Recombinant plasmid dna encoding the complete sequence of staphylokinase, escherichia coli sa 9325 - producer of recombinant protein

 

(57) Abstract:

The invention relates to the field of Microbiology and medical industry and can be used to create medicines, in particular, for the creation of recombinant forms of staphylokinase. The proposed recombinant DNA containing the full sequence staphylokinase and leader peptide with a block of 6 his-tag residues. The strain E. coli SA 9325 producing a recombinant protein containing the full sequence staphylokinase of S. aureus. The invention allows to obtain a highly active protein having fibrinolytic activity. The yield of recombinant protein was 20-26% of the total number of proteins. 2 S. p. f-crystals, 5 Il., table 1.

The invention relates to the field of Microbiology and medical industry and can be used to create medicines.

The creation of high-strain-producer of staphylokinase will allow you to get the most powerful, compared to the existing national Academy of Sciences of and thrombolytic analogues, a drug with a high affinity for fibrin, low immunogenicity, and relatively cheap way of biotechnological production on the basis of Escherichia coli.

In connection with the above properties staphylokinase many researchers have developed strains-producers for the recombinant analogue of this protein (EP 0077664, 1983; EP 052252, 1993; EP 0563385, 1992; EP 0721982, 1996: WO 93/13209; WO 95/27048; WO 96/21016).

Closest to the technical essence and the achieved result is recombin, 1983).

Known recombinant DNA contains a portion of the sequence of staphylokinase and leader peptide and produced on the basis of E. coli.

However, the use of incomplete sequence staphylokinase can lead to a weakening of the enzymatic activity of Sak in relation to plasminogen.

The present invention is the creation of recombinant plasmid DNA containing the sequence staphylokinase, with activity similar to that of the natural variant of this protein can be a simple process of isolation and purification. Another object of the invention is the creation of producer strain on the basis of E. coli.

The problem is solved by the described recombinant plasmid DNA containing the full sequence staphylokinase and leader peptide with a block of six his-tag residues.

The problem is solved also by the creation of producer strain SA9325.

To obtain the recombinant DNA was used vector pQE30.

Synthesized primers flanking the gene sak, containing sites of restriction endonucleases that are necessary to clone the sequence amplicon synthesized in the composition of the and S. aureus on the resulting primers amplicon synthesized, bearing in its composition sak gene.

Cloned synthesized sequence of a gene sak in the composition vector plasmids pQE30. Then the DNA of the recombinant plasmid was transformed into E. coli cells, and from the obtained transformants was selected clone with optimal staphylokinase activity. After that producing strains was introduced gene repressor (lac 1) lactose operon, providing in this system inducibility synthesis of the cloned protein.

The result is a hybrid protein containing the leader sequence with a block of 6 his-tag residues and sequence of the Sak gene encoding the full staphylokinase (Fig. 1).

Spatial structure staphylokinase shown in Fig. 2.

An example implementation of the invention

Synthesis of amplicons sak gene

From the database Entez retrieved 10 sequences of the gene of staphylokinase as chromosomal and phage origin. Four of them also contained the sequence surrounding regions.

All 10 of the obtained sequences were processed in the program DNASIS to evaluate the conservativeness of the study of the a (chromosome or DNA converting phage). A high degree of homology of the nucleotide sequences of the gene sak and adjacent regions demonstrates the conservatism of this region of the genome, therefore to obtain the cloned gene could be used PCR method.

Based on these data, conducted the calculation of the optimal DNA sequences flanking the gene sak, which could be used as primers for the future synthesis of an amplicon. For each of the primers (right and left from the gene) have calculated the optimal "replacement" nucleotides for introduction of restriction sites necessary for cloning the amplicon plasmid pQE30 (see tab. A).

As can be seen from Fig. 3, the replacement of 5 nucleotides in the "left" primer provides the appearance of a restriction site for the endonuclease BamHI, and replacing 2-"right" - for enzyme Pst1. The selection of these restricted due to the presence of sites of their restriction in polylinker vector plasmids pQE30. These primers were synthesized by PCR.

Polymerase chain reaction was performed in standard buffer: 50 mM KCl, 10 mM Tris-HCI, pH of 9.0, 2.5 mM MgCl2.

PCR was performed using as a matrix gene sac DNA of six strains of S. aureus that had staphylokinase activity. As Cold Spring Harbor, NY.

