The method of producing biomass recombinant strains of escherichia coli bacteria enriched polypeptides with the properties of human cytokines

 

(57) Abstract:

The invention is intended for the biosynthesis of cytokines on human factors tumor necrosis alpha and beta, granulocyte and granulocyte-macrophage colony-stimulating factor) by recombinant strains of E. Li SG20050/pTNF322, E. coli SG20050pLT21, E. coli SG20050/pGGF8 and E. coli SG20050/p280GM. To obtain seed with a high content of plasmadynamic cell inoculum treated with ampicillin at a concentration of 400 to 500 µg/ml for 2-3 hours In the fermentation process in the environment add ampicillin to a concentration of 100 μg/ml Ampicillin added every 1-2 h of culture growth. The invention reduces segregation instability of recombinant strains and increases the content of the target proteins in the biomass. 5 C.p. f-crystals, 1 Il., 6 table.

The invention relates to biotechnology and Microbiology and is a method of obtaining biomass of recombinant strains of E. coli containing plasmid DNA encoding the biosynthesis of cytokines with the properties of factors of tumor necrosis (TNF-alpha and TNF-beta), granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) human and bearing as a selective marker gene bla (ostannih cytokines: TNF-, TNF, G-CSF and GM-CSF, for their detailed research and application in medical practice.

Cytokines are proteins produced mainly activated cells of the immune system and are mediators of cell-cell interactions in the immune response, hematopoiesis, inflammation, and intersystem interactions. Traditionally cytokines are divided into several groups: interleukins, interferons, tumor necrosis (TNF), colony stimulating factors (CSF) and others In the body they are interconnected, forming a single solid cytokine network [1,2]. All this makes the perspective a detailed study of these proteins and its application in medicine [3-7] and, as a consequence, the actual development of methods for their preparation.

Known methods for producing cytokines, based on the use of cultural liquid producing their cell lines [8-13]. However, these methods are time-consuming, difficult scalable, does not allow to obtain proteins in large quantities; and only the technique of recombinant DNA resulted cytokines in amounts available for large-scale research and application in medical practice. Methods preparation of recombinant cytokines microbiologist One of the first fundamental stages of technology for proteins is the stage of producing biomass cells recombinant strain-producer. Usually, competent cells of strain-recipient (often cells of Escherichia coli) transformed with the plasmid carrying the gene of the target protein and selective genetic marker (usually a gene of resistance to the antibiotic). Transformants are grown on medium containing the antibiotic and then multiply the transformed cells in the fermenter with the purpose of producing biomass. Often when growing cells need to carry out the induction of the promoter for the gene expression of the target protein using a special inductors or fever [15,19-22] , which leads to more complicated and expensive manufacturing techniques. The most technologically advanced, when the target protein is expressed during cell cultivation continuously, without induction [16,17, 24-32]. However, practice shows that the process of obtaining high-quality biomass recombinant strains producing highly unstable, the content of the target protein in it is small (from 3% to 10% of the total cellular proteins), as well as greatly varies from experience to experience [10,22,24,30,32] . The main cause of instability biomass productivity is the segregation of plasmids and selective advantage in the speed of propagation of cells containing plasmid [33-37]. To improve the quality of biomass recombinantly and used to obtain the supernatant most productive of them, as inoculum to use culture in the first third to mid - logarithmic phase of growth, increase the selective pressure on the cells during fermentation by increasing in the concentration of antibiotic selection during culture growth optimum temperature for the synthesis of the desired metabolite. The first two recommendations of the authors are undisputed and are applicable for all investigated recombinant strains of E. coli that produce TNF-, TNF-, 2 - interferon. However, the increase in the concentration of the antibiotic to enhance selective pressure used successfully in [35] for culturing E. coli strain SG20050/pIFN16 producer leukocyte interferon 2, it is not always reasonable, because it can lead to a sharp decrease in the rate of crop growth and reduced biomass yield [34] . The temperature change in cultivation of recombinant strains can lead not only to changes in the level of synthesis of the target protein, but also to change its conformational state [37,38]. This option is in each case requires a special investigation.

The closest (prototype) of the claimed method is a method of producing biomass recombinant strain of E. coli bacteria enriched polypeptide with svojstvennoe content in biomass in 2,0-2,5 times is achieved by selection of highly productive clones of cells transformed to obtain seed, of obtaining seed in a dense environment with the addition of a second antibiotic is chloramphenicol order to increase amplification of plasmids and biomass harvesting cells in the late logarithmic phase of growth culture. The combination of these techniques allows to obtain the biomass content of the target protein 10-16% of the amount of cellular proteins. The disadvantages of the method include low manufacturability at the stage of obtaining inoculum (inoculum), because getting it on a solid nutrient medium hard scales, and narrow specialized receiving increasing plasmid DNA in strain-producer, suitable only for improved recombinant strains containing plasmid gene for resistance to chloramphenicol.

