LT3948B - Polypeptide, plasmids, method for the preparation of plasmids, method for the preparation of polypeptide, thrombolytic material and applicatio of polypeptide - Google Patents

Abstract

Polypeptides of the general formula Ser-X1-X2-rscuPA<143-411> (1) in which X1 and X2 are defined amino acids or lower peptide bridges, X2 can also be a single bond, and rscuPA<143-411> is the unglycosylated sequence region of amino acids 143 (Glu) to 411 (LeuOH) from scu-PA 54 k, which can be used as plasminogen activators are described. To obtain these compounds, plasmids which are described in detail as such or in terms of their isolation were produced and expression thereof in suitable bacterial hosts provides, in each case in high yields, an expression product which does not act as plasminogen activator but whose protein-chemical conversion then leads to the single-chain compounds of the formula I which are valuable as plasminogen activators. <IMAGE>

Description

According to this description, novel polypeptides are potent activators of plasminogen and are therefore used with thideLe success as thrombolytic agents. Plasminogen activators play an important role in the treatment of fibrin-derived thrombi in blood vessels such as myocardial infarction, pulmonary artery embolism, limb vein and arterial thrombosis, etc. Approximately since 1950 Beginning in the early 1990s, human urine contains at least one proteolytic enzyme that converts plasminogen into plasmin, that is, an enzyme that breaks down fibrin into soluble polypeptides while dissolving fibrin thrombi. This plasminogen activator was called urokinase, after which it appeared that various forms of urokinase actually exist, the smallest known urokinase precursor since 1970, the prourokinase. This prourokinase has a single chain and is also called scu-PA (urokinase-like single chain plasminogen activator). This scu-PA consists of 441 amino acid residues and is naturally glycosylated with 302 amino acid residues. The molecular weight (M _r ) of this scu-PA is reported in the literature at approximately 54,000 daltons.

Following the discovery in 1977 of a urokinase proferment in human tissue culture, by Nolan et al., Biochim. Biophys. ), Wijngaards et al. (Thrombosis Res. 42 (1986), pp. 749-760) and Pijken et al. (Thrombosis Res. 42 (1986), pp. 761-768) reported the existence of a 30kD prourokinase (30 kilodaltons) in monkey tissue culture for its purification and fibrinolytic properties. Also in 1986, Strump et al. (J. Biol. Chem. 261, p. 1712017126) reported that the human tumor CALU-3 (ATCC, HTB 55) supernatant contains a single chain prourokinase with a molecular weight (M _r ) of about 32,000. daltons and was designated scu-PA32k to distinguish them from high molecular weight prourokinase (scu-PA 54k). This scu-PA 32k corresponds to a scu-PA 54k lacking the N-terminus of 143 amino acids (Ser ¹ to Glu ¹⁴³ ), glycosylated being the same amino acid residue 302 (Asn.).

The published European application no. 247,674 Al describes the production of scu-PA32k from culture medium CALU-3 and from CHF (Chinese Hamster Ovary) cells transfected with plasmids pSVscu-PA-32k and pSVSDHFR. Although there is mention of the possibility of using genetically modified microorganisms to obtain scu-PA 32k, no further description or corresponding example is provided.

In European application no. No. 92,182 A2 describes, among other things, the production of DNA encoding low molecular weight urokinase having a molecular weight of about 33,000 Daltons and the corresponding plasmids, as well as their expression in E. coli K12 294 (ATCC, 31,446) non-glycosylated protein with molecular having a mass of about 33,000 Daltons, with the order of fig. 2A amino acid residue sequence corresponding to region 133 to 411 of the scu-PA 54k protein.

Some of the non-glycosylated product containing amino acid residues 136-411 was described by Gunz-Iev et al., 1987 (Thrombosis and Hemostasis, 58 (1987), p. 216).

In the published European patent no. 312,941 A2 discloses scu-PA 32k structured polypeptides having prourokinase activity, which in this case have an additional region of 136 to 143 amino acid residues, or individual domains in which the 156 amino acid residue (Arg) is substituted, if necessary (depending on the nature of Ngal), and production thereof by expression on the appropriate plasmids in E. coli K12 JM105 cells, during which the expression of these peptides occurs, the product accumulates in intracellular inclusion bodies.

The present invention provides novel polypeptides of the general formula I or according to the block diagram of FIG. Use of plasminogen activators in ointment 1:

Ser-X ₁ -X ₂ -rscu-PA ¹⁴³ " ⁴¹¹ (I), wherein rscu-PA is a non-glycosylated amino acid sequence region of 143 (Glu) to 411 (LeuOH) from scu-PA 54k,

X ₂ is one of the following amino acid residues: Asn, Pro or Ser and

X ₂ is a single bond or Glu, Pro-Glu, Pro-Pro-Glu or Giu-Leu-His-Leu-Leu-Gln-Val-Pro-Ser-Asn.

Ideally, X ₄ is Asn or especially Ser. Ideally, X ₂ is a simple bond either Glu, Pro-Glu or Pro-Pro-Glu.

Preference is given to compounds having the general formula (I):

Ser-Ser-Pro-Pro-Glu-rscu-PA ^{143 411} (la) wherein e rscu-PA ^{143 41 1 has} the same meaning as given above in formula (I).

