Natural and recombinant thrombin inhibitors, their preparation and use

 

(57) Abstract:

The invention relates to the field of biotechnology and biochemistry, and can be used in medicine. From the saliva of the bug Triatoma pallidipennis isolated protein with a molecular mass (180003000) and the value of PI 4,5-5,2, possessing properties of a thrombin inhibitor. Using an oligonucleotide probe synthesized based on the results of the determination of N-terminal amino acid sequence selected natural protein derived DNA fragments encoding its precursor and active form. These DNA fragments are included in the expression vector, which is used for transformation of strain E. Li. By culturing the obtained recombinant strain of a completed expression of recombinant analogue of a thrombin inhibitor from the saliva of the bedbug. After chromatographic purification methods both forms (natural and recombinant) polypeptide, as well as their mixture can be used as a drug for the treatment of thrombosis, unstable angina, arteriosclerosis, etc., both directly and as part of farbkomposition. 3 Il., table 2.

The invention relates to proteins, inhibitors of thrombin from the saliva of blood-sucking in mammals.

Therefore, the use of a thrombin inhibitor should lead to success in the treatment of thrombosis. Still the most important known inhibitors of thrombin are antithrombin III-heparin and hirudin. Antithrombin III is a located in the plasma protein with a molecular weight of 58 KD (kilodaltons) Antithrombin III first binds to heparin, which is a polysaccharide. Then the complex of antithrombin III-heparin binds to thrombin and inhibits the thrombin. Forms a very stable complex of thrombin with antithrombin III and antithrombin III is cleaved by thrombin. Along with thrombin-antithrombin III also inhibits other semipretioase, such as factor XA (Pratt, C. W., and Church, G. C. (1991) "Fntithrombin: Structure and Function", Seminars in Hematology 28: 3-9).

From the leech Hirudo medicinalis secrete protein hirudin. He has a molecular weight of about 7 KD and bound through ionic interactions with thrombin. It was specific for thrombin (Johnson, P. H., and others (1989) "Biochemistry and Genetic engineering of hirudin" the prior art, looking for other inhibitors, which have a different mechanism of action or increased activity.

The invention offers a natural or synthetic derived proteins that are inhibitors of thrombin and stand out from the saliva of insects, sucking the blood of mammals.

According to the invention preferred proteins that emit from predatory clone riatoma pallidipennis.

Proposed according to the invention, the proteins may be of natural origin. Proteins get that clear saliva. You can also select proteins from the salivary glands or to select synthesizing protein cells of the salivary gland and cultivate. Produced by this cell culture supernatant is collected and processed. The supernatant of the cell culture clear and proposed according to the invention the protein is isolated and enriched. All stages of the enrichment, isolation and purification are part of the invention. The preferred stage of enrichment, isolation and purification, in which proposed according to the invention, the proteins can be used for pharmaceutical purposes. So, reach cleanings 50% of the thrombin inhibitor, based on the entire protein, preferably 85%, more than the obtaining includes not only proteins, emit riatoma pallidipennis, but also proteins that can be synthesized in other insect species. So, along with proteins, which are secreted riatoma pallidipennis and which belong to the group of most preferred preferred other proteins that originate from riatoma infestans, riatoma dimidiata, riatoma maculata, Rhodnius prolixus, Panstrongylus megistus and Panstrongylus infestans.

You can also offer according to the invention the proteins to obtain synthetically. Such a way of obtaining true protein synthesis according to J. M. SEWART AND J. D. YOUNG, San Franzisco, 1969, and J. MEIENHOFER, Hormonal Proteing and Peptides, so 2, S. 46, Academic Press (New York) 1973, and E. SCHODER and K. LUBKE, The Peptides, T. 1, Academic Press (New York), 1965. To synthetically produced proteins also referred recombinant proteins that get by known methods. Depending on the host body proposed according to the invention, the proteins may be glycosylated or, if they are synthesized in prokaryotes, deglycosylation.

The inhibiting function set in different test systems. In examples 2-4 describes the conventional testing methods.

Proposed according to the invention the proteins determined in the saliva of insects that suck the blood of mammals. These proteins usually sintesio rotini not limited to this method of acquisition and allocation. On the contrary, together with them, covers all synthetically produced, proposed according to the invention, the inhibitors of thrombin, which is found in saliva and which can be distinguished from it.

N-terminal sequence of the Mature protein

The invention covers the next natural or synthetically produced protein, which is an inhibitor of thrombin and stands out from the saliva of insects that suck the blood of mammals, preferably from riatoma pallidipennis.

a) the protein as an active protein contains the following N-terminal sequence:

Ala-Glu-Gly-Asp-Asp-Cys-Ser-Leu-Glu-Lus-Ala-Met-Gly-Asp-Phe-Lys-Pro-Glu-Glu-Phe-Phe...

or b) the protein as the active protein has allelic modifications specified above in paragraph (a) N-terminal amino acid sequences, one or two amino acids N-terminal amino acid sequence substituted subjected to deletions or insertions, while not significantly impact on the activity of the active protein, or

in) and the protein as the active protein is post-translational update N-terminal sequences of PP (a) and (b) that is not affected significantly by the next protein, which is an inhibitor of thrombin and

g) which as an active Mature protein includes one of the following sequences:

I) the sequence identified as 1 (the sequence of Protocol 1);

II) the sequence identified as 2 (sequence Protocol 2);

III sequence, identified as 3 (sequence Protocol 3); and

IV) a sequence that is identified as 4 (sequence Protocol 4);

or

d) which as an active Mature protein contains allelic modification of the one specified above in paragraph (g) amino acid sequence, and at least one amino acid amino acid sequence substituted subjected to deletions or insertions, with substantially no effect on the activity of the active protein;

or

e) which as an active Mature protein includes post-translational modification of one of the sequences PP d) and e), which did not significantly affect the activity of the active protein.

