RU2326168C1 - RECOMBINANT PLASMID DNA pIF TREN CODING HUMAN POLYPEPTIDE OF ALFA-2b INTERFERON AND POLYPEPTIDE OF Escherichia BACTERIA RACE coli-PRODUCER OF HUMAN ALFA-2b INTERFERON - Google Patents

Abstract

FIELD: gene engineering.

SUBSTANCE: invention relates to gene engineering and may be used for production of recombinant polypeptide of human alfa-2b interferon. Recombinant plasmid DNA is constructed in vitro. It includes synthetic gene of human alfa-2b interferon, tandem of A2 and A3 promoters from the early stage of bacteriophage T7 and synthetic section - translation enhancer. All these elements determine constitutive biosynthesis of target protein in transformed by this DNA race of Escherichia coli SGK25 /pIF TREN with high yield.

EFFECT: high yield.

2 cl, 5 dwg, 5 ex

Description

The invention relates to biotechnology, in particular to genetic engineering, and is a recombinant plasmid DNA constructed in vitro that contains a synthetic human interferon alpha-2b gene, a tandem of A2 and A3 promoters from the early region of the T7 bacteriophage and a synthetic translation translation enhancer that causes the biosynthesis of the polypeptide human interferon alpha-2b, as well as a strain of Escherichia coli, the producer of this polypeptide.

Human interferon alpha-2b (IFα2b) is one of at least 13 representatives of the family of human leukocyte interferons [1, 2] - proteins that differ in amino acid composition but have similar biological activity. Human interferon alpha-2b is a polypeptide of 165 amino acids in length and with a calculated mass of about 18,000 Da. This is one of the main immunomodulators produced mainly by human leukocytes in response to a viral infection. It exhibits antiviral, antiproliferative and stimulating NK cells activity [3]. It can be used as medications for the treatment of various viral infections, including influenza, hepatitis, as well as in combination therapy of cancer.

Known methods for producing leukocyte interferon-alpha human, based on the use of activated lymphocytes [4], as well as recombinant alpha interferon obtained by microbiological synthesis [5]. The disadvantage of these methods is the low yield of the target product and, as a consequence, the high cost of IFα preparations.

Plasmid constructs and strains of E. coli created on their basis are produced, which produce human IFα2 with a rather high yield [6, 7].

Closest to the claimed technical solution (prototype) is the method described in [7]. Strain E. coli SS5 contains recombinant plasmid DNA pSS5 encoding recombinant human leukocyte interferon-alpha2, the expression of which is controlled by lactose, T7 phage and tryptophan promoters. The disadvantage of plasmid pSS5 and strain based on it is the lack of optimization of the transcription process of the recombinant interferon gene. Of the three promoters that control the biosynthesis of interferon, the T7 phage promoter is repressed in the absence of an inducer (i.e., under growing biomass), and the other two are not strong enough to ensure maximum expression of the target protein.

An object of the invention is to increase the level of biosynthesis of human interferon alpha-2b polypeptide and the creation of a more productive producer strain.

The problem is solved by constructing the plasmid pIF TREN, encoding the constitutive synthesis of the human interferon alpha-2b polypeptide, and the Escherichia coli strain SGK25 / pIF TREN, which ensures the synthesis of this polypeptide with an expression level of at least 40% of the total cellular protein. A high constitutive level of synthesis of the target polypeptide is ensured by the fact that the pIF TREN plasmid is highly copy-rich, contains the tandem of strong A2 and A3 promoters from the early region of the T7 bacteriophage and the synthetic plot - translation enhancer (TREN) of the T7 bacteriophage 10 gene. Recombinant plasmid DNA pIF TREN encoding a human interferon alpha-2b polypeptide is characterized by the following features:

has a molecular weight of 2.40 Md (3.788 kbp);

encodes the amino acid sequence of mature human interferon alpha-2b;

consists of KpnI / HindIII, a fragment of the plasmid pTNF331 [8], 3.170 kbp in length, containing the tandem of the A2 and A3 promoters from the early region of the T7 bacteriophage, the lambda phage transcription terminator, the β-lactamase bla gene and the replication initiation ori site; KpnI / ClaI - fragment of the plasmid pTrcTEGFb with a size of 0.09 kbp, including a plot of the translation enhancer TREN [9] and the ifh α2b gene flanked by the ClaI and HindIII restriction sites;

contains the tandem of promoters A2 and A3 from the early region of the T7 bacteriophage, a synthetic translation enhancer of the TREN gene 10 of the T7 bacteriophage, the synthetic ifn α2b gene, the lambda phage transcription terminator, the β-lactamase bla gene, which determines the resistance of ampicillin to the transformed pIF TREN plasmid, or replication unique recognition sites by restriction endonucleases having the following coordinates: EcoRI - 1 and 111, Kpn I - 246, Cla I - 336 and 856, PstI - 816 and 3030, HindIII - 849.