The novel compounds of formula (I) have a strong specificity in lysing fibrin clots,

i.e., they activate plasminogen in the presence of fibrin but do not cleave or very weakly circulate plasminogen to convert it to plasmin. This prevents the dissolution of fibrin clots by preventing the cleavage of unwanted coagulation factors and other proteins, as in the case of plasmin this is less pronounced when nonspecific plasminogen activators are used. The novel compounds of formula (I), unlike scu-PA 54k or its non-glycosylated protein moiety (zaruplase), exhibit an increase in specific amidolytic activity of approximately 1.7-fold (measured using the chromogenic substrate pyro-GluArg-p-nitroanilide after transfer to double-stranded plasmin). activator with plasmin). When measured directly on fibrinolytic activity on a fibrin-agar film, the new compounds of formula (I) have a potency of 2.5 to 3-fold (lysed area per milligram of protein) compared to scu-PA 54k. In addition, in contrast to scu-PA 54k or zaruplase, novel compounds of formula (I) are unable to bind to specific cellular urokinase receptors and to be deactivated. In this way, they circulate for a longer period of time and enhance therapeutic activity. Stronger specific activity and increased on-site biosynthesis results in more reliable murmur therapy with lower doses and fewer side effects such as bleeding.

For effective and reliable thrombolysis, particularly in the presence of arterial thrombi, particularly in the presence of myocardial infarction, a therapeutically effective single dose to an adult is limited to 15 mg of the compound of formula (I). For comparison, the work of Bode et al. (Am. J. Cardiol. ¢ 21 (1988) pp. 971-973): After administration of 74 mg of glycosylated scu-PA 54k, successful thrombolysis occurs in only 55% of cases and it should be noted that this known product according to Bode et al. . (Circulation 81 (1990) 907, p. 910) lyses the fibrin clot almost nonspecifically at the high doses required and, according to data from many tyriLT 3948B suppressors, these high doses produce marked adverse effects on the hemostatic system.

The novel compounds of formula (I) are obtained by recombinant DNA technology. For this purpose, novel plasmids were created, expressed in suitable bacterial hosts, and after processing of a product obtained with a high yield that does not act as plasminogen activators, the novel compounds of formula (I) were obtained.

As in most cases, by expressing eukaryotic proteins in bacteria, the compounds of formula (I) are denatured in the cells in the cells, which are denoted herein as probaltim, which, after intermediate processing, must be re-assembled with the correct tertiary structure of the desired product. The amount of plasminogen activator thus obtained in the compounds of formula (I) can be determined, for example, by the addition of plasmin to the corresponding double-stranded, non-glycosylated, low molecular weight urokinase derivative, which is subsequently measured for enzymatic activity. , a measure of quantity. But, of course, it is also possible to determine the yield of probaltim or the level of expression of the compound of formula (I) and thereby the desitometry of an electrophoregram containing all the proteins produced.

The plasmids of the present invention are characterized in that their operon contains a regulated, in this case synthetic, promoter acting as a ribosomal linkage, a ShineDalgarnase sequence, a start codon, a synthetic structural gene of the compound of formula (I) and a 5 'direction. the smallest strand breaking agent in the structural gene. Preferably there are two successive chain breaking agents, in particular the trpA chain terminating agent and / or the tetA / orfL chain nuLT 3948B pulling agent from ThlO (cf. Schollmeier et al., Nucl. Acids Res. 13 (1985) pp. 4227- 4237).

The regulated promoter is the Trp promoter or the 5 Tac promoter.

In the controlled plasmid region of the invention, the gap between the Shine-Dalgarno sequence and the start codon is 6-12, preferably 8-10 nucleotides.

In the construction of the present invention, coding amino acid triplets are inserted into the plasmid structural gene having the base sequence set forth in the table. In addition, not all triplets can be replaced in the structural gene

Amino acid arginine leucine valine glycine

Triptych

CGT

CTG

GTT

GGT

On the other hand, when constructing a structural gene, it is advisable to avoid the following codons wherever possible (and not all triplets may be substituted in the structural gene):

A codon to avoid

ATA

GTC

CCC

AAG

AGG, AGA, CGG, CGA

GGA, GGG

Amino acid isoleucine valine proline lysine arginine glycine

Due to this choice of codons in the structural gene and due to the above-mentioned features in the controlling part, the possibility of secondary structures between the structural gene and the controlling region is greatly reduced.

To obtain synthetic structural genes encoding compounds of formula (I), nucleotides of 40 to 80 base sequences are first synthesized. It is appropriate to synthesize them at 1 µM per carrier according to Adams et al. J. Am. Chem. Soc. 105 (1983) p. 661 -663 using a DNA synthesizer (Mode II Biosearch Model 8600, New Brunswick Scientific Co.). Commercially available β-cyanoethyl-protecting diisopropylaminophosphoramidites of the required deoxyribonucleosides are used as monomeric synthons. After removal of the carrier, the final tritiated-protected chains are sterile desalted by gel filtration and purified, followed by fractional reversal of the first phase by preparative thin layer chromatography. The basic product is detritilated and the next two purifications are carried out by preparative thin layer chromatography to obtain the required pure oligonucleotide and its purity is checked by gel electrophoresis (PAGE).

Groups 4-9 of these single nucleotide chains produce fragments of about 200 nucleotides in length because of the phosphorylation of the terminal oligonucleotides at the 5'-end and the overlapping of the corresponding additional chains with the terminal oligonucleotides. The resulting overlapping fragments, which can be cloned and have doubled chains, are inserted into suitable linear carriers. The following principle of operation follows from the following examples.

