Recombinant plasmid dna pes3-7 encoding peptide with sequence of human granulocyte colony-stimulating factor (g-csf) and strain escherichia coli bl21(de3)/pes3-7 as producer of human recombinant g-csf

FIELD: genetic engineering, in particular production of human granulocyte colony-stimulating factor.

SUBSTANCE: Recombinant plasmid DNA pES3-7 with molecular weight of 3.63 MDa (5907 b.p.) is constructed. Said DNA consists DNA Ndel/Notl-fragment containing sequence of recombinant G-CSF artificial gene, β-lactamase gene; and plasmid pET22b(+) DNA Ndel/Notl-fragment containing promoter and terminator of T-RNA-polymerase transcription, amplifier of 17 phage 10 gene translation. Plasmid pES3-7 contains as genetic marker β-lactamase gene which determines resistance of E.coli cells transformed with plasmid pES3-7 to ampicillin, and unique restriction endonuclease recognition sites existing on the next distance to the right from Ndel-site: Xbal - 38 b.p.; Hpal - 1332 b.p.; Pstl - 4065 b.p.; Pvul - 4190 b.p.; Xhol - 5363 b.p. Obtained plasmid is used in transformation of Escherichia coli cells to produce strain E.coli BL21(DE3)/pES3-7 as subproducer of recombinant G-CSF. Method of present invention makes in possible to produce recombinant G-CSF with high yield (20-30 % based on total cell protein content).

EFFECT: simplified method for production of recombinant G-CSF with high yield.

2 cl, 2 dwg, 2 ex

 

The invention relates to biotechnology, in particular genetic and protein engineering, and can be used to obtain a polypeptide with the sequence of recombinant granulocyte colony-stimulating factor human.

The polypeptide sequence granulocyte colony-stimulating factor human (G-CSF), synonyms: colony stimulating factor-3, pluripoietin, belongs to the protein family of colony stimulating factors including granulocyte-macrophage colony-stimulating factor and macrophage colony-stimulating factor. G-CSF is secreted activated monocytes, macrophages and neutrophils. G-CSF person is a glycoprotein with a molecular weight of about 20-25 kDa. The peptide part of G-CSF is provided in two versions: 174 and 177 amino acid residues derived from alternative splicing of the messenger RNA. Both contain 5 cysteine amino acid residues and two intramolecular disulfide bridge. G-CSF is a hematopoietic growth factor that stimulates the growth of bone marrow cells at later stages of differentiation, significantly increasing the number of leukocytes, mainly neutrophils in peripheral blood.

[Nagata, S., Tsuchiya m, Asano s, Kaziro Y., Yamazaki T., Yamamoto O., Hirata Y., Kubota N., Oheda M., Nomura, H., Ono, M. // Molecular cloning andexpression of cDNA for human granulocyte colony-stimulating factor. Nature, 1986, v.319, p.415-418. Nagata, S., Tsuchiya m, Asano, S., Yamamoto O., Hirata Y., Kubota N., Oheda M., Nomura, H., Yamazaki T. // The chromosomal gene structure and two mRNAs for human granulocyte colony-stimulating factor. EMBO I., 1986, v.5(3), p.575-581.]

G-CSF exerts its activity through binding to specific receptor [Fukunaga R., Seto Y., Mizushima, S., Nagata S. // Three different mRNAs encoding human granulocyte colony-stimulating factor receptor. Proc. Natl. Acad. Sci. USA, 1990, v.87, p.8702-8706].

Dosage forms G-CSF (synonyms - Neupogen (Phoffman-La Roche Ltd.) and Granocyte (Aventis)) are widely used in medicine for the treatment of neutropenia in patients receiving cytostatic drug for treatment nemieloidnykh malignant diseases; for edema side effects mielosupressivne chemotherapy for the treatment of hereditary periodic and malignant neutropenia; to reduce the duration of neutropenia and its clinical consequences in patients who are preparing for a bone marrow transplant.

