Recombinant dna, coding human granulocyte colony-stimulating factor (g-csf) and recombinant plasmid pas017, providing g-csf synthesis in cells of escherichia coli

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of biotechnology, namely to recombinant obtaining G-CSF, and can be used for production of G-CSF in cells of E.coli. For effective production of protein in cells of E.coli G-CSF-coding DNA sequence is optimised. On the basis of obtained optimised DNA sequence plasmid pAS017, also including NdeI/BamHI-fragment of DNA of pETM-50 vector and having physical map, presented on the drawing, is constructed.

EFFECT: invention provides effective production of protein in cells of Ecoli.

2 cl, 1 dwg, 1 tbl, 3 ex

 

The technical field to which the invention relates.

The invention relates to biotechnology, in particular genetic engineering, and can be used in microbiological industry to develop cost-effective and easily scalable methods for obtaining recombinant granulocyte colony-stimulating factor human (G-CSF). Available:

- synthetic gene G-CSF, the nucleotide sequence of which is optimized for expression in Escherichia coli cells and is characterized by the sequence SEQ ID No. 1;

recombinant plasmid pAS017, providing a high level of expression of G-CSF in Escherichia coli cells and containing the gene for G-CSF with the sequence SEQ ID No. 1 under the control of the promoter of the gene 10 of bacteriophage T7 and T7 terminator.

The level of technology

Colony-stimulating factors - the group of hematopoietic proteins that stimulate the proliferation and differentiation of hematopoietic cells from pluripotent stem cells. Specific for granulocytes and macrophages are the factors G-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF). G-CSF-specific enhances the production of neutrophils, performs a protective function of the body in combating bacterial and fungal infections. As a medical drug G-CSF is use for rehabilitation of cancer patients with neutropenia, caused by chemotherapy and radiation. Gene G-CSF person located on the chromosome 17 locus q21-q22, consists of 5 exons and 4 introns [1]. Expression of G-CSF, which increased in certain tumor lines, due to alternative splicing leads to the formation of two isoforms of the protein size 207 and 204 amino acids. The first 30 amino acids of both proteins are signal sequences. The Mature protein size 174 amino acids has a higher biological activity than Mature protein other isoforms size of 177 amino acids. Although natural G-CSF and O-glycosylated at residue Thr-133, this modification does not affect the biological activity and stability of the protein. Non-G-CSF obtained in cells of E. coli, as active as protein from mammalian cells [2-4].

Production of biologically active recombinant G-CSF in commercial scale is a complex task and requires the creation of effective recombinant strains and cell lines producing this protein. A number of patents describe methods of producing G-CSF in yeast cells, mammalian, bacteria, and methods protein purification to a homogeneous state [5-10].

The closest analogues of this invention can be considered genetic structure disclosed in the patent of the Russian Federation 2260049 [5] or patent of the Russian Federation 2321424 [9]. In the first data is about obtaining plasmids and expression in .oli contained partially optimized sequence, encoding G-CSF, with the release of 20-30% of the target product (of the total cellular protein). However, this high yield of protein found only in cultures with a low density, as used in the selection of ampicillin with a high density of cells is rapidly destroyed by cellular enzymes violation of the selective environment. In the patent [9] solved the preparation of recombinant vector for the expression of DNA G-CSF with partial optimization of codons in E. coli, which uses tryptophan promoter, which is also known in the prior art as significantly limiting the process of obtaining cultures with high values of optical density (25-30 O.E. and more), which represent additional opportunities to improve the overall yield of the target product.

The present invention solves the problem of constructing recombinant plasmids, providing the ability to scale the process to increase the overall yield of the target protein (effect) due to the use of SEQ ID NO:1 in combination with vector-a carrier that does not contain elements that would limit the suitability of the system for high-density cultures. The achievement of the technical result is confirmed by the fact that at a density of 25-30 per unit of output protein using the plasmids according to the invention (see example 3) is 1.5 g/l ku is Tory, 1.5-2 times higher in comparison with [9].

