DD282709A5 - PROCESS FOR MICROBIAL PRODUCTION OF RECOMBINANT SEROTYPE C STREPTOKINASE - Google Patents

Abstract

The invention relates to a process for the improved microbial production of recombinant serotype C streptokinase (short name: SKC) in gram-positive and gram-negative bacterial producer strains. Serotype C streptokinase is a thrombolytic agent introduced in human medicine. The invention aims to produce a recombinant streptokinase which is identical to that formed by the wild strain Steptococcus equisimilis H46A in an economical manner. The technical problem assigned to this purpose is solved by using {recombinant serotype C streptokinase; Protein SKC; thrombolytic agent; bacterial producer strains; Expression plasmid of the pMLS family; Expression plasmid pMLS10; Derivative of a multicopy shuttle vector; native expression signals of the skc gene}

Description

For this 3 pages illustrations

Field of application of the invention

The invention relates to a method for improved microwave production of recombinant serotype C streptokinase in bacterial producer strains, wherein the product formed in each process variant with the. Streptococcus equlslmilis H46A is identical to streptokinase. Streptokinase is a thrombolytic agent introduced in human medicine

 The field of application of the invention lies in microbiological-pharmaceutical research and industry.

Characteristic of the known state of the art

For the production of recombinant serotype C streptokinase in bacterial producers, methods are known in which strains of the genera Escherlchia, Streptococcus and Bacillus are used as producers (Malke et al., Mol. Gen. Genet., 196, 366-363, patents DD 249037, DD 249493 and DD 250546). The producer strains carry here recombinant serotype C streptokinase specifying plasmids, which are derived from the pMF and the pSM plasmid family {see also H. Malke, FEMS Lett. 11.27 to 31.1981). As preferred prototypes of skc-recombinant (carrier) plasmids from these families, the vectors pMF2, pMF5 and pSM752 have proven over the years. The following disadvantages exist for the recited methods for the production of recombinant serotype C streptokinase:

- The previously used recombinant, serotype C streptokinase specifying plasmids have a relatively high molecular weight and are present in the respective host cell only in a low copy number.

The use of the recombinant bacterial production strains obtained according to the previous methods yielded in each case only relatively low yields of serotype C streptokinase.

Object of the invention

The invention aims to produce a recombinant serotype C streptokinase, which is identical to that formed from the wild strain Streptococcus equisimilis H46A, in a more economical manner.

-3- 282 709 Explanation of the essence of the invention

The invention is based on the object to describe a genetic engineering microbiological process, with the help of heterologous bacterial producer strains after transformation with new expression plasmids recombinant, in o.g. Meaning authentic serotype C streptokinase (short name: SKC) can be obtained in high yields. According to the invention this object is in its basic feature using standard operations of genetic engineering. DNA linkage and the submerged fermentation of recombinant bacterial microorganisms in such a way that both Gram-negative and Gram-positive bacterial strains after their transformation with a skc-recombinant expression plasmid, which is a derivative of a Streptococcus-Escherichia coli multicopy shuttle vector and on the other hand a DNA which is regulated by its native expression unit and carries the DNA coding for the amino acid sequence of the protein C-streptokinase (shorthand: gene skc), and after completion of the fermentation becomes the gene product

- Released from the grown cells using gram-negative recombinant bacterial producer strains or

- isolated from the culture fluid using gram-positive recombinant bacterial producer strains.

In the further embodiment of the basic feature of the present technical solution, in each of its variants for the construction of the new skc-recombinant expression plasmid to be provided according to the method - these DNA molecules are referred to as plasmids of the pMLS family - on the one hand a polylinker-bearing Streptococcus Escherichia coli. multicopy shuttle plasmid vector of minimized molecular size, preferably one such polylinker-bearing multicopy shuttle vector of the pLM family. pLM expression vectors of this label, such as the shuttle plasmids pLM2 and pLM3, have become available as a result of a recent process invented central institution. Each of these pLM vectors contains a polylinker which has cleavage sites for restriction enzymes as common as EcoRI, BamHI, Salt and PstI.

