention, primers are produced with the aid of the known thrombin inhibitor obtained from the saliva-intestinal punctate from Dipetalogaster maximus, with which a cDNA library for the proteins of the stomach was produced. Using this cDNA library, further primers were synthesized, with which the complete sequence was then found.
Das Auffinden der einen Thrombininhibitor codierenden DNA-Sequenz im Magen von Dipetalogaster maximus hängt entscheidend von der Entwicklung eines geeigneten Primers ab. Zunächst wurden Primer auf der Basis des bekannten Thrombininhibitors Rhodniin synthetisiert. Mit diesem Primer konnte jedoch keine Amplifikation einer einen Thrombininhibitor codierenden DNA aus dem Magen von Dipetalogaster maximus erreicht werden. Überraschenderweise wurde gefunden, daß eine Primer-Sequenz, die basierend auf dem thrombinhemmenden Protein aus dem Speichel-Darm-Punktat von Dipetalogaster maximus synthetisiert wurde, zur Amplifikation des Thrombininhibitors aus dem Magen von Dipetalogaster maximus verwendet werden konnte.Finding the DNA sequence encoding a thrombin inhibitor in the stomach of Dipetalogaster maximus depends crucially on the development of a suitable primer. First, primers were synthesized based on the well-known thrombin inhibitor rhodniin. With this primer, however, none Amplification of a DNA encoding a thrombin inhibitor from the stomach of Dipetalogaster maximus can be achieved. Surprisingly, it was found that a primer sequence that was synthesized based on the thrombin-inhibiting protein from the saliva-intestinal punctate from Dipetalogaster maximus could be used to amplify the thrombin inhibitor from the stomach of Dipetalogaster maximus.
Obwohl oft mittels heterologer DNA-Sequenzen die korrespondierenden DNAs der untersuchten Organismen identifiziert und amplifiziert werden können, konnte keine Amplifikation der Thrombininhibitor-DNA bei D. maximus mit den von der Rhod- niin-Sequenz abgeleiteten Primern erzielt werden. Die bekannten Aminosäuresequenzabschnitte des Thrombininhibitors aus dem Speichel-Darm-Punktat von D. maximus boten überraschenderweise die Grundlage für die Konstruktion neuer Primer. Obwohl die genaue Aufeinanderfolge dieser Aminosäuresequenzabschnitte nicht bekannt war (mit Ausnahme der N-terminalen Sequenz) konnten Primer abgeleitet werden, die zu einer Ampli- fikation der entsprechenden DNA aus dem Magen von D. maximus führten. Eine weitere Schwierigkeit, die bei der Konstruktion der Primer zu überwinden war, liegt in der Degeneriertheit des genetischen Codes begründet.Although the corresponding DNAs of the examined organisms can often be identified and amplified by means of heterologous DNA sequences, no amplification of the thrombin inhibitor DNA in D. maximus could be achieved with the primers derived from the rhodium sequence. The known amino acid sequence sections of the thrombin inhibitor from the saliva-intestinal punctate from D. maximus surprisingly provided the basis for the construction of new primers. Although the exact sequence of these amino acid sequence sections was not known (with the exception of the N-terminal sequence), primers could be derived which led to an amplification of the corresponding DNA from the stomach of D. maximus. Another difficulty that had to be overcome in the construction of the primers lies in the degeneracy of the genetic code.
Jeder Organismus besitzt einen speziellen Codongebrauch ("Codon-Usage") , der festlegt mit welcher Wahrscheinlichkeit eine Aminosäure von unterschiedlichen Codons verschlüsselt wird. Bisher war noch keine DNA-Sequenz von einem Thrombininhibitor bei Dipetalogaster maximus und somit kein Codon-Usage bekannt . Für die Herstellung der Primer mußten nun alle möglichen Basenkombinationen in Betracht gezogen werden, was im Falle des Einbaus von mehreren Basenambiguitäten die Spezifi- tät der Primer (stark) herabsetzt. Durch die Konstruktion von mehreren Primern mit verschiedenen Basenvariationen und durch die Optimierung der PCR-Bedingungen auf 1 min Denaturierung bei 94,5°C, 1 min Annealing bei 55°C und 3 min Extension bei 72°C konnte jedoch eine Amplifikation der cDNA eines Thrombininhibitors aus dem Magen von D. maximus innerhalb von 29 Zyklen erreicht werden.Each organism has a special codon usage ("Codon Usage"), which determines the probability with which an amino acid is encoded by different codons. So far, no DNA sequence from a thrombin inhibitor in Dipetalogaster maximus and thus no codon usage was known. For the preparation of the primers, all possible base combinations had to be taken into account, which reduces the specificity of the primers (strongly) if several base ambiguities are installed. However, by constructing several primers with different base variations and by optimizing the PCR conditions for 1 min denaturation at 94.5 ° C, 1 min annealing at 55 ° C and 3 min extension at 72 ° C, an amplification of the cDNA was possible Thrombin inhibitors from the stomach of D. maximus can be achieved within 29 cycles.
Die aus dem Magen von Dipetalogaster maximus isolierte DNA- Sequenz besitzt die in dem beigefügten Sequenzprotokoll unter SEQ ID NO. 1 angegebene Sequenz oder eine davon abgeleitete Sequenz, die unter stringenten Bedingungen mit dieser Sequenz hybridisiert. Sie codiert einen neuen Thrombininhibitor, der sich sowohl in der Sequenz als auch in seinen Eigenschaften von den bisher bekannten Thrombininhibitoren unterscheidet. Der Inhibitor aus Dipetalogaster maximus besteht aus drei re- petitiven Abschnitten, wie vorstehend angegeben, und jede dieser Domänen enthält die fett gedruckten konservierten Sequenzen:The DNA sequence isolated from the stomach of Dipetalogaster maximus has that in the attached sequence listing under SEQ ID NO. 1 specified sequence or a sequence derived therefrom which hybridizes with this sequence under stringent conditions. It encodes a new thrombin inhibitor which differs from the previously known thrombin inhibitors both in sequence and in its properties. The Dipetalogaster maximus inhibitor consists of three repetitive sections, as indicated above, and each of these domains contains the conserved sequences in bold:
in den vorderen Abschnitten der drei Domänen: Domäne I VCGSDGNTYSNPCMLNC Domäne II VCGSDGNTYSNPCMLTC Domäne III VCGTDGRTYPNICVLKC in den hinteren Abschnitten der drei Domänen: Domäne I VCGDDQITYLNLCHLEC Domäne II VCGDDEITYRNLCHLEC Domäne III VCGTDGKTYGNLCMLGC.in the front sections of the three domains: Domain I VCGSDGNTYSNPCMLNC Domain II VCGSDGNTYSNPCMLTC Domain III VCGTDGRTYPNICVLKC in the back sections of the three domains: Domain I VCGDDQITYLNLCHLEC Domain II VCGDDEITYRNLCHLEC Domain III VCGTDGKTYGNLCMLGC.
Die Existenz von jeweils 11 Cysteinen an gleichen Positionen und in gleichen Abständen zueinander in den Domänen DI, DU und DIII läßt auf die Ausbildung von Disulfidbrücken schließen. Jede Domäne enthält ferner zwei Strukturbereiche (in den vorstehenden Sequenzen fett gedruckt) , die untereinander eine gewisse Ähnlichkeit aufweisen und die fett gedruckte Sequenz als Konsensussequenz aufweisen. Aufgrund der hohen Homologie der Domänen DI und DU zu der Aminosäuresequenz eines aus dem Speichel-Darm-Punktat von D. maximus isolierten natürlichen Thrombininhibitors sind diese Domänen direkt mit der Aktivität des Thrombininhibitors verbunden, wie auch experimentell bestätigt wurde. Diese Domänen können als separate Sequenzbestandteile in andere Thrombininhibitoren cloniert werden bzw. in anderen Thrombininhibitoren entsprechende Segmente ersetzen, wodurch noch potentere Thrombininhibitoren erhalten werden können. Die Domäne wird hierzu mit einer Sequenz bzw. einem Sequenzabschnitt eines anderen Thrombininhibitors, z.B. eines Exoside-Inhibitors, ligiert, in einen geeigneten Vektor cloniert und exprimiert .The existence of 11 cysteines at the same positions and at the same distance from each other in the domains DI, DU and DIII suggests the formation of disulfide bridges. Each domain also contains two structural regions (printed in bold in the above sequences) which are somewhat similar to one another and have the sequence printed in bold as a consensus sequence. Due to the high homology of the domains DI and DU to the amino acid sequence of a natural thrombin inhibitor isolated from the saliva-intestinal punctate of D. maximus, these domains are directly linked to the activity of the thrombin inhibitor, as was also confirmed experimentally. These domains can be cloned as separate sequence components into other thrombin inhibitors or replace corresponding segments in other thrombin inhibitors, as a result of which even more potent thrombin inhibitors can be obtained. For this purpose the domain is linked to a sequence or a sequence section of another thrombin inhibitor, e.g. of an exoside inhibitor, ligated, cloned into a suitable vector and expressed.