Abbreviations used in the present invention have the following meanings:

op pairs of bases DE52 anionite Whatman firm EDTA ethylenediaminetetraacetic acid IPTG i zopropiΙ-β-D-thiogalactopyranoside PA plasminogenic activator PAGE electrophoresis in a polyacrylamide gel PU Ploug units SDS sodium dodecyl sulfate

S.D. - Seguenz Shine-Dalgarno sequence tris-HCl trishydroxymethylaminomethane hydrochloride

Tween-80 polyethylene oxide (20) sorbitan monooleate TMAC tetramethylammonium chloride

According to the present invention, the commercial plasmid pBR 332 (4363 bp) is selected when constructing plasmids. Most often, the nic / bom region and / or the tetracycline resistant gene is removed. This does not require the complete removal of the tetracycline resistance gene, but such modifications are sufficient that the plasmid does not confer resistance to the tetracycline-transformed bacterial strains. These tools (removing the nic / bom region and the smallest single region of the tetracycline-resistant gene) result in the so-called safe plasmid.

In accordance with the present invention, preference is given to constructing a plasmid from the modified pBR 332 plasmid as described above (or others mentioned below), the following operation being the insertion of a synthetic polylinker sequence between EeoRI and the HindIII cleavage site. This polylinker sequence (see Fig. 3) has defined cleavage sites between their bases of choice, except for the sequence between Xbal and NdeI, which has a ribosomal binding site (Shine-Dalgarno sequence) from B. subtilis Xyl operon 5'-AAGGAG -3 '(Wilheim et al., Nucl. Acids Res. 13 (1985) pp. 5717-5722) and the identified fragment between SD sequence and start codon ATG. After S.D. - GAAAT-based sequences from Wilheim et al. and connected to CATATG at the NdeI clipping site. In this way, the spacing between S. D. - sequence and ATG is 8 base pairs. The distance between individual cleavage sites in the polylinker sequence should be 20 nucleotides in length, which in turn facilitates the removal of intermediate fragments.

The polynucleotide sequence introduces cleavage sites into the plasmid which - purposefully introduced after transcription of the terminating agent - facilitate the insertion of successive structural gene sequences for compound I, preferably starting at the C-terminus of the protein, i.e. the resulting structural gene. insertion of a '-gal as well as, for example, a synthetic isolated from another plasmid, or a commercial Trp * or Tac promoter.

Similarly, construction of plasmids from other known commercial plasmids having single EcoRI and HindIII restriction sites, such as: pHC9, pHC12, pHC13, pHC18, pHC19 and others, can be started.

Thus, for example, plasmid pGR Tac 06 is obtained (see Example and Figure 8). This plasmid encodes a compound of formula I wherein X _x represents Asn and X ₂ represents: Glu-LeuHis-Leu-Leu-Gln-Val-Pro-Ser-Asn (see Figure 9). This compound is hereinafter referred to as PA / 06.

This sequence is encoded by the Nde I and Xba I nucleotides for which the 54k N-terminal codons of the scu-PA protein were freely selected. This sequence is terminated at Leu-Leu, the Pst I intersection.

io

This PA / 06 protein, expressed, for example, in E. coli cells, unexpectedly exhibited high fibrinolytic activity when screened on a fibrin-agar film.

If the plasmid pGR Tac 06 replaces the Tac promoter between Eco R I and Xba I with the synthetic Trp promoter (see Fig. 10), the plasmid pGR Trp 06 is obtained.

Transformed with pGR Trp 06, E. coli cells K12JM 103 (ATCC 39 403), after induction with indolacrylic acid, produce an intercellular protein that exhibits fibronolytic activity upon cell fibrin-agar screening after cell lysis and protein structure repair.

According to the present invention, the other plasmids can be obtained from the engineered plasmids described above, for example, by means of Eco R I and Hind III, a synthetic structural gene with all regulatory elements and inserted into another bacterium of the Enterobacteriaceae family. E. coli, a plasmid that has been linearized for this purpose and truncated from Eco R I and Hid III. In principle, in the same way, but using Eco R I and Xba I, it is possible to replace the plasmids of any of the present invention, such as the Trp 'promoter with the Tac promoter (and vice versa).

According to the present invention, a plasmid elongated between the ShineDalgarn sequence and the start codon ATG can be obtained as described above by constructing a plasmid wherein said gap is, for example, 8 nucleotides crossed with Nde I, filled at the cleavage sites and clipped to the cut ends. a plasmid according to the present invention having a distance between SDs - The sequence and start codon contain 10 nucleotides.

In constructing the structural genes encoding compounds of Formula I, nucleotide sequences having a methionine codon at the 5 'end, followed by a serine codon, are selected. Express it on this. structural gene, due to the unstable nature of the Met-Ser bond, yields compounds of Formula I with serine at the amino acid Ngale. Suitable structural genes encoding junkur's formula I, corresponding 5'-ends (protein, born of formula

I, the N-terminus corresponds to the Ser-X ₁ -X ₂ region and the encoded but non-protein methionine is indicated in parentheses):

(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 Gi u - ATG AGC AGC CCA CCA GAA (Met) Ser Asn Glu Leu His ATG AGC AAT GAA CTT CAT

Leu

CTG

Leu

CAG

Gln Vai

CAA GTT

Pro

CCA

Ser ASn TGC AAC this from

As noted, the bacteria transformed with the find plasmids exhibit high expression levels. Finding suitable hosts for transformation is done in known ways. The plasmid expression of the present invention is best suited to bacteria of the E. coli or related Enterobacteriaceae family such as Pseudomonas, Salmonella, Enterobacter, Klebsiella or Serratia strains.