The technical object of the present invention is to stabilize the biosynthesis of groups of human cytokines (TNF-, TNF-, G-CSF and GM-CSF) and increase their percentage content in the biomass of recombinant strains containing as a selective marker gene-lactamase (resistance to ampicillin, Apr).

The problem is solved by processing the seed ampicillin in high concentrations (400-500 µg/ml) to destroy nekobus The essence of the proposed method of biomass production of recombinant strains of E. coli consists of the following:

The plasmid containing the target gene cytokine (pTNF311 or pLT21 or pGG8 or 280GM), transform competent cells of Escherichia coli SG20050, transformants pokasivaut on solid selective medium (Ap, 50-150 µg/ml) for 16-18 h at a temperature of 30-32oC. Colonies of transformants examined for the content of the target protein. The most productive sow in liquid selective medium (Ap, 50-150 µg/ml) and grown to one-third the mid - logarithmic phase of growth. Then in the culture, add ampicillin to a concentration of 400-500 µg/ml and incubated for 2-3 hours Obtained by culture of putting the fermenter with a selective nutrient medium (Ap, 100 μg/ml), and cells grown to late logarithmic phase of growth. During fermentation periodically in 1-2 hours add ampicillin at a concentration of 100 µg/ml of medium. At the end of the logarithmic phase of growth, the cells are harvested by centrifugation and used for allocation of cytokine immediately or after storage at -70oC.

Analysis of inoculum (inoculum) on the content plasmadynamic cells (Apr) showed that their number varies from experience to experience, from 60 to 80% (table. 1), i.e., about 20 to 40% of the population of cells at the stage of obtaining poseuses in the fermentation process, displace plasmodiidae cells, which will lead to low-quality biomass.

To remove besplatnih cells from the supernatant using the processing ampicillin in elevated concentrations of 400-500 µg/ml for 2-3 hours it has been shown (PL.2) that after such treatment besplatnye E. coli cells SG20050 (Apsalmost completely lose their viability. Analysis of the culture fluid by protein electrophoresis shows that the cells lyse (proteins go into the culture fluid) (Fig. 1). The capacity for growth of transformed E. coli cells after this treatment completely preserved (PL.2).

Analysis of the cell population of inoculum (inoculum) on the content plasmadynamic cells after treatment by high concentrations (400-500 μg/ml) ampicillin showed that their number increases and is not less than 90% (data table. 1).

Due to the fact that selection for ampicillin resistance is not effective, because the plasmid cells synthesize - lactamases, which, as secretively protein, ampicillin destroys the environment, with the aim of increasing selective pressure during fermentation and suppress growth Apscells appears icillin. It is experimentally shown that it is advisable to do this at the introduction of culture in the logarithmic growth phase, every 1-2 hours

New in comparison with the method of the prototype features are: seed treatment with recombinant strains of ampicillin at a concentration of 400-500 µg/ml for 2-3 hours to remove besplatnih cells and the fraction is added to the environment of ampicillin at a concentration of 100 µg/ml in the fermentation culture in experimentally chosen time to reduce the segregation of plasmids and accumulation besplatnih cells.

The combination of these features allows to increase the yield of the target protein in the biomass, to stabilize the process of biosynthesis and constantly receive high-quality biomass recombinant strains: E. coli SG200-50/pTNF311 (producer TNF-), E. coli SG20050/pLT21 (producer TNF-), E. coli SG20050/pGGF8 (producer G-CSF) and E. coli SG20050/p280GM (producer GM-CSF) containing the target proteins in an amount not less than 10% of the total cellular proteins. The content of TNF - biomass averaged 1.5 times, TNF- - 4 times, G-CSF - 2-3 times and GM-CSF - 8 times.

Graphic materials:

Fig. 1. Analysis of the culture fluid (without cells) in the presence of cellular proteins after incubation of E. coli cells SG20050 with s specific enriched with cytokines (TNF-, TNF - G-CSF and GM-CSF) biomass of recombinant strains of E. coli SG20050 containing a plasmid with the genes of the above cytokines and gene-lactamase (bla gene) as a selective marker.

Example 1. Obtaining transformed cells, the selection of highly productive clones and growing them in liquid selective medium

Transformation of competent E. coli cells SG20050 plasmid DNA pTNF311 [40], or pLT21 [24], or pGGF8 [30], or p280GM [32] carried out the calcium method, as described previously [41]. The selection of highly productive clones were carried out as described in method-prototype [39]. Clones with the highest content of the target protein is used to produce inoculum. Their pokasivaut in L-broth containing 100 μg/ml ampicillin at 32oC to mid-logarithmic phase of growth (D550= 0,5-0,6).

The analysis of the received cells on the content plasmadynamic cells carried out by the method described in [42]. The data obtained are presented in table. 1 (column 2).

As the table shows, the percentage of Aprcells in seeding material ranges from 60 to 85%.

Example 2. The effect of ampicillin on the viability of the recipient E. coli SG20050 and recombinant E. coli strain SG20050/pGGF8.

Investigated the influence of Replicant G-CSF, the data presented in table.2 and Fig. 1.