Die Serin-Protease Thrombin ist ein wichtiger, multifunktio- neller Bestandteil des hämostatischen Systems. Thrombin katalysiert nicht nur die Bildung des Fibrins, sondern aktiviert verschiedene Gerinnungsfaktoren und die Blutplättchen, besitzt einen direkten Einfluß auf das Gefäßendothel und ver- mittelt nichthämσstatische zelluläre Effekte. Weiterhin sind auch neuronale Funktionen des Thrombins bei der Gehirnentwicklung bekannt . Ein Einfluß von Thrombin auf die Bildung und Entwicklung von Krebs und Sekundärtumoren wurde auch beschrieben. Diese Funktionen des Thrombins waren und sind Ausgangspunkt für die Isolierung und Modifizierung effektiver Thrombinhemmstoffe für die Antithrombin-Therapie . Mit der Neukombination von spezifischen Sequenzen verschiedener Thrombininhibitoren soll eine noch effektivere (festere) , spezifischere und selektivere Bindung an das Thrombin und somit eine starke Hemmung des Thrombins erreicht werden. Weiterhin kann durch die Neukombination von ThrombininhibitorSequenzen mit spezifischen Bereichen anderer Inhibitoren von Koagulationsfaktoren ein multifunktioneller Inhibitor geschaffen werden, der gleichzeitig mehrere Enzyme des Gerin- nungssapparates hemmen kann. Spezifische Mutationen der Thrombininhibitorsequenz können die Spezifitat des Inhibitors für Thrombin ebenfalls erhöhen. Beispielsweise kann das Argi- nin im reaktiven Seitenloop der vorderen Domänenbereiche des erfindungsgemäßen Inhibitors durch ein Histidin ausgetauscht werden. Während Arginin auch eine typische Spaltstelle für die Serinprotease Trypsin darstellt, gilt bei dem spezifischen Thrombininhibitor Rhodniin das Histidin als spezifische Thrombinspaltstelle. Durch den Aminosäureaustausch kann somit die Spezifitat des erfindungsgemäßen Inhibitors für Thrombin erhöht werden. Die Isolierung und Charakterisierung solcher neuen Blutgerinnungshemmstoffe ist für die Anwendung gerin- nungshemmender Proteine zur Diagnostik, Therapie und Prophylaxe Thrombin-mediierter Erkrankungen unumgänglich. Beispielsweise kann der gesamte vordere Abschnitt der Domäne I bzw. II, der für die Bindung an das' aktive Zentrum verantwortlich ist, mit einem Exoside-Inhibitor (wie Triabin) gekoppelt werden. Somit ist eine doppelt feste Bindung an das Thrombin möglich. Durch Ligationen können die Sequenzen miteinander verbunden werden, in den Expressionsvektor pGEX-5X-l cloniert, anschließend exprimiert und isoliert werden. Möglich ist auch eine Kopplung des C-terminalen Bereichs des Hirudins, der ebenfalls für die Bindung an die Anionen-Exo- side des Thrombininhibitors verantwortlich ist, mit den vorderen Bereichen der Domänen I bzw. II. Die Domänen I und II des erfindungsgemäßen Thrombininhibitors aus D. maximus sind untereinander sehr identisch, unterscheiden sich jedoch sehr von der Domäne III. Nun könnte durch die Kopplung der vorderen Bereiche der Domänen I bzw. II mit dem hinteren Bereich der Domäne III eine veränderte Aktivität des aus der Domäne III exprimierten Proteins erhalten werden.The serine protease thrombin is an important, multifunctional component of the hemostatic system. Thrombin not only catalyzes the formation of fibrin, but also activates various coagulation factors and platelets, has a direct influence on the vascular endothelium and averages nonhematic static cellular effects. Furthermore, neuronal functions of thrombin in brain development are also known. An influence of thrombin on the formation and development of cancer and secondary tumors has also been described. These functions of thrombin were and are the starting point for the isolation and modification of effective thrombin inhibitors for antithrombin therapy. With the new combination of specific sequences of different thrombin inhibitors, an even more effective (firmer), more specific and selective binding to the thrombin and thus a strong inhibition of the thrombin is to be achieved. Furthermore, by combining thrombin inhibitor sequences with specific areas of other inhibitors of coagulation factors, a multifunctional inhibitor can be created which can simultaneously inhibit several enzymes of the coagulation apparatus. Specific mutations in the thrombin inhibitor sequence can also increase the specificity of the inhibitor for thrombin. For example, the arginine in the reactive side loop of the front domain areas of the inhibitor according to the invention can be replaced by a histidine. While arginine is also a typical cleavage site for the serine protease trypsin, histidine is considered to be a specific thrombin cleavage site for the specific thrombin inhibitor rhodniin. The specificity of the inhibitor according to the invention for thrombin can thus be increased by the amino acid exchange. The isolation and characterization of such new anticoagulants is essential for the use of anticoagulant proteins for the diagnosis, therapy and prophylaxis of thrombin-mediated diseases. For example, the entire front portion of domain I or II, which is responsible for binding to the ' active center, can be coupled with an exoside inhibitor (such as triabin). Double binding to thrombin is thus possible. The sequences can be connected to one another by ligations, cloned into the expression vector pGEX-5X-1, then expressed and isolated. It is also possible to couple the C-terminal region of hirudin, which is also responsible for binding to the anion exoside of the thrombin inhibitor, with the front regions of domains I and II. Domains I and II of the thrombin inhibitor according to the invention D. maximus are very identical to each other, but are very different from domain III. By coupling the front regions of domains I and II with the rear region of domain III, an altered activity of the protein expressed from domain III could be obtained.
Die vollständige cDNA für das erfindungsgemäße Protein mit thrombinhemmender Wirkung ist in dem Plasmid pV/6 enhalten. Dieses Plasmid wurde am 24.02.1998 bei der Deutschen Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) , Mascheroder Weg lb, D-38124 Braunschweig unter der Hinterlegungsnummer DSM 12033 gemäß den Bedingungen des Budapester Vertrags hinterlegt.The complete cDNA for the protein according to the invention with thrombin-inhibiting activity is contained in the plasmid pV / 6. This plasmid was deposited on February 24, 1998 with the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ), Mascheroder Weg lb, D-38124 Braunschweig under the deposit number DSM 12033 in accordance with the terms of the Budapest Treaty.
Die cDNA kann in an sich bekannten Expressionssystemen, wie z.B. prokaryotisehen Expressionssystemen (Das Glutathione-S- transferase- (GST) -Gene Fusion System ist z.B. ein System für die Expression, Reinigung und Detektion von Fusions-Proteinen in E. coli unter Verwendung von pGEX-Plasmid-Vektoren) oder eukaryotischen ExpressionsSystemen (z.'B. Transfektion von Hamster-Nieren-Zellen mit Expressionsvektoren, die die DNA für den Thrombininhibitor enthalten, und anschließende Detektion und Reinigung der gebildeten Proteine mittels CAT-Elisa (Boehringer Mannheim, Cat.No. 1363727)) exprimiert werden. Das Protein kann in an sich bekannter Weise isoliert und gereinigt werden. Das so erhaltene Protein eignet sich als solches oder zusammen mit anderen Wirkstoffen zur Konfektionierung in Präparaten zur Diagnostik, Prophylaxe und Therapie Thrombin-mediierter Erkrankungen. Diese Präparate können nach auf dem Gebiet der Arzneimittelformulierung bekannten Verfahren hergestellt werden.The cDNA can be used in expression systems known per se, such as, for example, prokaryotic expression systems (the glutathione-S-transferase (GST) gene fusion system is, for example, a system for the expression, purification and detection of fusion proteins in E. coli using pGEX plasmid vectors) or eukaryotic expression systems (e.g. ' transfection of hamster kidney cells with expression vectors which contain the DNA for the thrombin inhibitor, and subsequent detection and purification of the proteins formed by means of CAT -Elisa (Boehringer Mannheim, Cat.No. 1363727)) can be expressed. The protein can be isolated and purified in a manner known per se. The protein obtained in this way is suitable as such or together with other active ingredients for packaging in preparations for diagnosis, prophylaxis and therapy of thrombin-mediated diseases. These preparations can be made by methods known in the pharmaceutical formulation art.
Die folgenden Figuren erläutern die Erfindung näher:The following figures explain the invention in more detail:
Die Figur 1 zeigt ein 219 bp großes cDNA-Fragment von pPCRDip34 (+ abgeleitete Aminosäuresequenz) , das zum Durchmustern der λgtlO-cDNA-Bank aus dem Magen von D. maximus verwendet wurde .Figure 1 shows a 219 bp cDNA fragment from pPCRDip34 (+ deduced amino acid sequence) which was used to screen the λgtlO cDNA library from the stomach of D. maximus.
Die Figur 2 zeigt die Ergebnisse der Expression des Thrombin- inhibitorgens in verschiedenen Geweben aus D. maximus : Bahn 1+3 : mRNA aus Speicheldrüsen Bahn 2+4: mRNA aus MagenFIG. 2 shows the results of the expression of the thrombin inhibitor gene in different tissues from D. maximus: lane 1 + 3: mRNA from salivary glands lane 2 + 4: mRNA from stomach
Die Figur 3 zeigt die Hemmung der aktivierten partiellen Thromboplastinzeit durch den natürlichen Thrombininhibitor aus dem Speichel von D. maximus (Dipetalin aus D. maximus) , durch den rekombinanten Inhibitor aus D. maximus = Domäne II = r-Dipetalin und durch rekombinantes Hirudin (= r-Hirudin) .FIG. 3 shows the inhibition of the activated partial thromboplastin time by the natural thrombin inhibitor from the saliva of D. maximus (dipetaline from D. maximus), by the recombinant inhibitor from D. maximus = domain II = r-dipetal and by recombinant hirudin (= r-hirudin).
Die Figur 4 zeigt die Hemmung der Thrombingerinnungszeit durch den natürlichen Thrombininhibitor aus dem Speichel von D. maximus (Dipetalin aus D. maximus) , durch den rekombinanten Inhibitor aus D. maximus = Domäne II = r-Dipetalin und durch rekombinantes Hirudin (= r-Hirudin) .FIG. 4 shows the inhibition of thrombin coagulation time by the natural thrombin inhibitor from the saliva of D. maximus (dipetaline from D. maximus), by the recombinant inhibitor from D. maximus = domain II = r-dipetaline and by recombinant hirudin (= r- Hirudin).