The most suitable hosts are E. coli, e.g.

E. coli GRT-I strain and in particular E. coli K12 subgroup strains such as: E. coli GRT-I strain and especially E. coli K12 subgroup strains such as:

E. coli K12 JM101 (ATCC 33876), E. coli K12 JM103 (ATCC 39403), E. coli K12 JM105 (DSM 4162), E. coli K12 MM294 (ATCC 33625), E. coli K12 (ATCC 31446) or E. . coli K12 DHI (ATCC 33849).

In E. coli expression systems containing the Tac promoter, induction can be accomplished, for example, by the addition of lactose or glucose, but preferably by β-Dtiogalactopyranoside (IPTG). If the plasmid contains the Trp promoter, induction is best done with indolacrylic, indolacrylic acetic or indolacrylic propanoic acids. It is also to be understood that induction can be accomplished with other known inductors.

After induction and at a certain cell density, the cells are centrifuged and the pellet suspended in saline, homogenized and the cells disrupted. After repeated centrifugation, a probalt of formula I remains in the precipitate along with cell debris. Probalt is dissolved in the resulting hydrochloride solution and subjected to a redox-oxidation system (e.g., oxidized and reduced glutathione) to give the protein its native conformation, i.e., compounds of formula I. These compounds remain in the reaction mixture and can be purified by conventional chromatography followed by lyophilization).

For analytical purposes, it is convenient to work as follows: the transformed plasmids of the present invention are transformed into E. coli by inducing protein expression at the required cell suspension density. After the required time of fermentation, the suspension was centrifuged in portions and cells lysed with lysozyme (1 mg lysozyme in 1 ml 50 mM Tris-HCl buffer, pH 8.0, with 50 mM EDTA and 15% sucrose). The homogenized cells are solubilized in 4-5 M saline, diluted to 1.2 M guanidine hydrochloride, and the reducing agents (glutathione, mercaptoethanol or cysteine) are added with air and left for several hours to recover the protein structure (cf. Winkler et al. Bioche. , 1986, pp. 4041-4045). The single-chain compounds of formula I thus obtained are converted, after the addition of plasmin, to the corresponding double-chain polypeptide derivatives, the activity of which is determined with the chromogenic substrate pyroGlu-Gly-Arg-p-nitroanilide, which is cleaved only by double-chain active urokinases. This activation of the compound of Formula I is performed with a plasmin solution in 50 mM Tris-HCL buffer with 12 mM sodium chloride, 0.02% Tween 80 at pH 7.4 at 37 ° C for several hours. The ratio of compound to plasmin should be from about 100 to about 1500 per mole of plasmin, expressed in moles, or from 8000 to 36000 with 1, based on units of enzyme activity. Activity was determined in 50 mM Tris-HCl buffer with 0.36 mM sodium chloride at pH 8.8 with 0.36 mM aprotinin (for plasmin attenuation) and 0.27 mM substrate at 37 ° C. Depending on the amount of the compound of formula I, after incubation for 5 to 60 minutes, the reaction is stopped by the addition of 50% acetic acid and the optical density is measured at 405 nm. In this assay, the increase in optical density at 0.05 units at 405 nm per minute, according to the substrate manufacturer (Kabi Vitrum, Sweden), corresponds to 25 units of Ploug urokinase per ml of standard solution.

The figures of the invention show:

FIG. 1: Schematic diagram of compounds of Formula I, indicating required disulfide bridges

FIG. 2a and 2b: Construction of plasmid pBF 158 from the commercial plasmid pBR 332. The nic / bom domains and the major part of the tetracycline resistance gene are sequentially deleted.

FIG. 3: Synthetic polylinker sequence.

FIG. 4: Construction of plasmid pBF 158-01. An insert is provided in the plasmid PBF 158 of the synthetic polylinker sequence between EcoR I and Hind III cleavage sites.

FIG. 5: Construction of plasmid pBF 158-01. The Cla I to Hind III fragment is replaced by the corresponding fragment T from plasmid pRT 61 (see Jorgensen et al., J. Bacteriol. 138, 1978, pp. 705-714), which contains the tetA / orf L chain terminating agent from Tn10 (see Sohollmeier). and others Nucl. Acids Res. 13, 1985, pp. 4227-4237).

FIG. 6a-6e: Nucleotide Sequences of Synthetic Fragments of a Coding Gene (Their Insertion to Constitute a Structural Gene for Compounds of Formula I according to the present invention into plasmid pBF 158-01, T is described in Example IId and shown in Figures 7a-7c).

FIG. 7a-7c: Construction of plasmid pBF-02 T-pBF 158-06 by inserting oligonuleotide fragments M4-M8 starting from the C-terminus of plasmid pBF 158-01 T.

