Non-polypeptide with properties of prourokinase, method thereof, recombinant plasmid dna for expression of the polypeptide and its preparation

 

(57) Abstract:

The invention relates to biotechnology and allows you to get the polypeptides used as plasminogen activators having high specific amylolyticus and fibrinolytic activity. Upon receipt of the polypeptide with properties of prourokinase spend the transformation of E. coli with recombinant plasmid comprising a DNA fragment encoding the target product. Select transformants. Cultivate them in optimal accumulation of recombinant polypeptide conditions. Separate and hold the reactivation of the expressed product. Upon receipt of plasmids for expression of the polypeptide in the cells of Enterobacteriaceae unite operon regulated promoter sequence binding to the ribosome Shine-Dalgarno initiating codon, the DNA fragment encoding the target polypeptide, and one or two terminator, available for the specified fragment. For combining use plasmid pBR 322 from which the removed nic / bom region and/or part resistant to the tetracycline gene. In the latter between sites E coRI and Hind III enter a multiple cloning site, designed for embedding terminator of transcription, DNA fragment, the code is 143-411. rscu RA 143-411 represents the region of the sequence of amino acids 143 (Glu-411 (LeuOH) from scu PA 54K. x1means one of the amino acids Asn, Pro, or Ser, and x2represents a simple bond or Glu or Pro - Glu or Pro - Pro - Glu or Glu - Leu - His - Leu - Leu - Gln - Val - Pro - Ser. 4 C. and 7 C.p. f-crystals, 20 ill.

In the invention of new polypeptides are highly effective activators of plasminogen and so with great success can be used as thrombolytic agents.

Plasminogen activators play an important role in the treatment due to fibrin blockages of blood vessels, for example, in myocardial infarction, pulmonary embolism, etc.

Approximately since the early 50-ies, it is known that in human urine contains at least one proteolytic enzyme that promotes the conversion of plasminogen to plasmin, i.e., in an enzyme that causes the breakdown of fibrin into soluble polypeptides and, accordingly, the dissolution is caused by fibrin occlusions. This plasminogen activator was called urokinase, which as it turned out later exists in various forms, including in the form of a precursor known about since the mid 70-ies of Imogene prourokinase, he has dniproremont scu-PA consists of 411 amino acids in nature picolitres amino acid residue at position 302. Molecular weight (Mr) scu-PA is indicated in the literature as approximately of 54,000 daltons.

In 1977 Nolan and others (Biochim, biophys. Acta, 496, 384-400) identified in cultures of human cells of proferment urokinase, molecular weight which corresponded to the molecular weight of the known low molecular weight form of urokinase (31.500 daltons), and later Wijngaards and others (Thrombosis Res., 42, (1986), 749-760) and Pijken and others (Thrombosis Res., 42, (1986), 761-768) reported in cultures of cells of the monkey prourokinase size 30 kilodaltons, cleaning and fibrinolytic properties. In 1986. Stump et al. reported (J. Biol. Chem., 261, 17120-17126) that in the supernatant fluids cell line tumor human CALU-3 (ATSC, NTV 55) contains the single-stranded PUK, which has a molecular weight (Mr) about 32.000 daltons, and to distinguish from high-molecular prourokinase ("scu-PA 54k) was labeled "scu-PA 32k"/ Form scu-PA 32k corresponds scu-PA 54k without 143 N - terminal amino acids (Ser1to Glu143and glycosylases in the same position.

Published under N 247674 Al European patent application describe getting scu-PA 32k from the culture medium of the cell line CALU-3 (ATSC, NTV 55) and from the culture medium transfection plasmids pSVscu-PA-32k and pSV5DHFR CHO - cells (cells of the ovary eticheski modified microorganisms.

In European patent application 92182 A2 (1983, C 12 N 15/00) described getting encodes a low molecular weight urokinase (mol. weight. about to 33,000 daltons) DNA and the corresponding plasmids and their expression in E. coli K12 294 (ATS, 31446) with the formation of replicationmanager protein with a molecular weight of approximately of 33,000 daltons and there with the specified sequence of amino acids, which corresponds to the region 133 - 411 amino acid residue scu-PA 54k.

On some properties deglycosylation product, which includes amino acids 136-411, reported in 1987. Gunz-lev and others (Thrombosis and Haemostasis, 58 (1987), 216)

Polypeptides with the activity of prourokinase scu-PA 32k, which contain complementary to native form region sequence 136 - 143 residue or its fragments, and in which, if necessary (depending on the nature of the N - Terminus) amino acid at position 156 (Arg) changed their receipt by the appropriate expression plasmids in E. coli K12 JM105 and subsequent conversion of the initially formed intracellular Taurus inclusion described in published European patent 312941 A2.

The invention relates to a new use in therapy as activators of plasminogen polypeptides following General form means deglycosylation region amino acid sequence from 143 (Glu) to 411 (LeuOH) balance scu-PA 54k, X1- Acn, Pro or Ser

X2or a simple link, or Glu or Pro-Glu or Pro-Pro-Glu or Glu-Leu-His-Leu-Leu-Gln-Val-Pro-Ser-Asn.

Preferably X1is Asn or Ser, particularly preferably Ser. X2preferably means or a simple link, or Glu, or Pro-Glu or Pro-Pro-Glu.

