RU2326169C1 - RECOMBINANT PLASMID DNA pA3GF CODING POLYPEPTIDE OF HUMAN GRANULOCYTE COLONY-STIMULATING FACTOR, AND Escherichia BACTERIA RACE coli-PRODUCER OF HUMAN GRANULOCYTE COLONY-STIMULATING FACTOR POLYPEPTIDE - Google Patents

Abstract

FIELD: gene engineering.

SUBSTANCE: invention can be used for production of recombinant polypeptide of human granulocyte colony-stimulating factor. Recombinant plasmid DNA is constructed in vitro. It includes synthetic gene of human granulocyte colony-stimulating factor, strong constitutive promoter A3 from the early stage of bacteriophage T7 and synthetic section - translation enhancer (TREN) of gene 10 in bacteriophage T7. This DNA in combination with high copy number of plasmid and optimisation of cultivation conditions ensures constitutive biosynthesis of target protein in transformed by this DNA race of Escherichia coli SGK25/pA3GF with high yield.

EFFECT: constitutive biosynthesis of target protein in cells; high yield.

2 cl, 4 dwg, 7 ex

Description

The invention relates to biotechnology, in particular to genetic engineering, and is an in vitro-engineered recombinant plasmid DNA containing a synthetic gene of a granulocyte colony stimulating human factor, A3 promoter from the early region of the T7 bacteriophage and a translation enhancer synthetic region causing the biosynthesis of the human granulocyte colony polypeptide biosynthesis as well as a strain of Escherichia coli, the producer of this polypeptide.

Human granulocyte colony stimulating factor (GCSF) is a glycoprotein weighing about 19 kDa, belonging to the α-helical cytokine family and produced by macrophages, fibroblasts and endothelial cells with appropriate stimulation. It has a wide spectrum of biological activity and is one of the main factors of hematopoiesis. GCSF induces the differentiation of progenitor cells into neutrophils, stimulates the proliferation of neutrophilic granule cells and the activity of mature neutrophils [1-2]. GCSF as a medicine reduces the duration of neutropenia after bone marrow transplantation for leukemia and intensive chemotherapy of cancer, promotes the rapid recovery of granulocytes after irradiation, and also increases resistance to viral and infectious diseases [3-5]. These properties determine the widespread use of G-CSF in the treatment of malignant tumors and other diseases. Known methods for producing natural granulocyte colony-stimulating human factor from cell lines producing this cytokine, as well as recombinant G-CSF from higher eukaryotic cells [6-8], however, the yield of the target product using these methods is extremely low. A much more effective way to obtain GCSF is microbiological synthesis using recombinant DNA. Using this approach allows you to create optimal conditions for bacterial expression of the recombinant gene, significantly reduces the cost of obtaining the final product while maintaining its biological properties, since it is known that the polysaccharide chain of the GCSF glycoprotein is not necessary for its biological activity [9]. Known methods for producing recombinant G-CSF by microbiological inducible synthesis in Escherichia coli cells [10], however, the use of expensive inducers reduces the processability and increases the cost of the final product.

Closest to the claimed technical solution (prototype) is the method described in [11]. The E. coli strain SG20050 contains recombinant plasmid DNA pGGF8 encoding a recombinant human granulocyte colony stimulating factor (rh-G-CSF), the expression of which is controlled by the tandem tryptophan promoters. The disadvantage of plasmid pGGF8 and the strain based on it is the insufficiently high yield of the final product — about 10% of the total cellular protein. This, apparently, is explained by the insufficiently efficient transcription process, since the promoters of the tryptophan operon are not constitutive and provide only conditionally constitutive biosynthesis when grown on a rich medium.

An object of the invention is to increase the level of biosynthesis of a polypeptide of a granulocyte colony stimulating human factor and the creation of a more productive producer strain.

The problem is solved by constructing the plasmid pA3GF, which provides constitutive synthesis of the polypeptide of the granulocyte colony stimulating human factor, and the Escherichia coli strain SGK25 / pA3GF, synthesizing this polypeptide in an amount of not less than 25% of the total cellular protein, which is 2.5 times higher than in the prototype . A high constitutive level of synthesis of the target polypeptide is ensured by the fact that the pA3GF plasmid is highly copied, contains a strong constitutive A3 promoter from the early region of the T7 bacteriophage and a synthetic plot - translation enhancer (TREN) of the T7 bacteriophage 10 gene.