Die Figur 5 zeigt die Fibrinogengerinnungszeit durch den natürlichen Thrombininhibitor aus dem Speichel von D . maximus (Dipetalin aus D. maximus) , durch den rekombinanten Inhibitor aus D. maximus = Domäne II = r-Dipetalin und durch rekombinantes Hirudin (= r-Hirudin) .FIG. 5 shows the fibrinogen clotting time by the natural thrombin inhibitor from the saliva of D. maximus (dipetaline from D. maximus), through the recombinant inhibitor from D. maximus = domain II = r-dipetaline and through recombinant hirudin (= r-hirudin).
Die Figur 6 zeigt die Hemmung der Eearingerinnungszeit durch den natürlichen Thrombininhibitor aus dem Speichel von D. maximus (Dipetalin aus D. maximus) , durch den rekombinanten Inhibitor aus D. maximus = Domäne II = r-Dipetalin und durch rekombinantes Hirudin (= r-Hirudin) .FIG. 6 shows the inhibition of Eearin coagulation time by the natural thrombin inhibitor from the saliva of D. maximus (dipetaline from D. maximus), by the recombinant inhibitor from D. maximus = domain II = r-dipetaline and by recombinant hirudin (= r- Hirudin).
Die Figur 7 zeigt die Restriktionskarte des 1,439 kb EcoRI- Fragmentes von pV/6, welches in die EcoRI-Schnittstelle des Polylinkers von pBluescript KS kloniert wurde. Dieses Fragment trägt eine 1,035 kb cDNA, welche für einen Thrombininhibitor aus dem Magen von D. maximus codiert und in der Figur schraffiert dargestellt ist.FIG. 7 shows the restriction map of the 1.439 kb EcoRI fragment from pV / 6, which was cloned into the EcoRI site of the polylinker from pBluescript KS. This fragment carries a 1.035 kb cDNA which codes for a thrombin inhibitor from the stomach of D. maximus and is shown hatched in the figure.
Die Isolierung der erfindungsgemäßen DNA wird nachstehend beispielhaft erläutert. Beispiel 1 -The isolation of the DNA according to the invention is explained below by way of example. Example 1 -
Isolierung der RNA und Synthese der cDNAIsolation of the RNA and synthesis of the cDNA
Der Magen von zwei Raubwanzen wurde bei -70°C gefroren, zerschnitten und in 1 ml auf 45°C vorgewärmten Stammpuffer (200 mM NaCl, 200 mM Tris-HCl pH 7,5, 1,5 mM MgCl2, 2% SDS) überführt, der 25 μl Rnase/Protein-Abbaulösung (INVITROGENE Corporation) enthielt. Zur Zerkleinerung des Gewebes wurde diese Suspension mehrmals durch eine Spritze mit einer Nadel der Größe 21 geleitet und anschließend in einem 45°C warmen Wasserbad 30 min inkubiert. Unlösliches Material wurde durch 5minütige Zentrifugation bei 5000 UpM abgetrennt. Das Lysat wurde durch Zugabe von je 63 μl 5M NaCl-Stammlösung pro ml Lysat auf eine Salzkonzentration von 0,5 M eingestellt. Nach dem Scheren der DNA mittels einer sterilen Spritze mit einer Nadel der Größe 18 bis 20 wurde eine Oligo (dT) -Zellulose- Tablette zugegeben, die sich nach 2 min aufgelöst hatte. Der Ansatz wurde 20 min bei Raumtemperatur geschüttelt, danach die Oligo (dT) -Zellulose 8 min bei 5000 UpM pelletiert, vorsichtig in 1,3 ml Bindepuffer (500 mM NaCl, 10 mM Tris-HCl pH 7,5, in DEPC versetztem Wasser) gelöst und anschließend durch Zentrifugation erneut pelletiert. Diesen Waschschritt fünfmal wiederholen. Das Pellett zum Schluß in 0,3 ml Endvolumen Bindepuffer lösen. Der Ansatz wurde in ein Spin-Röhrchen/Zentri- fugenröhrchen-Set (INVITROGENE Corporation) gegeben und 10 Sekunden bei Raumtemperatur und 6000 UpM zentrifugiert . Die Zellulose im Spin-Röhrchen wurde mit Bindepuffer gewaschen und anschließend 10 Sekunden bei 6000 UpM zentrifugiert. Dieser Waschschritt wurde fünfmal wiederholt. Die Nicht- PolyA+-RNA wurde durch zweimalige Zugabe von 200 μl Waschpuffer (250 mM NaCl, 10 mM Tris-HCl pH 7,5, in DEPC-Wasser) und Zentrifugation für 10 Sekunden bei 6000 UpM abgespült. Das Spin-Röhrchen wurde nun in ein neues Zentrifugenrδhrchen gesetzt und die PolyA+-RNA durch zweimalige Zugabe von je 100 μl Elutionspuffer (10 mM Tris-HCl pH 7,5 in DEPC-Wasser) und anschließende Zentrifugation von der Zellulose abgelöst. Die PolyA+-RNA wurde durch Zugabe von 10 μl Glykogen (2 mg/ml DEPC-Wasser) , 30 μl 2 M Natriumacetat und 600 μl 100%igem Ethanol ausgefällt, in einer gekühlten Zentrifuge 15 min bei 16000 UpM zentrifugiert und in 10 μl Elutionspuffer resuspendiert. Die PolyA+-RNA-Isolation ist auch mit anderen kommerziell erhältlichen Kits möglich.The stomach of two predatory bugs was frozen at -70 ° C, cut and placed in 1 ml of stock buffer preheated to 45 ° C (200 mM NaCl, 200 mM Tris-HCl pH 7.5, 1.5 mM MgCl 2 , 2% SDS) transferred containing 25 ul RNase / protein degradation solution (INVITROGENE Corporation). To shred the tissue, this suspension was passed several times through a syringe with a size 21 needle and then incubated in a 45 ° C. water bath for 30 minutes. Insoluble material was separated by centrifugation at 5,000 rpm for 5 minutes. The lysate was adjusted to a salt concentration of 0.5 M by adding 63 μl of 5M NaCl stock solution per ml of lysate. After shearing the DNA using a sterile syringe with a size 18 to 20 needle, an oligo (dT) cellulose tablet was added, which had dissolved after 2 min. The mixture was shaken for 20 min at room temperature, then the oligo (dT) cellulose was pelleted for 8 min at 5000 rpm, carefully in 1.3 ml binding buffer (500 mM NaCl, 10 mM Tris-HCl pH 7.5, in water added with DEPC ) dissolved and then pelleted again by centrifugation. Repeat this washing step five times. Finally, dissolve the pellet in 0.3 ml final volume of binding buffer. The mixture was placed in a spin tube / centrifuge tube set (INVITROGENE Corporation) and centrifuged for 10 seconds at room temperature and 6000 rpm. The cellulose in the spin tube was washed with binding buffer and then centrifuged for 10 seconds at 6000 rpm. This washing step was repeated five times. The non-PolyA + RNA was rinsed by adding twice 200 μl wash buffer (250 mM NaCl, 10 mM Tris-HCl pH 7.5, in DEPC water) and centrifugation for 10 seconds at 6000 rpm. The spin tube was then placed in a new centrifuge tube and the PolyA + RNA was detached from the cellulose by adding 100 μl of elution buffer (10 mM Tris-HCl pH 7.5 in DEPC water) twice and then centrifuging. The PolyA + RNA was precipitated by adding 10 μl glycogen (2 mg / ml DEPC water), 30 μl 2 M sodium acetate and 600 μl 100% ethanol, centrifuged in a cooled centrifuge at 16000 rpm for 15 min and in 10 μl Elution buffer resuspended. PolyA + RNA isolation is also possible with other commercially available kits.
Die isolierte mRNA wurde zur Synthese einzelsträngiger cDNA, bei Verwendung von AMV Reverser Transkriptase, in Polymerase- Ketten-Reaktion (PCR) -Amplifikationen eingesetzt (cDNA Cycle® Kit) . 10 ng mRNA in einem Gesamtvolumen von 11,5 μl Wasser wurden mit 1 μl Oligo dT-Primer (0,2 μg/μl) gemischt, 10 min bei 65°C inkubiert und danach 2 min bei Raumtemperatur belassen. Es wurden 1 μl Rnase-Inhibitor, 4 μl 5xRT-Puffer, 1 μl 100 mM dNTPs, 1 μl 80 mM Natriu pyrophosphat und 0,5 μl AMV Reverse Transkriptase (10 Einheiten/μl) zugegeben und der Ansatz 60 min bei 42°C inkubiert. Die RNA-cDNA-Hybride wurden durch 2minütige Inkubation bei 95°C und anschließende Plazierung der Probe auf Eis denaturiert. Die cDNA-Synthese wurde durch Zugabe von 0,5 μl AMV Reverser Transkriptase zum denaturierten Ansatz und erneuter Inkubation bei 42°C für 60 min mit anschließender Denaturierung wiederholt. Je 2 μl des Re- aktionsmixes, der einzelsträngige cDNA enthält, wurden mit lxPCR-Puffer, 2 mM dNTPs, 1 E Taq-Polymerase und 1 μM der Oligonukleotide DIPETALOLG1 und DIPOU3 sowie DIPETALOLG1 und DIPOU6 direkt zur Amplifikation der cDNA mittels PCR eingesetzt. Das Gesamtvolumen des PCR-Ansatzes betrug 50 μl .The isolated mRNA was used for the synthesis of single-stranded cDNA, using AMV reverse transcriptase, in polymerase chain reaction (PCR) amplifications (cDNA Cycle ® Kit). 10 ng mRNA in a total volume of 11.5 μl water were mixed with 1 μl oligo dT primer (0.2 μg / μl), incubated for 10 min at 65 ° C. and then left at room temperature for 2 min. 1 μl RNase inhibitor, 4 μl 5xRT buffer, 1 μl 100 mM dNTPs, 1 μl 80 mM sodium pyrophosphate and 0.5 μl AMV reverse transcriptase (10 units / μl) were added and the mixture was run at 42 ° C. for 60 min incubated. The RNA-cDNA hybrids were denatured by incubating at 95 ° C. for 2 minutes and then placing the sample on ice. The cDNA synthesis was repeated by adding 0.5 .mu.l AMV reverse transcriptase to the denatured mixture and incubating again at 42 ° C. for 60 min with subsequent denaturation. 2 μl each of the Action mixes containing single-stranded cDNA were used directly with lxPCR buffer, 2 mM dNTPs, 1 U Taq polymerase and 1 μM of the oligonucleotides DIPETALOLG1 and DIPOU3 as well as DIPETALOLG1 and DIPOU6 for the amplification of the cDNA by means of PCR. The total volume of the PCR mixture was 50 μl.