In a preferred embodiment, the polypeptide has the formula

Ser-Ser-Pro-Pro-Glu-rscu-PA143-411(Ia)

where

rscu-PA143-411has the same meaning as above.

The new compounds of formula I show a pronounced specificity of lysis fibrinous clots, i.e. activate plasminogen in the presence of fibrin, but not turn or slightly turn the circulating plasminogen to plasmin. Thus is achieved that fibrinous clots dissolve, while undesirable in the treatment of the destruction of coagulation factors and other proteins by plasmin is not to the same extent as when using non-specific plasminogen activators. New polypeptides of the formula I, in contrast scu-PA 54k or deglycosylation protein part ("Sarup is using a chromogenic substrate Glu-Gly-Arg-p-nitroanilide after conversion into double-stranded plasminogen activators in the presence of plasmin). If measured directly fibrinolytic activity on fibrinogenesis plate, the new compounds of formula I in comparison with scu-PA 54k show increased 2.5-3 times the efficiency (the area of the zone of lysis in mm2on mg). In addition, new polypeptides of the formula I in contrast to the scu-PA 54k or Saruplase cannot reach specific binding cellular receptors, urokinase and deactivated. Thus, they are more a long time are in circulation, exerting their therapeutic effect.

Thanks stronger compared to conventional fibrinolytic by means of specific fibrinolytic action and due to better opportunities biosynthesis in place actions that achieve more reliable Listeria at lower doses and with fewer side effects, in terms of the bleeding.

For effective and reliable thrombolyse when occlusions in blood vessels, especially in the myocardial infarction, therapeutically effective single dose for adult patients is approximately 15 mg of the compounds of formula I. For comparison, you can lead, for example, data from Bode and others (Am. J. Cordiol. 61, (1988), 971-973), according to which PR is e'er high doses was essentially non-specific and could be followed according to many researchers, harmful side effects on the hemostatic system.

Valuable new compounds of formula I receive recombinant DNA technology. For this we have created new plasmids of the present invention, the expression of which in a suitable bacterial hosts and subsequent procainamidesee transformation obtained with high yields inactive product provides new compounds of formula I.

As in most cases the expression eukaryotically proteins in bacteria, polypeptide chains of the compounds of formula I form in the host cell in the form of so-called "inclusion bodies", which are referred to here as "the precursor protein". After an interim allocation of this primary product proteinkinases by must be returned to the correct tertiary structure of the desired product. Thus obtained compound of the formula I can then be translated (for example, the addition of plasmin) in the corresponding double-stranded deglycosylation low molecular weight derived urokinase, enzymatic activity which can serve as a measure for the formed amount of the compounds of formula I. to Determine the "output" of the protein precursor or the compounds of formula I and thereby effect the Les.

The plasmids according to the invention in the preferred embodiment are characterized in that their operon is regulated in this case, a synthetic promoter that acts as the binding site of ribosomes Shine-Dalgarno sequence, a start codon, a synthetic structural gene that encodes a compound of formula I, followed by a terminator. Most preferably the inclusion of two consecutive terminators, in particular A trp and/or otet A/orf-of Nh10 (cp. Schollmeier and others, Nucl. Acids Res., 13, (1986), 4227-4237). As a regulated promoter, in particular, can be used Trp-promoter or Tac-promoter.

The plasmids according to the invention the distance between the Shine-Dalgarno sequence and the start codon is usually 6 to 12, preferably 8 to 10 nucleotides.

When designing introduced into the plasmids according to the invention the structural gene embed it triples the preferred encoding the corresponding amino acids shown below (in the structural gene for these codons should not be submitted simultaneously for all amino acids).

Amino acid - Codon

Arginine - CGT

Leucine - CTG

Valine - GTT

Glycine - GGT

On the other hand, when constructing a structural gene price the lot (again these codons should not be introduced simultaneously for all amino acids).

Amino acid - Codon

Isoleucine - ATA

Valine - GTC

Proline - CCC

Lysine - AAG

Arginine - AGG, ACA CGG, CGA

Glycine - GGA, GGG

Because of this choice of codons for specific amino acids in the structural gene and thanks to the above characteristics of a controlling area substantially preventing the formation of stable secondary structures between the structural gene and regulatory region.

To obtain the coding of the compounds of formula I is a synthetic structural gene primarily synthesize single-stranded oligonucleotide segments of size 40-80 bases. It is advisable to get them in quantities of 1 µmol by the method of stationary phase Adams and others J. Am. Chem. Soc., 105, (1983), 661-663, using DNA synthesizer (Mode 11 Biosearch 8600 firm, New Brunswick Scientifc Co).

Teams 4-9 these oligonucleotide chains then get denitive fragments with a length of approximately 200 nucleotides formed fragments then insert in appropriate Linearisation media. Further, the principle follows from the following examples.

Used in this application abbreviations have the following meanings:

bp: base pair;

DE 52: aminoalkenes company Whatman;

EDTA: the on;

PAGE: polyaryletherketones;

PU: Ploug units;

SDS: sodium dodecyl sulphate;

S. D. - Sequenz: Shine-Dalgarno sequence;

Tris-HCl: trihydroxystilbene hydrochloride;

Tween-80: polyethylene oxide (20) servicemanual;

TMAC: Tetramethylammonium.