FIG. 8: Plasmid pGR Tac 06 obtained from pBF 158-06

T, Inserting a Tac promoter between EcoRI and Xbal (See Example 1)

FIG. 9:

FIG. 10th

FIG. 11th

FIG. 12th

The nucleotide sequence inserted between the Nde I and Sac I cleavage sites in pGR Tac 06 and the corresponding amino acid sequence. The amino acid numbering is the same as in FIG. 1.

Nucleotide sequence of the synthetic Trp ~ promoter.

The compounds of formula I or their probal compounds constructed according to FIG. Figure 9: Expression dependence of expression level of transformed pGR Tac 06 plasmid with Tac promoter in E. coli cells by inducing IPTG at time 0. Reported activities in Ploug units per ml of culture fluid at 578 nm using chromogenic assay substrate (o ----- o) after plasminogen digestion and no digestion (· - - - ·). The indicated activities are the average of 4 fermentations.

Nucleotide sequences and corresponding amino acid sequences for compounds of formula (Met) -Ser-X _L -X ₂ -rscu? A ^{143 411} . Methionine, indicated in parentheses, is clipped in an E. coli cell.

FIG. 13th

Dependence on the expression level of the compounds of formula I or their probes transformed with pGR 318, pGR 319 or pGR 327, that is, with the Trp promoter in E. coli K12 JM 103 cells. Induction was performed at a time equal to 0 with indole acrylic vuma Ploug acid. in units

Report the presence of Akt1 ml culture medium with an optical density of 1, measured at 578 nm, using a chromogenic substrate (o ---- o) after plasminogen digestion and without digestion (· --- ·) ·

The examples provided use commercial enzymes in experiments (cf. Nachr. Chem. Techn. Nab. 35, 1987, p. 939).

The culture media used in the examples are also commercial.

Example 1:

The expression plasmid pGR Tac 06 encoding compounds of formula I according to FIG. 9, with a synthetic Tac promoter.

A. Construction of plasmid pBF 158

i) Deletion of the nic / bom region between 2207 and 2263 bases from plasmid pBR 322 (Pharmacy, No. 27-4902, 4363 bp) (see Winnacker, Genes and Clones, VCH Vainhaim, 1985, p. 296 ), as follows (see also Fig. 2): The plasmid pBR 322 is cut with Nde I at 2998 base and the plasmid is thereby linearized. The adhesive ends are obfuscated by a filling reaction (see Maniatis et al., Molecular Cloning, Cold Spring Harborlab, 1982). It is then cleaved at base 2068 with PvuII, and both adhesive ends of plasmid pBR 322 are ligated in a conventional manner with T4 ligase. It is then transformed into competent E. coli K12 JM 103 (ATCC 39403) cells (see Hanahan, DNA Cloning, T.I, IRL Press, Oxford, 1985, pp. 109-135). Transformed cells are cultured in medium containing 150 pg / ml ampicillin. From the resulting clones, those which have the plasmid pBF 157, which is different from the parent plasmid pBR 322, are selected.

230 nucleotides: a-Pst-BspMII, b-Pstl-Ball, c-Pstl-Aval fragments. Both single PvuII and NdeI cleavage sites are not present in pBF 337 in pBF 157.

i) The major part of the tetracycline resistance gene is removed from plasmid pBF 157 by cleavage of EcoPV-NruI and ligation of the sticky ends. Transformation into E. coli K12 JM 103 and digestion medium containing 150 µg / ml ampicillin results in the selection of clones containing the plasmid pBF 158. This plasmid is 787 nucleotides smaller than pBF 157 and is different. it has no BamHI clipping site. Transformed strains do not confer resistance to tetracycline in plasmid pBF 158.

B. Construction of plasmid pBF 158-01, insertion of a synthetic polylinker sequence

A 31 nucleotide fragment is excised from plasmid pBF 158 EcoPI and HindIII and inserted into this site by the multicloning site shown in FIG. 3. After transformation into E. coli K12 JM103 cells and culturing medium containing 150 µg / ml ampicillin, clones are selected which contain plasmid pBF 15801. This plasmid differs from pBF158 in that it has additional single cleavage sites on Xbal, NdeI, EagI, KpnI, Spel and has a second PstI cleavage site (Fig. 4).

Between Xbal and NdeI is the Shine-Dagarno sequence from the Xyl operon

B. subtilis (see Ihelm et al., Supra).

Construction of C. pBF 158-01, T plasmid, insertion of a transcription chain terminating agent.

Plasmid pBF 158-01 deletes the Clal-HindIIIII polylinker sequence fragment and replaces it with Clal LT 3948B.

A HindIII fragment from pRT 61 (see Jorgensen et al., Supra).

Following transformation with E. coli K12 JM 103 cells and 5 digestion media containing 150 µg / ml ampicillin, clones containing the plasmid pBF are selected.

158-01 T. This plasmid differs from pBF158-01 in that it is larger by the 297 nucleotide Clal-HindIII fragment (Figure 5).

0

D. Construction of plasmids pBF 158-02 T-pBF 158-06 T, insertion of the M4-M8 synthetic fragments into the coding gene.

All synthetic fragments are described in FIG. 6a-6e. They are introduced into fragments of 200 nucleotides into appropriate vectors. For this purpose, vectors with known cleavage sites are enzymatically quenched, cut and electrophoresed on agarose, electroeluted and purified via DE 52 cellulose to separate them from the fragments to be removed. This is followed by ligation with T4 ligase with the appropriate synthetic double-stranded fragment, transformed into E. coli K12 JM 103 (Figs. 7a-7c) and selection in medium containing ampicillin.