DIPETALOLG1 5' TTY CAR GGN AAY CCN TGY GAR TG 3 'DIPETALOLG1 5 'TTY CAR GGN AAY CCN TGY GAR TG 3'
DIPOU3 5' RTC AGA NCC GCA AAC 3 'DIPOU3 5 'RTC AGA NCC GCA AAC 3'
DIPOU6 5' CAA AGC TTR TCN GAN CCR CAN AC 3 'DIPOU6 5 'CAA AGC TTR TCN GAN CCR CAN AC 3'
R = A, GR = A, G
N = A, G, T, CN = A, G, T, C
Y = C, TY = C, T
Die spezifischen Oligonukleotide DIPETALOLG1, DIPOU3 und DIPOU6 wurden von der Proteinsequenz eines aus dem Speichel- Darm-Punktat von Dipetalogaster isolierten Thrombininhibitors abgeleitet. DIPETALOLG1 wurde von der Sequenz FQGNPCEC, DIPOU3 von VCGSD und DIPOU6 von QSFSDPHT abgeleitet. Die entsprechenden Sequenzinformationen wurden der Firma EUROGENTEC mitgeteilt, die die Synthese der Primer übernahm.The specific oligonucleotides DIPETALOLG1, DIPOU3 and DIPOU6 were derived from the protein sequence of a thrombin inhibitor isolated from Dipetalogaster's saliva intestinal punctate. DIPETALOLG1 was derived from the sequence FQGNPCEC, DIPOU3 from VCGSD and DIPOU6 from QSFSDPHT. The corresponding sequence information was communicated to EUROGENTEC, which took over the synthesis of the primers.
2 μl der so erhaltenen PCR-Produkte wurden in herkömmlicher Weise mittels 2 E T4-DNA-Ligase, 50 ng Vektor pCR2.1 in lxLigationspuffer in pCR2.1 cloniert. Die Ligation erfolgt bei 14°C für 12 h. Die Vermehrung der so erhaltenen Plasmide erfolgte durch ihre Transformation in kompetente TOP10F'- E. coli-Zellen. Je 2 μl der Ligationsansätze wurden zu je 50 μl kompetenten Zellen gegeben, vorsichtig gemischt, 30 min auf Eis inkubiert, danach 30 Sekunden bei 42°C inkubiert und anschließend 2 min auf Eis belassen. Nach Zugabe von 450 μl SOC-Medium und Kultivierung der Transförmationsansätze bei 37°C und 225 UpM für 1 h wurden die Bakterien auf LB-Agar, der 50 μg/ml Ampicillin und X-Gal enthält, ausplattiert und 15 h bei 37°C kultiviert.2 μl of the PCR products obtained in this way were cloned in a conventional manner using 2 U T4 DNA ligase, 50 ng vector pCR2.1 in lx ligation buffer in pCR2.1. The ligation is carried out at 14 ° C for 12 h. The plasmids obtained in this way were propagated by their transformation into competent TOP10F'-E. coli cells. 2 μl each of the ligation mixtures were added to 50 μl competent cells, mixed carefully, incubated on ice for 30 min, then incubated at 42 ° C. for 30 seconds and then leave on ice for 2 min. After adding 450 μl SOC medium and culturing the transformation batches at 37 ° C. and 225 rpm for 1 h, the bacteria were plated on LB agar containing 50 μg / ml ampicillin and X-Gal and for 15 h at 37 ° C. cultured.
Die clonierten PCR-Produkte wurden unter Verwendung kommerziell erhältlicher T7-Sequenzier-Kits nach dem Didesoxy-Ket- tenabbruchverfahren von Sanger (Proc. Natl . Acad. Sei., USA 74: 5463-5467) sequenziert. Die PCR-Amplifikationen wurden mit PCR Core Reagentien - Gene Amp durchgeführt; die Bedingungen betrugen 1 min., 94,5°C Denaturierung, 1 min. 55°C Annelierung, 3 min., 72°C, Extension, Dauer 29 Zyklen.The cloned PCR products were sequenced using commercially available T7 sequencing kits according to the Sanger dideoxy chain termination method (Proc. Natl. Acad. Sei., USA 74: 5463-5467). The PCR amplifications were carried out with PCR core reagents - Gene Amp; the conditions were 1 min., 94.5 ° C. denaturation, 1 min. 55 ° C annealing, 3 min., 72 ° C, extension, duration 29 cycles.
Beispiel 2Example 2
Herstellung und Durchmustern der cDNA-BankPreparation and screening of the cDNA library
Ausgangspunkt für die Herstellung einer cDNA-Genbank war die aus dem Magen von D. maximus isolierte mRNA.The starting point for the preparation of a cDNA library was the mRNA isolated from the stomach of D. maximus.
2 bis 5 μg mRNA wurden zuerst zur Synthese doppelsträngiger cDNA eingesetzt (mittels Copy Kit cDNA Synthese-System) nach dem Gubler-Hoffmann-Verfahren (Gene 25: 263, 1987). Die mRNA wurde mit 5 μl Oligo dT-Primer und destilliertem Wasser bis zu einem Endvolumen von 32,5 μl gemischt. Zur Denaturierung der RNA-Sekundärstruktur wurde der Ansatz 10 min bei 65°C erhitzt und 2 min bei Raumtemperatur belassen. Es wurden 1 μl Placental Rnase Inhibitor, 10 μl 5xRT-Puffer, 2 μl 100 mM dNTP, 2,5 μl 80 mM Natriumpyrophosphat und 2,0 μl Reverse Transkriptase zugegeben und alles vorsichtig gemischt . Der Reaktionsansatz wurde durch kurze Zentrifugation am Boden des Reaktionsgefäßes gesammelt, 60 min bei 42°C inkubiert und anschließend für 2 min auf Eis abgekühlt. Nach Zugabe von 166,5 μl Second-Strang-Puffer (191 mM KCl, 4,5 mM MgCl2, 15 mM NH4S04) , 12,5 μl BSA (1 mg/ml), 5 μl 10 mM ß-NAD, 2,5 μl 1 M DTT, 10 μl Rnase H (0,3 E/μl) , 1 μl E. coli DNA Ligase und 2 , 5 μl DNA Polymerase wurde der Ansatz vorsichtig gemischt und nacheinander 90 min bei 15°C und 30 min bei Raumtemperatur inkubiert. Die cDNA-Synthese wurde durch Erhitzen des Ansatzes 10 min bei 70°C, 2 min Inkubation bei Raumtemperatur und 2 min Abkühlen auf Eis gestoppt. Die Erzeugung glatter Enden der cDNA wurde durch Zugabe von 1 μl T4-DNA-Polymerase (10 E/μl) und Inkubation für 10 min bei Raumtemperatur erreicht. Anschließend wurden 2 μl 0,5 M EDTA zum Abstoppen der Reaktion zugegeben. Mittels 250 μl Phenol/- Chloroform wurde der Ansatz gereinigt. Die obere wäßrige Phase, welche die doppelsträngige cDNA enthält, wurde vorsichtig abgenommen und mit 5 μl Mussei-Glykogen (2 mg/ml) die DNA ausgefällt. Es wurden 250 μl 4 M Ammoniumacetat und 1 ml 100%iges Ethanol zugegeben, der Ansatz 15 min auf Trockeneis gestellt und danach 15 min bei 4°C und 15000 UpM zentrifugiert. Das cDNA-Pellet wurde mit 500 μl 80%igem Ethanol gewaschen und der Waschüberstand vollständig abgenommen.2 to 5 μg mRNA were first used for the synthesis of double-stranded cDNA (using a copy kit cDNA synthesis system) according to the Gubler-Hoffmann method (Gene 25: 263, 1987). The mRNA was mixed with 5 μl oligo dT primer and distilled water to a final volume of 32.5 μl. To denature the RNA secondary structure, the mixture was heated at 65 ° C. for 10 minutes and left at room temperature for 2 minutes. There were 1 ul placental RNase inhibitor, 10 ul 5xRT buffer, 2 ul 100 mM dNTP, 2.5 ul 80 mM sodium pyrophosphate and 2.0 ul reverse Transcriptase added and everything mixed gently. The reaction mixture was collected by brief centrifugation at the bottom of the reaction vessel, incubated at 42 ° C. for 60 min and then cooled on ice for 2 min. After adding 166.5 μl second-strand buffer (191 mM KCl, 4.5 mM MgCl 2 , 15 mM NH 4 S0 4 ), 12.5 μl BSA (1 mg / ml), 5 μl 10 mM ß- NAD, 2.5 μl 1 M DTT, 10 μl RNase H (0.3 U / μl), 1 μl E. coli DNA ligase and 2.5 μl DNA polymerase, the mixture was mixed gently and successively for 90 min at 15 ° C. and incubated for 30 min at room temperature. The cDNA synthesis was stopped by heating the mixture for 10 min at 70 ° C., 2 min incubation at room temperature and 2 min cooling on ice. The generation of blunt ends of the cDNA was achieved by adding 1 μl of T4 DNA polymerase (10 U / μl) and incubating for 10 min at room temperature. Then 2 ul 0.5 M EDTA was added to stop the reaction. The mixture was purified using 250 μl phenol / - chloroform. The upper aqueous phase, which contains the double-stranded cDNA, was carefully removed and the DNA was precipitated with 5 μl of Mussei glycogen (2 mg / ml). 250 μl of 4 M ammonium acetate and 1 ml of 100% ethanol were added, the mixture was placed on dry ice for 15 minutes and then centrifuged for 15 minutes at 4 ° C. and 15,000 rpm. The cDNA pellet was washed with 500 μl of 80% ethanol and the washing supernatant was removed completely.