All allelic modifications, which include substitutions, deletions and/or insertions of up to 30 amino acids, oerle up to 20 amino acids, more preferably up to 10 amino acids, more preferred deletions, substitutions and/or insertions of one, two, three, four, five, six, seven, eight or nine amino acids.

Sequence of a Mature protein with a signal sequence

A further form of implementation of proposed according to the invention the protein is the protein which consists of the signal sequence and Mature, proposed according to the invention protein;

W) and the protein has one of the following sequences:

I) the sequence identified as 5 (Protocol sequence 5),

II) the sequence identified as 6 (a sequence of Protocol 6),

III) a sequence that is identified as 7 (a sequence of Protocol 7), and

IV) a sequence that is identified as 8 (a sequence of Protocol 8).

or

C) and the protein includes allelic modification of one of the above in paragraph (g) amino acid sequence, and at least one amino acid amino acid sequence substituted subjected to deletions or insertions, without adding significantly to the promotion of one of the sequences of p. p. W) and C), which did not significantly affect the activity of the active Mature protein.

All allelic modifications, which include substitutions, deletions and/or insertions of up to 32 amino acids belong to the group of proposed according to the invention proteins. Preferred deletions, substitutions and/or insertions of up to 21 amino acids, more preferably up to 11 amino acids, more preferred deletions, substitutions and/or insertions of one, two, three, four, five, six, seven, eight or nine amino acids.

Most preferred proposed according to the invention, the proteins are recombinant proteins. While proteins can be glycosylated.

Proposed according to the invention proteins include Mature proteins and the corresponding protein precursors, which are formed from the signal sequence and the sequence of the Mature protein. When this signal sequence is preceded by a sequence of the Mature protein. The Mature protein begins with the above N-terminal sequence of p. a) Signal sequence necessary for the implementation of the endoplasmic reticulum. the t also referred to as cDNA or DNA which encodes the Mature thrombin inhibitor,

AA) and cDNA or DNA encodes one of the following amino acid sequence:

I) the sequence identified as 1 (the sequence of Protocol 1),

II) the sequence identified as 2 (sequence Protocol 2),

III) a sequence that is identified as 3 (sequence Protocol 3), and

IV) a sequence that is identified as 4 (sequence Protocol 4);

or

BB) with cDNA or DNA encodes allelic modification of one of the amino acid sequence of p. AA),

moreover, at least one amino acid amino acid sequence substituted subjected to deletions or insertions, substantially without affecting the activity of the active protein. Allelic modifications are defined above in paragraph "Sequence of a Mature protein".

Further, the invention encompasses cDNA or DNA which encodes an inhibitor of thrombin,

centuries) and cDNA or DNA contains one of the following nucleotide sequences:

I) the Sequence identified as 9 (sequence Protocol 9),

II) the sequence ID is a sequence of Protocol 12);

or

gg) and cDNA or DNA includes allelic modification of one of the nucleotide sequences of p. C), and at least one nucleotide substituted, subject to deletions or insertions, while not affecting significantly the activity of the protein, which is encoded by allelic modification of the nucleotide sequence of the p. IV).

All DNA constructs also apply to listed, proposed according to the invention sequences, if exchange such nucleotides, which are based on the degenerate code, encode the same amino acid. The exchange of such nucleotides obvious, and the corresponding amino acids known from any book on biochemistry (R. Nnippers, 1982, 3rd edition, Moleculare Genetik, ed. George Thieme).

All allelic modifications that lead to changes in amino acid sequence, relate to the invention, and these modifications include substitutions, deletions and/or insertions of up to 30 amino acids. Preferred deletions, substitutions and/or insertions of up to 20 amino acids, more preferably up to 10 amino acids, more preferred deletions, substitutions and/or insertions of one, two, three, four, five, six, seven, eight or deviatio with the coding, the Mature protein sequence, the signal sequence. This signal sequence is a cDNA Bank; possible other signal sequences that allow expression and secretion of the protein.

Thus, the invention encompasses cDNA or DNA which encodes a thrombin inhibitor with a signal sequence,

DD) and cDNA or DNA contains one of the following nucleotide sequences:

I) the sequence identified as 13 (sequence Protocol 13),

II) the sequence identified as 14 (a sequence of Protocol 14),

III) a sequence that is identified as 15 (sequence Protocol 15), and

IV) a sequence that is identified as 16 (sequence Protocol 16);

or

it) and cDNA or DNA includes allelic modification of one of the nucleotide sequences and DD), and at least one nucleotide substituted, subject to deletions or insertions, substantially without affecting the activity of the Mature protein, which is encoded, including its signal sequence, allelic nucleotide modification is by the activity of the protein, in order to determine whether allelic modification to the group of proteins according to the invention, it is always necessary to measure the Mature protein, also if the signal sequence. If you want to specify the signal sequence, it is always necessary to measure the function of the protein, which is obtained after removal of the signal sequence.