5 i). Insertion of fragment M8 between BamHI and Clal in pBF 158-01 T

Plasmid pBF 158-02 T was obtained.

Oligonucleotide 019 contains the Cgal sequence of a compound of formula I.

Behind the stop codon TAA is the NdeI cleavage site followed by additional transcriptional terminus trpA chain termination agent sequences (see Shristie et al., P.N.A.S. 78, 1981, pp. 4180-4184) up to Clal.

ii) Insertion of fragment M7 between SpeI and BamHI in pBF 158-02 T.

Plasmid pBF 158-03 T was obtained.

iii) Insertion of fragment M8 between KpnI and Spel in pBF 158-04 T.

Plasmid pBF 158-04 T was obtained.

iv) Insertion of fragment M5 between EagI and KpnI in pBF 158-04 T.

Plasmid pBF 158-05 T was obtained.

v) Insertion of fragment M4 between SacI and EagI in pBF 158-05 T.

Plasmid pBF 158-06 T was obtained.

From the resulting clones i) - v), one is selected which contains the appropriate fragment with the verified regular sequence.

E. Construction of plasmid pGR Tac 06, insertion of the Tac promoter

Plasmid PBF 158-06 T is cleaved with Eco I and Xba I. The 26 nucleotide fragment is removed by preparative electrophoresis on an agarose gel, the remainder of the plasmid is eluted by electroelution and purified by DE 52.

An Eco I and Xba I fragment containing the Tac * promoter is isolated from plasmid ptacSDT (DSM 5018). For this, the plasmid ptac SDT is cleaved with Ecol and Xba and the fragment is separated on preparative electrophoresis agarose gel. The fragment was warmed to 65 ° C in ammonium acetate - SDS buffer, pH 8.0, eluted from mechanically shredded polyacrylamide and purified by repeated extraction with phenol-saturated 1M TRISHC1 buffer, pH 8.0.

Both fragments obtained are ligated in the usual manner to T4 ligase and transformed into E. coli K12 JM 103. After culturing in media containing 150 µg / ml ampicillin, clones which produce probaltim, PA / 06 of formula I according to the invention, are selected. FIG. 9. These clones contain the plasmid pGR Tac 06.

(f) Expression test

(i) Fermentation

Transformed E. coli K12 JM 103 cells in plasmid pGR Tac 06 medium containing: 38 mM ammonium sulfate, 56 mM phosphate buffer pH 7.0, 1 mM magnesium sulfate, 1% yeast extract, 1% glucose and 150 mg / L ampicillin. Induction of probalt PA / 06 is performed with 0.5 mM IPTG.

Prior to and every hour after induction, a total of 6 ml of cell suspension is withdrawn for 6 hours so that the optical density (OD) at 578 nm is equal to 1 and the expression level is determined.

(ii) Reconstitution of the probaltim, its cleavage to the corresponding PA / 06 double bond compound and measurement of its PAactivity.

After centrifugation, the cells are lysozyme-solubilized, followed by treatment of the cell lysate as described above.

The level of expression of PA / 06 or its probaltim was determined by measuring the activity of the double-stranded plasminogen activator obtained from PA / 06 (obtained after probalt restoration). The results obtained are shown in FIG. 11. This activity is present only after plasmin cleavage (see Figure 11), so that PA / 06 (or its probalt) is present with a single chain.

Example 2

Construction of the expressing plasmid pGR Trp 06

Described by De Boer et al., PNAS, 80, 1983, p. The 21-25 Trp promoter sequence is up to the Xba I site at the 5 'end of the 5'-AATTCTGAAAT-3' sequence.

The result is an EcoRI cleavage site (Fig. 10, fragment M1). Separate chains 021 and 021A are assembled and fused to the EcoRI and XbaI fragment from plasmid pGR tac 06. After transformation into E. coli K12 JM 103 cells and cultures containing 150 mg / ml ampicillin, clones containing plasmid pGR Trp are selected. 06. The plasmid pGR Trp 06 differs from the above precursors in that it transformed E. coli strain bacteria with the addition of indolacrylic acid to produce probaltim in PA / 06.

Example 3

Construction of the expression plasmid pGR 318

Plasmid pGR Trp 06 was cleaved with NdeI and Sacl. The resulting 58 bp fragment, which is a polylinker sequence is isolated by preparative electrophoresis on an agarose gel, the remainder of the plasmid is electroeluted and the fragment is purified via DE 52 cellulose.

This fragment contains a synthetic oligonucleotide 18

5'-TATG AGC AAT GAG CT-3 '

AC TGG TTA C

Conventionally ligated with T4 and transformed into E. coli K12 cultured in ampicillin-containing medium. Clones are selected which, upon addition of indolacrylic acid, produce probaltimic compound PA 318. These clones contain the plasmid pGR 318, which encodes compounds of formula I, PA 318. The amino acid sequence of these compounds is shown in FIG. 12th

B. Expression test

After transformation, the plasmid pGR 318 of E. coli K12 JM 103 is fermented in the same manner as described in Example 1. The induction is carried out with 62 mg of indoyl acrylic acid per liter of enzyme medium at 578 nm when the cell suspension reaches 2-3 optical units.

