DD250546A1 - METHOD FOR THE FERMENTATIVE MANUFACTURE OF A STREPTOKINASE - Google Patents

Abstract

The strain Streptococcus sanguis (Challis) (pSM 752), (DSM 3140, ZIMET 10959), which produces streptokinase, a unique streptokinase which can be obtained therefrom by fermentation, the preparation thereof and its pharmaceutical use, especially for the treatment of thromboembolic occlusions, are described. The fermentation is carried out in known steps using a Streptococcus strain of serological group H, preferably the strain Streptococcus sanguis (Challis) (pSM 752) (DSM 3140), which is capable of expression of a streptokinase gene after transformation with the plasmid pSM 752 obtained by gene manipulation. The result is a unique streptokinase with a molecular mass of about 42,000.

Description

Field of application of the invention

The invention relates to a process for the preparation of a streptokinase by means of submerged fermentation. The field of application of the invention lies in the microbiological-pharmaceutical industry.

Streptokinase is a therapeutic agent introduced in human medicine. This protein catalyzes the conversion of the enzymatically inert plasminogen of the blood plasma to the enzymatically active plasmin, which solubilizes the fibrin network of blood clots by limited proteolysis. This fibrinolytic reaction is based on the use of streptokinase, a major therapeutic in the treatment of thromboembolic occlusions (venous and arterial thrombosis, thrombophlebitis, pulmonary embolism, brain emboli, etc.).

Characteristic of the known technical solutions

For the production of streptokinase, submerged fermentation methods are known in which strains from the group of pathogenic streptococci of the serological groups A, C and G, but preferably strains of the genus Streptococcus from the serological group C, for example strains of the species Streptococcus equisimilis, as producers be used. Streptokinases prepared in this way are marketed, for example, under the trade name Awelysin (see patent specification DD Π1 096), Streptase or Kabikinase

 The following disadvantages are characteristic of these fermentation processes for producing streptokinase:

- The streptokinase former used so far provide high levels of bacterial toxins, eg. B. Streptolysin 0, Streptolysin S.

- From the previous microwave manufacturing process, the active ingredient streptokinase can be obtained only after extensive purification steps in pyrogen-free form.

- The currently used streptokinase-formers had to do even with conventional genetic methods carried out measures to increase the synthesis performance so far, as no knowledge of the genetics of the builders in general and the genetic basis of streptokinase synthesis in particular could be used.

Since the presentation by H.Mal'ke at the 35th GATM Conference, Reinhardsbrunn (GDR), October 1983, a genetic engineering method for streptokinase is known, which contains in detail:

a) a partial method for the isolation of a streptokinase (short: skc) gene from the chromosomal DNA of the donor strain S. equisimilis H46A and for the first cloning of this gene in E. coli phage vectors, a number of skc-carrying phage clones, including one designated AL47E skc Clone, were recovered;

b) then a partial method for constructing a primary skc ⁺ recombinant plasmid (plasmid pMF1), which is inserted into the unique Hind HI site of E. by inserting the 7.4 kb skc ⁺ fragment obtained after partial Hind HI digestion of clone AL47E skc. coli plasmid pBR322 is produced,

and based on that

c) partial procedure for the construction of further derived skc ⁺ recombinant plasmids for E. coli, Streptococcus (serol group H) and B. subtilis, it being proved that after transformation with such skc ⁺ recombinant plasmids prepared in accordance with the method, the respectively assigned strains of these heterologous Bacterial genera are capable of producing streptokinase on a qualitative scale.

Among other things, the shuttle plasmid pSM752 has become available through the said production process, a bifunctional chimera between pBR322 and the streptococcal plasmid pSM7 (see H.Malke, FEMS Microbiology Letters 11 [1981] p. 27ff.), In the PstI site of pBR322 contains a skc-carrying fragment and has the potency to synthesize a streptokinase by both selected E. coli strains, e.g. B. HB101, as well as selected Streptococcus (group H) strains such. S. sangius (Challis) 6.

The method outlined above is limited to the provision of a group of recombinant plasmids carrying a skc gene and a group of related transformed bacterial strains which are indicated to produce a streptokinase on a qualitative scale.

Object of the invention

The aim of the invention is to produce a novel, definitely unique spectrokinase by fermentation.

Explanation of the essence of the invention

The invention has for its object to describe a technologically simple fermentation process for a new streptokinase, which makes it possible to produce this product using a heterologous, previously known as streptokinase-formers microorganism of readily available and cheap starting materials as a unique component.

According to the invention, this object is achieved in that under sterile culture conditions in a liquid nutrient medium with carbon and nitrogen sources and mineral salts a heterologous, new streptococcal strain of the serologic group H, preferably a strain of the species Streptococcus sangius and in particular the strain S. sangius (Chalis) ( pSM752) into which a streptokinase gene from the donor strain S. equisimilis H46A has been inserted by means of genetic engineering methods.