Further, the invention encompasses binding molecules (e.g. peptides or their derivatives), single-chain proteins (Signale Ghain Proteins), antibodies or antibody fragments, domains which specifically recognize Mature, proposed according to the invention the protein. If it is cleared, proposed according to the invention the protein, the expert from easy to obtain monoclonal antibodies. In this case, applying the known method of köhler and Milstein and further implemented options. In particular, according to the traditional method, the mouse repeatedly subjected to immunization with purified protein selected cells of the spleen and connect with a suitable tumor cells. Hybrids then selectionyou.

The proteins according to the invention can be isolated from saliva predatory bugs Triatoma pallidipennis. Cleaning is carried out at the above-described amino acid sequence. They have a molecular weight of approximately 180003000 Yes (see example 6). Isoelectric point is at pH 4,5-5,2, when used as described in example 8 method.

The proteins according to the invention inhibit the action of thrombin in blood coagulation and activation of platelets and amiloliticheskoi test. Test systems are described in examples 2, 3, 4 and 9. Proteins inhibit clotting) at a concentration of 8 nmol/l when the concentration of the thrombin of 1.27 nmol/L. They inhibit induced by thrombin platelet aggregation up to 100% at a concentration of 15 ng/ml, This concentration corresponds, when used in the concentration of thrombin is 0.06 IU/ml=0,812 pmol/ml (IU= international unit), the molar ratio of thrombin to the protein according to the invention, is approximately 1: 1. In contrast, the proteins according to the invention in amiloliticheskoi test inhibit the activity of thrombin at the ratio of thrombin to the protein according to the invention, equal to 1.87, only about 50%. The proteins according to the invention in a concentration of 35 nmol/l increase thrombin (1 IU/ml) in 5 times.

The proteins according to the invention is specific for thrombin. Other semipretioase (for example, factor XA or trypsin) when 40-fold excess of the truth is Yu invention is a vector which contains a proposed according to the invention cDNA or DNA, then suitable promoter and, if necessary, suitable enhancer. Can also be enabled signal sequence. The vectors are described in detail in European publications EP - 0480651, EP - 0462632 and ER - 01731777

Another form of the invention is eukaryotic or prokaryotic cell host, which is transformed using the proposed in the invention of the vector.

Allelic modification

Most large deletions, insertions and substitutions do not cause, apparently, no significant changes in the characteristics of the protein according to the invention. As it is difficult to predict the exact effect of the substitution, deletion or insertion, it is necessary to compare the function of the modified protein with the function of the protein according to the invention. Used for this purpose, methods are specified, for example, in examples 2-4 and 9.

As the standard is the protein according to the sequence that is identical to 14, as well as protein, which is purified according to example 1 and purification method of example 1 for comparative protein.

The genetic code is degenerate, which means that the largest part of the amino acid to which Leonidov not change the amino acid sequence. So, probably allelic modifications occur in DNA, and may have an impact again on the amino acid sequence.

cDNA or DNA sequences that encode proteins according to the invention, can be modified by conventional methods to obtain the variants proposed in the invention of proteins, which essentially have the same activity, as described and characterized proteins of the invention. The activity is measured as described in examples 2-4 and 9.

Amino acids that can be overridden, as shown in table 1, substantially without affecting the function of the protein. In each individual case due to test on the activity you need to decide what effect the change has on the protein function.

Function or immunological identity change significantly when selected substituents, which upon substitution of less conservative than those shown in table 1 amino acids. Such a significant change can be achieved through substitution with amino acids, which are more diverse in structure and functional groups. Significant changes have the consequence that changes the three-dimensional structure which changes need to take into account the interaction between charges and hydrophobic chains.

Mutations are defined by homology (similarity) in respect of second Comparators proteins. The terms "homology" covers similar amino acids (for example, see table 1) and the gaps in the amino acid sequence (homology = similarity). Proposed according to the invention proteins have amino acid sequences which have a homology of at least 80%, preferably 90%, more preferably 95%, and preferably 98% proposed according to the invention structures, which are determined by the sequences of p. p. g) or g) (identical sequence 1-8) and which are obtained after purification according to example 1.

As mentioned above, the invention also includes modifications of the DNA or cDNA. These modified sequences hybridizing in stringent conditions with the DNA sequences that encode proposed according to the invention proteins (see sequence in PP AA), BB) and (DD)). cDNA or DNA sequences include nucleotide sequences that possess identity, including a short (up to 15 nucleotide deletions and insertions at least 70%, preferably 82%, more preferably 90%, prepost is in)). Identity, including short deletions and insertions (up to 15 nucleotides) can be identified by hybridization, which is described by R. KNIPPERS, Molekulare Genetik, 1982, 3-e ed. Georg Thime, Stuttgart - New York.

Posttranslational modification

Under the above-mentioned post-translational modifications understand the changes that appear during or after the broadcast. These include glycosylation, formation of disulfide bonds, chemical modification of amino acids, as, for example, sulfation, which is described in connection with hirudin (J. W. FENTON (1989) "Thrombin Interactions with Hirudin", Seminars un Thrombosis and Hemostasis, 15; 265-268).

Glycosylation is an essential function of the endoplasmic reticulum and/or Golgi-apparatus. Sequence and branching of the oligosaccharide are formed in the endoplasmic reticulum and changes in Golgi-apparatus. Oligosaccharides may represent an N-linked oligosaccharides (United due to asparagine), or-United oligosaccharides (serine-, threonine -, or hydroxylysine-related). Form of glycosylation depends on producing cell type and kind, from which the corresponding cell type. On the extent and type of glycosylation can be influenced by substances, protein.