To construct the plasmids according to the invention is mainly used by commercial plasmid pBR 322 (4363 Bp.). Preferably it is removed nic/bom-area and/or a gene of resistance to tetracycline. While complete removal of the named gene is not required. It is sufficient if it is in such an extent that the plasmid no longer gives resistance to tetracycline transformed with its capacity of bacteria strains. Thanks to these measures (removal nic/bom-region and at least part of the gene of resistance to tetracycline are the so-called "high security plasmid".

The preferred strategy for constructing the plasmids according to the invention on the basis of the modified plasmid pBR 322 (or other named following the well-known plasmid) provides as a next operation introduction synthetic multiple cloning site between the cleavage sites EcoRI and Hind III (Fig. 4). Space decomposition with the ribosome (Shine-Dalgarno sequence) of the Xyl operon 5'-AAGGAG-3' B. Subtilis (Wilhelm and others, Nucl. Res., 13, (1985), 5717-5722) and the necessary distance between S. D. sequence and the start codon ATG. Following S. D. sequence of bases GAAAT associated with Nde l-sites CATATG. Thus, the distance between S. D. sequence and ATG is 8 base pairs. The distance between the individual cleavage sites of the multiple cloning site had to have a length of at least about 20 nucleotides, resulting in easier removal of the intermediate fragments.

By using the site multiple cloning in plasmid injected sites that may then be used for the introduction of a separate sequences of the structural gene encoding the compound of formula I, preferably starting from the C-Terminus of the protein, i.e., the 3'-end of the DNA chain derived structural gene, as well as for installation, for example, synthetic or isolated from other plasmids, or commercial Trp or Tac-promoter.

The same can also be as basic plasmid to use other known forms, which have a unique Eco R1 and Hind III sites, such as, for example, Fig 9, Fig 12, Fig 13, Fig 18, Fig 19 and others.

In particular, obtain plasmid pGR Tac 06 (example 1 and Fig.the sequence Glu-Leu-His-Leu-Leu-Gln-Val-Pro-Ser-Asn- (Fig. 16).

This connection is denoted below as "PA/06".

This sequence encode from nucleotides between Nde 1 and Xba 1, which were randomly selected codons for the N-end amino acids scu - PA 54k. This sequence of amino acids interrupt from Leu-Leu for Pst 1 slice.

Expressed, for example, in E. coli protein PA/06 after re-laying circuit shows an unexpectedly high fibrinolytic activity in the sample on the fibrin-agar plate.

If the plasmid pGR Tac 06 replace the Tac-promoter between sites Eco R1 and Xba 1 synthetic Trp promoter (Fig. 17), we obtain plasmid pGR Trp 06.

Transformed using pGR Trp 06 cells of E. coli K12 JM 103 (ATCC 39 403) after induction intracrinology acid also produce the protein, which, after lysis of the cells and renaturation of Taurus-on exhibits fibrinolytic activity in the sample on the fibrin-agar plate.

Other plasmids according to the invention can be obtained from the above the extraction Eco R1 and Hind III insert with a synthetic structural gene and all units regulation and embed it in another, especially capable of Autonomous replication in E. coli, plasmid, pre-Ec split the torus on the Tac-promoter (and Vice versa) in one of the plasmids according to the invention.

The plasmids according to the invention with an elongated distance between the Shine-Dalgaruo sequence and the start codon ATG can be obtained due to the fact that is constructed as described above plasmid, in which the said distance is, for example, 8 nucleotides, cut Nde 1 increasing sequence and then are ligated formed new ends with the formation of the plasmids according to the invention, in which the distance between S. D. sequence and the start codon is, for example, 10 nucleotides.

When designing the structural genes that encode the compounds of formula I, prefer such sequences of nucleotides are in 5'-position are codon for methionine, followed by a codon for serine. When the expression of that structural gene gain due to instability due Met-Ser corresponding compound of formula I with a serine in the N-terminal amino acids. Suitable 5'-ends of the structural genes for compounds of formula I are the following:

(Met) Ser Asn

ATG, AGC AAT

(Met) Ser Asn Glu-

ATG, AGC AAT GAA

(Met) Ser Pro Pro Glu-

ATG-AGC CCA CCA GAA

(Met) Ser Ser Pro Pro Glu-

ATG, AGC AGT CCA CCA GAA

(Met) Ser Asn Glu Leu His Leu Leu Gln Val Pro Ser

Asn-ATG-AGC AAT GAA CTT CAT CTG CAG CAA GTT CCA TGC

AAC

Transformer competent organisms hosts is carried out in a known manner. For the expression plasmids according to the invention is particularly suitable organisms-hosts are strains of the species E. coli and related Enterobacteriaceae, such as strains of Pseudomonas, Salmonella, Enterobacter, Klebsiella or Serratia.

Preferred organisms hosts are strains of the species E. coli, in particular E. coli GRT-1, and especially strains of E. coli subgroups K12, as, for example, E. coli K12 JM101 (ATCC 33876), E. coli K12 JM103 (ATCC 39403, E. coli K12 JM105 (DSM 4162), E. coli K12 294 MM (ATCC 33625), E. coli K12-ATCC 31446 or E. coli K12 DHI (ATCC 33849).

