DD245444B5 - Process for the preparation of a new staphylokinase with heterologous producers - Google Patents

Description

For this 6 pages drawings

Field of application of the invention

The invention relates to the production of genetically engineered recombinant plasmids carrying a staphylokinase gene and their introduction into various heterologous bacterial hosts, which as a result of these manipulations can be advantageously used to produce the novel staphylokinase by submerged fermentation.

The field of application of the invention is thus mainly in the microbiological-pharmaceutical research and industry.

Staphylokinase (SAK) is a protein that is produced by Staphylococcus aureus strains, usually after lysogenic conversion, and excreted into the culture medium. Heterologous we call according to the invention such Staphylokinaseproduzierenden bacteria that do not belong to the genus Staphylococcus and naturally have the ability to produce SAK, not possess, but can acquire them by targeted genetic manipulation.

Staphylokinase converts the fibrinolytically inactive plasminogen of the blood plasma to plasmin, which dissolves blood clots by limited proteolysis of the fibrin network (Lack and Glainville, in: Methods in Enzymology XIX, 706-714 [197O]). This is the only known property of this bacterially formed protein.

Activation of plasminogen to plasmin is also effected by a number of other proteins (streptokinase, urokinase, tissue plasminogen activator), which are currently in use. alsthrombolytic therapeutics are already being used in clinical medicine. The possible main application of staphylokinase is therefore also in human medicine, to some extent but also in veterinary medicine.

Characteristic of the known technical solutions

Staphylokinase (SAK) can be obtained from the culture supernatant of certain Staphylococcus aureus strains. SAK-producing strains are generally lysogenic for temperate bacteriophages whose presence enables the cell to staphylokinase-form (lysogenic conversion) (Winkler et al., J. Gen. Microbiol 39: 321-333 [1965]).

However, it is known in this connection that S. aureus strains produce a number of extracellular proteins which are substantially responsible for the high pathogenicity and virulence of these bacteria (Rogolsky, Microbiol Rev 43: 320-360 H 979]). , This variety of toxic products and the associated problems for industrial-scale fermentations have so far made it impossible to obtain SAK in sufficient quantity and quality to be able to examine its suitability as a therapeutic agent.

By means of standard genetic engineering methods (summarized, for example, in Maniatis, Fritsch, Sambrook: Molecular Cloning - a laboratory manual, Cold Spring Harbor Laboratory, 1982), a gene for the production of staphylokinase from the DNA of staphylococcal phage S0-C has recently been isolated on the plasmid pBR322 in the Gram-negative bacterium Escherichia coli (Sako et al., Mol. Gen. Genetics 190: 271-277 [1983]). The E. coli strains constructed in this way were able to produce staphylokinase, although the SAK protein formed was not secreted or only to a small extent into the surrounding culture medium. Digestion or osmotic shock treatment of the cells was necessary for the quantitative recovery of staphylokinase (Sako et al., Mol. Gen. Genet 190: 271-277 (1983); Sako, Eur. J. Biochem. 149: 557-563

The staphylokinase gene cloned in this method has been subjected to a primary structural analysis. The DNA sequence of this gene from S0-C and the amino acid sequence of this staphylokinase derived therefrom are present (Sako and Tsuchida, Nucleic Acids Research 11: 7679-7693 [1983]).

Object of the invention

The invention aims to provide heterologous bacterial hosts for the production of this protein after introduction of a previously isolated gene encoding a novel staphylokinase.

Explanation of the essence of the invention

The invention has for its object to develop a genetic engineering process for the isolation and cloning of a staphylokinase gene and for the subsequent production of heterologous bacterial hosts for the fermentative production of the gene product.

According to the invention, this object is achieved in its basic feature as follows:

From the DNA of Staphylococcus aureus phage DNA fragments are first isolated, which contain the characterized by the nucleotide sequence of Fig. 1 a gene for a new staphylokinase (SAK gene). DNA fragments of this characterization are then transferred to an E. coli cloning vector and the resulting progeny subjected to screening for SAK * recombinant vectors, hereinafter referred to as SAK screening (step I).

From each one of the SAK ^T recombinant (primary) vectors obtained in step I, fragments with functional SAK gene are taken in the next step and for obtaining SAK ⁺ recombinant secondary plasmids each on a vector plasmid, which component of a heterologous bacterial host vector -Systems is subcloned, wherein the derivatives resulting in this step are again subjected to a SAK screening (process step II).

Each SAK ⁺ recombinant plasmid obtained in steps I and II is then transformed into heterologous bacterial producer strains (step III), and each heterologous bacterial producer strain genetically engineered in this manner is used in the last step (step IV) for submiculturation to obtain the Staphylokinase characterized by the amino acid sequence according to Fig. 1 b.

In a further embodiment of this basic feature, the problem of the invention is realized by the following experimental sub-steps:

1. isolation of DNA of gene-donating phage

As a donor for the SAK gene according to Fig. 1, a temperate Staphylococcus aureus phage of the serological group F, preferably a phage from the amount 0W46,0L80,0L55,042 D, is used in step I of the method.

In a preferred embodiment of the method, the gene donor used in the above amount is the latter phage 042 D, a known typing phage. This bacteriophage is known to convert to the formation of staphylokinase upon lysogenization of susceptible S. aureus strains.

In order to isolate its DNA, the group F-gene donor selected in each case, and also the preferably used phage 042 D, are propagated and enriched on a sensitive host strain of the species S. aureus, preferably on the strain S. aureus W57, using the known soft agar layer technique.

After extraction of the phages, preferably the 042 D phage, from the soft agar layer, there is a phage lysate with a titer of at least 10 ¹⁰ pfu / ml. To separate cell debris and further concentrate the bacteriophages, the lysate is subjected to a cycle of low and high speed centrifugations. The potentially SAK gene-carrying DNA is released from the concentrated phage lysate by addition of a detergent and freed of protein in a manner known per se by means of phenol and chloroform extractions. Finally, the phage DNA prepared in this manner is subjected to centrifugation in the EB-CsCl density gradient, so that purified and concentrated phage DNA, preferably the 042 D, is available for the subsequent experimental steps.