Proteins often form covalent bonds in the chains. These disulfide bridges are obtained between the two cysteine. When this protein is specifically. Disulfide bridges stabilize the three-dimensional structure of proteins.

Further, the amino acids can be modified as described in international publication WIPO 91/10684. Protein can also be sulfated. This change is described in connection with hirudin.

The isolation and preparation of proposed according to the invention of proteins

The invention further encompasses a method of obtaining a offer in the invention of proteins, comprising the following stages:

culturing the host cell which is transformed with a vector, which contains a proposed according to the invention cDNA or DNA; and

isolation and purification of the protein or proteins.

Proteins are preferably purified according to example 1. However, there might also be other methods of isolation and purification: Methods of Enzymology, I. 182: Guide to Protein Purification, ed. Murray P. DEUTSCHER, Academic Press, 1990; Protein Purification Application-A Practical Approach, ed. E. L. V. RRIS and S. ANGEL, IRL-Press, 1990; Protein Purification, Prunciples and Practice Ropect SCOPES, Springer Verlag 1982; and Protein Purification, Principles, High Pesolution Methods and Applications, ed. H. - C. JANSON AND L. RYDEN, VCH publishers, 1989.

Idelay, cleanse at least when using one column and then concentrated. The preferred column chromatography or adsorption chromatography.

Moreover, the invention relates to a method of purification protein according to the invention, the method comprises the following steps:

Applying saliva on "Superose 12 HR column and elution and the new drawing on SN-activated Sephorose column that is pre-bound thrombin, and elution.

Purification is described in detail in example 1.

Use as medicines

The proteins according to the invention have pharmacological effects and therefore applicable as a pharmaceutical biologically active substances. The invention also covers medicinal product, which contains one of the suggested according to the invention proteins or their mixture. Further, the invention relates to a pharmaceutical composition that contains one proposed according to the invention the protein or mixture proposed according to the invention proteins, in the presence of pharmaceutically compatible and acceptable compounds and carriers. The invention also encompasses pharmaceutical Compay the compound and pharmaceutically acceptable salt or pharmaceutically acceptable carrier.

In particular, proposed according to the invention the proteins, according to the Protocols decryption sequences 1-4, have inhibitory effects on the activity of thrombin.

The inhibition activity of thrombin can be detected in the test for coagulation (see example 2), in the test for platelet aggregation (see example 3), in amiloliticheskoi the text (see example 4) and by measuring thrombin time (see example 9). The preferred test system is the measurement of thrombin time.

Proposed according to the invention proteins detect the lengthening of thrombin time at concentrations 10-66 nmol/l (the concentration of thrombin 1 IU/ml). At a concentration of 58 nmol/l is 9-fold elongation. Higher concentrations applicable without disturbing the test system. Thus, the proposed according to the invention proteins applicable at concentrations of 10-200 nmol/L.

The results of the tests according to this test in vitro show that the proposed according to the invention, the proteins can be used as a drug or medical treatment. These test results can be transferred to the test system in vitro system in vivo, as in the case of test rombosis and Hemostasis 15: 293-301). The proteins of the invention can therefore be used for treatment and prevention of thrombosis, unstable angina, or arteriosclerosis, or to prevent re-occlusion of vessels after RTSA/MOUTH (angioplasty with balloon catheter), or to prevent blood clotting during hemodialysis. The proteins of the invention can be used as antithrombotic and/or antiarteriosclerotic medicines in the case of mammals, especially humans, for the treatment of thrombotic and/or arteriosclerotic ailments. They can occur at jerking pain due arteriosclerotic aggregation (platelet), tissue destruction of the endothelium, such as sepsis, in the case of grafts or unstable angina. They can also be used in order to avoid a new occlusion after treatment of myocardial infarctions and/or fibrinolysis or angioplasty. When the protein of the invention can be entered before, during and/or after insertion of the catheter.

The invention further includes:

(I) the use of one of the proteins according to the invention or mixtures thereof for the preparation of drugs for the treatment of thrombosis, unstable angina or avertive blood during hemodialysis;

(II) a method of treating thrombosis, unstable angina or atherosclerosis to prevent re-occlusion of vessels after RTSA/MOUTH or thrombosis or to prevent blood coagulation in the case of hemodialysis, the method includes the introduction of a number of protein according to the invention, which suppresses the disease, and the protein is administered to a patient who needs such medicinal products;

(III) a pharmaceutical composition for treating thrombosis, unstable angina or arteriosclerosis, or to prevent re-occlusion of vessels after RTSA/MOUTH or thrombolyse, or to prevent blood clotting during hemodialysis, and this composition includes one proposed according to the invention, a protein or a mixture thereof and at least one pharmaceutically acceptable carrier and an additive.

For therapeutic effects fit different doses. They depend, for example, used from protein, owner, species introduction, and the kind and severity of treatable conditions.

In General, however, satisfactory results in animals are to be expected when daily doses are values within the 2-2000 mcg per kg of body weight.ena body, when using purified according to example 1 protein. For example, this dose would be introduced in the form of partial doses up to four times per day. Daily dose in the case of short-term treatment of acute thrombus may is 20-2000 μg per 1 kg of body weight, i.e., to be higher than doses for chronic treatment, equal 2-200 μg per 1 kg of body weight.