The flow of expression in E. coli expression systems, which contain the Tac-promoter, can be facilitated, for example, the addition of lactose or glucose challenge, but preferably by the addition of isopropyl- -D-thiogalactopyranoside (IPTG). However, if there plasmid Trp-promoter, the induction is preferably intracrinology, intracellulare or intracrinology acid. Of course, you can also use other known inducers.

After induction and achieve a certain density cell culture centrifuged, and the precipitate from the centrifugation after suspension in water-salt solution is transferred to a homogenizer. After re-centrifugation to precipitate falls precursor compounds folorida protein is translated in the solution and processed "redox system" (containing for example, reduced and oxidized glutathione), thereby restoring the natural conformation, i.e., the formation of the desired compounds of formula I. the Latter is in this case in the reaction mixture in dissolved form and can be isolated in pure form by using conventional cleaning operations (for example, chromatography and subsequent freeze-drying).

For analytical purposes fermented transformed with this plasmid strains E. coli and after reaching the set optical density of cell suspension induce a suitable inducer of the expression of the corresponding original protein. After an appropriate period of fermentation selected aliquots and precipitated from these cells then destroy by using lysozyme (1 mg lysozyme in 1 ml of 50 mm Tris-HCl buffer solution, pH 8.0, 50 mm EDTA and 15% sucrose). The homogenate was subjected to lysis of the cells is dissolved in 4 - 5 M guanidine hydrochloride and after dilution by addition of a reducing agent (glutathione, mercaptoethanol, or cysteine concentrations up to 1.2-m of guanidine hydrochloride for a few hours is subjected in the presence of oxygen renaturation (see also e.g., Winkler and others in Biochem. 25, (1986), 4041 - 4045). Thus obtained one the polypeptide, the activity of which is determined then by using a chromogenic substrate pyroGlu-Gly-Arg-p-nitroanilide, split only double-stranded active urokinase. This activation of the compounds of formula I are produced by plasmin in 50 mm Tris-buffer with 12 mm sodium chloride, 0.02% of Tween 80 at pH 7.4 and 37oC. the Ratio of the test substance to plasmin should be about 100 - 1500 to 1 in terms of both molarity or approximately 8.000 - 36.000 to 1, in terms of units of enzyme. Test-incubation occurs in 50 mm Tris-buffer with 38 mm sodium chloride at a pH of 8.8 in the presence of 0.36 μm Aprotinin (for inhibition of plasmin) and 0.27 mm substrate at 37oC. depending on the content in the sample of compounds of formula I after 5 - 60 minute incubation stop the reaction by addition of 50% acetic acid and measure the extinction at 405 nm. According to the manufacturer of the substrate (the cubie Vitrum, Sweden) with this principle, the change in the extinction of 0.05 per minute at 405 nm corresponds to the activity of urokinase 25 Ploug units/ml standard solution.

In Fig. 1 shows a diagram of the structure of the compounds of formula I, indicating necessary to maintain the conformation of the disulfide bridges of Fig.2 and 3 construction of plasmids cycline; in Fig.4 is the nucleotide sequence synthetic multiple cloning site; Fig.5 - construction of the plasmid pBF 158-01 shown embed a synthetic multiple cloning site in the plasmid pBF 158 between the cleavage sites Eco RI and Hind III; Fig.6 - construction of the plasmid pBF 158-01, fragment Cla I - Hind III replace the corresponding fragment of the "T" from the plasmid pRT 61 (see Jorgensen and others, J. Bacteriol., 138, 1978, 705-714), which contains A tet/orf-terminator from TP (see Sohollmeier and others, Nucl. Acids Res., 13, (1985), 4227-4237); in Fig.7-11 - of a nucleotide sequence of the synthetic fragment encoding gene (their insertions in the formation of the structural gene for compounds of formula I in plasmid according to the invention, for example pBF 158-01, T described in example 1d and shown in Fig.12-14 of Fig.12-14 - construction of the plasmid pBF 158-02 T - pBF 158-06 by embedding oligonucleotide fragments M 4 - M 8; Fig. 15 - plasmid pGR Tac 06, obtained from pBF 158-06 T Tac insertions - view between the restriction sites Eco RI and Xba I (example 1), Fig.16 is the nucleotide sequence between restriction sites NleI and Sac I in pGR Tac 06 and deduced the amino acid sequence, the numbering of the amino acids corresponds to Fig.1; Fig.17 is a nucleotide sequence synthetic Thr-ol is transformed using plasmids pGR Tac 06 the E. coli strain JM 103, under the control of the Tac-promoter, depending on the time of IPTG induction at time O. the Specified determined using the chromogenic substrate activity in Ploug units per 1 ml of fermentation medium with an optical density of 1 at 578 nm after (----) and without ( ---) effects of plasmin before the test. Values represent average values from fermentation mixtures, Fig.19 - nucleotide sequences and deduced amino acid sequence of the compounds of formula (Met)-Ser-X1-X2-rscu PA143-411methionine in parentheses hatshepsuts in E. coli, Fig.20 levels of expression for compounds of formula I or their precursors in E. coli strain K12 JM 103, transformed pGR 318, pGR 319 or pGR 327, i.e. control Thr-promoter. The induction took place at the time Of intracrinology acid. Contains a specific using a chromogenic substrate activity in Ploug units per 1 ml of fermentation medium with an optical density of 1 at 578 nm after (----) and without ( ---) effects of plasmin before the test.