2. Cloning of potentially SAK gene-carrying DNA fragments in an E. coli cloning vector (part of method step I)

The purified Staphylococcus aureus (GruppeF) phage DNA, preferably the purified 042 D DNA, is first treated with enzymes that cleave DNA at specific recognition sites. Preferably restriction enzymes and in particular the restriction enzymes CIaI, Hpall or EcoRI are used for this purpose. As a result of this treatment, the phage DNA, preferably the 042 D DNA, is each resolved into a characteristic number of fragments varying in size from 0.5 to about 10.0knp.

In parallel with the treatment of the potentially SAK gene-bearing phage DNA, the selected E. coli cloning vector is linearized in preparation for the desired conversion with a restriction enzyme for which there is a singular cleavage site on the vector and which simultaneously generates such ends, which are compatible with those of fragments of donor phage DNA.

As the E. coli cloning vector, either an E. coli phage vector, preferably a λ phage, or an E. coli plasmid vector is provided. From the totality of the E. coli plasmid vectors, a vector is selected from the subset of the amplifiable E. coli plasmid vectors, preferably an amplifiable E. coli plasmid vector, for the purpose according to the invention

a) a CoIEI or a p15A replication origin and

b) in addition at least two antibiotic resistance markers and in one of these markers at least one singular Restrictase cleavage site

having.

A common vector with CoIE 1 replication origin satisfying these criteria is plasmid pBR 322; a vector with ρ 15 A replication origin corresponding to these criteria is plasmid pACYC184. The latter is used in a preferred embodiment of the method as E. coli cloning vector.

An amplifiable E. coli plasmid vector of the above label is linearized for the intended conversion with a restriction enzyme whose unique cleavage site on that vector is located so that insertion of foreign DNA leads to inactivation of one of its two antibiotic resistance markers and again simultaneously generates such ends that are compatible with those of fragments of donor phage DNA.

When using the E. coli plasmid vector pBR322 this linearization is carried out with CIaI; when using the E. coli plasmid vector pACYC 184, d. H. in the preferred embodiment of the process, EcoRI or CIaI is used for linearization.

Such linearized E. coli cloning vector DNA, preferably such linearized E. coli plasmid vector DNA and in particular such linearized pACYC 184 DNA, is provided with the above-prepared S. aureus phage DNA, preferably with fragmented 042 D-DNA, now mixed so that the latter DNA fragments are present in excess. By addition of the enzyme DNA ligase, preferably T4 DNA ligase, the phage DNA fragments with the linearized E. coli vector, in particular with the linearized pACYC184 vector plasmid, in a conventional manner in a random manner to recombinant Vectors, preferably to circular DNA molecules (recombinant plasmids) linked.

The resulting mixture of recombinant E. coli phages or recombinant E. coli plasmids, in particular the resulting mixture of recombinant pACYC184 derivatives, is subsequently used to produce, in a manner known per se, a Gram-negative host strain, preferably an E. coli . coli strain and in particular the strain E. coli 294, to infect or transform. The resulting transformants are subjected to a screening in the next step, with which the successful establishment of recombinant phages or plasmids in the respective recipient strain can be determined. In order to be able to keep the description of the inventive solution also sufficiently clear, the subsequent description of the essence of the invention in its further embodiment from now on will comprise only one basic variant of the method, namely those in which the primary cloning of potentially SAK gene carrying 042 D fragments in an E. coli plasmid vector. This basic variant is based on the design scheme according to Fig.2. For the successful establishment of the plasmids in each selected E. coli strain (see above) is selected by addition of an antibiotic, preferably by the addition of tetracycline or chloramphenicol, to the culture medium (prescreening). Testing the plasmid-carrying transformant colonies for resistance to the respective other antibiotic, d. H. either against chloramphenicol or against tetracycline, allows the differentiation between transformants with reconstituted pACYC184 plasmid (= resistant) or those with recombinant 042 D-DNA-carrying plasmids (= sensitive).

3. Detection of transformers with recombinant plasmids carrying the staphylokinase gene

Those E. coli transformants identified as such by recombinant plasmids as a result of screening are now spread on indicator plates containing casein in the form of skimmed milk as well as human plasminogen in addition to agar (or agarose) and yeast extract. Transformants that are capable of staphylokinase production due to their recombinant plasmids form a clear lysis zone caused by the caseinolytic activity of the SAK-activated plasminogen. Nonspecific or protease-responsive reactions are disrupted by parallel testing of the same transformants on indicator plates to which no human plasminogen has been added. SAK positive transformants (SAK ⁺ transformants) do not form lysis sites on such plates. In this assay system, the E. coli strain used for cloning does not show nonspecific background activity.

4. Characterization of recombinant SAK plasmids

From several transformant clones which form staphylokinase, the plasmids are obtained in a manner known per se in so-called rapidscreening processes in small amounts and subjected to rapid analysis with restriction enzymes,

in particular with CIaI, Hpall and EcoRI. For further work, among the recombinant plasmids, the one with the smallest SAK ⁺ -042 O-DNA insert is selected. This plasmid was named pDB 3. It carries an approximately 7.0-knot 042 D fragment flanked by Clal sites

 5. Subcloning of the staphylokinase gene into E. coli plasmid vectors

The DNA of the recombinant SAK plasmid pDB3 is preparatively isolated from E. coli strain 294 (pDB3) in a manner known per se in a larger amount and purified by dye density gradient centrifugation. First, the pDB3 plasmid DNA is cleaved with a series of different restrictases, the intersections of which are preferably mapped in the 042 D-DNA insert by comparative analysis in a manner known per se. Among the restriction enzymes used, a pair is selected, preferably the EcoRI and EcoRV enzyme pair, which breaks down the 042 D insert of pDB3 into two nearly equally sized subfragments. Linkage of each of these EcoRI-EcoRV subfragments with an EcoRI or Pvull and EcoRV-cut E. coli vector plasmid, preferably with pACYC184 or with pBR322, by treating the vector fragment mixture with the enzyme T4 DNA ligase for the preparation of new recombinant plasmids with shortened 042D insert. These plasmids are used in a manner known per se to transform an E. coli host strain, preferably strain E. coli 294. The selection of plasmid-carrying transformants and the detection of transformants with recombinant plasmids, including those carrying the SAK gene, is carried out in the manner already described (see Part 3).