Die doppelsträngige, glattendige cDNA wurde zur Herstellung einer Genbank in λgtlO-Bakteriophagen mittels handelsüblicher Kits, wie beispielsweise dem cDNA-Rapid-Adaptor-Ligation-Mo- dule, dem cDNA-Rapid-Cloning-Module-λgtlO und λ-DNA-Packaging Module, ligiert. Die Dipetalogaster maximus cDNA-Bank wurde nach Plattieren von etwa 25000 rekombinanten Phagen auf Nitrocellulosemembra- nen unter hoch stringenten Bedingungen durchgemustert, wobei als Sonde ein 220 bp großes subcloniertes PCR-Fragment , das die cDNA des Trombininhibitors aus D. maximus enthielt, verwendet wurde. Die DNA-Insertionen wurden aus positiven λ-Pha- gen herausgeschnitten und nach Standardverfahren in pBlue- script cloniert.The double-stranded, smooth-ended cDNA was used to produce a gene bank in λgtlO bacteriophages using commercially available kits, such as, for example, the cDNA rapid adapter ligation module, the cDNA rapid cloning module λgtlO and λ DNA packaging module , ligated. The Dipetalogaster maximus cDNA library was screened after plating approximately 25,000 recombinant phages on nitrocellulose membranes under highly stringent conditions, using a 220 bp subcloned PCR fragment containing the cDNA of the D. maximus trombin inhibitor. The DNA inserts were cut out of positive λ phages and cloned into pBluescript according to standard methods.
Die Hybridisierung wurde wie folgt durchgeführt :The hybridization was carried out as follows:
1. Hybridisierungslösung wurde nach Vorschrift von Sambrook et al . (1982), versetzt mit 5% Dextransulfa , hergestellt.1. Hybridization solution was according to the instructions from Sambrook et al. (1982), mixed with 5% dextran sulfa.
2. Die Sondenmarkierung erfolgte mittels Random Prime La- beling. Maximal 50 ng Sonden-DNA wurde in einem Endvolumen von 11 μl destilliertem Wasser 5 min bei 95°C denaturiert und 10 min auf Eis geschockt. Nach Zugabe von je 2 μl 0,5 M dNTPs, 15 μl Random-Prime-Puffer (0,67 M HEPES, 0,17 M Tris-HCl, 10 mM MgCl2, 33 mM 2-Mercapto- ethanol, 1,33 mg/ml BSA, 18 OD260 E/ml Oligodesoxyribonu- kleotid-Primer, pH 6,8), 16 μl destilliertem Wasser,2. The probes were marked using random prime labeling. A maximum of 50 ng probe DNA was denatured in a final volume of 11 μl distilled water for 5 min at 95 ° C. and shocked on ice for 10 min. After adding 2 μl 0.5 M dNTPs, 15 μl Random Prime buffer (0.67 M HEPES, 0.17 M Tris-HCl, 10 mM MgCl 2 , 33 mM 2-mercaptoethanol, 1.33 mg / ml BSA, 18 OD 260 U / ml oligodeoxyribonucleotide primer, pH 6.8), 16 μl distilled water,
1 μl Klenow-Polymerase (3 E/μl) , 1 μl spezifischer Thrombininhibitor-Primer (1,5 pmol) und 1 μl [α- P] dATP (10 μCi/μl) wurde der Reaktionsansatz 3 bis 5 h bei 37°C inkubiert. Die Reaktion wurde durch Zugabe von 5 μl Stop-Puffer (0,2 M Na2EDTA, pH 7,5) und 50 μl Dextran- blau-Lösung (0,2% SDS, 0,5 M EDTA, 1% Dextranblau) ge- stoppt und die freien Nukleotide über eine Sephadex-G50- Säule abgetrennt .1 μl of Klenow polymerase (3 U / μl), 1 μl of specific thrombin inhibitor primer (1.5 pmol) and 1 μl of [α- P] dATP (10 μCi / μl) was the reaction mixture for 3 to 5 h at 37 ° C. incubated. The reaction was started by adding 5 μl stop buffer (0.2 M Na 2 EDTA, pH 7.5) and 50 μl dextran blue solution (0.2% SDS, 0.5 M EDTA, 1% dextran blue) - stops and the free nucleotides are separated on a Sephadex G50 column.
3. Die Hybridisierung erfolgte über Nacht bei 68°C im Was- serbad.3. The hybridization took place overnight at 68 ° C. in the water bath.
4. Es wurden Nitrozellulosemembranen eingesetzt.4. Nitrocellulose membranes were used.
SequenzanalyseSequence analysis
Die DNA-Sequenzierung der Plasmidclone wurde nach dem Dides- oxy-Kettenabruchverfahren nach Sanger unter Verwendung des T7The DNA sequencing of the plasmid clones was carried out according to the Sides dideoxy chain termination method using the T7
Sequenzier TM Kits durchgeführt. Es wurde die in SEQ ID NO. 1 angegebene Sequenz erhalten.Sequencing TM kits performed. It was the in SEQ ID NO. 1 given sequence received.
Beispiel 3Example 3
Klonierung und Sequenzierung der cDNA für ein Protein mit thrombinhemmender Wirkung aus Dipetalogaster maximusCloning and sequencing of the cDNA for a protein with thrombin-inhibiting activity from Dipetalogaster maximus
Die Aminosäuresequenzen FQGNPCEC und VCGSD des Thrombininhibitors aus dem Speichel von D. maximus wurden zur Herstellung von Sinn- und Antisinn-Primern für die PCR und für die Sequenzierung verwendet. Die PCR-Amplifikation der cDNA aus dem Magen von D. maximus unter Verwendung dieser Primer führte zur Isolierung von fünf PCR-Fragmenten, die in pCR2.1 cloniert wurden. The amino acid sequences FQGNPCEC and VCGSD of the thrombin inhibitor from the saliva of D. maximus were used to prepare sense and antisense primers for PCR and for sequencing. PCR amplification of the cDNA from the stomach of D. maximus using these primers resulted in the isolation of five PCR fragments that were cloned into pCR2.1.
Die Sequenzierung der PCR-Fragmente zeigte, daß die Plasmide pPCRDip32, pPCRDip34 und pPCRDip38 das gleiche Fragment enthielten. Ein Sequenzvergleich des 219 bp großen Fragments mit den zwei anderen Fragmenten zeigte eine hohe Ähnlichkeit. Die cDNA von pPCRDip35 umfaßt das gesamte 219 bp-Fragment und noch weitere Nukleotide. Im Gegensatz dazu enthält die cDNA von pPCRDip39 nur die ersten 57 Basenpaare des 219 bp-Fragments und weitere Regionen, die mit der Sequenz des Thrombininhibitors Rhodniin homolog sind. Ein Vergleich der abgeleiteten Aminosäuresequenz des 219 bp großen Fragments mit der Proteinsequenz von Rhodniin zeigte eine 53,7%ige Identität zwischen den beiden Thrombininhibitoren. Daher wurde das 219 bp große Fragment von pPCRDip34 als homologe Sonde zum Absuchen der cDNA-Bank aus dem Magen verwendet. Um die vollständige cDNA für den Thrombininhibitor zu isolieren, wurde die λgtlO-cDNA-Bank aus dem Magen von D. maximus unter Verwendung des 219 bp großen Fragments als Sonde unter stringenten Hybridisierungsbedingungen abgesucht (Figur 1) . 6 der 25 positiven λ-Klone wurden durch Restriktionsspaltung und Southern Blot Analyse untersucht. Die Regionen, die vermut- lieh die cDNA des Thrombininhibitors enthielten, wurden sub- cloniert und sequenziert.Sequencing of the PCR fragments showed that the plasmids pPCRDip32, pPCRDip34 and pPCRDip38 contained the same fragment. A sequence comparison of the 219 bp fragment with the two other fragments showed a high degree of similarity. The cDNA of pPCRDip35 comprises the entire 219 bp fragment and still further nucleotides. In contrast, the cDNA of pPCRDip39 contains only the first 57 base pairs of the 219 bp fragment and further regions that are homologous to the sequence of the thrombin inhibitor rhodniin. A comparison of the deduced amino acid sequence of the 219 bp fragment with the protein sequence of rhodniin showed a 53.7% identity between the two thrombin inhibitors. Therefore, the 219 bp fragment of pPCRDip34 was used as a homologous probe for screening the cDNA library from the stomach. In order to isolate the complete cDNA for the thrombin inhibitor, the λgtlO cDNA library from the stomach of D. maximus was screened using the 219 bp fragment as a probe under stringent hybridization conditions (FIG. 1). 6 of the 25 positive λ clones were examined by restriction cleavage and Southern blot analysis. The regions that are If the cDNA contained the thrombin inhibitor, they were subcloned and sequenced.