Applied examples of the implementation of restrictase are commercially available (see, for example, Nachr. Chem. Teohn. The Nab. 35, 939, 1987).

Used in examples of the nutrient medium also available for sale.

When the role of the Tac-promoter.

a) Construction of the plasmid pBF 158.

From the plasmid pBR 322 (pharmacy, N 27-4902, 4363 bp) remove the nic/bom-region, which is located between the bases 2207 and 2263 (see Winnacker, Gene und kione, VCH, Weinheim, 1985, page 298), as follows (cf. also Fig. 2 and 3): pBR 322 split Nde I at the base 2998 and thereby linearizing. Protruding ends are filled and get blunt ends (see Maniatis and other "Molecular Cloning", Cold Spring Harbor/lab, 1982). Then make the cut with PvuII the basis 2068, and both ends of the remaining part of the pBR 322 conventional method are ligated by T4-ligase. Then legirovannoi mixture transform competent cells of E. coliK12 Jm 103 (ATCC 39403) (see Hanahan, "DNA Cloning" v. I IRLPress, Oxfrd, 1985, 109-135). Transformed cells were cultured in medium in the presence of 150 μg of ampicillin/ml of the obtained clones, choose those that contain the plasmid pBF 157, different from the original plasmid pBR 322 smaller (230 nucleotides),

a) > PST - BspM II, b) > PST - BaI I-, c) > PST - Ava I-fragments. Both unique restriction site (Pvu II and Nde I), characteristic of pBR 322, the plasmid pBF 157 no. ii) From pBF 157 remove most of the gene of resistance to tetracycline treatment Eco RVu Nru I and the subsequent crosslinking of the formed ends. After transformation of E. coli K12 JM 103 and culturing cells on medium containing 150 μg of ampicillin/IU than pBF 157, and is characterized, moreover, by the absence of BamHI-site. Transformation of bacterial strains using pBR 158 gives the last resistance to tetracycline.

b) Design of pBF 158-01, insert a synthetic multiple cloning site.

From pBF 158 processing Eco P1 x Hind III to remove the fragment size 31 nucleotide and "cut" enter the sequence synthetic multiple cloning site (Fig.4). After transformation ligiously a mixture of E. coli K12 JM 103 and cultivation in medium with 150 μg of ampicillin/ml choose those clones that contain the plasmid pBF 158-01. The latter differs from the pBF 158 additional unique restriction sites Xba I, Nde I, Sac I, Eag I, Kpn. Spe I and the second site Pst I (Fig.5).

Between Xa I and Nde I is a Shine-Dalgarno sequence of the operon XVI B. subtilis (see Ihelm and others cited place)

c) Constructing pBF 158-01, T, embedding terminator of transcription.

From pBF 158-01 remove Cla I-Hind III fragment of website clone and modify the Cla I - Hind III fragment from pRT 61 (see Jorgensen and others cited), which contains A tet/orf terminator from Tn 10 (Schollmeier, etc. cited place)

After transformation of the strain of E. cli K12 JM 103 and cultivation in medium with 150 µg of ampicillino) ClfI - Hind III fragment (Fig.6).

d) the Consistent inclusion of synthetic fragments of M 4 V - 8 for encoding gene, construction of the plasmid pBF 158-02T - pBF 158-06T.

All synthetic fragments described in Fig.7-11. They are administered as fragments with a length of about 200 nucleotides in the corresponding vectors. For this purpose, the split vectors corresponding restrictase and electrophoresis in agarose, electroelution and purification via DE 52 is separated from the deleted fragment. After that carry out ligation with an appropriate synthetic double-stranded fragment using T4 ligase, transformation of E. coli K12 Jm 103 (Fig.12-14) and the selection contains ampicillin environment.

I) Ligation of the fragment of the M8 between BamHI and Cla I in the plasmid pBF 158-01 T.

Formed plasmid pBF 158-02 T.

Oligonucleotide 019 corresponds to the C-terminal sequence of the compounds of formula I.

Behind the stop codon TAA is the site Nde I and sequence trpA terminator (see Shristie etc. P. N. A. S. 78, (1981), 4180-4184) to Cla I.

II) Ligation of the fragment M7 between the Spe I and BamHI plasmid pBF 158-02 T.

Formed plasmid pBF 158-03 T.

III) Ligation of the fragment of the M6 between Kpn I and Spe I, the plasmid pBF 158-04 T.

Formed pBF 1pBF 158-05 T.

V) Ligation M4 between the Sac I and Eag I plasmid pBF 158-05 T.

Formed pBF 158-06 T.

From the obtained clones I - V is chosen such that different from earlier forms built a new snippet in proven correct orientation

e) Constructing a plasmid pGR Tac 06, insert a Tac promoter,

Plasmid pBf 158-06 T restricion Eco RI and Xba I. the Resulting fragment length 26 nucleotides separated by preparative electrophoresis in agarose gel, the rest of the plasmid elute Electrosila and then purified via DE 52.

From the plasmid ptac SDT (DSM 5018)allocate Eco RI - Xba I fragment, which contains the Tac-promoter. For this ptac SDT restricion Eco RI and Xba I and separated fragment preparative electrophoresis in agarose gel. Fragment elute by heating to 65oC in acetate-ammonium buffer with SDS, pH 8.0 out mechanically crushed polyacrylamide and purified by repeated extraction with phenol, saturated with 1 M tricom, pH 8.