As a result of the preferred embodiment of this subcloning I. order, there is an SAK-producing E. coli strain, in particular E. coli 294 strain (pDB12), which contains a recombinant pBR322 derivative, hereinafter referred to as plasmid pDB 12. carries with about 2.5 knp large 0 42 D-DNA insert.

As a result, pDB 12-DNA is now isolated in a known manner in a larger amount, purified and characterized with respect to the location of restriction sites in the (SAK ⁺ ) -042 D-DNA insert. The enzymes used in particular include the restrictase Rsal.

Rsal fragments of the plasmid pDB 12 are subsequently prepared in a manner known per se and preferably those Rsal fragments which are approximately 1.0knp in size are isolated. These Rsal fragments, one of which originates from the 042 D-DNA insert of plasmid pDB 12, are transformed in the manner already described with an E. coli vector plasmid, preferably with plasmid pACYC184 or pBR322 previously separately linearized with the EcoRV restriction gene , linked (subcloning 2nd order). Transformation of an E. coli host strain, preferably strain E. coli 294, results in the recovery of a transformant clone which is capable of producing staphylokinase due to the presence of a novel recombinant plasmid, designated plasmid pDB17, as previously described. After preparative isolation of plasmid DNA, the structural characterization of pDB 17 is carried out in a manner known per se by mapping the location of restriction sites for various enzymes. Thereafter, the plasmid pDB 17 is 4.1 knp and carries an approximately 1.0 knp piece of the 042 D DNA insert with complete information for the formation of staphylokinase; at the same time, it has suffered a deletion of approximately 1.25knp, which comprises the entire structural gene of the tetracycline resistance of the parent vector pBR322 (see also Fig. 3). β. DNA sequence analysis of the 042 D-DNA insert of pDB 17

From purified pDB17 plasmid DNA fragments are cut out in a manner known per se preparative amounts and isolated, containing the entire (SAK ⁺ ) -042 D-DNA insert or most of it, preferably those fragments with the restriction enzymes Hpall or Hinfl can be won.

Subsections of these 042 D fragments from pDB 17, which are preferably generated with such small-cutting restrictases as Taql, Sau ЗA, AluI and Haelll, or optionally also the complete Hpall fragment (see Fig. 4) are in the following in As is well known, the double-stranded replicative form of E. coli phages M 13mp9 or M 13mp18 which have been linearized with the restriction genes Accl or Hincll are randomly linked using the enzyme T4-DN A ligase. These recombinant DNA molecules are then used in a manner known per se for the transfection of a suitable E. coli host strain, preferably of the strain E. collTGI. Phage plaques derived from recombinant M 13mp phage can subsequently be recognized in a manner known per se and used to obtain high titre phage preparations. The single-stranded DNA of the recombinant phage is then isolated in a manner known per se and used after attachment of a known DNA oligonucleotide primer as a template for the synthesis of a complementary strand. The addition of didesoxynucleotide triphosphates to the synthesis reaction leads to the randomly discontinued termination of the synthesis of the complementary strand and allows the DNA sequence of the 042D insert of the recombinant M 13 phage to be determined in a manner known per se.

Computer-aided evaluation of the individual sequences of 042 D-DNA inserts of individual recombinant M13 mp phage clones furthermore allows the total DNA sequence of the 042 D-DNA insert from the recombinant plasmid pDB 17 to be assembled in a manner known per se, thus allowing the determination the DNA sequence of the staphylokinase gene of phage 042 D. This sequence is shown in Fig. Lain a form that allows the best possible comparison with the DNA sequence of the S0-C-Stapnylokinasegens.

The comparison shows: In the section from position 1 to position 951 there are 11 differences (indicated by underlines in the sequence shown in Fig. 1a) between 042D and S0-C-SAK genes. These differences concern either single base pair Replacements or insertions. In the section from position 952, the sequence of the 042 D fragment deviates completely from the sequence of the S0-C fragment.

In the coding sequence of the gene for the enzyme staphylokinase there are 3 positional differences (no difference is located in the signal peptide sequence, by the way). Of these 3 positional differences, one is not phenotypically expressed, i. that is, it does not induce any change in the corresponding amino acid sequence (so-called silent mutation). The two other positional differences in the staphylokinase-encoding sequence, however, each involve an amino acid exchange (a conservative exchange, but also an exchange between amino acids with significantly different properties).

Taking into account the latter, the enzyme encoded by the 042D SAK gene is thus to be classified as a new staphylokinase.

7. Cloning of the SAK gene in Gram-positive bacteria

For the two gram-positive bacterial genera Streptococcus and Bacillus there are, among others, a family of plasmid cloning vectors available as natural shuttle vectors for autonomous replication in both

Genera are capable. These vectors, referred to as pGB plasmids, carry erythromycin and / or chloramphenicol resistance genes that allow the selection of plasmid-bearing cells. From the pGB vector family, for the subcloning of the SAK gene according to the method, ie for the subcloning of this gene in gram-positive hosts, preferably the plasmid pGB3631 is selected, which carries a short polylinker and an erythromycin (MLS) resistance gene and carries approx 6.1knp is big. This plasmid is recovered in a preparative amount, purified and subsequently cleaved with the restriction enzymes BamHI and EcoRI in the polylinker region. Parallel to this, starting from the recombinant E. coli plasmid pDB 12 obtained according to the method, a SAK gene-carrying DNA fragment (about 2.5 knp) is obtained, which is also flanked by cleavage sites for the restrictases BamHI and EcoRI. The linkage of this fragment with the pGB3631 vector is carried out in a manner known per se by the enzyme T4 DNA ligase. The recombinant plasmids thus constructed are used to detect in parallel suitable host strains of the genus Streptococcus and Bacillus, preferably strains of the species S. mflleri var. Anginosus or S. sanguis (in particular strain S. sanguis Challis57) and B. megaterium or B. subtilis ( in particular strain B.subtilis GB500), in a manner known per se. Streptococcus and Bacillus cells, which have erythromycin resistance and are capable of producing SAK, carry the same new recombinant plasmid, designated pDB 15 (size approximately 8.9knp, see also Fig. 5). It is generally known for both Streptococcus and Bacillus cells that the proteins carrying the genetic information for protein secretion in the form of a signal peptide sequence segregate into the surrounding culture medium. The same situation could also be stated in the present inventive solution: The Streptococcus and Bacillus strains of the method transformed with the SAK ⁺ recombinant plasmid pDB 15 are capable of excretion of active SAK into the culture medium.