Disclosure of inventions

The task of obtaining a vector directing the synthesis of the target protein in a heterologous system, includes the following required steps:

a) the design of the structure gene of the target protein, suggesting optimal for transcription and translation of its coding sequence;

b) selecting host cells and create vector designs for gene expression of the target protein in a selected heterologous system;

C) optimization of the conditions of production of the target protein.

The goal is to get protein on the basis of G-CSF in E. coli cells with a high yield is achieved due to the fact that

1) method of oligonucleotide synthesis obtained gene G-CSF;

2) constructed a recombinant DNA plasmid DNA pAS017, containing the gene for G-CSF under the control of the promoter of T7 phage;

3) the transformation expressing the plasmid pAS017 cells of E. coli strain BL21(DE3) were obtained recombinant Escherichia coli strain BL21(DE3)/pAS017 characterized by a high level of induced synthesis and stable production of protein G-CSF.

The nucleotide sequence encoding a variant of a gene G-CSF-optimized for expression in E. coli cells kdonovan composition was obtained by the method of oligonucleotide synthesis.

To create the expression vector directing in the cells of E. coli protein G-CSF, was the used vector retm-50 [11].

Expressing recombinant plasmid pAS017 was obtained by sublimirovanny the coding sequence of a gene G-CSF in the vector retm-50 at the restriction sites NdeI/BamHI (drawing).

By transforming cells of Escherichia coli strain BL21(DE3) [12] constructed a plasmid pAS017, selection and cultivation of clones transformed with a high level of synthesis of the target protein was obtained recombinant Escherichia coli strain BL21(DE3)/pAS017 producing protein G-CSF. Synthesis of G-CSF in the resulting recombinant strain is under cultivation in conventional selective media with the addition of the inducer isopropyl-D-thiogalactoside (IPTG) or lactose.

Thus, the present invention includes 2 object:

The first object is a recombinant DNA which encodes a G-CSF and characterized by nucleotide sequence SEQ ID No. 1.

A second object of the recombinant plasmid pAS017 for synthesis of protein G-CSF in Escherichia coli cells and consisting of a DNA fragment with the sequence SEQ ID No. 1 and fragment of plasmid retm-50.

A brief description of the drawing is a Physical and genetic map of the vector pAS017

The position of the gene G-CSF, other elements of the vectors, unique restriction sites.

The implementation of the invention

When carrying out the invention in addition to the methods disclosed in detail in the following examples, used well known for the e specialists techniques, described in the manuals of molecular biology and genetic engineering [13].

Example 1. Design and construction of gene G-CSF

Design synthetic version of the gene G-CSF encoding a Mature polypeptide size 174 S.A. with additional N-terminal methionine residue, was performed using the program GeneDesigner [14]. This program allows on the basis of amino acid sequences to select the appropriate coding sequence optimized for expression in a selected organism, the host with the characteristic frequency of occurrence of codons and other characteristics that contribute to the efficient heterologous expression of a selected polypeptide. Fed thus the nucleotide sequence of the gene G-CSF, optimized for expression in E. coli cells, broke the oligonucleotides with a length of 40, for example, by using DNAworks [15] for the subsequent Assembly of a gene by recombinant PCR. When designing flanking primers to their 5'-end sequences were added to the sequence containing the sites of restricted NdeI and BamHI, to then create the expression constructs.

The Assembly of the synthetic gene was carried out in two stages using optimized PCR method for the production of synthetic genes [16] using the oligonucleotides listed in table 1.