A method of obtaining recombinant G-CSF, based on the preparation of recombinant G-CSF in transformed yeast cells, Saccharomyces cerevisiae [Gillis S., L. Urdal, Clevenger, W., Klinke R., Sassenfeld, H., Price, V., D. Cosman // Production of recombinant human colony-stimulating factors in yeast. Behring Inst. Mitt., 1988, p.1-7]. With this approach it is possible to receive G-CSF person in the correct conformation with good physiological activity. The disadvantages of this method is the reduction of the yield due to the de is radzie target protein by intracellular proteases.

Also known is a method of obtaining G-CSF, including expression in Escherichia coli cells, in which secretion by bacterial cells of recombinant G-CSF in the form of soluble protein in periplasmatic space [PCT application WO 01/00549, MKI C 12 N 15/70, publ. 2001]. The method provides the possibility of obtaining the correct conformation of the protein without refolding. The disadvantage is the expression of G-CSF with an additional peptide on the N-end to eliminate the toxic action of the protein, which necessitates the cutting of a specific protease of this peptide.

Known closest to the claimed method of receiving G-CSF, including expression in Escherichia coli cells, which consists in the rapid biosynthesis of the bacterial cells of recombinant G-CSF in the form of insoluble Taurus enable [RF patent №2113483, MKI C 12 N 15/27, publ. 1/21]. In this way carry out construction of recombinant plasmid DNA pGGF8 that encodes a constitutive synthesis of the polypeptide with properties of G-CSF human and Escherichia coli SG20050/pGGF8, providing a synthesis of this peptide with the level of expression of at least 10% of the total cellular protein. The disadvantage of this method is the length of the process (16 hours) constitutive expression, which reduces the manufacturability of the process.

The invention solves the problem of constructing plasmids determining the synthesis recombinante what about the protein, and the creation of highly productive bacterial strain-producer, allowing to obtain recombinant G-CSF with high yield (up to 30% of the total protein content of cells) and simplified technology (6 hours).

The problem is solved by constructing recombinant plasmid DNA pES3-7, having a molecular weight 3,63 MDA (5907 BP) and consisting of NdeI/NotI fragment of DNA plasmids 22b(+), containing the promoter and terminator of transcription of the T7-RNA polymerase, power broadcast of gene 10 of phage T7 gene β-lactamase, and NdeI/NotI fragment of DNA containing the sequence of the synthetic gene recombinant G-CSF. Plasmid pES3-7 contains as a genetic marker gene β-lactamase determining sustainability transformed with this plasmid to the cells of E. coli to ampicillin. Plasmid pES3-7 contains a unique recognition sites of restriction endonucleases that are located in the following distance to the right of the NdeI site: XbaI - 38 BP, HpaI - 1332 BP, PST - 4065 P.O., PvuI - 4190 P.O., XhoI - 5363 softwares For solving the problem is also used producing strains of Escherichia coli BL21(DE3)/pES3-7 containing recombinant plasmid DNA pES3-7 - producer of recombinant G-CSF and for synthesis of recombinant G-CSF in the amount of 20-30% of the total protein content of the cells.

The advantages of the invention are, first, to use the communicate with chemical-enzymatic synthesis of gene G-CSF widest possible set of codons, which is optimal for production of the protein in Escherichia coli; the location in which the synthetic gene eliminates the possibility of formation of synthesized on mRNA longest "studs", potentially inhibiting translation. Figure 1 shows the amino acid sequence of G-CSF and the corresponding nucleotide sequence of the gene: the top row is a natural cDNA G-CSF, the bottom line is synthesized artificial gene. Secondly, the application for recombinant protein biosynthesis optimal regulatory elements that control its expression: T7-Iac promoter to prevent basal gene expression prior to induction and a high level of transcription of the corresponding mRNA induction, highly efficient transcription terminator T7, and the block stop codons, excluding the biosynthesis of elongated variants of recombinant G-CSF. The advantages of the proposed strain of E.coli is to use bacteria phenotype OmpT Lon, which excludes the possibility of proteolytic cleavage of the synthesized de novo recombinant G-CSF and pollution emitted the most active protein by E. coli proteases.