Also, satisfactory results are to be expected when the protein of the invention is injected subcutaneously. Preferred target injection in the part of the body, which was formed clots.

Proposed according to the invention, the proteins can be entered by any conventional route, in particular in the form injectively solutions or suspensions.

The present invention relates to pharmaceutical compositions which include one proposed according to the invention, a protein or a mixture thereof and at least one pharmaceutically acceptable carrier or additive. Such compositions can prepare by known methods. Thus it is necessary to indicate Remington's Pharmacentical Science, 15-th ed. Mack Publishing Company, East Pennsylvania (1980).

The results of the tests are explained, based on Fig. 1-3, which, in particular, show the following.

Fig. 1: UDL is 2">

Fig. 2: Inhibition of the activity of thrombin in amiloliticheskoi test (Triatoma = thrombin inhibitor from Triatoma pallidipennis).

Fig. 3: Inhibition of the activity of thrombin in the test for clotting.

Example 1

Obtaining saliva of Triatoma pallidipennis and purification of the protein of the invention

Predatory bugs stir by mechanical stimulation of their probative to highlight their saliva. Saliva catch on the glass plate and is collected using a siliconized removing (pulling) Pasteur pipette.

Saliva is subjected to drying by freezing and dissolved in distilled water at a concentration of 2.5 mg/ml 2 ml of this solution is subjected to gel filtration through a column of "Superose 12 HR 16/50" (Pharmacia) in 10 mmol/l Tris-Hcl, pH 7.4, 0,0001% "Pluronic F 68". Active in the test for coagulation (see example 2) fractions are combined and applied to SN-activated Sepharose (Pharmacia) which according to the manufacturer associated pre-thrombin. The protein according to the invention binds to this is supplied by a thrombin column. The column is washed first with Tyrode-buffer (without serum albumin), then elute first with 10 mmol/l sodium acetate, pH 4.5, and then with 10 mmol/l glycine, pH of 2.5. The eluates set pH 7. 10 mmol/l test platelet aggregation (see example 3), in amiloliticheskoi test (see example 4) and prolong thrombin time (see example 9). The product contains no detectable in SDS-gel electrophoresis of impurities.

Example 2

The inhibition of the activity of thrombin in the test for clotting

On the plate for micrometrology, which is covered with albumin in the blood serum of cattle (0.1% in 0.1 mol/l NaHCO3pH 9,5), a pipette put 80 μl of 20 mmol/l S, pH 7.4; 0.15 mmol/l NaCl; 20 μl of 20 mmol/l l2; 100 ál of the diluted protein solution according to the invention (5-50 ng) and 20 μl of thrombin (0,03 IU=0,03 international units). After incubation for 2 min at 37oWith add 100 μl of fibrinogen (5 mg/ml) and incubated for 40 min at 37oC. Then measure the absorbance at 405 nm. 45 ng (= 8 nmol/l) of purified protein according to the invention completely inhibit the cleavage of fibrinogen. Under the same test conditions hirudin shows the same activity (complete inhibition with 8 nmol/l) (see Fig. 3).

Example 3

The inhibition of the activity of thrombin in test platelet aggregation

500 ál of filtered platelets (300000 /ml) together with the protein of the present invention (5-100 ng/ml) incubated for 1 min at 37

Example 4

The inhibition of the activity of thrombin in amiloliticheskoi test

On the plate for micrometrology 80 μl of 100 mmol/l Tris-Hcl, pH 7.4, 150 mmol/l NaCl and 0.03 IU (1.35 nmol/l), thrombin and 100 µl of the diluted solution of the protein of the invention (32-630 ng) incubated for 10 min at 37oC and then mixed with 100 μl (50 nmol) of the substrate S2238 (abi Vitrum). After incubation for 30 min at 37oTo measure the absorption at 405 nm. 630 ng (117 nmol/l) inhibit the activity of thrombin by about 50%. Hirudin at a concentration of 6 nmol/l inhibits the activity of thrombin to 85% (see Fig. 2).

Example 5

Determination of N-terminal amino acid sequence,

The purified protein according to the invention is sequenced in an automatic analyzer amino acid sequence (Applied Biosystems, Inc.) according to the manufacturer's instructions. The sequence of amino acids 1-21 (from N-end) is: la-Glu-Gly-Asp-Asp-Cys-Ser-Leu-Glu-Lys-Ala-Met-Asp-Phe-? -Pro-Glu-Glu-Phe-Phe. "?" means that with a full warranty is not identified. In the case of this amino acid with a certain probability we are talking about lysine.

Example 6

SDS-Gel electrophoresis and determination of molecular weight the run condition together with molecular weight markers (set for calibration during electrophoresis firm Pharmacia), as phosphorylase b (94 KD), albumin (97 KD), ovalbumin (43 KD), arbonic (containing tetravalent carbon)anhydrase (30 KD), tripcony inhibitor (20,1 KD) and lactalbumin (14.4 KD), put on a 12.5% SDS polyacrylamide gel, and after electrophoresis according to Laemmli (1970, Nature, 227, 680-685) stained with Coomassie brilliant blue. In a reduced state of the protein according to the invention during electrophoresis migrates only slightly higher trypsin. This corresponds to a molecular weight of approximately 21,000 Yes. In unrestored condition the protein according to the invention migrates between trypsinogen inhibitor and lactalbumin, which corresponds to a molecular weight of about 18000 Yes.