Table 1
The oligonucleotides used to assemble the synthetic gene G-CSF
No.SequenceP.N.
1ggacatATGACTCCTCTCGGTCCGGCGAGCT 31
2TTGAGCAGGA7AGGATTGCGGGAGAGAGCTCGCCGGACCGA40
3CGCAATCCTTCCTGCTCAAATGCCTCGAACAAGTTCGTAA40
4CGCACCGTCGCCCTGGATTTTACGAACTTGTTCGAGGCAT40
5AGGGCGACGGTGCGGCGCTGCAAGAAAAGCTCTGCGCAAC40
6CCGGGTGACACAGTTTGTAGGTTGCGCAGAGCTTTTCTTG40
7CTACAAACTGTGTCACCCGGAAGAGCTGGTTCTGCTGGGT 40
8GCCCACGGAATACCCAGAGAGTGACCCAGCAGAACCAGCT40
9TCTGGGTATTCCGTGGGCTCCGCTGTCTAGCTGTCCGTCT40
10ACCCGCGAGCTGCAGGGCCTGAGACGGACAGCTAGACAGC40
11CTGCAGCTCGCGGGTTGCCTCTCTCAGCTGCACTCTGGTC40
12GCAGACCCTGGTACAGGAACAGACCAGAGTGCAGCTGAGA40
13TCCTGTACCAGGGTCTGCTCCAGGCGCTCGAAGGCATCTC40
14CGAGGGTCGGACCCAGTTCCGGGGAGATGCCTTCGAGCGC40
15CTGGGTCCGACCCTCGATACCCTGCAACTGGACGTTGCCG40
16TTGCCAAATCGTCGTCGCGAAATCGGCAACGTCCAGTTGC40
17 GCGACGACGATTTGGCAACAGATGGAAGAACTCGGTATGG40
18GTCGGTTGCAGAGCAGGCGCCATACCGAGTTCTTCCATCT40
19CCTGCTCTGCAACCGACCCAGGGTGCAATGCCAGCGTTCG40
20CGCACGACGCTGGAACGCAGAGGCGAACGCTGGCATTGCA40
21GTTCCAGCGTCGTGCGGGTGGTGTTCTGGTTGCGTCTCAC40
22AACTTCGAGGAAAGACTGCAGGTGAGACGCAACCAGAACA40
23CTGCAGTCTTTCCTCGAAGTTTCTTACCGTGTCCTGCGTC40
24cggatcctaCGGCTGCGCCAGGTGACGCAGGAcacggtaag41

In the first stage, carried out PCR in a mixture of primers No. 1-24 using Taq polymerase under the following conditions:

Mixture for PCR (100 μl):

10 ál of 10x PCR buffer ("Fermentas");

10 μl of 2.0 mm of each dNTP;

55 μl of deionized water;

1 ál of DNA p is Limeray ("Fermentas");

1 µl of each primer No. 1-24 with a concentration of 5 μm.

Conditions for PCR: 94°, 5' (denaturation), 94°C, 30"; 50°C, 30"; 72°C, 40" (amplification), 25 cycles.

1 µl of the resulting mixture is used for the second PCR with primers # 1 and # 24 in the same conditions.

After re-amplification PCR mixture was analyzed by electrophoresis in 2% agarose gel and identify homogeneous fragment size of about 550 i.e. Fragment isolated from the gel using the set Wizard PCR Preps Kit (Promega, USA) according to the manufacturer's instructions.

Example 2. Construction of expression vector pAS017

The obtained PCR fragment with gene G-CSF embed in the composition of the vector retm-50, containing the promoter of T7 phage, providing a high level of expression of heterologous genes in E. coli strains synthesizing T7 polymerase [14]. For this purpose, 100 ng of PCR fragment obtained as described in example 1, hydrolyzing jointly by restrictase NdeI/BamHI and embed using T4 DNA ligase into the NdeI/BamHI vector retm-50.

Received ligase mixture transform competent cells of Escherichia coli strain XL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F proAB lacIqZΔM15::Tn10 (Tetr)] (Stratagene, USA) and the resulting kanamycin-resistant transformants analyzed by PCR screening with primers # 1 and # 24 (table 1) and select "positive" clones, forming a PCR fragment sizes 550 is p Several positive clones was checked by sequencing using the same primers and select the clone with the gene G-CSF without nonspecific PCR mutation, which is designated as pAS017.

Example 3. Determination of productivity of the producer strain Escherichia coli BL21(DE3)/pAS017

The obtained recombinant plasmid pAS017 transform the E. coli strain BL21(DE3) [F-, ompT, hsdSB (rB-, mB-), dcm, gal (DE3)]. Then, the transformed culture is plated on solid selective culture medium and incubated for 18 h at 37°C. the Choice of a culture of E. coli BL21(DE3) as the recipient strain for the production of G-CSF due to the fact that it synthesizes the RNA polymerase of phage T7, and also has reduced by activity, thereby increasing the yield of the target protein.