The setting reaction. For greater specificity of the reaction was used primitive "hot start": add Taq polymerase (half the volume of the buffer with MgCl2) heated to 95oC tubes with the other components of the reaction mixture (containing 10 pmol of each primer, 9 ál of buffer, 25 μm each deoxyribonucleotide and 5 µl of the DNA of Staphylococcus aureus). A fragment of the expected length was detected in all samples except the control, which contained the DNA of E. coli.

Next, we used all amplicons sak gene.

Cloning sak gene in multicopying vector under a strong promoter.

The restriction and ligation of DNA amplicon DNA vector was performed by standard methods (T. Maniatis, 1984). Scheme construction of recombinant plasmids is shown in Fig. 4.

Received legirovannye mixtures containing DNA recombinant plasmid was used to transform cells of the host strain E. coli SG13009.

The present system provides that the recipient strain SG13009 (NalS StrS rifS, lac -, ara - gal - mtl - F - recA+ uvr+) must contain the plasmid pREP4, carrying a gene repressor of Lac operon - lacl and marker resistance to kanamycin (gene - kan). The product of this gene inhibition is config gene. The effect of repression due to the fact that the promotor region pQE30 in addition to the promoter of phage T5 carries two lac operator sequences, which is the object of the action of protein-Lacl repressor. The action of the repressor in inducible systems is removed by the operation of the inductor, which, in this case, you may be lactose or IPTG.

The present system provides a high level of protein synthesis. In addition, the nucleotide sequence of the plasmid pQE30 contains additional codons for synthesis in the composition of the produced protein molecule 6th additional histidine (His). Vector pQE30 contains a marker for selection - bla gene, encoding resistance to ampicillin (ApR).

Obtaining strain-producer

At the first stage of transformation used the E. coli strain GC13009 without plasmid that encodes the repressor. Conditions constitutive synthesis of proteins facilitate the selection of clones Sac+.

Transformation was performed by the following method: the cells of the host strain SG13009 were grown to early log phase in L-broth (see j.Miller 1976), then besieged by centrifugation at 1 thousand rpm for 10 minutes at 4oC, resuspendable in 1/10 volume of buffer TSB (L-broth pH 6.1 with 10% PEG mol. weight 3,350, 5% DMSO and 20 die was poured into 0.1 ml and stored at -70oC.

As necessary, the competent culture used in the following way. To 0.1 ml of culture was added the required amount legirovannoi mixture or plasmid DNA and the mixture is incubated in an ice bath at 4oC 5-30 minutes Then added to 0.9 ml of TSB with 20 mM glucose, and incubated at 37oC for one hour, then were sown on plates with selective medium: L - agar containing 50 μg/ml ampicillin.

After one day of incubation grown ApR (ampicillinresistant) clones were once cleared on the selective medium and subjected to further analysis.

Analysis of transformants

The resulting transformants were tested on staphylokinase activity (Sak+). For this night culture of the studied clones grown in nutrient broth at 37oC, was applied on the solid nutrient agar in the form of drip rassimov and incubated over night. Since literature data indicate that the protein encoded by the gene sak, in E. coli cells is mainly found in periplasm cells (T. Sako, Eur. J. Biochem. 14. 557-563, 1985), Petri dishes with raised "drops" handled pairs of chloroform to increase the membrane permeability of bacteria. Next, lysed cells by the method of replicas carried on SUP>oC). For control in the same way on agar containing plasma, caused the suspension of cells not treated with vapors of chloroform. After a day of incubation made rassimov found that part of the investigated clones of transformants both handled and unhandled (i.e. living) formed around the drip sieving zone of lysis. This positive signal testified that transformants inherited intact copy of the sak gene encoding the active form of staphylokinase (protein plazmogeneratora). This fact suggests that some protein staphylokinase excreted by the cells of E. coli strain SG13009 in the external environment. Therefore, further testing of the studied clones phenotype Sak was carried out on the living, not the processed pairs of chloroform cells of Escherichia coli.

For quantitative comparison of protein in periplasm and the supernatant three from the obtained transformants were grown in liquid nutrient medium with the necessary controls of S. aureus and E. coli 13009. Cells were besieged by centrifugation, and the resulting adosados used for the quantitative determination of the protein. In addition, the suspension resuspending cells received drugs periplasmatic faction, which ycombinator protein in periplasmatic faction on the tracks 7,8,9. In supernatant tracks 1,2,3 protein almost not detected. On the tracks 4,5 - supernatant S. aureus, 6 - control strain E. coli/pQE30, 10,11 - periplasmatic fraction of S. aureus, 12 - periplasmatic fraction of E. coli/pQE30.