Clonierte cDNA-Fragmente aus verschiedenen λ-ClonenCloned cDNA fragments from different λ clones
Die Ergebnisse aus der Sequenzanalyse zeigen, daß die Plasmide pV/6, pH/2, pVIl/2 und pIII/3 die vollständige cDNA für nur einen Thrombininhibitor enhalten. Das Plasmid pIV/4 enthält nur einen Teil der cDNA. Die cDNA für den Thrombininhibitor aus dem Magen von D . maximus besitzt ein offenes Leseraster von 1032 Basenpaaren, die 344 Aminosäuren codieren. Die abgeleitete Aminosäuresequenz enthält 3 repetitive Regionen, was anzeigt, daß das Protein aus drei Domänen besteht. Aminosäuresequenzvergleiche dieser Domänen zeigten eine hohe Ähnlichkeit untereinander und mit der Peptidsequenz des Proteins aus dem Speichel von D. maximus . Die größten Ähnlichkeiten wurden zwischen den Domänen 1 und 2 aus dem Magen gefunden (Identität 86%) , ebenso zwischen dem nativen Protein aus dem Speichel und der Domäne 2 aus dem Magen. Beispiel 4The results from the sequence analysis show that the plasmids pV / 6, pH / 2, pVIl / 2 and pIII / 3 contain the complete cDNA for only one thrombin inhibitor. The plasmid pIV / 4 contains only a part of the cDNA. The gastric thrombin inhibitor cDNA from D. maximus has an open reading frame of 1032 base pairs that encode 344 amino acids. The deduced amino acid sequence contains 3 repetitive regions, indicating that the protein consists of three domains. Amino acid sequence comparisons of these domains showed a high degree of similarity with one another and with the peptide sequence of the protein from the saliva of D. maximus. The greatest similarities were found between domains 1 and 2 from the stomach (identity 86%), also between the native protein from saliva and domain 2 from the stomach. Example 4
Expression des Inhibitor-GensExpression of the inhibitor gene
Northern Blot AnalyseNorthern blot analysis
2 bis 3 μg mRNA aus dem Magen und den Speicheldrüsen wurden elektrophoretisch untersucht und auf Hybond-N+-Filter überführt, wobei ein Kapillartransfer verwendet wurde. Die Northern Hybridisierung wurde nach dem Verfahren von Church und Gilbert (Proc. Natl. Acad. Sei., USA 81: 1991-1995) durchgeführt. Die die mRNA enthaltende Membran wurde mit dem [α32- P]dATP markierten cDNA-Fragment des Thrombininhibitors aus D. maximus nach Standardverfahren hybridisiert . Die Analyse der mRNAs aus verschiedenen Geweben zeigte ein hybridisierendes 2,1 kb-Fragment für die RNA aus dem Magen. Keine Transkripte wurden für die RNA aus den Speicheldrüsen nachgewiesen. Dies zeigt die Expression des Thrombininhibitors in dem Magen aus D. maximus (siehe Figur 2) .2 to 3 μg mRNA from the stomach and salivary glands were examined electrophoretically and transferred to Hybond-N + filters using a capillary transfer. Northern hybridization was carried out according to the method of Church and Gilbert (Proc. Natl. Acad. Sei., USA 81: 1991-1995). The membrane containing the mRNA was hybridized with the [α 32 - P] dATP-labeled cDNA fragment of the thrombin inhibitor from D. maximus by standard methods. Analysis of the mRNAs from different tissues showed a hybridizing 2.1 kb fragment for the RNA from the stomach. No transcripts were detected for the RNA from the salivary glands. This shows the expression of the thrombin inhibitor in the stomach from D. maximus (see FIG. 2).
Beispiel 5Example 5
Expression von rekombinantem Dipetalin (= r-Dipetalin = Domäne II)Expression of recombinant dipetalin (= r-dipetalin = domain II)
Die Expressionsklone sind Escherichia coli JM 105 oder BL21, transformiert mit dem Vektor pGEX-5X-l (von PHARMACIA Biotech) , in dessen "multi cloning site" die DNA-Sequenzen der Domäne II des erfindungsgemäßen Dipetalins ligiert wurden. Die Anzucht der Expressionsklone erfolgt in flüssigem oder auf festem 2xYT-Medium, welches mit Ampicillin in einer Endkonzentration von 100 μg/ml versetzt ist.The expression clones are Escherichia coli JM 105 or BL21, transformed with the vector pGEX-5X-1 (from PHARMACIA Biotech), in the "multi cloning site" of which the DNA sequences of domain II of the dipetaline according to the invention were ligated. The expression clones are grown in liquid or on solid 2xYT medium, which is mixed with ampicillin in a final concentration of 100 μg / ml.
2xYT-Medium: 16 g/1 Trypton2xYT medium: 16 g / 1 trypton
10 g/1 Hefeextrakt 5 g/1 NaCl10 g / 1 yeast extract 5 g / 1 NaCl
- pH 7,0- pH 7.0
- für das Festmedium ist die Zugabe von 15 g/1 Agar erforderlich- For the solid medium, the addition of 15 g / 1 agar is required
Kultivierungsbedingungen (falls nicht anders angegeben) :Cultivation conditions (unless otherwise stated):
- 37°C, über Nacht- 37 ° C, overnight
- bei Flüssigkulturen 200 UpM- 200 rpm for liquid cultures
Für die Expression und Isolation des r-Dipetalin wird eine Einzelkolonie eines Expressionsklons (von einem frischen Bakterienausstrich) in 5 ml 2xYT-Medium mit Ampicillin über Nacht bei 37°C und 200 UpM kultiviert. Von dieser Vorkultur werden 1,5 ml in 150 ml fisches 2xYT-Medium mit Ampicillin überführt und diese Hauptkultur bei 30°C und 200 UpM bis zu einer optischen Dichte OD600 = 1,0 kultiviert. Durch Zugabe von 100 mM IPTG bis zu einer Endkonzentration von 0,75 M wird die Expression der Fusionsproteine (r-Dipetalin gekoppelt an Glutathion S-Transferase) eingeleitet. Es folgt eine weitere Inkubation der Ansätze bei 30°C und 200 UpM für 3 Stunden. Danach werden die Bakterien durch 15minütige Zentrifugation bei 4°C und 5500 E/min pelletiert. Der Überstand wird verworfen und die Bakterien in 50 μl lxPBS-Puffer (eiskalt) pro ml Kulturvolumen resuspendiert. Die Bakterienzellen werden mittels Ultraschall bei Verwendung eines Sonicaters zerstört . Hierzu wird der Ultraschallstab direkt in die Bakteriensuspension getaucht und zweimal 20 Sekunden Ultraschall angeschaltet. Nach Zugabe von Triton X-100 bis zu einer Endkonzentration von 1% wird der Ansatz 30 min leicht geschüttelt. Nach lOminütiger Zentrifugation bei 10000 UpM und 4°C wird der Überstand in einem neuen Zentrifugengefäß gesammelt. Die Abtrennung der Fusionsproteine von den übrigen Proteinen erfolgt mittels Glutathions-Sepharose 4B-Matrix plaziert in entsprechenden Säulen (PHARMACIA Biotech) . Die Fusionsproteine binden an die Matrix und alle anderen werden durch die Säule gespült. Nach dreimaligem Waschen der Matrix mit eiskaltem lxPBS-Puffer werden die Fusionsproteine mittels 900 μl eines spezifischen Elutionspuffers (10 mM reduzierte Gluta- thione in 50 mM Tris-HCl (pH 8,0)) von der Matrix abgetrennt. Das rekombinante Dipetalin wird danach von seinem Fusionspartner, der Glutathion S-Transferase, mittels 20 Einheiten Faktor Xa abgespalten und mittels HPLC gereinigt und aufkonzentriert. Es wird ein reines Protein erhalten, das der Sequenz der Domäne II entspricht.For the expression and isolation of r-dipetalin, a single colony of an expression clone (from a fresh bacterial smear) is cultivated in 5 ml of 2xYT medium with ampicillin overnight at 37 ° C. and 200 rpm. 1.5 ml of this preculture is transferred to 150 ml of fishy 2xYT medium with ampicillin and this main culture is cultivated at 30 ° C. and 200 rpm to an optical density OD 600 = 1.0. The expression of the fusion proteins (r-dipetalin coupled to glutathione S-transferase) is initiated by adding 100 mM IPTG to a final concentration of 0.75 M. A further incubation of the batches follows at 30 ° C. and 200 rpm for 3 hours. The bacteria are then pelleted by centrifugation at 4 ° C. and 5500 rpm for 15 minutes. The supernatant is discarded and the bacteria are resuspended in 50 μl lxPBS buffer (ice cold) per ml culture volume. The bacterial cells are destroyed using ultrasound when using a Sonicater. For this purpose, the ultrasound rod is dipped directly into the bacterial suspension and ultrasound is switched on twice for 20 seconds. After adding Triton X-100 to a final concentration of 1%, the mixture is shaken gently for 30 minutes. After centrifugation at 10,000 rpm and 4 ° C. for 10 minutes, the supernatant is collected in a new centrifuge tube. The fusion proteins are separated from the other proteins by means of a glutathione-Sepharose 4B matrix placed in appropriate columns (PHARMACIA Biotech). The fusion proteins bind to the matrix and all others are flushed through the column. After washing the matrix three times with ice-cold lxPBS buffer, the fusion proteins are separated from the matrix using 900 μl of a specific elution buffer (10 mM reduced glutathione in 50 mM Tris-HCl (pH 8.0)). The recombinant dipetalin is then split off from its fusion partner, the glutathione S-transferase, using 20 units of factor Xa and purified and concentrated by means of HPLC. A pure protein is obtained which corresponds to the sequence of domain II.