8. Identification of the heterogeneous host-derived SAK gene product

The following evidence clearly shows that the cloning experiments carried out led to the isolation of another staphylokinase gene, which in the heterologous hosts is so pronounced that a protein with characteristic substrate and immunospecificity is formed:

- The caseino- and fibrinolytic activity of all primary and subclones is dependent on the presence of human plasminogen.

- In the immunodiffusion test both staphylokinase obtained from culture supernatants of heterologous hosts and authentic staphylokinase from S. aureus strains yield identical precipitation lines with an antiserum produced against authentic staphylokinase (Fig. 6).

9. Synthesis of staphylokinase with heterologous hosts

As a result of the realization of the basic variant of the present process for producing a recombinant staphylokinase, there is a group of genetically manipulated heterologous bacterial strains which are now able to form staphylokinase. Specifically, the producer strains E. coli SK1590 (pDB3), E. coli SK1590 (pDB12), E.coli SK1590 (pDB17), E.coli 294 (pDB3), E.coli are named from this group 294 (pDB 12), E. coli 294 (pDB 17), S. sanguis Challis 57 (pDB 15) and B. subtilis GB500 (pDB 15) used for the synthesis of staphylokinase.

Each of these producers is cultivated at temperatures in the range of 25 ° C to 40 ⁰ C, preferably in the range of 33 ⁰ C to 37.5 ⁰ C, submerged in a medium, each with known nutrient sources until the end of the logarithmic growth phase, and the new recombinant staphylokinase formed is obtained by processing steps known per se, such as by concentration of the culture supernatant and cell disruption in the case of E. coli strains (see also Section E of the embodiment).

Notes on the deposit of microorganisms

In connection with the present process for the production of a new staphylokinase in heterologous producers is in the depository for microorganisms, Central Institute of Microbiology and Experimental Therapy of the Academy of Sciences, DDR - 6900 Jena, Beutenbergstr. 11, each a biologically pure culture of

Registration number

- Staphylococcus aureus bacteriophage 042 D-lysate ZIMET11193

Strain Bacillus subtilis GSB 26 (pGB 3631) ZIMET11137

Strain E. coli 294 (pDB 17) ZIMET11136

- Strain B. subtilis GB 500 ZIMET has been deposited.

Advantages of the invention

The process according to the invention makes it possible to obtain a new staphylokinase by submerged fermentation of bacterial species which do not belong to the genus Staphylococcus and do not form, like these, a multiplicity of toxic and antigenic products. It is of particular advantage that Gram-positive bacteria such as B. subtilis or S. sanguis can be used as a producer, since these bacteria excrete active staphylokinase in the surrounding culture medium.

This circumstance makes expensive and expensive digestion work, as they are necessary for the heterologous E. coli hosts, superfluous and considerably reduces the effort required for the purification of staphylokinase.

The levels of staphylokinase produced by B. subtilis are significantly higher than those found in other heterologous hosts. B.subtilis is a widely used microorganism in the fermentation industry, particularly for the production of bulk enzymes, which are among others. used in the food industry. B. subtilis strains are considered pathogenetically harmless.

The cloned staphylokinase gene and the knowledge of its primary structure provide the basis for targeted molecular manipulations to increase the product yield of the production strains.

Finally, the DNA structures controlling expression and secretion of staphylokinase are useful in further genetic engineering for the production of other proteins of eukaryotic or prokaryotic origin by heterologous bacterial hosts.

The following statements are intended to illustrate an example of the individual steps of the method. However, they do not contain any detailed information on standardized individual methods of genetic engineering (such as extraction of plasmid DNA, cleavage of DNA with restriction enzymes, insertion of genes into vector plasmids, transformation of bacterial recipient strains with plasmid DNA and DNA sequence analysis). Such methods are known to those skilled in the art and are described in detail in a number of publications, such as in:

- Methods in Enzymology, Vol. 65: Nucleic Acids (Part 1), Academic Press, 1980;

- Methods in Enzymology, Vol. 68: Recombinant DNA, Academic Press, 1980;

- Methods in Enzymology, Vol. 100, Academic Press, 1983;

"Molecular cloning, a laboratory manual" (T. Maniatis, E. R. Fritsch, J. Sambrook), Cold Spring Harbor Laboratory, 1982;

- "Advanced bacterial genetics, a manual for genetic engineering" R.W. Davis, D. Botstein, J.R. Roth, Cold Spring Harbor Laboratory, 1980.

A. Primary Cloning of the Staphylokinase Gene (SAK Gene)

A.1. Obtaining donor DNA from S. aureus phage 042 D

In order to obtain high-titer lysates of Staphylococcus areus phage 042 D, surface enrichment is carried out in the following manner: starting from a single colony of S. aureus strain W57, 5 ml of nutrient broth Il (NBII)

Pancreatic peptone (meat, gelatin, casein) 6.75 g / l

Protein hydrolyzate 1.75 g / l

Yeast extract 1.50 g / l

NaCl 5.00 g / l

(Sterilized at 121 ^° C. for 12 minutes, pH 7.2 ± 0.3)

inoculated and shaken for about 2 hours at 37 ⁰ C. This culture is used in the following to increase the phage 042 D by adding 0.5 ml of the W57 culture, 0.1 ml of a 042D lysate (about 10%) to 2 ml of liquefied and tempered soft agar (NBII + 0.7% agar) ⁷ pfu / ml) as well as 0.05 ml of a sterile 10OmM CaC ^ -solution and this is used after thorough mixing to overlay Nähragar II (NAH, NBII solidified with 1% agar) base layers in Petri dishes (0100 mm). After incubation of the plates overnight at 30 ⁰ C, the bacterial lawn is lysed in the upper layer confluent. 2 ml of NBII are added to each plate, then the soft agar top layer is carefully scraped off with a spatula and subsequently homogenized by repeated pipetting. The solid agar components and bacterial debris are separated by centrifugation (15 minutes, 6000rpm) and the decanted, phage-containing supernatants pooled. To suppress secondary bacterial growth, approximately 1 % to 2% chloroform is added to the lysate.

