Method for production of human gene engineered glucagone, recombinant plasmide dna per-gl, encoding autocatalitically cleavable hybrid protein forming human glucagone and strain of escherichia coli er-2566/per-gl as producer of said protein

FIELD: gene and protein- engineering, medicine, pharmaceutical industry.

SUBSTANCE: invention relates to recombinant plasmide DNA pER-Gl is constructed, which provides synthesis of hybrid polypeptide containing human glucagone and intein in Escerichia coli cells. Producer of said hybrid polypeptide is obtained by transformation of E.coli ER2566 with pER-Gl plasmide. Method for production of human recombinant glucagone includes providing and cultivation of hybrid polypeptide producer containing human glucagone and intein followed by isolation and cleavage of said hybrid protein.

EFFECT: increased yield of human recombinant glucagone; simplified technology.

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The invention relates to biotechnology, in particular genetic and protein engineering. It can be used to produce recombinant human glucagon.

Glucagon is a peptide hormone produced by the alpha cells of the islets of Langerhans, which is involved in carbohydrate and lipid metabolism. It is a physiological antagonist of insulin, as well as a stimulant of its secretion. These properties of glucagon determine its value as a medical drug, particularly in the treatment of hypoglycemia.

The human glucagon is a 29-membered polypeptide (I).

Natural glucagon is produced by extraction from the pancreas of pigs and cattle, while it is difficult to be separated from impurities by further purification. In connection with the proposed methods both chemical and microbiological synthesis of the polypeptide. In the world practice glucagon usually obtained by microbiological method, the hybrid protein, the leader of which provides the transport of the polypeptide into periplasm cells of Escherichia coli (Wen, S., Gan, R., Zhu, S. (2003) Curr. Environ., 47, 180-185), or using yeast Sacharomyces cerevisia (Oh, GH, Hahm, M.S., Chung, B.H. (1999) Protein Expr. Purif., 17, 428-434) with the subsequent release of the hormone from the culture medium. Biosynthesis of glucagon in the cytoplasm little use is conducted, because glucagon is easily cleaved by proteolytic enzymes, resulting in the yield of the target polypeptide is small, and in the biosynthesis of the hybrid protein requires the use of expensive sitespecifically proteases (Okamoto H, Iwamoto And, Tsuzuki H, Teraoka H, Yoshida N. (1995) J Protein Chem., 14, 521-6; Egel-Mitani M, Andersen AS, Diers , Hach M, Thim L, Hastrup S, Vad K. (2000) Enzyme Microb Technol., 26, 671-677).

The closest way to the claimed is receiving recombinant human glucagon in the composition of the hybrid protein with subsequent enzymatic cleavage (Shin, C.S., Hong, M.S., Kim, D.Y., Shin, H.C., Lee, J. (1998) Appl. Environ. Biotechnol., 49, 364-370), which, however, does not reach a high enough yield of the target product.

The invention solves the problem of obtaining highly productive recombinant bacterial strain-producer, allowing to obtain recombinant human glucagon with high yield and simplified technology.

The problem is solved due to the fact that:

1. Construct the expression plasmid DNA pER-Gl synthesized by cloning synthetic glucagon gene in the vector plasmid pTWIN1.

2. By transformation of plasmid DNA pER-Gl cells of Escherichia coli ER2566 get producing strains, cultivated it until the accumulation of hybrid protein IntGl that includes, along with the structure of recombinant human glucagon page is the established levels of intein, allowing aimed autocatalytic cleavage destroys the cells in the buffer solution and produce a calf enabled.

3. A hybrid protein IntGl separated in the form of Taurus inclusion. Sediment Taurus inclusion is dissolved in a buffer containing 2M urea, diluted and adjusted pH to 5.5-6, thereby inducyruya autocatalytic cleavage of the hybrid protein, and incubated for 24 h at 25°C. Further purification and analysis of recombinant human glucagon carried out by reversed-phase chromatography. Identification of the resulting recombinant human glucagon performed using mass spectrometry.

The technical result of the autocatalytic cleavage of the hybrid protein is the formation of glucagon, the output of which reaches 10% (relative to total protein of the cells) with the purity of the drug up to 95%.

In order to avoid difficulties associated with the cleavage of the recombinant hybrid protein with various proteases, such as enterokinase, factor X, and others, as well as to reduce the cost of this stage, we used the hybrid protein mini-dnaB intein from Synechocystis sp. (Wu, N., Xu, M.-Q., Liu, X.-Q. (1998) Biochim. Biophys. Acta, 1387, 422-4327) for directional autocatalytic rasselenia hybrid on the target polypeptide and the intein. For this purpose, we synthesized an artificial gene of glucagon structure to the which is shown in figure 1, and cloned it into a vector plasmid pTWIN1, containing the gene intein from Synechocystis sp. dnaB (Wu, H., Xu, M.-Q., Liu, X.-Q. (1998) Biochim. Biophys. Acta, 1387, 422-432.; Evans, J, T.C., Bermer, J., Xu, M.-Q., (1999) J. Biol. Chem., 274, 18359-18363), sites restricts NruI and BamHI. The obtained expression by plasmid pER-Gl, the structure of which is shown in figure 3, was used to transform cells of E. coli ER2566. Formed during the expression of the recombinant hybrid protein gene IntGl capable of autocatalytic cleavage in the human glucagon and intein. The output of glucagon high purity (98%) after reverse-phase chromatography reaches 10% relative to the total protein of the cell.