To determine the productivity obtained producer strain Escherichia coli BL21(DE3)/pAS017 grown on solid medium, colonies inoculant 50 ml of medium TV containing 50 μg/ml kanamycin, at the rate of 1 colony per 1 ml and grown in an orbital shaker with an amplitude of rotation 50 mm at a speed of 300 rpm and 37°C to an optical density And600around 4.0 PU, then make the inductor IPTG to a final concentration of 0.4 mm and continue cultivation to the stationary phase of growth (approximately 12 hours). The yield of recombinant protein assessed by electrophoresis in LTO-PAG. According to electrophoresis output GCSF is not less than 1500 mg/l at the end of the optical density of 25-30 PU

The list of cited sources

1. Avalos BR /Blood. 1996. V.88. P.761.

2. Welte K., Gabrilove J., Bronchud M.H., E. Platzer, G. Morstyn // Blood. 1996. V.88. P.1907.

3. Lu H.S., Boone T.C, L.M. Souza, Lai P.H. // Arch. Biochem. Biophys. 1989. V.268. P.81.

4. Asano, S. // Am.J.Pediafr. Hematol. Oncol. 1991. V.13. P.400.

5. A.G. Gabibov, Ponomarenko N., Vorobiev I.I., A.V. Demin, Martyanov, VA, Schuster A.M., Baratashvili DI, Miroshnikov A.I. RECOMBINANT PLASMID DNA PES3-7 ENCODING a POLYPEPTIDE WITH a SEQUENCE of GRANULOCYTE COLONY-stimulating FACTOR HUMAN, AND the ESCHERICHIA COLI STRAIN BL21(DE3)/PES3-7 - PRODUCER of RECOMBINANT GRANULOCYTE COLONY-stimulating FACTOR HUMAN. Patent for invention No. 2260049 from 24.07.2003.

6. Romanov V.P., Masarikova NI, Zhdanov V., Afinogenov G.N., Gladchenko T.N., Pustoshilova NM, Masycheva VI, Sinichkina S.A., Sandakhchiev PS, Goldberg ED, Digi A.M., Paganico NS a method of OBTAINING a RECOMBINANT GRANULOCYTE COLONY-stimulating FACTOR HUMAN. Patent for invention No. 2201962 from 19.03.2001.

7 Peter LE, Kryukov E.A., Shingareva L.N., Kirpichnikov M.P. RECOMBINANT PLASMID DNA pFGM17 ENCODING a POLYPEPTIDE GRANULOCYTE-MACROPHAGE COLONY-stimulating FACTOR HUMAN, AND BACTERIAL STRAIN Escherichia coli BL21(DE3)/pFGM17 PRODUCING POLYPEPTIDE GRANULOCYTE-MACROPHAGE COLONY-stimulating FACTOR HUMAN. Patent for invention No. 2271392 from 15.11.2004.

8. Black who made it happen IE, Pustoshilova NM, Rudenko Mrs x, Denisov L.A., maple AV, Sobolov DL METHOD of production, separation, PURIFICATION AND stabilization of RECOMBINANT GRANULOCYTE COLONY-stimulating FACTOR HUMAN, SUITABLE FOR MEDICAL USE, AND IMMUNOBIOLOGICAL TOOL BASED ON IT. Patent for invention No. 2278870 from 30.08.2004.

9. Jarocki SV, Chuvpilo S.A., Scrapin VI, Mogutov M.A., Yakovenko, A.R., PREPARATION of RECOMBINANT PLASMID DNA pSX70 ENCODING the SYNTHESIS of RECOMBINANT HUMAN GRANULOCYTE-COLONYSTIMULATING FACTOR (G-CSF), Escherichia coli SX70 - INDUSTRIAL STRAIN PRODUCER of RECOMBINANT HUMAN G-CSF AND a METHOD for INDUSTRIAL preparation of G-CSF. Patent for invention No. 2321424 from 30.10.2006.