The titer of the phage lysate is also determined by the Weigh agar layer technique. In this case, however, only 0.1 ml of a W57 culture and 0.1 ml of a suitable dilution of the phage lysate in addition to 0.05 ml 10OmM CaCl _{2 are} added to the soft agar. Lysates with a 042 D concentration of -10 ¹⁰ pfu / ml are suitable for further processing for DNA recovery. For phage DNA recovery, the lysate is first subjected to low-speed centrifugation (10 minutes, 5,000 rpm to 6,000 rpm) to remove cell debris. The phages are then sedimented by high-speed centrifugation (2 hours, 25,000 rpm, 4 ° C.). The phage pellet is taken up in vio of the starting volume (3.5 ml) of TE buffer (50 mM Tris-HCl, pH 7.5, 10 ml EDTA) and mixed with 0.25 ml of a 10% SDS solution. The mixture is then extracted twice with 1 volume of phenol-chloroform (2: 1, buffer saturated), once with 1 volume of chloroform-isoamyl alcohol (24: 1) and once with 1 volume of ether. The phage DNA is finally precipitated with 2.5 volumes of ethanol at -7O ⁰ C resumed in 500μΙ 1OmM Tris-HCl (pH 7.5). For further purification, the phage DNA is subjected to centrifugation in the dye density gradient. The purified 042D phage DNA is finally dissolved in 1OmM Tris-HCI (pH 7.5) in a concentration of about 1 mg / ml and stored at 4 ⁰ C or frozen at -20 ⁰ C. A.2. Cloning of 042 D-DNA fragments into an E. coli plasmid vector

A sufficient amount (5k to Юmd) of purified phage 042D DNA is digested separately with the restriction enzymes EcoRI, CIaI and Hpall in known manner into fragments (0.5knp to 10knp) having single-stranded 5 'ends. In parallel, DNA of the E. coli plasmid vector pACYC184 is linearized with either the enzyme CIaI or the enzyme EcoRI. 042 D-DNA fragments made with Hpall or CIaI have compatible single-stranded ends with the Clal-ionized pACYC184 vector (analogous to EcoRI). After linking the 042 D-DNA fragments with pACYC 184 using the enzyme T4 DNA ligase, the recombinant plasmids thus generated in vitro are introduced into the recipient strain Е.СОІІ294 by standard methods of transformation. The selection for transformants is carried out by the addition of 25pg / ml chloramphenicol (or 10pg / ml tetracycline), if pACYC184 plasmids are sought with Clal insert (or with EcoRI insert). A.3. Detecting transformants with SAK recombinant plasmids

The detection of transformants with SAK recombinant plasmids is based on the detection of the formation of staphylokinase. For this purpose, the staphylokinase property is exploited to effect the activation of human plasinogen to proteolytic (and fibrinolytic) active plasmin. The transformant clones are seeded on indicator plates for the purpose of testing for possible staphylokinase formation, having the following composition:

9 ml of a 1% agarose solution in 50 mM Tris-HCl (pH 8.1) 15 mM NaCl are temperature-controlled at 48 ° C., with 1 ml of sterile skimmed milk, 0.1 ml of a human plasminogen solution (1 mg / ml) and 0.2 ml a sterile 5% yeast extract solution and poured into Petri dishes (100mm 0). The transformants to be tested are supernatant on these indicator plates and incubated overnight at 37 ⁰ C. Staphylokinase formation is recognizable at a surrounding the transformant clone, sharply demarcated Lysehof. To exclude the formation of a nonspecific protease, the positive clones are then tested on the same indicator plates, but without the addition of human plasminogen. Staphylokinase itself has no proteolytic activity, so that in the absence of human plasminogen it is possible to discriminate between protease and staphylocinase production. From the cloning experiments with pACYC 184 as a vector, a total of 11 SAK-producing recombinant plasmids were produced, 7 of which were formed by insertion of Clal fragments, 3 by insertion of Hpall fragments and one by insertion of EcoRI fragments of phage 042 D.

A.4. Restriction analysis of the recombinant SAK plasmids

From the SAK-forming transformant clones which have been purified via single colony isolation, plasmid DNA is isolated in a known manner and subjected to restriction analysis. In the first step, the enzymes used for cloning are used. Among the recombinant plasmids, plasmid pDB3 is selected for more detailed restriction mapping since it contained the smallest 042 D-DNΑ insert, an approximately 7.0 knp sized ClaI fragment. This fragment is also detectable in all other recombinant plasmids obtained with CIaI and apparently carries the SAK coding sequences. Analysis of purified pDB3 plasmid DNA with further restriction enzymes (EcoRI, EcoRV, BglII, HindIII, Accl) allows the preparation of a restriction map of the plasmid pDB3 (see Fig. 2).

B. Subcloning of the SAK-coding sequences in E. coli

The subcloning of the SAK-coding sequences is carried out here in the plasmid vector pBR322 in strain E. coli294. As donor DNA, the DNA of the primary clone pDB3 is used. Subcloning occurs in two steps, which lead to the greatest possible narrowing of the SAK gene and form the basis for the DNA sequence analysis of the SAK gene.