The results of the analysis for further work was selected 1 clone, which showed the maximum zone of lysis.

For additional verification of the obtained results (i.e., the fact that the zone of lysis, educated living growing cells really are formed due to partial withdrawal of Sak protein in the external environment) in a liquid nutrient medium grown cells: the original clinical isolate of S. aureus, DNA which has served as a matrix for the synthesis of an amplicon, the strain of E. coli host-vector pQE30 and selected strain of transformant containing the recombinant plasmid with the cloned gene sak. In this experiment we determined the activity of staphylokinase in supernatant and periplasmatic fraction of cells that were received after the osmotic shock procedure (T. Sako, 1985). It was found that cells of strain-producer SA9-32 (this name has been selected clone) really produce partially active protein into the environment. The experiments were clearly visible zone lisitano evaluation of recombinant protein, contained in periplasmatic fraction of the investigated strain used MClCD - M4 (v 3.0 - Rev5) firm "Imaging research Inc".

It was found that the number of identified protein is 20-26% of the total number of proteins in cells. Quantitative variation can be explained by the different density of grown cell suspensions.

We created the system works in terms of constitutive protein synthesis. To create a system of inducible synthesis of plasmid DNA (pQE30) of the producer strain SA9-32 was transformed into cells of the recipient strain E. coli SG13009 carrying plasmid pREP4 with lacl gene, encoding a protein is a repressor of Lac operon (as described above).

Four of the obtained transformants selected on medium containing kanamycin (marker plasmid pREP4) and ampicillin, were tested staphylokinase activity in the presence and absence of inducer (IPTG). These clones were grown in liquid nutrient medium containing both antibiotics. Then Persiani in the same environment 1:10 and potroseni for 2 hours, then each sample was divided into two equal parts and one of them added IPTG at a concentration of 510-4and pokasivali another 1.5 - 2 hours at 37oC. Raised suspension nananne with the inductor, form a zone of lysis, indicating the presence of an active protein staphylokinase. At the same time, the clones grown in the absence IPTG, do not form zones of lysis, i.e., does not produce staphylokinase.

Obtained producing strains of Escherichia coli is characterized by the following features:

Morphological features. Cells are rod-shaped, gram-negative, risperadone.

Cultural characteristics. Cells grow well on simple nutrient media. On arape "Difco" cells form a smooth round colonies with smooth edges. With the growth in liquid nutrient medium intensive form a smooth suspension.

Physico-chemical characteristics. Cells grow in the temperature range of 8o40oC, the optimum pH of 7.4. As sources of nitrogen, carbon, and other necessary components are mineral salts, casein, peptone, yeast autolysate, etc.

Resistance to antibiotics. Cells are resistant to kanamycin and ampicillin.

The resulting strain is tested for stable preservation of the plasmid pQE30 and pREP4. It is shown that the plasmid pQE30 stably inherited in cells of strain SG13009, and plasmid pREP4 in the absence of selective pressure (kanamycin in the medium) lose is to grow the culture in the presence of the antibiotic kanamycin at a concentration of 20-25 mg/ml

Thus, the resulting strain producing a hybrid protein with staphylokinase activity and called E. coli SA9325.

In accordance with the above, the proposed invention has solved the problem of creating a recombinant DNA that encodes a protein having staphylokinase activity, which can be easily selected using metalhalide affinity chromatography due to the presence of 6 histidines in the top part. The coding sequence of staphylokinase shown in Fig. 5.

In addition, the invention solves the task of the producer strain of staphylokinase E. coli SA9325 that allows you to get 20 - 26% of the recombinant protein in relation to the total number of proteins.

1. Recombinant plasmid DNA encoding the complete sequence of staphylokinase Staphylococcus aureus c leader peptide consisting of six his-tag residues and having a nucleotide sequence shown in Fig.5.

2. The Escherichia coli strain SA 9325 producing a recombinant protein containing the full sequence staphylokinase Staphylococcus aureus c leader peptide consisting of six his-tag residues having the amino acid sequence shown in Fig.

 

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