Construction of the gene encoding G-CSF person, carried out on the basis of plasmids 22b(+). Artificial gene encoding G-CSF, flanked by sites restricts NdeI and NotI, get him the co-enzymatic synthesis of a set of oligonucleotide fragments and their subsequent Assembly and amplification using polymerase chain reaction (PCR). Before legirovaniem to generate sticky ends of amplificat and vector plasmid is treated with restrictase NdeI and NotI. Ligase mixture used to transform competent cells of E. coli DH5α. Selection of positive clones was carried out using PCR using specific primers followed restrictum the analysis of isolated plasmid DNA restrictase NdeI and NotI. The structure of the gene encoding the recombinant G-CSF as determined by the sequencing method Singer. It must fully comply with the nucleotide sequence of the source of artificial gene G-CSF (figure 1).

Recombinant plasmid DNA pES3-7 encoding a polypeptide G-CSF, characterized by the following features:

- has a molecular weight 3,63 MDA;

- encodes the polypeptide G-CSF;

- consists of: NdeI/NotI fragment of DNA plasmids 22b(+), containing the promoter and terminator of transcription of the T7-RNA polymerase, power broadcast of gene 10 of phage T7 gene β-lactamase, and NdeI/NotI fragment of DNA, containing an artificial gene G-CSF;

- has a unique set of features: the promoter and terminator of transcription RNA polymerase of bacteriophage T7, power broadcast of the gene 10 of bacteriophage T7; artificial gene encoding G-CSF; gene β-lactamase determining the stability of the transformed plasmid pES3-7 cells E. coli to ampicillin; the unique recognition sites of restriction endonucleases, located on the following distance to the right of the NdeI site: XbaI - 38 BP, Hpal - 1332 BP, PST - 4065 P.O., PvuI - 4190 P.O., XhoI - 5363 BP

To obtain strain-producer of recombinant G-CSF plasmid DNA pES3-7 is used to transform competent cells of Escherichia coli BL21(DE3) and direct the selection of clones that retain the level of biosynthesis of recombinant polypeptide is not lower than 20-30% of the total cellular protein for at least six consecutive passirovanny. To do this, the clones transformed with plasmid pES3-7 cells E. coli BL21(DE3) grown in rich medium (YT-, LB-broth and others) with the addition of ampicillin to 100 μg/ml and glucose solution up to 1% for 12-14 hours, inoculant a new portion of the nutrient medium at a ratio of 1:100, grow the culture to achieve an optical density of 1 PU, induce isopropylthio-β-D-galactoside, and raise another 3-6 hours. Obtaining from cells producing recombinant G-CSF includes the following stages: separation of bacteria from the culture medium, their destruction is one of the commonly used methods; washing buffer solutions Taurus inclusion of the water-soluble components of the cells; the solubilization and recovery of the target protein, its refolding and final cleaning.

Obtained producing strains of Escherichia coli BL21(DE3)/pES3-7 is characterized by the following features.

Morphological features: cell p is locquignol form, gram-negative, risperadone.

Cultural characteristics: the growth on agar LB medium colonies are round, smooth, dull, shiny grey, smooth edge. With the growth in liquid medium (minimal medium with glucose or YT-broth) intensive form a smooth suspension.

Physical-biological characteristics: the cells grow at temperatures from 4°C to 40°s at the optimum pH of from 6.8 to 7.5. As the source of nitrogen used as a mineral salt in ammonium form, and organic compounds in the form of peptone, tryptone, yeast extract, amino acids, etc. as a source of carbon use amino acids, glycerol, carbohydrates.

Antibiotic resistance: the cells are resistant to ampicillin (500 µg/ml), due to the presence of plasmid gene β-lactamase(bla).

The advantages offered by the producer strain is to use bacteria phenotype OmpT Lon, which excludes the possibility of proteolytic cleavage of the synthesized de novo recombinant G-CSF and pollution emitted the most active protein by E. coli proteases.

Cells of E. coli BL21(DE3)/pES3-7 are superproduction. When induction of isopropylthio-β-D-galactoside is effective biosynthesis of recombinant G-CSF, which accumulates in the cells in more than 20% of the total protein of bacteria.

On Phi is .1 presents the nucleotide sequence and encoded by its amino acid sequence NdeI/NotI fragment of plasmid pES3-7; figure 2 - physical map of the plasmid pES3-7.

The invention is illustrated by the examples.

Example 1.