B.1. Construction of plasmid pDB12

Approximately 20,000 of the DNA of the plasmid pDB3 are simultaneously cleaved with the restriction enzymes EcoRI and EcoRV, and the fragments are separated by electrophoresis in an agarose gel. Among the 5 fragments obtained, there are two approximately 2.5knp fragments flanked by EcoRI and EcoRV sites, which together span most of the 042 D DNA insert (see Figure 2). Using fow-melting agarose, these fragments are excised from the agarose gel, purified and used for insertion into the plasmid vector pBR322. The vector is also treated for this purpose with the enzyme pair EcoRI / EcoRV, and the insertion of the 042 D fragments is carried out according to standard methods. Transformants of strain E. coli 294 are selected by incubation in the absence of 40 μg / ml ampicillin. Transformant clones carrying a plasmid with 042 D insert are recognizable by testing for tetracycline resistance: insert-carrying plasmids no longer confer tetracycline resistance. The tetracycline-sensitive transformant clones are tested for the formation of SAK using the assay described in 1.3. Among the SAK-producing transformant clones, some are selected for preliminary characterization of their recombinant plasmids. Among them is the plasmid pDB 12, which is expected to carry a 2.5-knot 042 D insert flanked by EcoRI and EcoRV sites.

DNA purified from standard methods of plasmid pDB12 is subjected to extensive restriction analysis. The restriction enzymes EcoRI, EcoRV, HindIII, BglII, Bstll, Accl and Rsal are used for this purpose. Part of the restriction map of the plasmid pDB12 is shown in Fig.2

 B.2. Construction of plasmid pDB 17

Restriction analysis of the plasmid pDB 12 revealed the localization of an approximately 1 knp Rsal fragment in the range of 042D DNA insert present in pDB 12. This Rsal fragment is recovered in a preparative amount by electrophoretically separating approximately 1 μg of a pDB 12 × Rsal fragment mixture in the agarose gel and extracting the desired fragment from the gel by standard methods This Rsal fragment from the 042 D-DNA Insert of pDB 12 is then ligated into the EcoRV site of plasmid pBR322 in vitro, and after transformation of strain E. coli 294, clones with recombinant plasmids are identified by their resistance to ampicillin and its susceptibility to tetracycline. Plasminogen agar plates allow the detection of SAK-forming transformant clones, thus obtaining a transformant clone carrying the plasmid pDB 17. The structure of this plasmid is shown in Fig. 3. The plasmid pDB 17 is approximately 4, 1 knp and carries a 1.0 npt DNA insert, which is spontaneously deleterious during the cloning process ion, which has removed from the pBR322 vector a 1225np fragment which includes the entire region of the tetracycline residue gene.

C. DNA sequence analysis of the SAK gene

CA. Cloning Subchannels of the SAK Gene into M 13mp Bacteriophage Vectors Purified DNA of the SAK recombinant plasmid pDB 17 is recovered in a preparative amount. 10 to 200 g of this pDB 17 DNA are completely digested with the restriction enzyme Hpall and the fragments are separated by electrophoresis on a 1% agarose gel. With the aid of Iow-melting agarose, an approximately 1.1 knp fragment is selected in a known manner among the pDB 17 Hpall fragments and finely recovered. This 1.1-knp Hpall fragment from pDB 17 includes all of the pDB17 042 D DNA insert and is flanked by a few nucleotides of pBR322 vector DNA. Such purified Hpall-SAK fragment of pDB 17 is then in separate reactions (each about 1 \ ig DNA) with the enzyme Taql or a mixture of the enzymes AIuI + Haelll (BspRI) decomposed into further subfragments, in a known manner with the double-stranded replicative form of E. coli phage Mi3mp9 or M13mp18, previously linearized with either the enzyme Accl (ends compatible with Hpall and Taql) or Hincll (ends compatible with AluI and Haelll). Transfection of the E. coli strain TG1 then allows recognition of phage plaques derived from recombinant M13 phage (white plaques, as compared to the blue plaques caused by native phage) in a known manner. A number of such recombinant phage are recovered from each subcloning experiment and propagated in a manner known per se for DNA recovery. The single-stranded DNA of the recombinant phages is subsequently used for DNA sequence analysis according to the chain termination method (Sanger technique). C.2. DNA sequence analysis of the SAK gene of 042 D

The DNA sequence of the 042 D DNA inserts of the recombinant M13 bacteriophages is determined in the known manner according to the Sanger technique. Figure 4 shows the location and length of the identified partial sequences of the 042D insert of pDB 17. All DNA sequences are determined in at least two independent experiments, and most of the sequences of both DNA strands are determined to minimize the error rate to keep. The overall sequence of the 042 D-DNA insert of pDB 17 is partially assembled by means of a computer from the single sequences of the M 13 clones. It is shown in Fig. 1. Computer analysis of the sequence then allows the only possible positioning of a gene encoding a staphylokinase-sized protein, as shown in FIG.

D. Subcloning of the SAK gene in Gram-positive bacteria

In order to express the SAK gene in Gram-positive bacteria, it is transferred to a carrier plasmid which is replicable as a natural shuttle vector in both Streptococcus and Bacillus strains. As a carrier plasmid is

for this purpose, derived from a natural Streptokokkenplasmid vector pGB3631 (size about 6.1 knp, encoded for resistance to MLS antibiotics, carries a polylinker with different unique restriction sites, see Fig. 2) isolated in preparative amount in a known manner. Purified pDB 12 DNA was also used to obtain a 2.5knp fragment using preparative agarose gel electrophoresis using the restriction enzymes BamHI and EcoRI. This fragment spans the entire 042 D-DNA insert of pDB 12 and an approximately 200 np fragment of the Tet ^R gene from pBR 322. This isolated BamHI-EcoRI fragment from pDB12 becomes pGB3631 which also cleaves with this enzyme pair was inserted, in a known manner. After primary transformation of S. sanguis Challis 57 with the recombinant plasmid, SAK-producing transformant clones are identified using the assay already performed. These transformant clones contain the plasmid pDB15 whose structure is determined by standard restriction mapping and which is reproduced in detail in FIG.

