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

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

 

The invention relates to biotechnology, in particular genetic engineering, and is designed in vitro recombinant plasmid DNA containing the synthetic gene for granulocyte colony-stimulating factor human, A3 promoter of the early region of bacteriophage T7 and synthetic plot - power broadcast, causing the biosynthesis polypeptide granulocyte colony-stimulating factor human and Escherichia coli producing this polypeptide.

Granulocyte colony-stimulating factor human (g-CSF) is a glycoprotein with a mass of about 19 kDa, belonging to the family α-helical cytokines and produced by macrophages, fibroblasts and endothelial cells under appropriate stimulation. It has a wide spectrum of biological activity and is one of the main factors of hemopoiesis. G-CSF induces the differentiation of precursor cells into neutrophils, stimulates the proliferation of neutrophil coast of lozito and activity of Mature neutrophils [1-2]. As a medical drug g-CSF reduces the duration of neutropenia after bone marrow transplantation in leukaemia and intensive chemotherapy of cancer, promotes rapid recovery of granulocytes after irradiation, but also improves the color stability, the ü to viral and infectious diseases [3-5]. These properties cause a wide application of g-CSF in the treatment of cancer and other diseases. Known methods for producing natural granulocyte colony-stimulating factor human from cell lines producing this cytokine, as well as recombinant g-CSF from cells of higher eukaryotes [6-8], however, the yield of the target product when using these methods is extremely low. A much more efficient way of receiving g-CSF is a 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 the final product while preserving its biological properties, as it is known that polysaccharide chain glikoproteid g-CSF is not required for its biological activity [9]. Known methods for producing recombinant g-CSF microbiological inducible synthesis in Escherichia coli cells [10], however the use of expensive inductors reduces the adaptability of the process and increases the cost of the final product.

Closest to the claimed technical solution (prototype) is the method described in [11]. The strain of E.coli SG20050 contains recombinant plasmid DNA pGGF8 encoding recombi is based human granulocyte colony-stimulating factor (RH-g-CSF), the expression of which is under the control of tandem tryptophan promoter. The disadvantage of plasmids pGGF8 and strain based on it is not a high yield of the final product is about 10% of the total cellular protein. This is, apparently, due to inefficient transcription process, as the promoter of tryptophan operon are not constitutive and provide only conditionally constitutive biosynthesis when grown on rich medium.

An object of the invention is to increase the level of biosynthesis polypeptide granulocyte colony-stimulating factor people and creating more productive producer strain.

The problem is solved by constructing plasmids pA3GF providing constitutive synthesis of the polypeptide granulocyte colony-stimulating factor human and Escherichia coli SGK25/pA3GF synthesizing the polypeptide in an amount not less than 25% of the total cellular protein, which is 2.5 times higher than in the prototype. High constitutive level of synthesis of the target polypeptide is provided that plasmid pA3GF has a high kopiosto, contains a strong constitutive promoter A3 of the early region of bacteriophage T7 and synthetic plot - power broadcast (TREN) gene 10 of bacteriophage T7.

Recombinant plasmid DNA pAGF, encoding a polypeptide granulocyte colony-stimulating factor is characterized by the following features:

has a molecular weight of 2.44 Md (3,693 KBP);

encodes the amino acid sequence of the Mature granulocyte colony-stimulating factor human;

consists of EcoRI/HindIII fragment of plasmid pTNF331 [12] long 2,929 KBP containing the transcription terminator of phage lambda, the bla gene β-lactamase and plot ori of replication initiation; Kpnl/ EcoRI fragment of this plasmid size 0,136 KBP, including the A3 promoter of the early region of bacteriophage T7; Cloned/SalI fragment of plasmid pTEGFb [13] the size of 0,637 KBP containing the plot of the amplifier broadcast TREN and gcsf gene, encoding the amino acid sequence of the Mature form of g-CSF and flanked by the restriction sites ClaI and HindIII;

contains: A3 promoter of the early region of bacteriophage T7, synthetic amplifier broadcast TREN gene 10 of bacteriophage T7, synthetic gcsf gene, the transcription terminator of phage lambda, the bla gene β-lactamase, which determines the stability of the transformed plasmid pA3GF cells to ampicillin, plot ori of replication initiation; the unique recognition sites of restriction endonucleases, with the following coordinates:

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

A distinctive feature of the proposed plasmid to the regulations, that gcsf gene is under the control of a strong constitutive promoter A3 of the early region of bacteriophage T7, and to enhance the broadcast, we use a synthetic amplifier broadcast TREN, which gives a constitutive biosynthesis of the target protein with high yield.