On the other hand, in the further embodiment of the invention for the construction of the respective skc-recombinant pMLS expression plasmid, the required, regulated by its native expression unit gene skc from a recombinant carrier plasmid pMF or pSM family, such as learning plasmid pSM752 as 2438th bp large Sall-PstI fragment or as 2568bp PstI-PstI fragment taken. All skc recombinant carrier plasmids included. the streptokinase gene isolated from the chromosomal DNA of the serotype C donor strain Streptococcus equlsimllis H46A.

After these preparatory operations, the DNA fragment with the thus labeled gene skc is inserted by ligation into the polylinker of the pLM multicopy shuttle vector used in each case, and each in the subsequent screening step (is preferably carried out in an E. coli system). The novel DNA molecule recognized as a skc-recombinant multicopy shuttle expression plasmid of the pMLS family is provided according to the method for the transformation of bacterial recipient strains. For transformation with a pMLS shuttle expression plasmid thus obtained

- From the field of Gram-negative bacterial strains preferred recipient from the species Escherlchia coil and

- From the field of Gram-positive bacterial strains preferred recipient from the species Streptococcus sanguis, Streptococcus bactis and Bacillus subtilis

used, d. H. In each of its variants, in the present process, the production of SKC takes place by way of the synthesis of this protein by heterologously transformed producer organisms which by their nature are unable to form streptokinase.

For the genetic engineering process step, the above-discussed inventive solution is still present in a particular embodiment. This is characterized in its introduction step in that the plasmid pLM3 (molecular size 4,6kb, phenotype marker: erythromycin-lincomycin resistance gene) is used for expression plasmid construction from the collection of polylinker-bearing Streptococcus Escherichia coll multicopy shuttle vectors. This shuttle vector has proven to be advantageous for the expression of gene products by the increased copy number, in which it is present in each cell in Streptococcus and Bacillus, as well as its sufficient stability even in fermentations without selection pressure. In a ligation mixture, the 4.2 kb PstI-PstI main fragment of this vector is fused with the skc gene provided from the carrier plasmid pSM752 as 2568 bp PstI-PstI fragment; and in a screening step (performed in an E. coli recipient system) (after clones expressing both erythromycin resistance and SKC synthesis in the klasein / plasminogen overlay assay), the plasma products among such clones are Isolated DNA molecules in the 6.7 kb size class and established as a new skc-recombinant multicopy shuttle expression plasmid pMLS10 (see also Fig. 1).

In connection with the sub-step "construction of the expression plasmid pMLSIO" of the present technical solution it should be pointed out that on the occasion of earlier patent applications of the Central Institute in the depository for microorganisms of the GDR, Central Institute for Microbiology and Experimental Therapy of the AdW, Beutenbergstrasse 11, Jena, DDR 6900 already the following deposits

Name of the strain Registration number

Streptococcus sangius Challis 6 (pSM752) ZIMET10 95 lbs

Escherichia coli TG1 (pLM3) IMET11 308,

which make the recombinant starting plasmids pSM752 and pLM3 available to the skilled artisan.

In the preferred embodiment, the polylinker-bearing multicopy shuttle expressing plasmid pMLS10 obtained in the above method step is primarily useful in over-expressing the ability to synthesize recombinant serotype C streptokinase for Gram-positive heterologous bacterial pollen, preferably strains from those already mentioned Bacterial species from the genera Streptococcus üowie Bau.'Ss and in particular to the recipient Str. Sangius Challis 6, Str lactis. VIG1363 and 8. subtilis used GB500. The present after the respective transformation recombinant bacterial strains, in particular the recombinant strains