Plasmid DNA derived from SAK-positive transformant clones is subsequently used to construct further gram-positive SAK producer strains by known transformation procedures: thus, the strain Bacillus subtilis GB 500 (pDB 15) is recovered, which is capable of staphylokinase production and production. Secretion is capable. E. Synthesis of SAK in heterologous hosts; Identification of the recovered SAK gene product Hereinafter, the SAK synthesis will be carried out by the producers obtained according to the method

- S.-sanguis Challis 57 (pDB 15),

E. coll294 (pDB17) and

B. subtilitis GB500 (pDB 15).

E.1. Gene Expression in S.sanguis Challis 57 (pDB 15)

The producer strain Challis 57 S.sanguis (pDB 15) at 34 ⁰ C in a Hefeextraktnährboden (40 g / l yeast extract plus 4 g / l pancreatic casein peptone) with 1% glucose content, as well as 5 .mu.g / ml erythromycin addition at pH 7, 2 with stirring and without aeration bred.

To achieve increased staphylokinase yields, the pH is kept constant by automatic addition of 5M sodium hydroxide solution with simultaneous addition of appropriate amounts of 2.75 M sterile glucose solution.

The SAK formation occurs in the logarithmic growth phase. About 1 ^ g / ml staphylokinase was found serologically by radial immunodiffusion according to Mancini.

E.2. Gene expression in E. coil294 (pDB 17)

The formation of staphylokinase was in the above-mentioned producer strain in shake flasks (150 ml yeast dialysate medium in 500 ml round bottom flask) at 37 ⁰ C until the late logarithmic phase (5.5 hours) are detected. Later samples had no SAK activity.

After concentration of the culture supernatant and after cell disruption, only 0.75% staphylokinase per ml was found.

E.3. Gene expression in B.subtilis GB 500 (pDB 15)

The B. subtilis G B 500 (pDB15) producer strain is grown to 150 ml in 500 ml round bottom flasks at 37 ° C. in shake culture. As nutrient medium, either trypticase soy broth (30 g / l), Todd-Hewittbroth (30 g / l), yeast extract dialyzed (40 g / l) or casein peptone (20 g / l) with salt and glucose additives are selected, the culture in each case 5 kd / ті erythromycin be added.

The SAK formation occurs in the logarithmic growth phase. Up to 20 ^ g / ml of staphylokinase were found serologically by Mancini radial immunodiffusion.

Claims (48)