Both of the thus obtained fragment "sew" the usual way of T4-ligase and transformed ligiously a mixture of E. coli K12 JM 103. After culturing on medium containing 150 μg of ampicillin/ml select clones that after the addition IPTC produce protein-predshestvennitsej

Transformation using plasmid pGP Tac 06 cells of E. coli K12 JM 103 fermented in a medium consisting of 38 mm ammonium sulfate, 56 mm phosphate buffer pH 7.0, 1 mm magnesium sulfate, 1% yeast extract, 1% glucose and containing 150 mg of ampicillin per liter, and induce using 0.5 mm IPTC expression of precursor compounds PA/06.

Before induction and every hour after induction (6 h) is separated by centrifugation of the cells in the cell suspension (1 ml) with an optical density of 1 at 578 nm determine the level of expression.

II) the Laying of the protein chain connection PA/06, its cleavage to the corresponding docebocore connection and measurement of PA activity.

Separated by centrifugation cells destroy lysozyme, and the homogenate was subjected to lysis of cells treated further as described above.

The levels of expression for the connection of PA/06 or previous protein, identified by measurement of the activity of the corresponding double-stranded forms of plasminogen activator shown in Fig. 18. This activity is observed only in the presence of plasmin (see Fig. 18) therefore, the connection PA/06 (like its predecessor) is expressed as a single chain.

Primenet Thr promoter is transferred to the Xba I - site and is extended at the 5'end of the sequence 5'-AATTCTGAAT-3'. As a result of this site is formed Eco RI (Fig. 17, the fragment M I). A separate chain 021 021 and A collect and are ligated with restricciones Eco R1 and Xba I, the plasmid pGR Tac 06. After transformation of E. coli K12 JM103 and cultureware on medium containing 150 mg of ampicillin/ml select those clones that contain pGR Thr 06. Plasmid pGR Thr 06 differs from all the above vectors so that the transformed E. coli strains Express predstaleny protein compounds PA/06 after the addition intracrinology acid.

Example 3. a) Construction of plasmids pGR 318

Plasmid pGR Thr 06 cut with Nde I and Sac I. the Resulting fragment length 58 bp, which belonged to the multiple cloning site is separated by preparative electrophoresis in agarose gel, the rest of the plasmid elute Electrosila and then purified via DE 52.

This fragment are ligated with a synthetic oligonucleotide 18 5' - T ATG AGC AAT GAG CT-3'.

AC TGG TTA C are ligated in the usual way using T4 ligase, transforming a product of E. coli K12 and cultivated containing ampicillin environment. Select those clones that after the addition intracrinology acid produce aggregated protein for connection "PA 318". These to the TEW amino acids.

b) the Test expression.

E. coli K12 JM 103 after transformation with plasmid pGR 318 fermented as described in example 1. The induction is carried out at optical density of 2-3 at 578 nm, adding 62 mg intracrinology acid in 1 l of fermentation medium.

Before induction and every hour after induction during the entire 5 h cells 1 ml of cell suspension with an optical density of 1 at 578 nm is separated by centrifugation and used for testing norms expression.

After renaturation expressed protein (see example 1) determine amylolyticus activity connection PA 318 before and after treatment with plasmin. The results are presented in Fig. 20. Obviously reached the same level of expression and plasmid pGR Tac 06 and that amylolyticus activity can only be detected after cleavage of the plasmid. Therefore, the compound of formula I PA 318 is also expressed in E. coli K12 c pGR 318 in the form of single-stranded predecessor.

(C) isolation, purification and procainamidesee feature.

For separation, purification and procainamidesee characteristics are 1 l fermentation under controlled conditions of pH and temperature (pH 7.0, 37oC). These conditions allow the achievement of pageroot 50 ml of cell suspension, again suspended sediment cells in 40 ml of K2O and destroy in the high-pressure homogenizer. After centrifugation the precipitate, which contains all the inactive protein inclusion bodies were dissolved in 100 ml of 5 M guanidine hydrochloride, 40 mm cysteine, 1 mm CDTA (installed PH 8.0) and diluted with 400 ml of 25 mm Tris buffer solution, pH of 9.0. By accessing or oxygen reaction recovery conformation ends at approximately 12 o'clock

Connection PA 318 isolate and purify by chromatography. Analysis of the N-terminal sequence were proved, first, odnotsepochechnoi and, secondly, the correct structure of the N-end.

Example 4. a) Construction of plasmid for expression of pGR 319.

Plasmid pGR Thr 06 cleaved as described in example 3a, Nde I and Sac I and emit large fragment.

This fragment are ligated with a synthetic oligonucleotide 19 5' - TATG AGC AAT GAA GAG CT-3.

AC TCG TTA CTTT C in the usual way using T4 ligase, transforming a product of E. coli K12 and cultivated containing ampicillin environment. Choose these clones, which after the addition intracrinology acid produced by the initial protein for compounds of formula I "PA 319". These clones contain plasmids with expression.

E. coli K12 transformed using plasmids pGR 319, fermented, induce and then collect samples for testing as described in example 3b.

Isolation, purification and procainamidesee characteristic is carried out as described in example 3c.