Beispiel 6Example 6
Untersuchung der Aktivität des Proteins der Domäne IIInvestigation of the activity of the protein of domain II
1. Methodik zur Hemmung der Gerinnungsaktivi tät des Thrombins durch den r- Thrombininhibitor (r-Inhibitor) von D. maximus1. Methodology for inhibiting the coagulation activity of thrombin by the r-thrombin inhibitor (r-inhibitor) from D. maximus
Die Koagulationstests wurden in einem CL4-Koagulometer (Behnk-Elektronik, Norderstedt) bei 37°C durchgeführt. • Die Bestimmung der aktivierten partiellen Thrombopla- stinzeit (aPTT) wurde gemäß den Anweisungen des Herstellers (Boehringer Mannheim) durchgeführt (Figur 3) .The coagulation tests were carried out in a CL4 coagulometer (Behnk-Elektronik, Norderstedt) at 37 ° C. • The determination of the activated partial thromboplastin time (aPTT) was carried out according to the instructions of the manufacturer (Boehringer Mannheim) (FIG. 3).
• Zur Bestimmung der Thrombingerinnungszeit (TT) und der Fibrinogengerinnungszeit (FCT) wurden 50 μl Thrombin (5NIH-E/ml in 0,05 M Tris-HCl-Puffer pH 7,5 mit 0,154 M• To determine the thrombin clotting time (TT) and the fibrinogen clotting time (FCT), 50 μl thrombin (5NIH-E / ml in 0.05 M Tris-HCl buffer pH 7.5 with 0.154 M
NaCl und 1% Albumin) und 50 μl r-Inhibitor (73,5 nM) oder Tris-HCl-Puffer 2 min bei 37°C vorinkubiert . Die Messung der Gerinnungszeit wurde durch Zugabe von 100 μl Plasma (37°C) zur Bestimmung der TT oder 100 μl Fibrino- genlösung (5 mg/ml in Tris-HCl-Puffer mit 5 mM CaCl2) zur Bestimmung FCT gestartet . Die Zeit bis zur Gerinnselbildung wurde gemessen (Figuren 4 und 5) .NaCl and 1% albumin) and 50 μl r-inhibitor (73.5 nM) or Tris-HCl buffer for 2 min at 37 ° C. The measurement of the clotting time was started by adding 100 μl plasma (37 ° C.) to determine the TT or 100 μl fibrinogen solution (5 mg / ml in Tris-HCl buffer with 5 mM CaCl 2 ) to determine FCT. The time to clot formation was measured (Figures 4 and 5).
• Die Eearingerinnungszeit zur quantitativen Messung der direkten Antithrombinaktivität wurde nach Nowak und Bucha (Nowak G. und Bucha E., 1993, Thromb. Haemostas. 69: 1306) durchgeführt. 80 μl r-Inhibitor in 0,05 M Tris-HCl-Puffer (pH 7,5) mit 0,54 M NaCl wurden zu 200 μl Plasma gegeben und die Gerinnungsreaktion durch Zugabe von 20 μl Ecarinlösung (5 E/ml in 0,154 M NaCl/ 0,05 M CaCl2) gestartet (Figur 6).• The Eearin coagulation time for the quantitative measurement of direct antithrombin activity was carried out according to Nowak and Bucha (Nowak G. and Bucha E., 1993, Thromb. Haemostas. 69: 1306). 80 μl r-inhibitor in 0.05 M Tris-HCl buffer (pH 7.5) with 0.54 M NaCl was added to 200 μl plasma and the coagulation reaction by adding 20 μl ecarin solution (5 U / ml in 0.154 M NaCl / 0.05 M CaCl 2 ) started (Figure 6).
Als Ergebnis dieser Bestimmungen kann man feststellen, daß der rekombinante Thrombininhibitor von D. maximus eine ähnliche antikoagulierende Aktivität wie Hirudin und der natürliche Inhibitor von D. maximus aufweist. 2. Methodik zur Ki -Wert -BestimmungAs a result of these determinations, it can be seen that the D. maximus recombinant thrombin inhibitor has an anticoagulant activity similar to that of hirudin and the natural D. maximus inhibitor. 2. Methodology for determining the Ki value
Die Bestimmung der Dissoziationskonstante Ki erfolgte nach Stone S.R. und Hofsteenge J., 1986, Biochem. 25: 4622-4628. Der Test wurde bei 25°C in 0,05 M Tris-HCl-Puffer (pH 8,0), der 0,1 M NaCl, 0,1% PEG 6000, r-Inhibitor und H-D-Phe-Pip- Arg-p-Nitroanilin (S2238, Endkonzentration 50 μM) enthielt, durchgeführt. Der r-Inhibitor wurde in verschiedenen Endkonzentrationen (0 pM, 7,35 pM, 14,7 pM, 29,4 pM, 44,1 pM, 58,8 pM, 73,5 pM, 88,2 pM, 102,9 pM und 117,6 pM) zugesetzt. Die Reaktion wurde durch Zugabe des an aktiver Stelle titrierten Thrombins (Endkonzentration 77,5 pM) gestartet. Die p-Nitroanilin-Bildung wurde über 20 min bei 405 n aufgezeichnet .The dissociation constant Ki was determined according to Stone S.R. and Hofsteenge J., 1986, Biochem. 25: 4622-4628. The test was carried out at 25 ° C in 0.05 M Tris-HCl buffer (pH 8.0), the 0.1 M NaCl, 0.1% PEG 6000, r-inhibitor and HD-Phe-Pip-Arg- p-Nitroaniline (S2238, final concentration 50 μM). The r inhibitor was measured at various final concentrations (0 pM, 7.35 pM, 14.7 pM, 29.4 pM, 44.1 pM, 58.8 pM, 73.5 pM, 88.2 pM, 102.9 pM and 117.6 pM) added. The reaction was started by adding thrombin titrated at the active site (final concentration 77.5 pM). The p-nitroaniline formation was recorded over 20 min at 405 n.
Ergebnis der Ki-Wert-Bestimmung:Result of the Ki value determination:
Der r-Inhibitor von D. maximus wirkte unter den erfindungs- gemäßen experimentellen Bedingungen als langsamer und festbindender Thrombininhibitor. Der Ki-Wert wurde anhand der nichtlinearen Regression nach der Theorie der langsamen und festbindenden Inhibitoren berechnet. Der Ki-Wert des r-Inhibitors beträgt 49,3 x 10"15 M ± 22,28 x 10"15 M.The r-inhibitor of D. maximus acted as a slow and binding thrombin inhibitor under the experimental conditions according to the invention. The Ki value was calculated using the nonlinear regression according to the theory of slow and binding inhibitors. The Ki value of the r inhibitor is 49.3 x 10 "15 M ± 22.28 x 10 " 15 M.
3. Die Molekulargewichtsbestimmung3. The molecular weight determination
Das Molekulargewicht wurde mittels MALDI-TOF-Analyse (Matrix Assisted Laser Desorption Time-Of-Flight) bestimmt. Diese Analyse wurde an einem linear betriebenen "HP G2024A" -Gerät (Hewlett Packard) durchgeführt . Als Matrixlosung diente eine Stammlösung bestehend aus 20 mg Dihydroxyacetophenon (DHAP, Fluka) und 5 mg Ammoniumeitrat in 1 ml 80%igem Isopropa- nol/Wasser. 0,5 μl der Matrix und der 'r-Inhibitor wurden direkt auf dem MALDI-Target gemischt und unter reduziertem Druck auf der Probenvorbereitungsvorrichtung von "HP G2024A" getrocknet. Die Analyse wurde unter Verwendung von vorgegebenen Einstellungen für die Laserenergie und TOF-Parameter durchgeführt .The molecular weight was determined by MALDI-TOF analysis (Matrix Assisted Laser Desorption Time-Of-Flight). This analysis was carried out on a linearly operated "HP G2024A" device (Hewlett Packard). A stock solution consisting of 20 mg dihydroxyacetophenone (DHAP, Fluka) and 5 mg ammonium citrate in 1 ml 80% isopropanol / water. 0.5 ul of the matrix and the ' r inhibitor were mixed directly on the MALDI target and dried under reduced pressure on the "HP G2024A" sample preparation device. The analysis was carried out using predefined settings for the laser energy and TOF parameters.
Das Molekulargewicht für den r-Inhibitor von D. maximus beträgt 13 kDA.The molecular weight for the D. maximus r inhibitor is 13 kDa.
Es zeigt sich, daß die Domäne II für die Thrombininhibitor- wirkung des erfindungsgemäßen Proteins verantwortlich ist. Bereits die isolierte Domäne II besitzt thrombinhemmende Aktivität. Diese Domäne kann nun, sofern zweckdienlich und aus therapeutischen Gründen, z.B. immunologische Verträglichkeit, Retention im Organismus erforderlich ist, in andere Proteine cloniert werden, wobei die zusätzlichen Proteinstrukturen die Thrombininhibitorwirkung nicht ungünstig beeinflussen dürfen. It turns out that domain II is responsible for the thrombin inhibitory effect of the protein according to the invention. Domain II isolated already has thrombin-inhibiting activity. This domain can now, if appropriate and for therapeutic reasons, e.g. immunological tolerance, retention in the organism is required, be cloned into other proteins, the additional protein structures must not adversely affect the thrombin inhibitor effect.