1. A process for the preparation of a new staphylokinase with heterologous producers using parts of standard genetic engineering techniques of DNA recombination, characterized in that DNA fragments which contain the gene for a staphylokinase (SAK gene) characterized by the nucleotide sequence according to FIG. 1 a are transferred to an E. coli cloning vector and the resulting derivatives are screened for SAK ⁺ recombinant vectors (SAK Screening) (method step I), - From each one of the SAK ⁺ recombinant vectors obtained here extracts fragments with functional SAK gene and subcloned them to obtain SAK ⁺ recombinant secondary plasmids each on a vector plasmid, which is a component of a heterologous bacterial host vector system (method step II ) each SAK ⁺ recombinant plasmid obtained in steps I and II is transformed into heterologous bacterial producer strains (process step III) and - each such genetically engineered heterologous bacterial producer strain in Submerskultivierung for obtaining the characterized by the amino acid sequence according to Fig. 1 b staphylokinase used (step IV). 2. The method according to claim 1, characterized in that in step I as a donor for the SAK gene as shown in Fig. 1 selects a temperate Staphylococcus aureus phage of the serological group F and in the recovery of the potentially SAK gene-bearing DNA Fragments of phage lysates with a titer of at least 10 ¹⁰ pfu / ml emanates. 3. Provides a natural shuttle vector capable of autonomous replication in the gram-positive bacterial genera Streptococcus and Bacillus. 3. The method according to claim 2, characterized in that one selects as a donor for the SAK gene according to Fig. 1 a temperate phage from the group {0 W46,0 L80,0 L55,042 D}. 4. The method according to claim 3, characterized in that one selects as a donor for the SAK gene according to Fig. 1 the temperate phage 042D. 5. The method according to claim 1 to 4, characterized in that one cleaves the purified DNA of the temperate S. aureus (group F) phage with the restriction enzymes CIaI, Hpall or EcoRI to obtain the SAK gene according to Fig. 1 and the thus obtained fragment mixture for the conversion to an E. coli cloning vector provides. 6. The method according to claim 1 to 5, characterized in that one uses in step I as a cloning vector an E. coli phage vector. 7. The method according to claim 6, characterized in that selecting a λ phage in step I as the E. coli phage vector. 8. The method according to claim 1 to 5, characterized in that one uses in step I as a cloning vector an E. coli plasmid vector. 9. The method according to claim 8, characterized in that one uses in step I as an E. coli plasmid vector a vector from the group of amplifiable E. coli plasmid vectors. 10. The method according to claim 9, characterized in that one selects a plasmid with CoIE 1-Replikationsorigin as amplifiable E. coli plasmid vector. 11. The method according to claim 10, characterized in that one selects such plasmid with CoIE 1-Replikationsorigin as amplifiable E. coli plasmid vector, which has at least two antibiotic resistance markers and in one of these markers at least one singular Restriktase cleavage site. 12. The method according to claim 11, characterized in that the plasmid pBR322 is selected as the amplifiable E. coli plasmid vector. 13. The method according to claim 9, characterized in that selecting a plasmid mitp15A replication origin in step I as the amplifiable E. coli plasmid vector. 14. The method according to claim 13, characterized in that is selected as the amplifiable E. coli plasmid vector such a plasmid with p15A replication origin, which has at least two antibiotic resistance markers and in one of these markers at least one singular Restriktase cleavage site. 15. The method according to claim 14, characterized in that the plasmid pACYC 184 is selected as the amplifiable E. coli plasmid vector. 16. The method according to claim 6 to 10 and 13, characterized in that one linearizes the E. coli cloning vector for the transfer according to step I with a restriction enzyme, for which a) on the vector a singular cleavage site is present and which at the same time b) generates such ends that are compatible with those of fragments of donor phage DNA. 17. The method according to claim 6,11,14 and 16, characterized in that one linearizes the amplifiable E. coli plasmid vector for the transfer according to step I with a restriction enzyme, a) whose cleavage site is located on this vector so that an insertion of foreign DNA leads to the inactivation of one of its two antibiotic resistance markers and which simultaneously b) generates such ends that are compatible with those of fragments of donor phage DNA. 18. The method according to claim 12 and 17, characterized in that one linearizes the amplifiable E. coli plasmid vector pBR322 for the transfer according to step I with CIaI. 19. The method according to claim 15 and 17, characterized in that one linearizes the amplifiable E. coli plasmid vector pACYC 184 for the transfer according to step I with EcoRI. 20. The method according to claim 15 and 17, characterized in that one linearizes the amplifiable E. coli plasmid vector pACYC 184 for the transfer according to step I with CIaI. 21. The method according to claim 1,4,5 and 20, characterized in that the obtained in step I and in the subsequent SAK screening as SAK ⁺ recombinant recognized plasmid pDB3 (molecular size about 11,0knp) after determining the location of Restriction cleavage sites for the subcloning according to process step II as well as for process step III provides. 22. The method according to claim 1 and21, characterized in that restriction enzyme for the restriction of the plasmid pDB3 EcoRI and EcoRV used and obtained after use of these enzymes 2 subfragments, each containing part of the 042 D fragment of pDB3, for the subcloning according to step II provides. 23. The method according to claim 1, characterized in that one carries out in step II subcloning 1st, 2nd and 3rd order. 24. The method according to claim 1 and 23, characterized in that one provides for the subcloning I.Ordnung an E. coli vector plasmid. 25. The method according to claim 24, characterized in that is provided for the subcloning of the first order with EcoRI and Pvull cut E. coli vector pACYC 184. 26. The method according to claim 24, characterized in that one provides for the subcloning of the first order cut with EcoRI and EcoRV E. coli vector pBR322. 27. The method according to claim 1,22 and 26, characterized in that the obtained in a subcloning 1st order within step II and in the subsequent SAK screening recognized as SAK ⁺ recombinant secondary plasmid pDB 12 (molecular size 6.8 knp) after Determining the location of restriction sites for higher order subcloning and for step III. 28. The method according to claim 1,23 and 27, characterized in that one uses for the restriction analysis of the plasmid pDB 12, the restriction enzyme Rsal and obtained after use of this enzyme subfragments of about 1.0 knp size, which is part of the 42nd Contain D fragments of pDB 12, for the subsequent subcloning according to process step II provides. 29. The method according to claim 1 and 23, characterized in that one provides for subcloning 2nd order an E. coli vector plasmid. 30. The method according to claim 29, characterized in that one provides for subcloning the second order linearized with EcoRV (or with other smooth-ending enzymes) E. coli vector pACYC 184. 31. The method according to claim 29, characterized in that one provides for subcloning 2nd order with the EcoRV linearized E. coli vector pBR322. 32. The method of claim 1.28 and 31, characterized in that the obtained in a subcloning 2nd order within step II and recognized in the subsequent SAK screening as SAK ⁺ recombinant secondary plasmid pDB 17 (molecular size 4.10knp) step III provides. 33. The method according to claim 1,23 and 27, characterized in that the E. coli secondary plasmid pDB 12 contains an approximately 2.8knp BamHI-EcoRI subfragment which contains the 042 D fragment harbored by pDB 12, takes and provides for subcloning 3rd order according to process step II. 34. The method according to claim 1 and 23, characterized in that for the subcloning 35. The method according to claim 34, characterized in that one provides for the subcloning 3rd order a natural shuttle vector from the pGB plasmid family. 36. The method according to claim 35, characterized in that one provides for subcloning 3rd order the polylinker-carrying and with BamHI (or mitzu him compatible enzymes) as well as EcoRI-cleaved shuttle vector pGB3631. 37. The method of claim 1.33 and 36, characterized in that the obtained in the subcloning 3rd order within step II and recognized in the subsequent SAK screening as SAK ⁺ recombinant secondary plasmid pDB 15 (molecular size 8,9knp) for process step III provides. 38. The method according to claim 1,21,27 and 32, characterized in that one provides each of the SAK ⁺ - recombinant plasmids pDB3, pDB12 or pDB 17 for the transformation of E. coli producer strains. 39. The method according to claim 1 and 38, characterized in that one provides each of the SAK ⁺ - recombinant plasmids pDB3, pDB 12 and pDB 17 for the transformation of the strain E. coli SK1590. 40. The method according to claim 1 and 38, characterized in that one provides each of the SAK ⁺ - recombinant plasmids pDB3, pDB 12 and pDB 17 for the transformation of the strain E. coli294. 41. The method according to claim 1 and 37, characterized in that one provides the SAK ⁺ recombinant plasmid pDB 15 for the transformation of producer strains of the genera Streptococcus or Bacillus. 42. The method according to claim 1 and 41, characterized in that one provides the plasmid pDB 15 for the transformation of producer strains of the species Streptococcus milleri var. Anginosus. 43. The method according to claim 1 and 41, characterized in that one provides the plasmid pDB 15 for the transformation of producer strains of the species Streptococcus sanguis. 44. The method according to claim 43, characterized in that one provides the plasmid pDB 15 for the transformation of the strain Str. Sanguis Challis 57. 45. The method according to claim 1 and 41, characterized in that one provides plasmid pDB 15 for the transformation of producer strains of the species Bacillus megaterium. 46. The method according to claim 1 and 41, characterized in that one provides plasmid pDB 15 for the transformation of producer strains of the species Bacillus subtilis. 47. The method according to claim 46, characterized in that one provides plasmid pDB 15 for the transformation of the strain B. subtilis GB500. 48. The method of claim 1 and 38 to 47, characterized in cultivating the genetically manipulated heterologous bacterial producers at temperatures of 25 ⁰ C to 40 ⁰ C respectively until the end of the logarithmic growth phase and the new Staphylokinase formed by known processing steps wins.