St. Sanglus Challis 6 (pMLSI 0),

St lactls MG13G3 (pMLSIO) as well

B. "ubtilis GB500 (pMLSIO),

be in this embodiment in submerged culture (in the case of said pMI S10 recombinant Streptococcus strains) or in aerated Submerekultur (in the case of said pMLSIO-recombinant B. subtllls strain) in a BHI medium nlt Erythromycin · and optionally with Glucose additive for a period of preferably 24 hours at 36 ° C (in the case of Streptococcus strains) or at 3O ⁰ C (in the case of B ubtllis strain) incubated and the gene product SKC isolated from the culture fluid. Using a standard well plate assay based on the casein plasminogen technique, on the one hand, the SKC-authentic casein-cleavage effect of the gene product was verified and, on the other hand, a sufficiently accurate quantitative assessment of SKC formation was made in the selected culture period. As can be seen from FIGS. 2 and 3, the novel pMLSI O expression systems obtained in the above-described method in gram-positive heterologous bacterial producer organisms already under the exemplary cultivation conditions described above increase the level of recombinant serotype C streptokinase produced by 75%. up to 100% exceeded.

Ausführungsbelspiele

The following statements are intended to illustrate the individual steps of the method for the microwave production of recombinant serotype C streptokinase (SKC) using two examples. However, they do not contain any detailed information on the standard methods used in genetic engineering and microbiology (eg extraction of plasmid DNA, disruption of DNS rr.U restriction enzymes, characterization of plasmid DNA and plasmid DNA fragments by means of agarose gel). Electrophoresis, recovery of DNA fragments from agarose gels, insertion of gene sequences into vector plasmids, transformation of bacterial recipient strains with plasmid DNA, cultivation of microorganisms, etc.). Such methods are known to those skilled in the art and described in detail in a number of publications, for example, in: "Molecular Cloning, a Laboratory Manual" (T.Maniatis, E.F. Fritsch, J. Sambrook) Cold Spring Harbor Laboratory, New York, 1982.

example 1

1.1) Provision of vector DNA and skc gene

First, the required amounts of plasmid pLM3 DNA (about 5 μg) are isolated from a culture of E. coil JM101 (pLM3), purified and dissolved in TrisHCl buffer. Similarly, plasmid pSM752 DNA (about 10 μg) is isolated from a culture of E. coil JM101 (pSM752), purified and dissolved in TrisHCl buffer.

1.2) Construction of the Expression Plasmid pMLSIO

First, the plasmid pLM3 is digested with PstI. The resulting 2 fragments are separated in an agarose gel and a 4.2kb fragment is isolated and purified.

By digesting the plasmid id pSM76 with the restriction enzyme PstI and subsequent separation of the fragments obtained in the agarose gel, a 2568 b, -) large fragment is isolated and purified. This fragment contains the gene skc regulated by its native expression unit, including flanking DNA sequences.

The fragments thus obtained are added to a ligation mixture. From the obtained in this ligation reaction mixture, a recombinant plasmid is separated in such a way that transformed with samples of the mixture competent cells of the recipient strain E. coil JM101, Erythromycinresistente clones selected and these clones with a

Casein plasminogen softener can be overcoated. All clones forming lightening zones (halos) in this casein plasminogen agar are subsequently isolated and screened for DNA molecules of size 6.7 kb.

Plasmid DNA samples of such transformant clones are then subjected to digestion with the enzymes PstI, EcoRI and HindIII and then those pMLSI O-type plasmid DNA samples are identified (molecular size 6.7 kb; size of PstI digest Inserts to be determined is 2.5kb, see Fig. 1).

1.3) Transformation of the recipient strain SR sanguls challis 6 with the expression plasmid pMLSI 0

First, required amounts of the plasmid pMLSIO (about 5 \ ig) are obtained from a culture of the strain E. coil JM101 (pMLSIO). Samples of this plasmid DNA are then used to transform the recipient strain S. sanguis challis 6 in a manner known per se and to screen for erythromycin-resistant clones. These clones are then overlaid with a casein plasminogen soft agar and selected for clones that form farms in this soft agar. From such clones, plasmid DNA is isolated and subjected to digestion with PstI, EcoRI and HindIII. Following a restriction analysis, those pMLS10-type DNA samples are identified (molecular size 6.7 kb, the size of the insert to be detected by PstI digestion is 2.5 kb). In this way it was possible to verify that the transformant clones obtained in the previous sub-step actually contained the plasmid pMLSIO.