Resaturate connection "PA 319 and the level of expression are performed as described in example 3b. The results are shown in Fig. 20. It is seen that the expression level similar to the level of expression plasmid pCP Tac 06 and that amidopirina activity can be detected only after cleavage by plasmin. Therefore, PA 319 is expressed in E. coli K12 with plasmid pGR 319 (protein precursor), as a single polypeptide.

Example 5. A) Construction of plasmid for expression of pGR 327.

Plasmid pGR Thr 06 cut as described in example 3a, Nde I and Sac I and emit large fragment.

This fragment are ligated with a synthetic oligonucleotide

5' - TATG AGC AGT CCA CCA GAA GAG CT - 3'

AC TCG TCA GGT CCT CTT C

the usual way using T4 ligase and transformed product of E. coli K12. Breeding and selection of clones are produced as described in example 3a. Selected clones contain plasmid pGR 327, which encodes the compounds of formula I "PA 327" specified in fermented and experience level of expression as described in example 3b. The results are presented in Fig. 20. It is seen that the expression level compared to the level of expression plasmids pGR Tac 06 (about the same and that the activity can only be determined after cleavage by plasmin. Therefore, the connection "PA 327" is expressed E. coli K12 with plasmid pGR 327 (predecessor) as a single chain. Isolation, purification and procainamidesee characterization was carried out as described in example 3c.

Example 6. Plasmid pGR Thr 06 cut as described in example 3a, Nde I and SacI and emit large fragment.

This fragment are ligated with a synthetic oligonucleotide 46

5'-TATG AGC CCA CCA GAA GAG CT -3'

AC TCG GGT CCT CTT C

the usual way using T4 ligase and transformed product of E. coli K12 JM 105. Breeding and selection of clones are produced as described in example 3a. Selected clones contain plasmid pGR 336, which encodes a compound of formula I "PA 336" indicated in Fig. 19 the sequence of amino acids.

E. coli K12 JM 105 transform the plasmid pGR 336, then fermented and after induction determine the level of expression as described in example 3b. From the initially received protein get a connection PA 336 (the amino acid sequence shown in Fig. 19) as a single-chain protein. the 06.

1. Non-polypeptide with properties of prourokinase obtained in the E. coli strain transformed with the recombinant vector and named the vector comprises a DNA fragment encoding the polypeptide of General formula

Met - Ser - X1- X2- rscu PA 143 - 411,

where rscu RA 143 - 411 represents a region of the sequence of amino acids 143(Glu) - 411 (LeuOH) from scu RA 54;

X1one of the amino acids, Asn, Pro, and Ser;

X2- a simple link, or Glu, or Pro - Glu or Pro - Pro - Glu or Glu - Leu - His - Leu - Leu - Gln - Val - Pro - Ser - Asn,

showing compared to the scu RA 54 to higher specific amylolyticus activity, measured using the chromogenic substrate pyro - Glu - Gly - Arg - p - nitroanilide after conversion to double-stranded activators of plasminogen by plasmin and higher fibrinolytic activity, measured on fibrinogen plate and does not contain a N-terminal Met residue.

2. The polypeptide under item 1, characterized in that X1Is Asn or Ser.

3. The polypeptide under item 1, characterized in that X1- Ser.

4. The polypeptide under item 1, or 2, or 3, characterized in that X2means a simple link, or Glu, or Pro - Glu or Pro - Pro - Glu.

5. The polypeptide under item 1, characterized in that X
Ser - Ser - Pro - Pro - Glu - rscu - PA 143 - 411,

where rscu RA 143 - 411 - deglycosylation region amino acid sequence from 143 (Glu) to 411 (Leu-OH) of scu - PA 54 K.

7. Recombinant plasmid DNA for expression of the polypeptide with properties of prourokinase containing the fragment EcoRI - Hind III vector plasmid pBR 322, which includes a marker gene (AmpR) resistance to ampicillin, the sequence Trp or Tac promoter, a fragment of the sequence of site multiple cloning with unique restriction sites, which includes the sequence of the Shine - Dalgarno located between Xba I and Nde I site and at a distance of 8 - 10 p. O. from the start codon, a synthetic structural gene that encodes a polypeptide according to PP.1 - 6, and TrpA sequence and/or sequence tet A/orfL terminator from Tn 10.

8. DNA under item 7, characterized in that the promoter sequence is a synthetic Trp promoter sequence of nucleotides I, presented in C.

9. DNA under item 7, characterized in that the promoter sequence is Tac-promoter plasmid ptac SDT (DSM 5018).

10. The way to obtain a plasmid for the expression of polypeptide in the ti binding to the ribosome Shine-Dalgarno, the initiating codon, a DNA fragment encoding the target polypeptide, one or two terminators available for the specified fragment, characterized in that for Association using plasmid pBR 322 from which the removed nic/bom region and/or at least part resistant to tetracycline gene, in which between sites E coRI and Hind III enter a multiple cloning site sequence of nucleotides II, presented on pages designed for embedding terminator of transcription, DNA fragment encoding the polypeptide under item 1, and the synthetic trp promoter.

11. A method of obtaining a polypeptide with properties of prourokinase, including the transformation of E. coli with the recombinant vector containing the DNA fragment encoding the target product, the selection of transformation, culturing them in the optimal accumulation of recombinant polypeptide conditions, the Department and the reactivation of the expressed product, characterized in that the transformation strain of the recipient using the recombinant plasmid DNA under item 7.