A feature of the proposed plasmid construct is that the interferon gene is controlled by the tandem of strong constitutive promoters A2 and A3 from the early region of the T7 bacteriophage, and TREN uses a synthetic translation enhancer to enhance translation, which together provides constitutive synthesis of the target protein in high yield.

To obtain a producer strain of the human interferon alpha-2b polypeptide, competent cells of Escherichia coli strain SGK 25 [12], created at Biocad CJSC based on strain SG20050 [13], are transformed with the recombinant plasmid pIF TREN.

The obtained strain of Escherichia coli SGK25 / pIF TREN is characterized by the following features.

Morphological signs. The cells are small, rod-shaped, gram-negative, motile, 1 × 3-5 μm in size, non-spore-bearing.

Cultural signs. When growing on an agarized LB medium, the colonies are round, smooth, translucent, shiny, gray, the edge is even; the diameter of the colonies is 1-3 mm; pasty consistency. Growth in a liquid LB medium is characterized by uniform turbidity of the medium.

Physiological and biochemical characteristics. The cells of the producer strain can grow in the temperature range of 20-42 ° C, while the optimum is 37 ° C. The most favorable pH values for growth are in the range of 6.8–7.2. When grown under aerobic conditions, the culture can absorb nitrogen from both organic compounds (peptone, tryptone, amino acids, yeast extract), and ammonium salts. Carbon is absorbed in the form of carbohydrates, polyhydric alcohols (glycerin), amino acids.

Antibiotic resistance. Cells are resistant to ampicillin (up to 200 μg / ml), due to the presence of the beta-lactamase gene in the plasmid, as well as to streptomycin (25 μg / ml) and tetracycline (up to 50 μg / ml), associated, respectively, with the presence on the chromosome mutations rpsL and transposon Tn10.

The method, conditions and composition of the medium for storage of the strain. LB-broth with 15% glycerol, at a temperature of -70 ° C, in cryovials.

Significant differences of the proposed method from the prototype are the use of a more productive new strain and optimization of the conditions for its cultivation. The strain E. coli SGK25 / pIF TREN provides stable constitutive synthesis of the human interferon alpha-2b polypeptide in an amount of not less than 40% of the total cellular protein, which leads to a high technological process, because the yield of the recombinant polypeptide in the optimal cultivation mode is at least 1000 mg per liter of culture with a total activity of (3-4) 10 ¹¹ IU.

Figure 1 presents the physical map of the recombinant plasmid pIF TREN, figure 2 is the nucleotide sequence of the gene of human interferon alpha-2b; the initiating and terminating codons are shown in bold; the restriction enzyme sites are underlined: Hind III, PstI, Cla I; figure 3 is the amino acid sequence of the IFa2b polypeptide encoded by the recombinant plasmid pIF TREN; figure 4 is an electrophoregram of cell lysates of the recipient strain of E. coli SGK25 (lanes 1,2), the producer strain of E. coli SGK25 / pIF TREN (lanes 3-6) in a 12.5% polyacrylamide gel (M - protein markers of molecular weight, kDa: 14.5; 21.5; 31.0; 45.0; 66.0; 97.4); the arrow indicates the IFα2b polypeptide. Figure 5 presents the chromatogram of the isolated interferon alpha 2b obtained on a Waters chromatograph using a C4 Symmetry column (4.6 × 150) by reverse phase HPLC. The solid line indicates the gradient of the acetonitrile content in the elution buffer.

The invention is illustrated by the following examples.

Example 1. Amplification of the IFα2b gene with the simultaneous introduction of restriction sites ClaI and HindIII for subsequent cloning.