The proposed technical solution, use producing strains of Escherichia coli ER2566 containing plasmid DNA pER-Gl - superproducer hybrid protein IntGl that includes, along with the structure of recombinant human glucagon structure intein.

Use of recombinant plasmid DNA pER-Gl

- encoding the amino acid sequence of recombinant

human glucagon;

- having a molecular weight of 4.38 MDA;

- consisting of:

NruI/BamHI - DNA fragment commercial plasmids pTWIN1 (company New England Biolabs)containing the promoter and terminator of transcription of the T7-RNA polymerase, power broadcast of gene 10 of phage T7 gene β-lactamase gene mini-intein dnaB and NruI/BamHI fragment of DNA containing adaptirovano is to these sites the sequence of a gene recombinant human glucagon

- contains:

as a genetic marker gene β-lactamase determining the stability of the transformed plasmid pER-Gl cells of E. coli to penicillin antibiotics;

unique recognition sites of restriction endonucleases that are located in the following distance to the left of the site BamHI - Nrul - 118 BP, NdeI -: 773 BP, XbaI - 812 BP, EcoRV - 2847 BP, HpaI - 2903 BP

Construction of recombinant plasmid DNA pER-Gl provides a high level of expression of cloned into her hybrid protein gene IntGl containing at the 5'end of the gene modified intein dnaB connected with the genome of the human glucagon (figure 1). To construct plasmids used chemical approach to use for the expression of cloned structural gene optimal regulatory elements that control its expression.

The gene of the human glucagon get chemical-enzymatic synthesis.

End sections containing the corresponding vector sites restricts introduced by PCR using synthetic oligonucleotide primers PrGlu-int and PrGlu-ter (figure 1) and then gene clone in the vector plasmid pTWIN1.

Offer producing strains of Escherichia coli ER2566/pER-Gl has the following features:

Morphological features. Cells are rod-shaped, gram-negative, risperadone.

Cultural attribute is key. Cells grow well on simple nutrient media. During growth on agar "Difco" - 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 and biological characteristics. 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.

Resistance to antibiotics. Cells are resistant to penicillin antibiotics (up to 500 µg/ml).

Producing strains of E. coli ER2566/pER-Gl differs from strain-recipient E. coli ER2566 only in the presence of recombinant plasmid DNA pER-Gl, which gives it resistance to penicillin antibiotics.

Strains-producers obtained by transformation of competent cells E. coli ER2566 appropriate recombinant plasmid DNA.

Cells of E. coli ER2566/pER-Gl are superproduction. When induction of isopropylthio-β-D-galactoside is effective hybrid protein biosynthesis IntGl, which accumulates in the cells in more than 40% of the total protein of the cells in the form of inclusion bodies.

Image is eenie as follows. Construct recombinant plasmid DNA pER-Gl, for which the chemical-enzymatic synthesis of gene recombinant human glucagon amplified by PCR using synthetic oligonucleotide primers (figure 2), containing sites of restricted NruI (N-end of the gene, primer PrGlu-int) and BamHi (C - end of the gene, primer PrGlu-ter), the resulting DNA digested with appropriate restrictase and then are ligated with the cleaved by the same sites of vector plasmid pTWIN1.

Ligase mixture transform competent cells of E. coli ER2566 and plated on YT agar containing 50 µg/ml ampicillin or other penicillin antibiotic. The resulting clones analyzed by hybridization32P-labeled oligonucleotides A1 and B1 (figure 2), and hybridization of clones secrete plasmid DNA, which is subjected to restriction analysis using restricted Nrul and BamHl.

Producing strains of E. coli ER2566/pER-Gl grown in rich medium (YT-, LB-broth and others) (or induce isopropylthio-β-D-galactoside and again grow) up to the maximum density of the culture.

Figure 1 shows the structure of a gene recombinant human glucagon.

The invention is illustrated in the following examples.

Example 1.

Construction of recombinant plasmid DNA.

Chemical synthesis of oligonucleotides perform solid-phase FOSFA amidite method on 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). Use synthetic cycle standard phospholidine method.

For preparation of vector DNA plasmids pTWTN1 (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) with the restriction enzyme NruI (10% act.),and then 40 ál Bushra R (10 mm Tris-HCl, pH 8.5, 10 mm MgCl2, 100 mm KCl, 0.1 mg/ml BSA) with the restriction enzyme BamHI (10 edict.) for 1 h at 37°C. Vector fragment between 3.6 KBP after electrophoresis in 1% agarose gel electrophoretic move in the layer of DEAE-paper, then elute 1M NaCl and precipitate DNA from solution by ethanol.