Prior to and every hour after induction, a total of 5 ml of cell suspension is withdrawn for 5 hours so that the optical density (OD) at 578 nm is equal to 1 and the expression level is determined.

After reconstitution of the PA 318 probaltim (see Example 1), the collected samples were assayed for amidolytic activity after plasmin treatment before and 1 to 5 hours after induction. The results are shown in Figs. 13. It follows from these data that the same level of expression was achieved as with the plasmid pGR Tac 06 (but only for another probe) and that the amidolytic activity is only present after plasmin treatment. Therefore, PA 318 of formula I is produced in E. coli K12 with plasmid pGR 318 as a single-stranded probe.

C. Isolation, Purification and Characterization

To purify the protein, it is fermented in 1 liter volume, controlled by pH and temperature (pH 7.0, 37 ° C).

Under these conditions, cells can be grown to 20 optical units measured at 578 nm. After expression is complete (about 5-6 hours), 50 ml of cell suspension is centrifuged, the pellet is resuspended in 40 ml of water and digested in a high-pressure homogenizer. After centrifugation, the precipitate containing all inactive probaltim was dissolved in 100 ml of 5 M guanidine hydrochloride solution with 40 mM cysteine, 1 mM EDTA (pH 8.0) and diluted with 400 ml of 25 mM TRIS-HCl buffer, pH 9.0. It takes about 12 hours to recover the structure of the protein when exposed to air or flushed with oxygen.

PA 318 is isolated and purified by chromatography. In determining the N-terminus, it has been proved that the protein has a single strand and that the desired sequence is present at the N-terminus.

Example 4:

Construction of the pGR 319 expression plasmid

The plasmid pGR Trp 06 is cleaved as described in Example 3a with NdeI and SacI, and a large fragment is isolated.

This fragment contains a synthetic oligonucleotide 19

5'-TATG AG C AAT GAA GAG CT-3 ' AC TCG TTA CTT C usual way merging ligase T4 and transformed

E. coli K12 cultured in ampicillin-containing medium. Clones are selected, with the addition of indolacrylic acid, which produce probalt of PA 318. These clones contain the plasmid pGR 318, which encodes compounds PA 319 of formula I. The amino acid sequence of these compounds is shown in FIG. 12th

B. Expression test

E. coli K12, transfected with plasmid pGR 319, is fermented, induced, and assayed in the same manner as described in Example 3b.

PA 319 is isolated, purified and characterized in the same manner as in Example 3C.

Restoring the structure and expression level of probaltim of PA 319 is performed as in Example 3B. The results are shown in Figs. 13. It follows from these data that the same level of expression is reached as with the plasmid pGR Tac 06 and that the amidolytic activity is only present after plasmin treatment. Therefore, compound PA 319 of Formula I is produced in E. coli K12 plasmid pGR 318 as a single-stranded probe.

Example 5:

Construction of the expression plasmid pGR 327

The plasmid pGR Trp 06 is cleaved as described in Example 3a with NdeI and SacI, and a large fragment is isolated.

This fragment with synthetic oligonucleotides 27

5'-TATG AGC AG T CCA CCA GAG CT-3 ' AC TCG TCA GGT CCT CTT C normal by way of connection ligase T4 and trans formation

to E. coli K12. Selection and clone selection are performed as described in Example 3A. The selected clones contain the plasmid pGR 327, which encodes compounds PA 327_ of formula I. The amino acid sequence of these compounds is shown in FIG. 12th

B. Expression test

E. coli K12 transformed with plasmid pGR 327 is fermented and the expression level tested as in Example 3B. The results are shown in Figs. 13th

Restoring the structure and expression level of probaltim of PA 319 is performed as in Example 3B. The results are shown in Figs. 13. From these data, the same level of expression was reached as for the plasmid pGR Tac 06 and that the amidolytic activity is only present after plasmin treatment. Therefore, compound PA 327 of formula I is produced in E. coli K12 plasmid pGR 327 as a single-stranded probe. Isolation, purification and characterization were carried out in the same manner as in Example 3C.

Example 6:

The plasmid pGR Trp 06 is cleaved as described in Example 3a with NdeI and SacI, and a large fragment is isolated.

This fragment contains a synthetic oligonucleotide 36

5'-TATG AC AGC TCG CCA GGT CCA CCT GAA CTT GAG C CT-3 ' connected in the usual way ligase T4 and transformative to E. coli Q12 JM 105. Breeding and clones selection

are performed as described in Example 3A. The selected clones contain the plasmid pGR 336, which encodes the compounds, PA

336 of formula I. The amino acid sequence of these compounds is shown in FIG. 12th

E. coli K12 JM 105, transformed with plasmid pGR 336, is fermented and after induction assayed for expression levels in the same manner as in Example 3B. The primary probaltim yields a single chain compound PA 336 (amino acid sequence shown in Figure 12). The same level of expression was achieved as with the plasmid pGR Tac 06.