Amplification of the gene is carried out in a volume of 100 μl using the plasmid pSKIfn (JSC Biocad) containing the IFα2b gene as a template. The reaction mixture contains 100 ng pSKIfn, 50 pmol of primers

5'-TTAATATCGATATGTGTGATCTGCCGC and 5'-TATCTAAGCTTAAGTCGACTATTCC. dNTP mixture (0.2 mM each), 10 mM Tris-HCl, pH 8.8, 10 mM KCl, 2.5 mM MgSO ₄ , 2.5 units Pfu DNA polymerase (Stratagene) and 1 unit Taq DNA polymerase (Fermentas). Spend 25 cycles according to the scheme: 95 ° C / 40 sec, 50 ° C / 40 sec, 72 ° C / 1 min. The reaction products are analyzed by electrophoresis in a 1% agarose gel, a strip of about 530 bp in length. cut out, the DNA is extracted from the gel. The amplification product is treated with restriction enzymes ClaI and HindIII in accordance with the procedure described in [10], and isolated from agarose gel.

Example 2. Construction of expression plasmids pIF TREN.

30 μg of the plasmid pTrcTEGFb [9] was treated with restriction enzymes ClaI and Kpn I, a 90 bp fragment containing a TREN translation enhancer was isolated from an agarose gel. 5 μg of the plasmid pTNF331 [8] is treated with restriction enzymes Kpn I and HindIII and the linearized vector part is isolated from agarose gel. Next, 1 μg of the ClaI-Kpn I fragment, 1 μg of the linearized vector portion and 0.5 μg of the PCR fragment obtained in the previous step are ligated into 20 μl of the reaction mixture according to the standard procedure [10]. 5 μl of the reaction mixture was used to transform competent XL-1 Blue cells (Stratagene, USA). Transformants are plated on LB-arap containing 100 μg / ml ampicillin. Clone screening is performed by restriction analysis of ClaI, Kpn I and HindIII. The resulting recombinant plasmid was analyzed by HaeIII, Kpn I and HindIII. The structure of the cloned gene in the selected clones is confirmed by nucleotide sequence determination using the Cycle ReaderTM DNA Sequencing Kit (Fermentas, Lithuania), as described in [11]. The result is the expression plasmid pIF TREN (figure 1).

Example 3. Obtaining a producer strain of a human interferon alpha-2b polypeptide.

Recombinant plasmid DNA pIF TREN is transformed with competent Escherichia coli SGK25 cells [12] and, after growing recombinant clones on LB agar with ampicillin at 32 ° C, a human producer of human interferon alpha-2b polypeptide is obtained.

Example 4. Determining the productivity of a producer strain of a human interferon alpha-2b polypeptide.

To determine the productivity of the SGK25 / pIF TREN strain, cells of one clone are grown at 32 ° C in complex medium (LB or SB) in flasks with a medium volume of 40-100 ml / flask on a shaker at a rotation speed of 180 rpm for 16 hours. Samples for analysis are obtained by centrifuging the culture fluid at 6000 rpm for 5-10 minutes.

The content of recombinant IF α2b is determined in relation to the total cellular protein. For this, the cells are suspended in 100 μl of buffer containing 62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2.3% SDS, 5% mercaptoethanol, 0.001% bromophenol blue. The mixture is heated for 5 minutes in a boiling water bath and cooled to room temperature. Samples of 5.8 and 15 μl were subjected to electrophoresis in 12.5% SDS-PAGE. At the end of electrophoresis, the gel is stained with Coomassie R-250. After washing the dye, the gel is scanned and the results are mathematically processed using the Gel-Pro program.

According to the data obtained, the recombinant interferon α2b is at least 40% of the total protein of the cell (figure 4).

Example 5. Determining the productivity of a producer strain of a human interferon alpha-2b polypeptide when scaling the cultivation process.

To determine the productivity of the strain SGK25 / pIF TREN, biomass is produced in a 75 liter fermenter with a working volume of 50 liters. Inoculum is obtained according to the following scheme: cells of one clone (freshly obtained or stored in the Bank of cells at a temperature of -70 ° C) are incubated in 2-4 ml of LB medium for 4 hours, after which they are transferred to flasks.

For inoculation of the fermenter, 1 liter of seed is used, grown in flasks on LB medium with ampicillin for 8 hours at 32 ° C and 150 rpm until the late logarithmic growth phase. Cultivation in the fermenter is carried out on a rich complex medium with ampicillin (100 μg / ml) at a temperature of 32 ° C, without pH stating. The concentration of dissolved oxygen in the range of 40 (± 10)% of saturation is maintained by changing the speed of the mixer from 80 to 190 rpm and air supply from 10 to 14 l / min.

Fermentation is completed upon the transition of the culture to the stationary phase of growth, which corresponds to the optical density of 18-22 pu (λ = 560 nm).

Saturation is maintained by changing the speed of the mixer from 80 to 190 rpm and air supply from 10 to 14 l / min.