The mentioned microorganism S. sanguis (Challis) (pSM 752) is described in connection with an earlier patent application in the ZIMET depository for microorganisms, DDR, 6900 Jena, Beutenbergstr. 11, under registration number ZIMET 10959.

A thus characterized new group H streptococcal strain, preferably strain ZIMET 10959, is at a temperature of 25 ⁰ C to 40 ⁰ C, preferably from 3O ⁰ C, and at a pH in the range of pH 6.0 to pH 8, 0, preferably at pH 7.2, for a period of 10 hours to 36 hours, preferably 24 hours fermented.

The nutrient medium to be used contains as carbon source sugar, preferably glucose, as nitrogen source hydrolyzed casein, but preferably yeast autolysate, as well as common mineral salts and optionally vitamin supplements.

The sugar content during fermentation should be kept constant at a value in the range from 0.2% to 2%, preferably at the value of 1%. The yeast autolysate preferably used as the nitrogen source may have a nitrogen content of 0.1% to 0.8%, preferably 0.2 to 0.4%. The fermentation can be carried out in round bottom flasks of various contents, in glass fermenters and in V2A steel tanks.

The isolation of the novel unical streptokinase produced according to the method from the fermentation solution takes place in a manner known per se, for example according to the process described in the patent DD121 522.

The recovered new streptokinase activates human plasminogen to plasmin; it precipitates with rabbit specific antistreptokinase serum.

The resulting streptokinase has an isoelectric point in the range of 5.0 to 5.5 and a molecular weight of about

42000. (The comparative product Awelysiη, however, is characterized by a molecular weight of about 47,000).

The invention has the following advantages:

Obtained is a unique, by a gene-determined streptokinase product.

- Streptococcal toxins, such as hemolysins, are not co-formed in the present process.

embodiment

The streptococcal strain ZIMET 10959 is maintained on 2% agar with 3.7 g / 100 ml Brain heart infusion plus 5 / xg / ml erythromycin. Of these, 5 ml of yeast autolysate agar were inoculated as the 1st preculture. These were used after 20 hours of incubation at 37 ° C to inoculate the 2nd preculture of 250 ml of yeast autolysate agar. Both precultures contain 5 ^ g / ml erythromycin.

The culture solution consists of 40 g / l yeast autolysate in water according to patent DD111096. It is sterilized by autoclaving, followed by 1% glucose in the form of sterile 50% solution.

250 ml of preculture are used to inoculate 50 liters of the main culture.

Is cultured under stirring at 3O ⁰ C without addition of antibiotics. The pH of 7.2 and the glucose level of 1% is automatically regulated by pumping 20g / 100ml sodium hydroxide or 50g / 100ml glucose solution. The culture solution obtained after 24 hours contains 1 600 IU / ml streptokinase; their work-up is carried out on the basis of the patent DD121 522, wherein the concentration step is similar to the patent DD126342 with silica gel. In detail, the procedure is as follows:

50 liters of streptococcus culture are added to kill the cocci with 250 ml of 30% H ₂ O ₂ and stirred for 1 hour.

Subsequently, the cocci are separated. From the culture filtrate, the streptokinase is adsorbed by stirring in 1 kg of silica gel B1 (0.1 to 0.4 mm). The silica gel, washed intensively with water, is used to elute streptokinase with 5 liters of a solution consisting of 1% Na ₂ CO ₃ in 10% ethanol-water. From the eluate streptokinase is precipitated by the addition of 500 g NaCl and pH reduction to 4.0. The separated precipitate is dissolved in water at pH 8.0 (about 300 ml). The solution is stirred three times with freshly precipitated CaHPO ₄ -GeI at pH = 7.0 (about 4g). The streptokinase is precipitated therefrom with ammonium sulfate between 25% and 45% saturation. The separated ammonium sulfate precipitate dissolved in H ₂ O is finally fractionated on Sephacryl S200 (50 cm χ 5 cm column, 0.5 M NaCl, 0.01 M phosphate buffer, pH = 7.0) and the streptokinase-containing fractions are pooled.

Yield: 170 ml with 9.0 10 ⁶ IU yield and ca. 50000 IU / mg protein.

The characterization was carried out by sodium dodecyl sulfate electrophoresis according to WEBER, K. and OSBURN, M. (J. Biol. Chem.

244,4406 [1969]); In this case, a uniform substance was found with a molecular weight of about 42,000.

Claims (3)

1. A process for the fermentative production of a streptokinase, characterized in that For the fermentation, a streptococci strain of serological group H transformed with the plasmid pSM752 obtained by genetic engineering was used, and - After completion of the fermentation, the formed unical streptokinase with a molecular weight of about 42 000 isolated in a known per se 2. The method according to item 1, characterized in that a strain of the species Streptococcus sanguis transformed with the plasmid pSM752 is used for the fermentation. 3. The method according to item 1 and 2, characterized in that the strain Streptococcus sanguis (Challis) (pSM 752) ZIMET 10959 is used for fermentation.