1.4) Cultivation of the Recombinant Producer Strain sanguls Challis 6 (pMLSIO) and Proof of Expression To obtain SKC, the recombinant producer strain Str. Sanguis Challis 6 (pMLSIO) is used. The culture medium is BHI medium (DIFCO) at a concentration of 30 g / l. 2% glucose and 5 μg / ml erythromycin are added to the medium. The cultivation takes place as main culture.

First, a culture (100 ml) is incubated by inoculation with a single colony of the strain S. sanguis Challis6 (pMLS10) until reaching the late stationary phase (approximately 24 hours) as a stand culture at 36 ° C. Growth and SKC formation are measured by periodic sampling. The samples taken to determine SKC formation are centrifuged and the culture supernatant is passed to a standard well plate test. The SKC standard sample is purified SKC (strain Streptococcus equisimilis H46A) with an activity of 1500 U / ml. As a measure of the SKC activity of the individual samples, the diameter of the whitening zone (Hof) obtained in the perforated plate test is used. Under the conditions of this embodiment, about 1000 U / ml of SKC was contained in the culture supernatant obtained after 24 hours of cultivation (see Fig. 2).

Example 2

2.1) Transformation of the recipient strain Str. Lactli MG1363 with the expression plasmid pMLSIO

Required amounts of the expression plasmid pMLSIO constructed according to 1.1) and 1.2) are obtained from a culture of the strain E. coli JM101 (pMLSIO). Samples of this plasmid DNA are then used to transform the recipient strain Str. Lactis MQ1363 in a manner known per se and to screen for erythromycin-resistant clones. These clones are then overlaid with a casein plasminogen soft agar and selected for clones that form farms in this soft agar. From such clones, plasmid DNA is isolated and subjected to digestion with PstI, EcoRI and HindIII. Following a restriction analysis, those pMLS10-type DNA samples are identified (molecular size 6.7 kb, the size of the insert to be detected by PstI digestion is 2.5 kb). In this way it was possible to verify that the transformant clones obtained in the previous sub-step actually contained the plasmid pMLSIO.

2.2) Cultivation of the recombinant producer strain Str. Lactis MG1363 (pMLS10) and expression detection

To obtain SKC, the recombinant producer strain Str. Lactis MG1363 (pMLS10) is used. The culture medium is BHI medium (DIFCO) at a concentration of 30 g / l. 2% glucose and 5pg / ml erythromycin are added to the medium. The cultivation takes place as main culture.

. First, a culture (100 ml) by inoculation mi is: a single colony of the strain Str lactis MG1363 (pMLS10) until reaching the late stationary growth phase (about 24 hours) incubated as a static culture at 36 ⁰ C. Growth and SKC formation are measured by periodic sampling. The samples taken for the determination of the SKC formation are centrifuged and the culture supernatant is fed to the mentioned standard perforated plate test. Under the conditions of this embodiment, the culture supernatant obtained after 24 hours of culture contained about 750 U / ml SKC (see also Fig. 3).