Construction of recombinant plasmid DNA pES3-7.

The nucleotide sequence corresponding to gene G-CSF, get chemical-enzymatic synthesis. This theoretically calculated DNA sequence is divided into overlapping fragments of a size of about 50 BP Chemical synthesis of oligonucleotides corresponding to these fragments, perform solid-phase fostamatinib method using, for example, a DNA synthesizer ASM-102U (BIOSSET, Novosibirsk) with increasing oligonucleotide chain in the direction from the 3'end to the 5'-end using secure phosphamidon -5'-dimethoxytrityl-N-acyl-2'-deoxynucleoside-3'-O-(β-Tianeti-diisopropylamino)-phosphites activated by tetrazole. The synthesis is carried out in the scale of 0.5-0.7 µm, using as a carrier of porous glass (pore size 500 Å), to which 3'-succinate connection joining the first nucleoside link (load 20-30 µmol/g). The resulting oligonucleotides were subjected to 5'-terminal phosphorylation using T4 polynucleotide kinase (Fennentas, Lithuania). For this purpose oligonucleotides in 20 pmol mixed with the enzyme in an amount of 10 units in a buffer solution containing 50 mm Tris-HCl (pH 7.6 at 25° (C), 10 mm MgCl2, 5 mm dithiothreitol, 1 mm sperm is Dina, 0.1 mm ATP and 0.1 mm EDTA. The reaction of the lead 30 minutes, polynucleotide kinase inactivate by heating to 65°C for 10 minutes

Phosphorylated oligonucleotide mixed in equimolar ratio in 50 μl of buffer containing 20 mm Tris-HCl, pH 7,56, 10 mm MgCl2, 0.2 mm rATR, 10 mm dithiothreitol, heated to 65°C, slowly cooled to 37°C for one hour and add 10 EDUC. T4-DNA-ligase. The reaction ligating DNA spend 4 hours at 37°C. 0.1 μl of the obtained solution is used as template in polymerase chain reaction (PCR) in the presence of thermostable DNA polymerase Pfu and specific primers:

Spend 25 cycles of amplification (95°C, 20 s; 62°C, 40 s; 72°S, 60 s) for synthesis of full length DNA fragment containing the sequence of a gene G-CSF, flanked by recognition sites of restricted NdeI and NotI. The product of amplification hydrolyzing restrictase NdeI and NotI, purified by electrophoresis in 5% acrylamide the gel, the DNA band size of approximately 550 BP isolated from the gel by the method of electroelution and precipitate DNA from solution by ethanol.

For preparation of vector DNA plasmid pet-22b(+) (3 μg, 1 pmol) was worked up in 40 μl of Y buffer (33 mm Tris-acetate, pH of 7.9, 10 mm Mg-acetate, 66 mm K-acetate, 1, 0.5 mm DTT, 0.1 mg/ml BSA) restriction enzyme NdeI (10 edict.), precipitate the DNA with ethanol, dissolved in 40 μl Bushra R (10 mm t is IP-HCl, pH 8.5, 10 mm MgCl2, 100 mm KCl, 0.1 mg/ml BSA) and treated with restriction enzyme Notl (10 edict.) for 1 h at 37°C. the resulting DNA fragment size of 5.4 KBP after electrophoretic separation in 1% agarose gel, isolated from the gel by the method of electroelution and precipitate DNA from solution by ethanol.

1 μg of the obtained vector fragment are ligated with 2 pmol NdeI/NotI-fragment of 550 BP, containing the synthetic gene recombinant G-CSF, 10 μl of buffer (20 mm Tris-HCl, pH 7,56, 10 mm MgCl2, 0.2 mm rATR, 10 mm dithiothreitol) using 10 edict. T4-DNA-ligase for 12 hours at 10°C.

1 μl of ligase obtained mixture is used to electrotransformation competent cells E. coli BL21(DE3), which is carried out, for example, by using the apparatus for electrotransformation WITH 600 (VTH, USA), with the gap between the plates electroporation cuvettes 1 mm, and the discharge voltage of 1.4 kV. After transformation, the suspension of bacteria is mixed with a nutrient medium SOC, raise 1 hour at +37°and plated on Petri dishes with LB-agar containing 50 μg/ml ampicillin.