Recombinant plasmid DNA pA3GF encoding a polypeptide of a granulocyte colony stimulating human factor is characterized by the following features:

has a molecular weight of 2.44 Md (3.693 kbp);

encodes the amino acid sequence of a mature human granulocyte colony stimulating factor;

consists of EcoRI / HindIII - a fragment of plasmid pTNF331 [12] with a length of 2.929 kb containing a lambda phage transcription terminator, β-lactamase bla gene and replication initiation ori site; Kpnl / EcoRI - a fragment of the same plasmid with a size of 0.136 kbp, including the A3 promoter from the early region of the T7 bacteriophage; The kpnI / SalI fragment of the plasmid pTEGFb [13], size 0.637 kbp, containing the TREN translation enhancer region and the gcsf gene encoding the amino acid sequence of the mature form of G-CSF and flanked by the ClaI and HindIII restriction sites;

contains: A3 promoter from the early region of T7 bacteriophage, synthetic translation enhancer of TREN gene 10 of T7 bacteriophage, synthetic gcsf gene, lambda phage transcription terminator, β-lactamase bla gene, which determines the resistance of ampicillin transformed by pA3GF plasmid, replication initiation site ori; unique recognition sites by restriction endonucleases having the following coordinates:

EcoRI - 1 and 808, Kpn 1-136, Cla I - 227 and 771, PstI - 626 and 2945, HindIII - 352 and 764, Xbal - 190, 1292 and 1394.

A feature of the proposed plasmid construct is that the gcsf gene is controlled by the strong constitutive A3 promoter from the early region of the T7 bacteriophage, and TREN uses a synthetic translation enhancer, which together provides the constitutive biosynthesis of the target protein in high yield.

To obtain a producer strain of a polypeptide of a granulocyte colony-stimulating human factor, competent cells of Escherichia coli strain SGK 25 [14], created at Biocad CJSC based on strain SG 20050 [15], are transformed with recombinant plasmid pA3GF.

The resulting strain of Escherichia coli SGK 25 / pA3GF 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 strain of the subcendent can grow in the temperature range of 20-42 ° C, 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), respectively associated with the presence of the rpsL mutation in the chromosome 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.

The advantage of the proposed method from the prototype is a higher level of biosynthesis of the target protein as a result of using a more productive producer strain and optimizing the conditions for its cultivation. Strain E. coli SGK25 / pA3GF provides a stable constitutive synthesis of a polypeptide of a granulocyte colony stimulating human factor in an amount of at least 25% of the total cellular protein. This leads to a high efficiency of the process of obtaining the polypeptide, because the yield of recombinant polypeptide in the optimal cultivation mode on LB medium is at least 25 mg per liter of culture.

Figure 1 shows the physical map of the recombinant plasmid pA3GF, figure 2 shows the nucleotide sequence of the GCSF gene with adjacent regulatory elements, the initiating and terminating codons are in bold, the restriction enzyme sites are underlined: EcoRI, Kpn I, Hind III, PstI, Cla I; figure 3 is the amino acid sequence of the GCSF polypeptide encoded by the recombinant plasmid pA3GF; figure 4 - electrophoregram of lysates of cells of the recipient strain of E. coli SGK25 (lane 1), four arbitrary clones of the producer strain of E. coli SGK25 / pA3GF (lanes 2-5) 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 RF polypeptide - G-CSF.

The invention is illustrated by the following examples:

Example 1. Construction of an intermediate vector plasmid pA ₃ 31 containing the A3 promoter

Plasmid pTNF331 [12] containing the tandem of A2A3 promoters served as the starting material for the vector construction of pA ₃ 31. To cleave the A2 promoter, 10 μg of pTNF331 [12] is treated with restriction enzymes EcoRI and PstI according to the standard procedure [16]. The reaction products are separated by electrophoresis in a 1% agarose gel; stripes with a length of 748 bp and 2938 bp cut out, the fragments extracted from the gel and ligation in 20 μl of the reaction mixture according to standard methods. 5 μl of the reaction mixture was used to transform competent XL-1 Blue cells (Stratagene, USA). Transformants are plated on LB agar containing 100 μg / ml ampicillin. Clone screening is performed by restriction analysis of EcoRI and PstI. The result is a vector plasmid pA ₃ 31 containing the A3 promoter.