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

The resulting strain Escherichia coli SGK 25/ pA3GF characterized by the following features.

Morphological features. Cells are small, rod-shaped, gram-negative, motile, size 1×3-5 μm, risperadone.

Cultural characteristics. During growth on agar LB-medium colonies are round, smooth, translucent, shiny, grey, smooth edge, the diameter of the colonies 1-3 mm; the pasty consistency. Growth in LB liquid medium is characterized by the clouding of the environment.

Physiological and biochemical characteristics. Cells of strain producenta can grow in the temperature range 20-42°C, optimum is 37°C. the Most favorable for growth pH values are in the range of 6.8 to 7.2. When growing under aerobic conditions a culture can assimilate nitrogen as organic compounds (peptone, Tr is pton, amino acids, yeast extract), and ammonium salts. Carbon is assimilated in the form of carbohydrates, polyhydric alcohols (glycerin), amino acids.

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

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

The advantage of the proposed method from the prototype is a higher level of biosynthesis of the target protein resulting from the use of more productive producer strain and optimization of the conditions for its cultivation. The strain E. coli SGK25/pA3GF provides a stable constitutive synthesis of the polypeptide granulocyte colony-stimulating factor human in an amount not less than 25% of the total cellular protein. This results in high efficiency of the process of obtaining a polypeptide, since the output of the recombinant polypeptide in the optimal mode of cultivation in LB medium is not less than 25 mg per liter of culture.

Figure 1 shows the physical map of recombinant plasmid pA3GF, figure 2 is the nucleotide sequence of the gene g-CSF with an adjacent R is untranslated elements, initiation and termination codons are shown in bold, underlined sites restricts: EcoRI, Kpn I, Hind III, PST, Cla I; figure 3 - amino acid sequence of g-CSF polypeptide encoded by the recombinant plasmid pA3GF; figure 4 - electrophoregram lysates of cells of strain-recipient E. coli SGK25 (lane 1), four random clones producer strain E. coli SGK25/pA3GF (tracks 2-5) in a 12.5%polyacrylamide gel (M - protein molecular weight markers, in kDa: 14,5; 21,5; 31,0; 45,0; 66,0; 97,4; arrow specified polypeptide RH-g-CSF.

The invention is illustrated by the following examples:

Example 1. The construction of the intermediate vector plasmids RA331, containing the promoter A3

The source material for obtaining vector design RA331 was the plasmid pTNF331 [12], which contains tandem promoters AA. For wydalenia promoter A2 10 µg pTNF331 [12] treated with restrictase EcoRI > PST and by the standard method [16]. The reaction products separated by electrophoresis in 1%agarose gel; strip length 748 POI 2938 BP cut, the fragments extracted from the gel and objective legirovanie in 20 µl reaction mixture by standard procedures. 5 μl of the Reaction mixture used to transform competent cells XL-1 Blue (Stratagene, USA). Transformants plated on LB-agar containing 100 μg/ml ampicillin. Screening of clones spend restrictum analysis of the m EcoRI and > PST. The result is a vector plasmid RA331, containing the promoter A3.

Example 2. Construction of expression plasmids pA3GF

10 μg of Plasmid pTEGFb [13] is treated with a restriction enzyme SaIl, upon termination of the reaction mixture is heated 5 min at 90°for inactivation of the enzyme and complete the protruding 3'-ends by a standard method in the presence of dNTP and fragment maple DNA polymerase I [16]. DNA from the reaction mixture, precipitated with ethanol and treated with restriction enzyme Kpn I. the Fragment length of 633 BP, containing the amplifier broadcast TREN and gcsf gene isolated from the agarose gel. 5 μg of the Intermediate vector plasmids RA331 is treated with restriction enzyme HindIII, protruding sticky ends is completed, as described above, is then treated with restriction enzyme Kpn I and the linearized vector part isolated from the agarose gel. Next, 2 μg SaII-Kpn I fragment containing the amplifier broadcast TREN and gcsf gene and 1 µg lineinitialize vector parts are ligated in a 20 μl reaction mixture by standard procedures. 5 μl of the Reaction mixture used to transform competent cells XL-1 Blue (Stratagene, USA). Transformants plated on LB-agar containing 100 μg/ml ampicillin. Screening of clones spend restrictum analysis ClaI, Kpn I and HindIII. The resulting recombinant plasmid analyze HaeIII, Kpn I, > PST and HindIII. The structure of the cloned gene in dobranich clones confirmed by determining the nucleotide sequence set Cycle ReaderTM DNA Sequencing Kit (Fermentas, Lithuania) as described in the manual [17]. The result expression plasmid pA3GF (figure 1).