 

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FIELD: genetic and protein engineering, medicine, molecular biology, pharmacy.

SUBSTANCE: invention proposes a modified form of plasminogen urokinase type activator (activator) wherein amino acid sequence differs from that in the natural activator as result of replacing the sequence Arg-Arg-His-Arg-Gly-Gly-Ser in the composition of inhibitory loop for the sequence Arg-His-His-Ala-Gly-Gly-Ser and by replacing 24 N-terminal amino acids for the foreign sequence consisting of 16 amino acid residues. Invention proposes the constructed recombinant plasmid (pUABC 34) comprising DNA fragment that encodes a new activator. As result of transformation of E. coli K-12 JM109 cells with plasmid pUABC 34 the recombinant strain E. coli VKPM-8145 as a producer of a modified form of activator is obtained. This polypeptide is characterized by reduced sensitivity to effect of inhibitor PAI-1 and absence of some by-side effects in the complete retention of biological activity of the natural activator produced by the recombinant. This provides effective applying a new activator as a component of pharmaceutical compositions eliciting thrombolytic effect.

EFFECT: valuable medicinal properties of activator and pharmaceutical composition.

6 cl, 6 dwg, 8 ex

FIELD: molecular biology, medicinal industry.

SUBSTANCE: invention relates to a method for preparing recombinant double-stranded enzyme urokinase (ds-uAP). Method involves culturing cells CHO-Meissi subjected for genetic manipulations that are transfected stably with cDNA encoding pre-prourokinase in culturing medium containing alkanoic acids or their derivatives at temperature from 30°C to 37°C and the following separation the prepared ds-uAP for low-molecular and high-molecular forms. Also, invention involves recombinant ds-uAP, high-molecular ds-uAP and low-molecular ds-uAP in different stages of their preparing. Advantage of proposed method involves the development of method for preparing recombinant forms of mature double-stranded protein ds-uAP and its low-molecular and high-molecular forms.

EFFECT: improved preparing method.

19 cl, 4 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: invention relates to new staphylokinase derivatives which represent expression products in heterogeneous system of mutant genes obtained by local gene PCR-mutagenesis ← wild type enzyme and differ from respective native staphylokinase form in one or more amino acid substitutions between 104 and 113 amino acid residues that leads to formation of RGD or KGD sequence in said site. Obtained recombinant staphylokinase derivatives (RGD/KGD-Sak), as well as variants thereof having deletions of 1-16 amino acids from NH2-terminal combine properties of thrombolitic and antocoagulating agents and are characterized by decreased polymerization ability and immunogenicity in contrast to wild type enzyme.

EFFECT: new effective agents for thrombosis preventing and treatment.

25 cl, 3 dwg, 3 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of biotechnology and deals with recombinant plasmid DNA, which contains sequence of gene of mature staphylokinase Staphylococcus aureus with replacement of codons K74, E75 and R77 with triplets, which code Ala, of strain Escherichia coli MZ09 and method of obtaining recombinant protein, which contains sequence of gene of mature staphylokinase with replacement of codons K74, E75 and R77 with Ala-coding triplets. Essence of method includes recombinant plasmid DNA, which codes sequence of gene of mature protein of staphylokinase from Staphylococcus aureus staphylokinase with replacement of codons K74, E75 and R77 with Ala-coding triplets, which has nucleotide sequence, given in dwg.1. Invention also includes strain Esherichia coli MZ09, producent of recombinant staphylokinase, with sequence, given in dwg.1, as well as method of its obtaining.

EFFECT: invention makes it possible to obtain staphylokinase protein which possesses high fibrinogenic activity, with output of recombinant protein to 20% from total amount.

3 cl, 2 dwg

FIELD: biotechnologies.

SUBSTANCE: synthetic oligonucleotide primers and probes are disclosed to detect genomes of 1st, 4th and 16th serotypes of the bluetongue disease virus. Also the method is described to detect genomes of 1st, 4th and 16th serotypes of the bluetongue disease virus using such synthetic oligonucleotide primers and probes.

EFFECT: method makes it possible to perform determination of a serotype of the bluetongue disease virus with the help of PCR with a stage of reverse transcription in real time mode, and also to increase specificity and sensitivity, to reduce time to perform work aimed at determination of a serotype of the bluetongue disease virus in samples of an investigated biological material.

4 cl, 4 tbl

FIELD: biotechnology.

SUBSTANCE: protease is presented which has enhanced milk clotting activity, containing amino acid sequence at least 80 % identical to SEQ ID NO: 3, where the said protease has at least one mutation selected from the group consisting of: (a) substitution of glutamine, corresponding to glutamine at a position of 265 in SEQ ID NO: 3, with amino acid; and (b) replacement of glutamine, corresponding to glutamine at a position of 266 in SEQ ID NO: 3, with amino acid. DNA is described which encodes the said protease, the expression vector containing the said DNA, and the cell transformed with the said vector, designed for expression of the said protease. The method of production of protease having enhanced milk clotting activity is proposed, comprising culturing the said transformed cell in the cultural medium and isolation of protease from the cultural medium.

EFFECT: invention enables to obtain the protease with enhanced milk clotting activity.

16 cl, 2 dwg, 4 tbl

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