Claims

PATENTANSPRÜCHE PATENT CLAIMS
1. Rekombinante Desoxyribonukleinsäure (DNA) , die aus Dipetalogaster maximus isolierbar ist, ein Protein mit thrombinhemmender Wirkung codiert und eine Nukleotidsequenz entsprechend der in SEQ ID NO. 1 angegebenen Nukleotidsequenz oder eine davon abgeleitete Nukleotidsequenz, die unter stringenten Bedingungen mit der in SEQ ID NO. 1 angegebenen Nukleotidsequenz hybridisiert, aufweist.1. Recombinant deoxyribonucleic acid (DNA), which can be isolated from Dipetalogaster maximus, encodes a protein with thrombin-inhibiting activity and a nucleotide sequence corresponding to that in SEQ ID NO. 1 specified nucleotide sequence or a nucleotide sequence derived therefrom, which under stringent conditions with the in SEQ ID NO. 1 specified nucleotide sequence hybridized.
2. DNA-Sequenz, die nach Expression einer prokaryontisehen oder eukaryontisehen Zelle ein Protein mit thrombinhemmender Wirkung codiert, wobei die DNA-Sequenz ausgewählt ist aus:2. DNA sequence which, after expression of a prokaryotic or eukaryotic cell, encodes a protein with a thrombin-inhibiting effect, the DNA sequence being selected from:
(a) der Sequenz gemäß SEQ ID NO. 1 oder dem komplementären Strang dazu,(a) the sequence according to SEQ ID NO. 1 or the complementary strand to it,
(b) der DNA-Sequenz, die mit der Sequenz von (a) oder Fragmenten davon hybridisiert,(b) the DNA sequence that hybridizes with the sequence of (a) or fragments thereof,
(c) der DNA, die wegen der Degeneriertheit des genetischen Codes mit der Sequenz von (a) oder (b) hybridisiert, und ein Protein mit der gleichen Aminosäuresequenz codiert .(c) the DNA that hybridizes to the sequence of (a) or (b) due to the degeneracy of the genetic code and encodes a protein with the same amino acid sequence.
3. Vektor, enthaltend3. Vector containing
(a) DNA-Sequenzen zur Replikation des Vektors in E. coli , (b) DNA-Sequenzen zur Expression und Sekretion eines Proteins mit thrombinhemmender Wirkung in einem E. coli- Stamm, die einen Promotor einer Signalpeptid-Sequenz und gegebenenfalls einen Terminator codieren,(a) DNA sequences for replication of the vector in E. coli, (b) DNA sequences for the expression and secretion of a protein with thrombin-inhibiting activity in an E. coli strain, which encode a promoter of a signal peptide sequence and optionally a terminator,
(c) eine ein Protein mit thrombinhemmender Wirkung codierende DNA-Sequenz, die funktioneil mit den DNA-Sequenzen nach (b) verknüpft ist,(c) a DNA sequence coding for a protein with thrombin-inhibiting activity and which is functionally linked to the DNA sequences according to (b),
dadurch g e k e n n z e i c h n e t , daß die DNA-Sequenz nach (c) eine Nukleotidsequenz entsprechend der in SEQ ID NO. 1 angegebenen Nukleotidsequenz oder eine davon abgeleitete Sequenz, die unter stringenten Bedingungen mit der in SEQ ID NO. 1 angegebenen Nukleotidsequenz hybridisiert, aufweist.in that the DNA sequence according to (c) has a nucleotide sequence corresponding to that in SEQ ID NO. 1 specified nucleotide sequence or a sequence derived therefrom, which under stringent conditions with the in SEQ ID NO. 1 specified nucleotide sequence hybridized.
4. Vektor nach Anspruch 3 , dadurch g e k e n n z e i c h n e t , daß der Vektor ein Plasmid ist.4. Vector according to claim 3, characterized in that the vector is a plasmid.
5. Vektor nach Anspruch 4 , dadurch g e k e n n z e i c h n e t , daß der Vektor das Plasmid pV/6 ist und die Restriktionskarte gemäß Figur 7 aufweist.5. Vector according to claim 4, characterized in that the vector is the plasmid pV / 6 and has the restriction map according to FIG. 7.
6. Transformierter Wirtsorganismus, geeignet zur Produktion eines Proteins mit thrombinhemmender Wirkung, dadurch g e k e n n z e i c h n e t , daß der Wirtsorganismus ein E. coli-Stamm ist und mit einem Vektor nach Anspruch 3 transformiert worden ist. 6. Transformed host organism, suitable for the production of a protein with thrombin-inhibiting effect, characterized in that the host organism is an E. coli strain and has been transformed with a vector according to claim 3.
7. Protein mit thrombinhemmender Wirkung, dadurch g e k e n n z e i c h n e t , daß es eine Länge von 344 Aminosäuren besitzt, an den Positionen 13 bis 117, 125 bis 229 und 234 bis 342 die Domänen DI, DU und DIII mit den konservierten Sequenzen7. Protein with thrombin-inhibiting action, due to the fact that it has a length of 344 amino acids, the positions DI, DU and DIII with the conserved sequences at positions 13 to 117, 125 to 229 and 234 to 342
in den vorderen Abschnitten der drei Domänen: Domäne I VCGSDGNTYSNPCMLNC Aminosäuren 27 bis 43 Domäne II VCGSDGNTYSNPCMLTC Aminosäuren 139 bis 155 Domäne III VCGTDGRTYPNICVLKC Aminosäuren 248 bis 264in the front sections of the three domains: Domain I VCGSDGNTYSNPCMLNC amino acids 27 to 43 Domain II VCGSDGNTYSNPCMLTC amino acids 139 to 155 Domain III VCGTDGRTYPNICVLKC amino acids 248 to 264
in den hinteren Abschnitten der drei Domänen: Domäne I VCGDDQITYLNLCHLEC Aminosäuren 80 bis 96 Domäne II VCGDDEITYRNLCHLEC Aminosäuren 192 bis 208 Domäne III VCGTDGKTYGNLCMLGC Aminosäuren 305 bis 321in the back sections of the three domains: Domain I VCGDDQITYLNLCHLEC amino acids 80 to 96 Domain II VCGDDEITYRNLCHLEC amino acids 192 to 208 Domain III VCGTDGKTYGNLCMLGC amino acids 305 to 321
besitzt und von einer DNA-Sequenz nach einem der Ansprüche 1 oder 2 codiert wird.possesses and is encoded by a DNA sequence according to one of claims 1 or 2.
8. Protein nach Anspruch 7, dadurch g e k e n n z e i c h n e t , daß es die folgenden Eigenschaften besitzt:8. Protein according to claim 7, characterized in that it has the following properties:
(a) es hemmt die Gerinnungsaktivität des Thrombins mindestens so stark wie Hirudin,(a) inhibits the clotting activity of thrombin at least as much as hirudin,
(b) es besitzt eine dem r-Hirudin vergleichbare amidolyti- sche Thrombinhemmaktivität,(b) it has an amidolytic thrombin inhibitory activity comparable to r-hirudin,
(c) das Molekulargewicht liegt zwischen 11 und 15 kDa, (d) der Ki-Wert beträgt ca. 125 x 10'"15 M i 32,9 x 10"xs M.(c) the molecular weight is between 11 and 15 kDa, (d) the Ki value is approximately 125 x 10 '"15 M i 32.9 x 10 " xs M.
9. Protein nach Anspruch 8 , dadurch g e k e n n z e i c h n e t , daß es die folgende Sequenz besitzt:9. Protein according to claim 8, characterized in that it has the following sequence:
FQGNPCECPRALHRVCGSDGNTYSNPCMLTCAKHEGNPDLVQVHEGPCDEHDH DFEDTCQCDDTFQPVCGDDEITYRNLCHLECATFTTSPGVEVKHEGECHPETK FQGNPCECPRALHRVCGSDGNTYSNPCMLTCAKHEGNPDLVQVHEGPCDEHDH DFEDTCQCDDTFQPVCGDDEITYRNLCHLECATFTTSPGVEVKHEGECHPETK
EP99953879A 1998-10-22 1999-10-19 Gene for a thrombin inhibitor from the triatoma genus and protein with a thrombin-inhibiting effect Withdrawn EP1123400A1 (en)

Applications Claiming Priority (3)

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DE1998148785 DE19848785A1 (en) 1998-10-22 1998-10-22 New thrombin inhibiting protein useful for diagnosis, treatment and prevention of thromboembolic diseases, is obtained from Dipetalogaster maximus
DE19848785 1998-10-22
PCT/EP1999/007900 WO2000024898A1 (en) 1998-10-22 1999-10-19 Gene for a thrombin inhibitor from the triatoma genus and protein with a thrombin-inhibiting effect

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GB0101879D0 (en) * 2001-01-24 2001-03-07 Enzyme Res Lab Ltd Anticoagulants and their uses
DK2109457T3 (en) * 2007-02-12 2016-04-11 Csl Behring Gmbh THERAPEUTIC USE OF KAZAL TYPE SERINE PROTEASE INHIBITORS
CN109735554A (en) * 2019-02-01 2019-05-10 中山大学 It is a kind of for identifying the COI gene and method for identifying molecules of triatoma rubrofasciata

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DE4136513A1 (en) * 1991-11-06 1993-05-13 Basf Ag NEW THROMBIN INHIBITORIC PROTEIN FROM RUBBER BUGS
DE19504776A1 (en) * 1995-02-14 1996-08-22 Inst Molekulare Biotechnologie Thrombin-inhibiting protein

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Title
See references of WO0024898A1 *

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WO2000024898A8 (en) 2000-09-21
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