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

Recombinant plasmid DNA pA3GF transform competent cells of Escherichia coli SGK 25 [14] and after cultivation of recombinant clones on LB-agar with ampicillin at 32°get producing strains polypeptide granulocyte colony-stimulating factor human.

Example 4. Determination of productivity of the producer strain polypeptide granulocyte colony-stimulating factor human.

To determine the productivity of a strain of E.coli SGK25/pA3GF cells of the same clone grown at 32°C in LB medium in the flasks with medium volume 100 ml/flask on a shaker at a speed of 100 rpm for 16 hours. The growth estimate on the value of the optical density of the culture at a wavelength of 560 nm. Samples for analysis are obtained by centrifugation of the culture liquid at 6000 rpm for 5-10 minutes

The content of RH-g-CSF defined relative to total cellular protein. For this, cells suspended in 100 μl of buffer containing 62.5 mm Tris-HCl, pH 6.8; 10% glycerol; 2,3% SDS; 5% mercaptoethanol; about 0.001% bromophenol blue. The mixture is heated 5 min in a boiling water bath and cooled to room temperature. Sample the volume of 5.8 and 15 ál subjected to electrophoresis in a 12.5% SDS-PAG. After electrophoresis the gel stain using Kumasi R-250. After washing the dye gel scan and carry out mathematical processing of the results with the help of the software Gel-Pro".

According to the data obtained, the culture reaches OP 3,2-4,0 PU, recombinant g-CSF is 25-30% of the total protein of the cells (figure 4).

Example 5. Determination of productivity of the producer strain polypeptide granulocyte colony-stimulating factor in optimization of cultivation parameters.

To determine the productivity of a strain of E.coli SGK25/pA3GF use cells of the same clone, stored in the form of glycerin effluent at minus 70°C. To improve the productivity of the strain using a rich complex medium (SB) in combination with the optimal mode of aeration. The cultivation is carried out in flasks containing 40 ml of medium and 100 μg/ml ampicillin, in the rocking chair at a temperature of 32°and a rotational speed of 150 rpm for 17 hours. The growth estimate on the value of the optical density of the culture at a wavelength of 560 nm, the content of RH-g-CSF is determined according to the method described in example 4.

According to the obtained data, optimization of cultivation conditions, OP reaches 14,0-15,0 PU, in this case the content of recombinant g-CSF is (35-40)% of the total protein of the cell.

Example 6. Determination of productivity of the strain is - producer polypeptide granulocyte colony-stimulating factor human scale cultivation process.

To determine the productivity of a strain of E.coli SGK25/pA3GF spend time biomass in a 75-liter fermenter with a working volume of medium 50 HP Planting material was obtained as follows: cells from one clone (their or stored in a Bank of cells at a temperature of minus 70°incubated With 2-4 ml of LB medium for 4 h and then subcultured in the flask.

For inoculation of the fermenter using 1 l of inoculum grown in flasks in LB medium with ampicillin for 7-8 h at 32°C and 150 rpm until late logarithmic phase of growth. Culturing in the fermenter is performed on LB medium with ampicillin (100 μg/ml) at a temperature of 32°without pH strirovaniya. The concentration of dissolved oxygen in the range of 40 (±10)% of the saturation support by changing the rpm of the stirrer from 80 to 190 rpm and air flow from 10 to 14 l/min

Fermentation end at transition culture in the stationary phase of growth, which corresponds to the values of optical density of 3.0-3.5 PU (λ=560 nm).

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

1. Relative to total cellular protein.

Recombinant g-CSF extending t is not less than 30% of the total protein of the cell.

2. The yield of recombinant granulocyte colony-stimulating factor.

Output RF g-CSF is at least 25 mg of 3 g of biomass obtained from 1 l of culture liquid (QL).