Example 7

The protein according to the invention did not inhibit the serine protease factor XA and trypsin

The activity of factor XA and trypsin was measured using the following test. To 80 μl of 50 mmol/l Tris-Hcl, pH 8.0, 227 mmol/l NaCl to determine the factor Ha type of 0.004 IU (0,653 pmol) of factor XA (American Diagnoctica) and 0.5 μg (25 pmol) of the protein according to the invention, and to determine trypsin type of 0.004 IU (0.019 pmol) trypsin (Sigma) and to 0.016 μg (0,817 pmol) of the protein according to the invention and incubated 2 min at 37oC. After the addition of 0.05 µmol of substrate S2222 (Kabi Vitr, as mixtures without protein according to the invention, i.e., the protein did not inhibit the activity of either factor XA or trypsin.

Example 8

Isoelectric focusing

The protein according to the invention is applied to the gel for isoelectric focusing at pH 3-9 (Pharmacia) and focus together with standard proteins (calibration kit, pH 3-10, Pharmacia). Place focus protein according to the invention is between that of soy trypsinogen inhibitor (I. P.=4,55) and so-lactoglobulin A (I. P.=5,2).

Example 9

Lengthening of thrombin time

Thrombin time is a measure of the activity of exogenous thrombin, which is added to the test plasma. To 0.1 ml of plasma, add 50 ál of protein solution according to the invention in different dilutions (6-58 nmol) and 50 μl of diethylbarbituric-acetate buffer with pH 7.6 and incubated for 1 min at 37oC. After the addition of 0.1 ml of thrombin (3 IU/ml) measure the time of occurrence of coagulation (Biomatic 2000-coagulator company Sarstedt). The protein according to the invention, which is a concentration of 35 nmol/l, prolongs the clotting time as compared to the control mixture 5 times (see Fig. 1).

Example 10

Determination of internal amino acid pivot using 10% 2-mercaptoethanol (2 h at room temperature and under nitrogen atmosphere) and then injected into the interaction with 4-vinylpyridine (2 h at room temperature and under nitrogen atmosphere). After dialysis against 25 mmol/l Tris-Hcl, pH 8.5; 1 mmol/l add, the sample is mixed with 1 μg Lys C (Boehringer Mannheim) and incubated for 6 h at 37oC. This mixture after splitting applied to Supersper RP-18,4 μm, in column size 250 x 4 mm) Mz-analesen-technik, Mainz) and elute using a gradient of 0.1% FA in N2About to 0.08% TFA in 70% acetonitrile (HPLC firm WOUTERSE). Record the absorbance at 280 nm and 214 nm and the eluate fractionary. Fractions that correspond to the peak absorption, evaporated to dryness and treated with 30 μl of N2O. Individual fractions is sequenced on an automatic amino acid analyzer sequencing (Applied Biosystems Inc.) according to the manufacturer's instructions. This way, the following sequence (beginning, depending on the circumstances, from the N end; "?" indicates that unambiguously not identified):

1. la-Met-Gly-Asp-Phe-Lys-Pro-Glu-Glu-Phe-Phe-? -Gly-Thr-Arg(?)-Try-Leu-Ala (amino acids Arg questioned);

2. Gly-Phe-Thr-Gln-Ile-val-Glu-Ile-Gly-Tyr-Acn-Lys-;

3. Asn-Gly-Glu-Gln-Tyr-Ser-Phe-Lys-.

Example 11

Molecular cloning proposed according to the invention cDNA

If the sequence N-Terminus of the protein according to the invention known, it is possible to determine the corresponding nucleotide sequence (see WIPO 90/0786).

Two oligonucleotide primers get the fact that the nucleotide sequence is removed from partially defined above, amino acid sequence N-terminal full website offered according to the invention protein and one of its fragments. In this case, applying the following amino acid sequence (see below).

Matrix (template) consists of cDNA, which is derived from poly-A+-RNA, which is pre-allocated from the salivary glands of Triatoma pallidipennis (SAMBROCK, etc. : olekular Cloning (Chapter 7, S. 18-22, Cold Spring Harbor Laboratory Press, 1989).

Matrix starting part (sense primer):

5'-GC1 GA (A/G) GGI GA (C-T) GA(C/T) TG(C/T) TCI CTI GA (A/G) AA(A/G) GCI ATG GGI GA(C/T) TT-3'

Amatriciana starting part (antisense primer):

5'-TT(G/A)TT (G/A)TA ICC (G/A)AT (T/C)TC IAC (G/A)AT (T/C)TG IGT (G/A)AA-3

A = deskside the worth of 40 cycles. One cycle is as follows:

a) 2 min denaturirovannyj at 94oWITH,

b) 90 to hybridization at 52oAnd

a) 2 min elongation at 72oC.

Obtained as described above, the PCR product - breeding forms on an agarose gel lane. It is separated from the agarose gel, which has a low melting temperature, and transferred directly into PCR plasmid (starting gene) and subcloning. The method corresponds to the description of the Protocol of the manufacturer (R - TM Script SK(+) Cloning kit company Stratagen). DNA Sequence subjected to the insertion of the product corresponds to the sequence PSR-product and is determined as described SANGER and others, Proc. Natl. Acad. Sci. USA (1977) 74, 5463-5467.