DEFINITION OF INVENTION

Claims (27)

DEFINITION OF INVENTION 1. Novel polypeptides of general formula I: Ser-X ₁ -X ₂ -rscu-PA ¹⁴³ ' ⁴¹¹ (I), wherein rscu-PA ¹⁴³ ' ⁴¹¹ is a non-glycosylated amino acid sequence region of 143 (Glu) to 411 (LeuOH) from scu-PA 54k, X! - one of the following amino acid residues: Asn, Pro or Ser, and X ₂ is a single bond or Glu, Pro-Glu, Pro-Pro-Glu or Glu-Leu-His-Leu-Leu-Gln-Val-Pro-Ser-Asn. 2. The polypeptides of claim 1, wherein X 1 is Asn or Ser. 3. Polypeptides according to claims 1 and 2, characterized in that X! is Ser. 4. Polypeptides according to claims 1-3, wherein X ₂ is a single bond or Glu, Pro-Glu or Pro-Pro-Glu. 5. The polypeptides of claim 1, wherein X _x is Asn or Ser and X ₂ is a single bond or Glu, Pro-Glu or Pro-Pro-Glu and rscuPA ^{143 411} have the same meaning as in claim 1. 6. The polypeptide of claims 1 and 3, wherein the polypeptide has the formula (I a): Ser-Ser-Pro-Pro-Glu-r scu-PA ^{143 4 1 1} , wherein rscu-PA ^{14 41} has the same meaning as in (i). 7. A novel plasmid used to obtain the polypeptides of claims 1-6, wherein the operon has a regulated promoter, which acts as a binding site with a ribosomal Shine-Dalgarn sequence, a start codon, a synthetic structural gene for the polypeptides of claims 1-6 and 5. in the direction of the structural gene from one to two termination agents, and are used for expression of probaltymc in Enterobacteriaceae, preferably Escherichia coli strains. Plasmid according to claim 7, characterized in that the distance between the Shine-Dalgarn sequence and the start codon is 6-12 nucleotides, preferably 8-10 nucleotides. Plasmid according to claims 7 and 8, characterized in that the regulated promoter is a synthetic Trp or Tac promoter. 10. The plasmid of claim 9, wherein the regulated promoter is a synthetic Trp promoter having the nucleotide sequence shown in FIG. 10, and FIG. 10 is part of this paragraph. Plasmid according to claim 9, characterized in that the regulated promoter is the Tac * promoter of ptac SDT (DSM 5018). Plasmid according to claims 7-11, characterized in that the termination agent in the operon is a successive Trp A termination agent and / or a tet A / orf L termination agent from Th 10. Plasmids according to claims 7-12, characterized in that in the structural gene arginine is encoded by the tplet CGT, leucine-CTG, valine-GTT and / or glycine-GGT. Plasmids according to claims 7-13, characterized in that the structural gene has the nucleotide sequences shown in FIG. 6a-6e, and figs. 6a to 6e are part of this paragraph, and in particular one of the following nucleotide sequences: ATGAGCAAT ATGAGCAATGAA ATGAGCAGTCCACCAGAA ATGAGCCCACCAC-AA. Plasmids according to claims 7-14, characterized in that the structural gene is composed of the following nucleotide sequence: ATGAGCAAT ATGAGCAATGAA ATGAGCAGTCCACCAGAA ATGAGCCCACCAGAA. Plasmids according to claims 7 to 15, characterized in that the nucleotide sequence of the structural gene has a methionine codon at its 5 'end followed by a serine codon. Plasmids according to claims 7-16, characterized in that they contain an operon in plasmid pBR 322, in which the nic / bom region has been removed. 18. Plasmids according to claims 7-16, characterized in that they contain an operon in plasmid pBR 322, in which the tetracycline resistance gene pilLT 3948B has been deleted or partially so that the plasmid has no tetracycline resistance. Plasmid according to claims 7 to 18, characterized in that it contains an operon in plasmid pBR 322, in which the nic / bom region has been deleted and the portion of the tetracycline resistance gene has been deleted such that the plasmid has no tetracycline resistance. Plasmids according to claims 7-19, characterized in that they are pGR, Trp 06, PGR 318, pGR 319, pGR 336 with a synthetic structural gene under the control of the Trp promoter. 21. Plasmid according to claims 7-19, characterized in that it is as shown in the figure: 22. The method of generating plasmids according to claim 7, wherein the plasmid pBR 322 deleting the nic / bom region and / or at least a portion of the tetracycline-resistant gene between EcoRI and HindIIIII inserts has a polylinker sequence having the nucleotide sequence shown in FIG. 3 (FIG. 3 is a part of this paragraph) and by means of known methods inserts a transcription termination agent, separate synthetic structural gene sequences for compounds of formula I, preferably starting at the 3 'C terminus of the structural gene, and a synthetic Trp promoter. 23. A method of producing plasmids according to claim 7, characterized in that the EcoRI and HindIII fragment of plasmid obtained according to claim 22 is inserted into another plasmid capable of autonomous replication in bacteria of the enterobacteriacea family, preferably E. coli, as an expression cassette. 24. A method of producing a polypeptide according to claims 1-6, which comprises, by known methods, transforming bacterial strains of the enterobacteriacea family, preferably E. coli strains, into a plasmid according to claims 7-21, inducing expression of a structural gene, isolating a compound of formula I, probaltim, probe is dissolved and then, using the oxidation-reduction system, obtain the polypeptide according to claims 1-6. 25. The method of claim 24, wherein the plasmid is transformed with an E. coli strain, preferably E. coli K12. 26. A thrombolytic preparation comprising one of the compounds of claims 1-6 as an active duck plasminogen into an active compound. The use of a compound of the formula I as claimed in claims 1 to 6 for the preparation of a thrombolytic preparation with plasminogen activator as active ingredient.