Initial selection of clones containing the desired plasmid, carried out by the method of PCR clones using specific primers:

Spend 25 cycles of amplification (95°C, 20 s; 62°C, 40 s; 72°S, 60 s), followed by electrophoretic analysis of PCR products in 1% of Garonna gel in the presence of a PCR product with a length of about 550 BP The selected clones are used for growth in liquid medium and selection of plasmid DNA plasmid, which is examined for the presence of inserts using restriction endonucleases NdeI and NotI, followed by separation of the products of hydrolysis in 5% polyacrylamide gel. The final structure of the plasmids containing NdeI/NotI fragment of about 550 BP, confirmed by determining the nucleotide sequence of the method of sequencing by Tengeru. According to the sequencing select the plasmid, the nucleotide and corresponding amino acid sequences NdeI/NotI fragment which is completely identical to the originally planned (figure 1). Spend the transformation of E. coli cells BL21(DE3) selected plasmid, as described above, the loop transfer 5-10 colonies in 5 ml of liquid 2xYT medium containing 50 μg/ml ampicillin, for 2 h on a rocking chair with speed 190 rpm to turbidity And550of 0.7-0.8, selected aliquot of the culture for further analysis, add an inductor - isopropylthio-β-D-galactoside to a concentration of 0.2 mm and continue to grow for another 2 hours Equal aliquots of cell suspension taken up to make the inductor and after the completion of growth, centrifuged, separating the supernatant and analyze sediment cells by electrophoresis in SDS page. The appearance is clearly visible protein bands in the region of 20 kDa in the sample the samples after induction, the witness is there about the ability of the strain to synthesize recombinant G-CSF after induction of IPTG and fully confirms the correctness of the Assembly plasmids.

Example 2.

Getting a producer strain E. coli BL21(DE3)/pES3-7 with recombinant G-CSF and determine its productivity.

Producing strains of E. coli BL21(DE3)/pES3-7 is produced by transformation of competent cells E. coli BL21(DE3) with plasmid pES3-7, as described in example 1.

The producer strain E. coli BL21(DE3)/pES3-7 grown at 37°With 100 ml YT-broth (pH 7.0) with 50 mcg/ml ampicillin for 2 h on a rocking chair with speed 190 rpm to turbidity And550of 0.7-0.8, add isopropylthio-β-D-galactoside to a concentration of 0.2 mm and continue the process for another 4 h, or continue growing in the absence of inducer for 4 hours Every hour take a sample of 2 ml, And determine550and the amount of culture, corresponding to 1 ml And5501,0, centrifuged 5 min at 6000 Rev/min the Precipitated cells in 100 ál lyse buffer with dye bromophenol blue handle 20 with ultrasound, heated 3 min at 100°and samples of 1 μl used for electrophoresis in 15% SDS page. Gel stain, Kumasi R-250 according to standard methods and scan to determine the relative amount of protein in the band of the target protein. According to scan the contents of recombinant G-CSF is 20-30% of all cellular proteins.

1. Recombinant plasmid DNA pES3-7 encoding a polypeptide with a sequence of granulocyte colony-stimulating factor brow of the ESA, having a molecular weight 3,63 MDA (5907 BP), consisting of

NdeI/NotI fragment of DNA plasmids 22b(+), containing the promoter and terminator of transcription of the T7-RNA polymerase and the amplifier broadcast of gene 10 of phage T7;

NdeI/NotI fragment of DNA containing the sequence of the synthetic gene recombinant granulocyte colony-stimulating factor, is shown in figure 1, and contains

gene β-lactamase determining the stability of the transformed plasmid pES3-7 cells of E. coli to ampicillin, as a genetic marker

unique recognition sites of restriction endonucleases that are located in the following distance to the right of the NdeI site: XbaI - 38 BP, HpaI - 1332 BP, PST - 4065 P.O., PvuI - 4190 P.O., XhoI - 5363 BP

2. The Escherichia coli strain BL21(DE3)/pES3-7 containing recombinant plasmid DNA pES3-7, the producer of recombinant granulocyte colony-stimulating factor human.



 

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