BB) the Method of selection of cDNA Bank and the allocation proposed according to the invention cDNA clone

About 400,000 primary clones from a cDNA Bank from the salivary glands of Triatoma pallidipennis (cDNA library) transferred to nylon membranes (Pall Biosupport, East Hills, NY, USA) and subjected to screening as described by the manufacturer. In this case, applying bulleted sample, which is obtained using the above method, PCR-breeding. The method of selection carried out twice, following the conversion of DNA phage DNA-plasmid and spending the definition posledovatel is presented in the form of sequences, identical 13-16, and only missing (triplets) Triplika for amino acids (-17)(-9) when Ti 28 and Ti 45 and (triplet) Rriplika to amicalola - 18 when Ti 12.

Each of the two strands of the selected DNA is sequenced. Produced from the coding DNA amino acid sequence identical to the amino acid sequence, which was found previously with the destruction of Atmano proposed according to the invention purified protein.

The longest cDNA clone (Ti 12) begins with a TG nucleotides, which correspond to an incomplete start codon for methionine. This becomes apparent when taking into consideration the isolated clone (Ti 5), which is found in the case of the method of breeding with 600000 phages of the same cDNA-Bank used in the selection of Ti 12 Ti 28 and Ti 45.

Four cDNA sequences proposed in the invention protein selectionyou identify and is sequenced in accordance with the above methods. Only position 9 have differences in the resulting amino acid sequence. The differences are listed in table 2.

Identity and likeness of four Mature protein according to the invention is approximately 95-98% zavisimost is according to the invention plasmids and expression and purification obtained in E. Li protein

If a known DNA sequence, suitable promoters and, if necessary, suitable enhancers can contact the relevant DNA sequences, so then formed a suitable vector. (M. WIRTH, L. SCHUMACHER, AND H. HAUSER in Protein Glycosylation, Gellular Biotechnical and Analytical Aspects (ed. H. S. CONRAAT), so 15, 49-52, VCH publishers, Weinheim 1991); also J. KRATZSCHMAR, ETC. (1992), Gene, 116, 281-284. The expression of this vector potential in eukarytic (for example, Baby Hamster Kidney Cells). Further, DNA sequences can be embedded in a suitable prokaryotic vectors for expression in strains of E. Li.

a) Obtaining plasmids

(I) Advance information

The construction for the expression proposed according to the invention the recombinant protein in prokaryotes get those that use a commercially available plasmid RCC-2, which contains an IPTG-inducible trc-promoter. Suitable for the expression vector comprises the plasmid RCC-2, into which is inserted as the coding sequence of the offer and in accordance with the invention, a protein, and the signal sequence. The signal sequence is a modified signal sequence, which is reduced to the selected E. coli protein - ticlopidine "and".mu is formed ALAC/h (T. HAYANO (1991), Biochemistry 30, 3041-3048).

Coding sequences proposed according to the invention the protein is inserted into a plasmid expression, and thus the resulting design (ALAC/cph-protein) is used to transform the component E. coli jM 105 cells.

(II) Construction of plasmid PKK/h

Use the following pair of partially overlapping lap and additional oligodeoxynucleotides to copy the coding DNA for a signal sequence cyclophilin "and". When this is modified corresponding To the end ticlopidinesee sequence to form the optimal splitting for bacterial signal peptidases. The modification is that the last seven (triplets) riplika-end exchange on riplika that encode the following amino acid sequence: Phe-Ser-Ala-Ser-Ala-Leu-Ala (R. E. DALBEY and G. von HEIJNE (1992), TIBS, 17, 474-478).

Sense oligonucleotide sequence (Sense oligo):

< / BR>
Antisense oligonucleotide sequence (Antisens oligo)

< / BR>
After joining the starting sequence and subsequent additions subsidiary plots in the application Taj polymerase DNA fragments were cleaved with porosity RCC-2. NneI - site, which together with Hindlll site facilitates further subclavian cDNA-sites are underlined.

(II) Construction of plasmids using the proposed invention protein

Apply the following couple starting sequence (primer) to reproduce the coding sequence for the Mature protein according to the invention (Ti28= sequence identified as 2). While NheI and Hind III sites of incision, which is underlined, is necessary to insert in the plasmid PKK/h.

Sense oligonucleotide sequence (Sense oligo)

< / BR>
Animelove oligonucleotide sequence (Antisens oligo)

< / BR>
Carry out ten cycles, consisting of: 2 min at 94oC, 2 min at 30oWith and 2.5 min at 72oC, PCR (Polymerase Chain Reaktion) = chain reaction polymerase), and using 2 µg of matrix filaments (emplate). Multiplied DNA emit from viscoplasticity agarose gel, digested with NheI and HindIII and transferred to plasmid PKK/h, which is pre-digested with the same restriction endonucleases. Thus, the resulting structure are examined using the full sequencing of the coding DNA and flanking coding is with SAS2. Using 1 µg ALAC/h DNA that encodes the protein according to the invention.

b) Purification and characterization obtained using E. Li protein according to the invention

The culture of the transformed cells of E. coli mixed with IPTG (1 mmol/l) and incubated for 6 h at 37oC. (IPTG = isopropyl--D-thiogalactoside). Cells are separated by centrifugation and subjected to osmotic "shock" treatment with sucrose and then water). The thus obtained fraction periplasm inhibits the activity of thrombin in the test for coagulation (see example 2). Fraction with inhibitory activity is purified by ion exchange chromatography on DE-52 - cellulose and trombino affinity chromatography (see example 1).