10. Nissen TL, Andersen KV, Hansen K., Mikkelsen EM, Scamble HIERONYMITE G-CSF. Patent for invention No. 2290411 from 09.01.2001.

11. http://www.embl.de/pepcore/pepcore services/cloning/pdf/pETM-50.pdf

12. Studier, F.W. and Moffatt.B.A. (1986). Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189, 113-130.

13. Ausubel F.M., Brent R.G., Kingston R.E., Moore D.D., Seidman J.G., Smith J.A., Struhl K.A. Current Protocols in Molecular Biology. Massachusets General Hospital and Harvard Medical School, John Willey&Sons Inc., 1994.

14. Villalobos A, Ness JE, Gustafsson C, Minshull J, Govindarajan S. (2006) Gene Designer: a synthetic biology tool for constructing artificial DNA segments. BMC Bioinformatics. 2006 Jun 6; 7:285.

15. Hoover DM, Lubkowski J (2002). DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis. Nucleic Acids Res.30(10)e43.

16. Dong, Mao R, Li B, Q iu, Xu P, Li G. (2007) An improved method of gene synthesis based on DNA works software and overlap extension PCR. Mol. Biotechnol. 37(3):195.

1. Recombinant DNA encoding G-CSF, the nucleotide sequence of which is optimized for production of the protein in E. coli cells and is characterized by SEQ ID NO:1.

2. Recombinant plasmid pAS017 designed for the expression of G-CSF in E. coli cells, which consists of
NdeI/BamHI fragment of DNA containing the recombinant DNA according to claim 1, and
NdeI/BamHI-DNA fragment of the vector retm-50, which are interconnected according to the scheme shown in figure 1.



 

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

2 cl, 4 dwg, 7 ex

FIELD: medicine, endocrinology, pharmacy.

SUBSTANCE: invention proposes an agent eliciting antidiabetic activity. Agent represents nonglycosylated recombinant human granulocyte colony-stimulating factor. It differs from native preparation by absence of glycosyl group and the presence of additional methionine residue by its N-end. Effect of agent is associated with mobilization and migration of bone marrow mesenchymal stem cells, homing into pancreas. This results to reparation of insulin-producing activity of organ and normalization of the peripheral blood glucose level after monotherapy with recombinant human granulocyte colony-stimulating factor.

EFFECT: valuable medicinal property of agent.

3 tbl, 2 dwg, 1 ex

FIELD: molecular biology.

SUBSTANCE: invention relates to isolated DNA fragment encoding horse GM-CSF, and isolated horse GM-CSF protein. Also disclosed are vectors and various compositions containing thereof. GM-CSF is useful as adjuvant for horse vaccination as well as non-specific immunity stimulator in veterinary.

EFFECT: new compositions for gorse vaccination.

18 cl, 2 dwg, 1 tbl, 7 ex

FIELD: biotechnology, microbiology, genetic engineering.

SUBSTANCE: invention describes construction of recombinant plasmid DNA pFGM17 encoding constitutive synthesis of polypeptide of human granulocytic-macrophagal colony-stimulating factor (GM-CSF) and consisting of Kpn I/Eco RI-fragment of plasmid pSPF1 DNA and artificial DNA sequence encoding signal peptide of the protein Caf1 from Yersinia pestis, and also Kpn I/Eco RI-fragment of intermediate plasmid pSK-GM comprising the synthetic human gene GM-CSF. Escherichia coli cells are transformed with plasmid DNA pFGM17 and strain E. coli BL21(DE3)/pFGM17 is prepared that is a producer of human polypeptide GM-CSF. Invention provides enhancing technological effectiveness and economy of process for preparing recombinant FM-CSF due to excluding the induction stage in biosynthesis process and in simultaneous increasing the yield of the end product by 2 times. Invention can be used for preparing human granulocytic-macrophagal colony-stimulating factor.

EFFECT: valuable properties of plasmid DNA and microorganism strain.

2 cl, 4 dwg, 4 ex

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

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

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