Fermentation is completed upon the transition of the culture to the stationary phase of growth, which corresponds to the optical density of 18-22 pu (λ = 560 nm).

To isolate recombinant interferon, the culture fluid is separated and the resulting biomass is suspended in lysis buffer (LB) cooled to 4 ° C (TrisHCl, NaCl, EDTA, PMSF, pH 8.0) for 30 minutes. The resulting suspension is processed on a french-press "AVP 2000" in one cycle and collected in a container with ice LB. The cooled suspension is centrifuged, the supernatant (extract) is discarded, and the sediment of inclusion bodies is washed three times. The washed bodies are dissolved in a buffer to restore disulfide bonds, the solution is centrifuged, the supernatant is collected and added to the renaturation buffer (TrisHCl, EDTA, Triton X-100). At the end of renaturation, the solution is filtered through membranes with a pore size of 0.4 μm, applied to a column with an anion exchanger, the eluate is collected, pH fractionation is carried out and further purification is carried out on a cation exchanger.

The homogeneity of the isolated IFα2b according to HPLC is 92-95%. (figure 5).

Interferon activity is determined on MDBK cells by a standard method for inhibiting the cytopathic effect against 100 TCD ₅₀ of the vesicular stomatitis virus. OCO 01-1102 (10 ⁶ ME / ml) was used as the reference standard.

The content of recombinant IFα2b is determined in three different ways.

1. In relation to the total cellular protein.

Recombinant interferon α2b is at least 40% of the total protein of the cell.

2. By the output of recombinant interferon α2b. The yield of interferon α2b is at least 1000 mg from 20 g of biomass obtained from 1 l of culture fluid (QL).

3. According to the definition of biological activity.

The productivity of the proposed strain is (3-4) · 10 ¹¹ ME / 1 l QOL.

Thus, the claimed technical solution allows to obtain a polypeptide with properties identical to the properties of natural human interferon alpha-2b,

the biosynthesis of the polypeptide is constitutive and the level of its synthesis is at least 40% of the total cellular protein due to the fact that the interferon alpha gene is controlled by the tandem of the A2 and A3 promoters from the early region of the T7 bacteriophage in the high-copy plasmid, and protein translation is increased due to synthetic broadcast amplifier. All this under the selected cultivation conditions can significantly increase the manufacturability and efficiency of the process for producing recombinant interferon alpha-2b while increasing the yield of the target product by 1.25 times (25%) compared with the prototype.

Information sources

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6. Kravchenko VV, Gileva I.P., Sandakhchiev L.S., Petrenko V.A., Korobko V.G., Dobrynin V.N.// Recombinant plasmid DNA pIF16 encoding a polypeptide with the properties of interferon α2 human and E. coli strain SG20050 is a producer of a polypeptide with the properties of human interferon α2. RF patent No. 2054041, 6 C12N, 15/21, С12Р, 21/02. BI No. 4, 1996.

7. Cherepanov P. A., Mikhailova T. G., Cherepanov P. P., Martinenko D. L., Shevchuk A. A., Fedyukin BC, Nikolaev T. M., Tolkachev B. B., Sventitsky E. N., Urakov N.N., Kalinin Yu.T., Denisov L.A., Tyagotin Yu.V. et al. Recombinant plasmid DNA pSS5 encoding the synthesis of recombinant human alpha-2b interferon, Escherichia coli SS5 strain - producer of recombinant human alpha-2b interferon and a method for producing interferon alpha-2b. RF patent No. 2165455, 7 C12N 15/21, C12N 1/21, C12P 21/02, C12R 1:19, 2001.

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Claims (2)

1. Recombinant plasmid DNA pIF TREN encoding a human interferon alpha-2b polypeptide mol. m. 2.40 Md (3.778 kbp), consisting of KpnI / HindIII - a fragment of plasmid pTNF331 3.170 kbp long, containing the tandem of promoters A2 and A3 from the early region of the T7 bacteriophage, lambda phage transcription terminator , β-lactamase bla gene and replication initiation ori site; KpnI / ClaI - fragment of the plasmid pTrcTEGFb with a size of 0.09 kbp, including the plot of the translation enhancer TREN and IFa2b gene, flanked by restriction sites Clal and HindIII; containing unique recognition sites by restriction endonucleases having the following coordinates: EcoRI - 1 and 111, Kpn I - 246, Cla I - 336 and 856, PstI - 816 and 3030, HindIII - 849. 2. The bacterial strain Escherichia coli SGK25 / pIF TREN is a producer of the human interferon alpha-2b polypeptide.

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