Purified fraction has the same inhibitory activity as the protein of saliva. It has identical behavior in SDS-polyaryletherketones (see example 6). Further, on the N - terminal amino acid sequence coincides with Mature protein of saliva. 50811AWOM I XXOO + P 25.11.93.2

1. Natural protein having the properties of a thrombin inhibitor isolated from the saliva of the bug Triatoma pallidipennis and has the following N-terminal amino acid sequence: Ala-Glu-Gly-Asp-Asp-Cys-Ser-Leu-Glu-Lys-Ala-Met-Gl Recombinant Mature protein, having the properties of a thrombin inhibitor and has a deduced amino acid sequence of 1, or 2, or 3, or 4, with the molecular weight and the pH value of the isoelectric point of the natural protein under item 1.

3. The DNA fragment encoding the Mature protein having the properties of a thrombin inhibitor, a nucleotide sequence 9, or 10, or 11, or 12.

4. The DNA fragment encoding the precursor of a Mature protein by p. 2, with nucleotide sequence 13, or 14, or 15, or 16.

5. Vector pKK/cph for expression of the protein containing the DNA fragment encoding the signal sequence, a modified to-end cyclophilins sequence so that the last of the seventh the balance-end is replaced by the sequence Phe-Ser-Ala-Ser-Ala-Leu-Ala, and the DNA fragment under item 3, subcloned between the Nco I and Hind III sites of the plasmid PKK 233-2.

6. Recombinant Escherichia coli strain JM 105 transformed by the expression vector pKK/cph - producer of the protein having the properties of a thrombin inhibitor.

7. A method of obtaining a Mature protein having the properties of a thrombin inhibitor, involving the cultivation of the producer strain E. coli transformed with the vector of expr is that the expression vector contains a DNA fragment under item 3.

8. The isolation and purification of thrombin inhibitor obtained in E. coli, characterized in that a) the culture of transformed E. coli bacteria is mixed with isopropyl--D-thiogalactoside, b) bacterial cells are separated by centrifugation and subjected to osmotic shock and then) obtained fraction periplasm purified by ion exchange chromatography on cellulose DE-52 and trombino affinity chromatography.

9. The isolation and purification of thrombin inhibitor from the saliva of Triatoma pallidipennis, characterized in that a) excite bugs (Triatoma pallidipennis) by mechanical stimulation of their probative to highlight their saliva, b) catch the saliva on the glass plate and is collected by a Pasteur pipette, in) collected saliva dried by freezing and dissolved in distilled water at a concentration of 2.5 mg/ml, g), 2 ml of this solution is filtered on a gel-filtration column d) active clotting fractions are combined and applied to SN-activated column pretreated with thrombin, e) the column was washed with Tyrode-buffer and then elute first buffer (10 mmol/l Na acetate, pH 4.5) and then 10 mmol/l glycine, W) the resulting eluate containing the inhibitor in glycine faction, neutralize to pH 7.

10. Drug their mix.

11. The pharmaceutical composition inhibiting thrombin, characterized in that it contains one of the proteins under item 1 or 2 or a mixture thereof in the presence of pharmaceutically compatible and acceptable additives or carriers.

12. A method of obtaining a drug that inhibits thrombin for the treatment of thrombosis, or unstable angina, or arteriosclerosis, or to prevent re-occlusion of vessels after angioplasty with balloon catheter, or to prevent blood clotting during hemodialysis, characterized in that one of the proteins on the PP.1 and 2 or their mixture is used in the amount of 2-2000 μg per 1 kg of body weight.

Priority points:

04.12.1992 on PP.7-9 and 12;

12.02.1993 on PP.1 and 2;

17.08.1993 on PP.3 and 4;

25.11.1993 on PP.5, 6, 10, and 11.

 

Same patents:

The invention relates to biotechnology, in particular genetic and protein engineering, and solves the problem of obtaining highly productive recombinant bacterial strain-producer of polynucleotide-phosphorylase

The invention relates to biotechnology and relates to amino acid sequencesandabedini polypeptide with the activity of nitrilimines and DNA fragments encoding the polypeptide

The invention relates to structures of nanometer size used to construct the microscopic and macroscopic structures

The invention relates to the complementarity determining regions (CDR, hypervariable regions) and variable regions (V regions) of murine monoclonal antibodies to human interleukin-8 (IL-8), human/mouse chimeric antibody to human IL-8, and reconstructed human antibodies, and region, defining a complementary variable region, a human light chain (L-chain) and variable regions of the heavy chain (H-chain) of the person replaced the CDR of mouse monoclonal antibodies to human IL-8

The invention relates to molecular biology and biotechnology

The invention relates to biotechnology

The invention relates to biotechnology and can be used in the selection of organisms, for example plants

The invention relates to new DNA compounds and clanroyden vectors recombinant DNA coding for new Imogene forms of human protein C

The invention relates to biotechnology, in particular genetic and protein engineering, and solves the problem of obtaining highly productive recombinant bacterial strain-producer of human proinsulin
The invention relates to biotechnology and can be used in Microbiology, immunology and veterinary medicine

The invention relates to biotechnology, in particular genetic and protein engineering, and allows you to get uridine-phosphorylase E

The invention relates to genetic engineering and medicine, in particular to a new polypeptide
Up!