Example 2. Construction of expression plasmids pA3GF

10 μg of the plasmid pTEGFb [13] was treated with the restriction enzyme SaIl; after the reaction was completed, the mixture was heated for 5 min at 90 ° C to inactivate the enzyme and the protruding 3'-ends were completed using the standard method in the presence of dNTP and the Klenov fragment of DNA polymerase I [16]. DNA from the reaction mixture was precipitated with ethanol and treated with restriction enzyme Kpn I. A 633 bp fragment containing the TREN translation enhancer and the gcsf gene were isolated from agarose gel. 5 μg of Intermediate vector plasmid pA ₃ 31 was digested with HindIII, protruding sticky ends were finished as described above, then digested with Kpn I and the linearized vector portion was isolated from agarose gel. Next, 2 μg of the SaII-Kpn I fragment containing the TREN translation enhancer and the gcsf gene and 1 μg of the linearized vector portion are ligated into 20 μl of the reaction mixture according to standard procedures. 5 μl of the reaction mixture was used to transform competent XL-1 Blue cells (Stratagene, USA). Transformants are plated on LB agar 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, PstI 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 [17]. The result is the expression plasmid pA3GF (figure 1).

Example 3. Obtaining a producer strain of a polypeptide of a granulocyte colony stimulating human factor

Recombinant plasmid DNA pA3GF is transformed with competent Escherichia coli SGK 25 cells [14] and, after growing recombinant clones on LB agar with ampicillin at 32 ° C, a human granulocyte colony stimulating factor polypeptide producing strain is obtained.

Example 4. Determination of the productivity of a strain producing a polypeptide of a granulocyte colony stimulating factor person.

To determine the productivity of the E. coli strain SGK25 / pA3GF, cells of one clone were grown at 32 ° C in LB medium, in flasks with a medium volume of 100 ml / flask, on a shaker at a rotation speed of 100 rpm for 16 hours. Growth is estimated by the value of the optical density of the culture at a wavelength of 560 nm. Samples for analysis are obtained by centrifuging the culture fluid at 6000 rpm for 5-10 minutes.

The content of rh-G-CSF 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 culture reaches OD values of 3.2-4.0 pu, recombinant G-CSF is 25-30% of the total protein of the cell (figure 4).

Example 5. Determining the productivity of a producer strain of a polypeptide of a granulocyte colony stimulating human factor in optimizing culture parameters.

To determine the productivity of the E. coli SGK25 / pA3GF strain, cells of one clone stored in the form of glycerol effluents at minus 70 ° C are used. In order to increase the productivity of the strain, a rich complex medium (SB) is used in combination with an optimal aeration mode. Cultivation is carried out in flasks containing 40 ml of medium and 100 μg / ml of ampicillin, on a shaker at a temperature of 32 ° C and a rotation speed of 150 rpm for 17 hours. Growth is estimated by the value of the optical density of the culture at a wavelength of 560 nm, the content of rh-G-CSF is determined by the method described in example 4.

According to the data obtained, when optimizing the cultivation conditions, the OD reaches 14.0-15.0 pu, while the content of recombinant G-CSF is (35-40)% of the total protein of the cell.

Example 6. Determining the productivity of a strain producing a polypeptide of a granulocyte colony stimulating human factor when scaling the cultivation process.

To determine the productivity of the E.coli strain SGK25 / pA3GF, biomass is produced in a 75-liter fermenter with a working volume of 50 l. 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 minus 70 ° C are incubated in 2-4 ml of LB medium for 4 hours, after which they are transferred to flasks.

For sowing the fermenter use 1 liter of seed grown in flasks on LB medium with ampicillin for 7-8 hours at 32 ° C and 150 rpm until the late logarithmic growth phase. Cultivation in the fermenter is carried out on LB 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 optical density values of 3.0-3.5 p.u. (λ = 560 nm).

The content of recombinant granulocyte colony stimulating factor is determined in two ways.

1. In relation to the total cellular protein.

Recombinant GCSF is at least 30% of the total protein of the cell.

2. The output of the recombinant granulocyte colony stimulating factor.

The yield of rh-G-CSF is at least 25 mg from 3 g of biomass obtained from 1 l of culture fluid (QL).

Example 7. Isolation and characterization of a recombinant colony stimulating factor human polypeptide

Isolation and purification of recombinant GCSF is carried out according to the scheme described previously [18].

The isolated recombinant polypeptide is characterized in two ways: by determining the N-terminal amino acid sequence and by the specific biological activity of rh-G-CSF in vivo.

1. The N-terminal amino acid sequence is determined using a Protein / Peptide Sequencer (model 477A) from Applied Biosystems (USA). Identification of PTH-amino acids was carried out on a 120A PTH Analyzer Analyzer from Applied Biosystems (USA). The recombinant G-CSF preparation has the following N-terminal structure of the molecule: Met + Thr ProLeuGly ..., i.e. corresponds to the structure of the N-terminus of natural human G-CSF with an additional methionine residue that does not affect its biological properties [7, 10].

2. The specific biological activity of rh-G-CSF is determined in vivo in mice of the CBA / CaLac line, which are injected subcutaneously with the substance of rh-G-G-CSF at a dose of 125 μg / kg 24 hours daily for four days after cytostatic injection.