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

Isolation and purification of recombinant g-CSF is conducted according to the scheme described previously [18].

Selected recombinant polypeptide is characterized in two ways: by definition, the N-terminal amino acid sequence, and the specific biological activity of RH-g-CSF in vivo.

1. N-terminal amino acid sequence determined on the appliance Protein/Peptide Sequencer (model A) the company Applied Biosystems (USA). Identification of FGF-amino acids was performed on the analyzer 120A PTH Analyzer company Applied Biosystems (USA). Recombinant g-CSF has the following structure N-Terminus of the molecule: Met+Thr ProLeuGly..., i.e. corresponds to the structure of the N-end natural human g-CSF with additional methioninol balance without affecting its biological properties [7, 10].

2. Specific biological activity of RH-g-CSF determine in vivo in mice CBA/CaLac, which after 24 hours after injection of the cytostatic agent is injected subcutaneously drug substance RH-g-CSF at a dose of 125 μg/kg, daily for four days.

Gemostimuliruyuschee (granulocytopoiesis the General) activity of RH-g-CSF was 840-1000% relative to control.

Thus, the claimed technical solution allows to obtain a recombinant polypeptide with the structure and properties identical to the structure and properties of natural granulocyte colony-stimulating factor human; biosynthesis polypeptide constitutive and the level of its synthesis is not less than 25% of the total cellular protein due to the fact that g-CSF gene is under the control of a strong promoter A3 of the early region of bacteriophage T7 in the composition vysokonapornoj plasmids, and protein translation increases due to synthetic amplifier broadcast. All this under selected culture conditions can significantly increase the adaptability and efficiency of the process of production of recombinant granulocyte colony-stimulating factor human while increasing the yield of the desired product 8 times in comparison with the prototype.

Sources of information

1. S.K. Kashyap, 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. P 15-18.

6. U.S. patent No. 4833127, CL AC 37/02, 1989.

7. European patent No. 215126, CL 12N 15/00, 1987.

8. European patent No. 220520, CL 12N 15/00, 1987.

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

10. Souza L.M., Boone T.C., Gabrilove J., Lai P.H., K.M. Zsebo, Mudrok D.C., Chazin V.R., Bruszewski J., Kenneth H.L., Chen, K.K., Barendt J., Platzer, E., Moore, M.A.S., Mertelsmann R, Welte K. // Science. 1986. V.232(4746). P.61-65.

11. Korobko VG, Shingareva L.P., Peter LE, Petrenko L.A., Pustoshilova N.M.// Patent RF 2113483 C1, 1996.

12. Shingareva L.N., Sagaidak, L.N., Turkish R.L., Nedospasov S.A., Esipov, D.S., Korobko VG// Bioorgan, chemistry. 1996. T(4). S-251.

13. Shingareva L.N., Kashyap S. Kaliev, Peter LE, Petrenko L.A., Pustoshilova NM, Sinichkina S.A., Korobko VG // Biotechnology. 1998. V.6. Pp.24-35.

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

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

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

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

18. Rospatent №2004126196/15(029366) from 30.08.2004.

1. Recombinant plasmid DNA pA3GF encoding a polypeptide granulocyte colony-stimulating factor human molecular weight of 2.44 Md (3,693 KBP), consisting of EcoRI/HindIII fragment of plasmid pTNF331 length 2,929 KBP containing the transcription terminator of phage lambda, the bla gene β-lactamase and plot ori of replication initiation; Cloned/EcoRI fragment of this plasmid size 0,136 KBP, including the A3 promoter of the early region of bacteriophage T7; Cloned/SalI fragment of plasmid pTEGFb size 0,637 KBP containing the plot of the amplifier broadcast TREN and gcsf gene, encoding the amino acid sequence of the Mature form of g-CSF and flanked by restriction sites Clal and HindIII;

containing a unique recognition sites of restriction endonucleases, with the following coordinates:

EcoRI - 1 and 808, Kpn I - 136, Cla I - 227 771, PST - 626 and 2945, HindIII - 352 and 764, b1 190, 1292 and 1394.

2. The bacterial strain Escherichia coli SGK25/pA3GF producing polypeptide granulocyte colony-stimulating factor human.



 

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The invention relates to biotechnology and can be used in the production of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) in industrial scale

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

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