As can be seen from the table.2, after incubation of the recipient cells (Apsin the medium with antibiotic (up to 400 µg/ml) for 1 h, individual colonies retain the ability to grow under the plating on a medium without selective pressure, whereas after 3 hours of incubation, growth was not observed. At the same time, after incubation of transformed cells (Aprin the same conditions their ability to grow practically does not change.

It is evident from Fig. 1 shows that the treatment of the recipient cells with the antibiotic in the culture fluid start to be detected in significant amounts of cell proteins of E. coli at concentrations of ampicillin above 400 µg/ml, indicating lysis of the cells. The data obtained indicate that these concentrations of antibiotic is suppressed growth besplatnih cells, but they can remain in their native state, and after inactivation ampicillin-lactamases able to intensive growth. After treatment of the cell population ampicillin at a concentration of 400-500 µg/ml for 2-3 hours, you can expect the death besplatnih cells.

Example 3. Obtaining seed with a high content of Aprcells.

From Catalogne flask with a volume of L-broth (200 ml) and the dose of ampicillin 100 µg/ml Pokasivaut culture at a temperature of 32oC and the speed of rotation of the rocking 150-160 rpm until the first third of the mid - logarithmic phase of growth (D550=0.5 to 0.6). Then hold incubation with ampicillin at a concentration of 400-500 µg/ml for 2-3 hours

In table. 1 (column 4) presents the results of the analysis of the obtained cell population. As can be seen from the table. 1, processing inoculum antibiotic at a concentration of 400-500 µg/ml for 3 h enhances Aprcells, and their content reaches 90%.

Example 4. Fermentation without fractional add ampicillin.

The fermentation is carried out in katalozhnyh flasks with medium volume of 250 ml or fermenter "Ultraform" displacement of 4 liters at a temperature of 31-32oC. culture medium (L - broth containing Ap at a concentration of 100 μg/ml of the Speed of rotation of the rocking 150-160 rpm Speed of rotation of the agitator in the fermenter from 100 to 200 rpm, the air supply of 0.5-1.0 l/min to 1 l of medium. Seeding using seed material obtained as described in example 3. The volume of inoculum 1% (V/V) of the volume of the medium for fermentation. Fermentation time 9-10 hours to Finish fermentation in the late logarithmic phase of growth. The culture fluid is cooled to 5-10oC holding cells during fermentation are presented in table. 3-6 (columns 1-3).

The content of the target protein in the biomass varies from experience to experience, and is the amount of cellular protein in %: TNF- - 10-15%, TNF - 1-3%, G-CSF - 1.0 to 4.0%, GM-CSF - 1-4%.

Example 5. Fermentation with a fractional addition of ampicillin.

The fermentation is carried out as described in example 4, except that the fermentation process every 1-2 h of growth add to the environment ampicillin concentrations up to 100 µg/ml of Data on the content plasmadynamic cells in the fermentation process are presented in table. 3-6 (columns 4-7). The target proteins in the biomass are always present in the amount not lower than 10% of the total cellular proteins: TNF - 15 -25%, TNF - 10-16%, G-CSF - 10-12%, GM-CSF - 10%.

Thus, the proposed method for obtaining biomass of recombinant strains of E. coli SG20050 containing the bla gene as a selective marker, stabilizes the process of biosynthesis of 4 target proteins, cytokines, to eliminate the "blank" industrial fermentation and to improve their content in biomass: TNF - 1.5 times, G-CSF and GM-CSF in 2-3 times. The content of TNF - in the biomass is not lower than in the method prototype, but the process of obtaining inoculum versatile and can be easily scaled.

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1. The method of producing biomass recombinant strains of Escherichia coli bacteria enriched polypeptides with properties of cytokines person, including the transformation of competent cells of Escherichia coli with plasmids containing the genes for cytokines person, plating on selective medium containing ampicillin, obtaining inoculum of pre-selected high yielding clones, fermentation and harvesting of biomass, characterized in that the transformation of the competent cells is performed with plasmids containing the bla gene as a selective marker, and to obtain a supernatant with a high content of plasmidfactory cells are seed treatment with ampicillin at a concentration of 400 to 500 µg/ml for 2 to 3 hours and fractional addition of this antibiotic in the fermentation process every 1 to 2 h of culture growth.

2. The method according to p. 1, characterized in that as the strain of the recipient using the bacterial strain Escherichia coli SG 20050.

3. The method according to p. 1, characterized in that the transformation to/P> 4. The method according to p. 1, characterized in that the transformation of the competent cells is performed with plasmid pLT21 containing the gene for tumor necrosis factor - human (TNF-).

5. The method according to p. 1, characterized in that the transformation of cells is performed with plasmid pGGF8, containing the gene for granulocyte-colony stimulating factor human.

6. The method according to p. 1, characterized in that the transformation of the competent cells is performed with plasmid p28GM, containing the gene for granulocyte-macrophage colony-stimulating factor human.

 

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