Hemostimulatory (granulocytopoiestimulating) activity of rh-G-CSF was 840-1000% relative to the control.

Thus, the claimed technical solution allows to obtain a recombinant polypeptide with a structure and properties identical to the structure and properties of a natural granulocyte colony stimulating human factor; the biosynthesis of the polypeptide is constitutive and its synthesis level is at least 25% of the total cellular protein due to the fact that the G-CSF gene is controlled by the strong A3 promoter from the early region of the T7 bacteriophage in the high-copy plasmid, and protein translation is increased due to the synthetic translation enhancer . All this under the selected cultivation conditions can significantly improve the manufacturability and efficiency of the process of obtaining a recombinant granulocyte colony stimulating human factor while increasing the yield of the target product by 8 times in comparison with the prototype.

Information sources

1. Kashyap S.K., Korobko V.G. // Current Science. 1994. V. 67. P.995-1001.

2. Nagata S., Tsuchiya M., Asano S., Yamamoto O., Hirata Y., Kubota N., Oheda M., Nomura H., Yamazaki T. // EMBO J. 1986. V. 5. P. 575-581.

3. Nagata S. // Bioassays. 1992. V. 10. P.113-117.

4. Tahakashi K., Taniguchi S., et al // Acta haematol. 1991. V.86. P.95-98.

5. Kobayashi Yu., Okabe T., Urabj A., et al // Jap. J. Cancer Res. 1987. V. 78. R 15-18.

6. US patent No. 4833127, CL. AK61K 37/02, 1989.

7. European patent No. 215126, cl. C12N 15/00, 1987.

8. European patent No. 220520, class. C12N 15/00, 1987.

9. Oheda M., Hasegawa M., Hattori K., Kuboniwa H., Kojima T., Orita T., Tomonou K., Yamazaki T., Ochi N. // J. Biol. Chem. 1990. V.265 (20). P.11432-11435.

10. Souza LM, Boone TC, Gabrilove J., Lai PH, Zsebo KM, Mudrok DC, Chazin VR, Bruszewski J., Kenneth HL, Chen KK, Barendt J., Platzer E., Moore MAS, Mertelsmann R., Welte K. // Science. 1986. V.232 (4746). P.61-65.

11. Korobko V.G., Shingarova L.P., Petrovskaya L.E., Petrenko L.A., Pustoshilova N.M. // RF Patent 2113483 C1, 1996.

12. Shingarova L.N., Sagaidak L.N., Turetskaya R.L., Nedospasov S.A., Esipov D.S., Korobko V.G. // Bioorgan, chemistry. 1996.V.22 (4). S.243-251.

13. Shingarova L.N., Kashyap S.K., Petrovskaya L.E., Petrenko L.A., Pustoshilova N.M., Sinichkina S.A., Korobko V.G. // Biotechnology. 1998.V.6. S.24-35.

14. Passport of the microorganism strain Escherichia coli SGK25. VKPM number V-8686.

15. Trisler P., Gottesman S. // J. Bacteriol. 1984. V.160 (1). P.184-191.

16. Sambrook J., Fritsch E., Maniatis T. // Molecular Cloning. A Laboratory manual. 2 ^nd ed. Cold Spring Harbor, NY 1989.

17. CycleReader DNA Sequencing Kit # K1711, Fermentas. Sequencing Protocol.

18. Rospatent No. 2004126196/15 (029366) dated 08/30/2004.

Claims (2)

1. Recombinant plasmid DNA pA3GF encoding a polypeptide of a granulocyte colony stimulating human factor with a molecular weight of 2.44 Md (3.693 kbp), consisting of EcoRI / HindIII - a fragment of plasmid pTNF331 with a length of 2.929 kbp containing the terminator lambda phage transcription, β-lactamase bla gene and replication initiation ori site; KpnI / EcoRI — a fragment of the same plasmid 0.136 kbp in size, including the A3 promoter from the early region of the T7 bacteriophage; The kpnI / SalI fragment of the pTEGFb plasmid of 0.637 kbp containing the TREN translation enhancer region and the gcsf gene encoding the amino acid sequence of the mature form of G-CSF and flanked by the Clal and HindIII restriction sites; containing unique recognition sites by restriction endonucleases having the following coordinates: EcoRI - 1 and 808, Kpn I - 136, Cla I - 227 and 771, PstI - 626 and 2945, HindIII - 352 and 764, Xba1 190, 1292 and 1394. 2. The bacterial strain Escherichia coli SGK25 / pA3GF is the producer of the polypeptide of human granulocyte colony stimulating factor.

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