For the preparation of fragment spend gene amplification by PCR using as template a plasmid with an artificial genome of glucagon (0,01 µg / sample), and as primers synthetic oligonucleotides A1 and B1 (60 pmol each). PCR is carried out in a DNA-amplifier, buffer rastv the re, consisting each of four dNTP at a concentration of 0.5 mm and 5 units of the act. Taq-DNA-polymerase, as follows: denaturation 1 min at 94°C, annealing 30 seconds at 60°With elongation of 40 sec at 72°C, 30 cycles of PCR. After that the reaction mixture deproteinizing chloroform, evaporated to dryness, the residue was dissolved in 20 μl of water, then break down the same restrictases that were used in the preparation of the vector and produce the target fragment from the agarose gel. Figure 1 shows the nucleotide sequence and encoded by its amino acid sequence of glucagon person.

The obtained synthetic fragment from the genome of recombinant human glucagon in the amount of 2 pmol was added to a solution of 1 μg of the above described vector fragment in 10 μl of buffer (20 mm Tris-HCl, pH 7,56, 10 mm MgCl2, 0.2 mm rATP, 10mm dithiotreitol) and are ligated with 10 educt-DNA-ligase for 12 hours at 10°C.

An aliquot of the reaction mixture used to transform competent cells of E. coli ER2566. Transformants plated on plates with YT-agar containing 50 μg/ml ampicillin. Screening of recombinants carried out by using hybridization of colonies in situ with32P-labeled oligonucleotide A1 (figure 2). From hybridization of clones secrete DNA plasmid pER-Gl and analyzed by endonuclease NruI and BamHI. Physical map of plasmid pER-Gl made the Lena figure 3.

Example 2.

Getting a producer strain E. coli ER2566/pER-Gl and determine its productivity.

Producing strains of E. coli ER2566/pER-Gl is obtained by transformation of competent cells E. coli ER2566 the plasmid pER-Gl, as described in example 1. Cells of E. coli ER2566, bearing plasmid, the structure of which is confirmed by the data analysis (see example 1)are superproduction.

The producer strain E. coli ER2566/pER-Gl 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 6 h, or continue growing in the absence of inducer for 6 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 sec ultrasound, heated 3 min at 100°and samples of 1 μl used for electrophoresis in 15% SDS-PAG. Gel stain, Kumasi R-250 according to standard methods and scanned using a densitometer Shimadzu CS-930.

Example 3.

Getting a hybrid protein IntGl, its autocatalytic cleavage and secretion of recombinant human glucagon.

After the fermentation the cells producing the hybrid protein IntGl (biomass) is separated by centrifugation (5000 g, 20 min, 4° (C)destroy on ultrasonic disintegrator and produce by centrifugation (15000 g, 45 min) calf inclusion. Sediment Taurus inclusion is dissolved in a buffer containing 2M urea, diluted and adjusted pH to 5.5-6, thereby inducyruya autocatalytic cleavage of the hybrid protein, and incubated for 24 h at 25°C. Further purification of recombinant human glucagon carried out by reversed-phase liquid chromatography. Analysis of the resulting product is carried out by means OF HPLC. Fractions with protein IntGl not less than 95% unite and lyophilization recombinant glucagon is 10 mg from 100 g of cells or 10% of the total protein of the cells.

Identification of the resulting recombinant human glucagon carried out using MALDI-mass spectrometry mass spectrometer Vision 2000. The received signal recombinant glucagon corresponds to a calculated value of the mass 3485 Yes.

1. Recombinant plasmid DNA pER-Gl for synthesis of a hybrid polypeptide containing glucagon person and intein, in Escherichia coli cells, having a molecular weight of 4.38 Hmm, consisting of

NruI/BamHI fragment DNA plasmids pTWIN-1;

NruI/BamHI fragment containing DNA adapted to these sites the sequence of a gene recombinant human glucagon, is shown in figure 1; and

containing as a genetic marker gene β-lactamase determining the stability of the transformed plasmid pER-Gl cells of E. coli to penicillin antibiotics; unique recognition sites of restriction endonucleases that are located in the following distance to the left of the site BamHI - NruI - 118 BP, NdeI - 773 BP, XbaI - 812 BP, EcoRV - 2847 BP, HpaI - 2903 BP

2. The Escherichia coli strain ER2566/pER-Gl, producing a hybrid polypeptide containing glucagon person and intein obtained by transforming cells of Escherichia coli strain ER2566 recombinant plasmid DNA pER-Gl according to claim 1.

3. A method of obtaining a recombinant human glucagon, comprising transforming cells of Escherichia coli expression plasmid DNA that encodes a hybrid protein of the human glucagon, culturing the obtained strain, destruction of bacterial cells in buffer solution using ultrasound, followed by separation and splitting of the hybrid protein and the isolation and purification of the target product, characterized in that as Escherichia coli cells using cells of Escherichia coli strain ER2566, as plasmid DNA using DNA pER-Gl according to claim 1, selection and cleavage of the hybrid protein of glucagon is carried out by the Department of Taurus include, dissolving them in a buffer containing 2M urea, dilution and delivery the resulting solution to a pH of 5.5 to 6.0, followed encubierta is receiving 24 h at 25° With, the target product was then purified by reversed-phase chromatography.



 

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