Claims (16)

1. A process for the microwave production of recombinant serotype C streptokinase (short: SKC) by means of recombinant bacterial microorganism using standard DNA engineering and submerged fermentation operations, characterized in that - with a skc-recombinant expression plasmid, which is a derivative of a Streptococcus Escherichia coli multicopy shuttle vector and a regulated by their native expression unit, for the amino acid sequence of the protein C-Streptokinase encoding DNA (in short: gene skc), transformed microorganisms cultivated and - The gene product released from the grown cells or isolated from the culture fluid. 2. The method according to claim 1, characterized in that one uses for the construction of the respective skc-recombinant expression plasmid a Polylinker-bearing Streptococcus Escherichia coli multicopy shuttle plasmid vector of minimized molecular size. 3. The method according to claim 2, characterized in that one uses for the construction of the respective skc-recombinant expression plasmid a polylinker-bearing Streptococcus Escherichia coli multicopy shuttle vector of the pLM family. 4. The method according to claim 3, characterized in that one uses the polylinker-carrying multicopy shuttle vector pLM 3 for the construction of the respective skc-recombinant expression plasmid. 5. The method according to claim 1 to 4, characterized in that one takes the required for the construction of the respective skc-recombinant expression plasmid, regulated by its native expression unit gene skc from a recombinant carrier plasmid of the pMF or the pSM family. 6. Method according to claim 5, characterized in that the gene skc regulated by its native expression unit is taken from the carrier plasmid pSM752. 7. The method according to claim 6, characterized in that one takes from its native expression unit renulierte gene skc the plasmid pSM752 as a 2438 bp Sall-Pstl fragment. 8. The method according to claim 6, characterized in that one removes the gene regulated by its native expression unit skc the plasmid pSM752 as 2568 bp Pstl-Pstl fragment. 9. The method according to claim 1,3 and 5, characterized in that one - Inserted from a skc-recombinant pMF or pSM carrier plasmid, regulated by its native expression unit gene skc inserted into the polylinker of a pLM multicopyshuttle-Vaktors and Each pMLS family skc recombinant expression plasmid provides for the transformation of bacterial recipient strains. 10. The method according to claim 3, 6 and 9, characterized in that one - Inserted from the skc-recombinant carrier plasmid pSM752, regulated by its native expression unit gene skc inserted into the polylinker of a pLM multicopy-shuttle-Vaktors and Each pMLS family skc recombinant expression plasmid provides for the transformation of bacterial recipient strains. 11. The method according to claim 4, 8 and 10, characterized in that the gene skc taken from the carrier plasmid pSM752 as 2568 bp PstI-PstI fragment is inserted into the polylinker of the multi-'opy shuttle vector pSM3 and the obtained skc recombinant expression plasmid pMLSIO (molecular size 7.3 kb; phenotype marker: erythromycin-lincomycin resistance gene) provides for the transformation of bacterial recipient strains. 12. The method according to claim 1,9 or 10, characterized in that one each providing a Gram-negative bacterial strain for transformation with a skc-recombinant pMLS expression plasmid, and - releases the gene product after culturing from the grown, transformed microorganism cells. 13. Method according to A: \ apruch 1, 9 or 10 and 12, characterized in that - For the transformation with a skc-recombinant pMLS expression plasmid each provides a bacterial strain of the species Escherichta coil and << - releases the gene product after culturing from the grown, transformed microorganism cells. 14. The method according to claim 1, 9 or 10, characterized in that - For the transformation with a skc-recombinariten pMLS expression plasmid each provides a gram-positive bacterial strain and - The gene product isolated after incubation of the transformed cells from Kultu! fluid. 15. The method according to claim 1,9 or 10 and 14, characterized in that one - To transform with a skc-recombinant pMLS expression plasmid in each case a gram-positive bacterial strain from one of the species Streptococcus sanguls,
Streptococcus lactis or
Bacillus subtilis
provides and - The gene product is isolated after incubation of the transformed cells from the culture fluid. 16. The method according to claim 11 and 15, characterized in that one - for transformation with the skc-recombinant expression plasmid pMLSIO one bacterial strain each from the collection St. Sanguis Challis 6,
Str lactis M G1363 or
B. subtilis GB500
provides and after incubation of the transformed strains St. Sanguis Challis 6 (pMLSIO),
Str lactis MG 1363 (pMLSIO) or
B. subtilis GB500 (pMLSIO)
isolated the gene product from the culture fluid.