RU2099421C1 - Method of preparing the human recombinant interleukin-3, recombinant plasmid dna p3pteil3 encoding the human recombinant interleukin-3, strain of bacterium escherichia coli - a producer of the human recombinant interleukin-3 - Google Patents

Abstract

FIELD: biotechnology, genetic protein engineering. SUBSTANCE: method is based on the use of new recombinant plasmid DNA p3PTEIL3 where an artificial gene of the human interleukin-3 is under control of three promoters tandem (P8, Ptac and Pt7) and its expression is carried out after induction with isopropylthiogalactoside and the use of the strain-producer E. coli VKPM B-6968 that provides high level of biosynthesis of the end protein (up to 30% of total cellular protein). Recombinant plasmid has tandem of promoters P8, Ptac and Pt7, two lac-operators, enhancer of translation of gene-10 of phage T7, terminator of transcription of phage fd, gene of β-lactamase and an artificial gene of the human interleukin-3. Strain Escherichia coli VKPM B-6968 is obtained by transformation of competent cells E. coli BL21(DE3) ekfpvbljq p3PTEIL3. Strain E. coli VKPM B-6968 is cultured on rich medium, the grown cells were disrupted in buffer solution and the human recombinant interleukin-3 is isolated from inclusion bodies followed by its renaturation by the known method. EFFECT: improved method of preparing. 3 cl, 3 dwg

Description

 The alleged invention relates to biotechnology, in particular to genetic and protein engineering. It includes an in vitro recombinant plasmid DNA p3PTEIL3, which determines the biosynthesis of recombinant human interleukin-3 (rhIL3), E. coli / p3PTEIL3 strain producing rhIL3, and a method for producing recombinant interleukin-3 based on the above recombinant DNA and strain producing.

Human interleukin-3 (hIL3) glycoprotein produced by T-lymphocytes is a mediator of the growth and differentiation of cells of various lines. It plays an important role in the regulation of hematopoiesis and the immune response [1, 2] hIL3 is a non-specific hematopoietin that acts on the precursors of all blood cells and even on mature cells [3] These properties of hIL3 determine its use as a hematopoietic regulator. Due to the low availability of natural hIL3, the use of recombinant hIL3 (rhIL3), which, despite the absence of glycosylating groups in the structure, has the same biological activity as natural hIL3, is of great importance [4, 5]
Earlier, to obtain rhIL3 by microbiological synthesis in E. coli, the expression plasmid pTE21L3 [6] was already constructed with which a high level of rhIL3 biosynthesis was achieved in some E. coli strains. However, a highly productive strain during prolonged cultivation or during many passages lost its productivity due to plasmid segregation due to the excessive level of constitutive biosynthesis of the recombinant protein.

A more stable system preserving a high rhIL3 yield was obtained in the construction of the expression plasmid pTOTE2IL3 using a biosynthesis scheme with transcription induction in the late stages of producer culture growth [7] In this plasmid, transcription was initiated with the tandem of two P3 promoters of phage fd and Ptac controlled by two lac-operators (7). Moreover, a high level of rhIL3 biosynthesis was achieved in E. coli JM109 and E.coli TGI strains, however, their productivity was insufficient due to either slow cell mass accumulation (JM 109) or plasmid recombination instability (in gesA + TGI strain) [7]
The proposed group of inventions allows to obtain recombinant human interleukin-3 in a stable producer strain with a high yield, allowing efficient purification using a simple technology.

For this purpose, a new expression plasmid construct was used, containing an additional phage T7 RNA polymerase (Pt7) promoter in E. coli strain BL21 (DE3), whose chromosome contains the T7 RNA polymerase gene under the control of the inducible Ptac promoter [8]
The proposed method for producing interleukin-3 is that p3PTYIL3 recombinant plasmid DNA is transformed with Escherichia coli strain BL21 (DE3), the resulting Escherichia coli strain BL21 (DE3) / p3PTEIL3 is cultured in a rich medium, the grown cells are destroyed in a buffer solution and rhIL3 protein is isolated .

The proposed recombinant plasmid DNA p3PTEIL3 is characterized by the following features:
encodes the amino acid sequence of the rhIL3 protein;
has a pier. m. 2.34 MDa;
contains:
the tandem of the P8 promoters of phage fd and Ptac, two lac operators, the P7 promoter, the translation enhancer of the T7 phage 10 gene, the interleukin-3 gene and the fd phage transcription terminator;
as a genetic marker, the β-lactamase gene that determines the resistance of E. coli cells transformed with plasmid p3PTEIL3 to penicillin antibiotics;
unique restriction endonuclease recognition sites located at the following distance to the right of the BamHI site:
XbaI 325bp, EcoRV 361bp, Ndel 365 and 1292 bp, HpaI 411 bp, Hind III-545 bp, EcoRI 774 bp, Bg III 1128 bp, PstI 2527 bp.

 The construction of recombinant plasmid DNA p3PTEIL3 provides a high level of gene expression of interleukin-3.

 The proposed producer strain Escherichia coli BL21 (DE3) / p3PTEIL3 is characterized by the following features.

 Morphological signs. The cells are rod-shaped, gram-negative, non-spore.

 Cultural signs. Cells grow well on simple nutrient media. When growing on Difco agar, the colonies are round, smooth, cloudy, shiny gray, the edge is even. When growing on liquid media (on a minimal medium with glucose or YT-broth) they form an intense smooth haze.

Physico-biological signs. Cells grow at a temperature of from 4 ^o C to 40 ^o C with an optimum pH of 6.8 to 7.5. As a source of nitrogen, both mineral salts in ammonium form and organic compounds in the form of peptone, tryptone, yeast extract, amino acids, etc. are used. Amino acids, glycerin, carbohydrates are used as a carbon source.

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

 The strain producing E. coli BL21 (DE3) / p3PTEIL3 differs from the strain of the recipient E. coli BL21 (DE3) only in the presence of recombinant plasmid DNA p3PTEIL3, which gives it resistance to penicillin antibiotics.

 The producer strain is obtained by transformation of competent E. coli BL21 (DE3) cells with the corresponding recombinant plasmid DNA.

 E. coli BL21 (DE3) / p3PTEIL3 cells are a producer of rhIL3 protein. When isopropylthio-b-D-galactoside is induced, an effective biosynthesis of the protein occurs, which accumulates in the cells in the form of inclusion bodies, and its yield is more than 30% of the total bacterial protein.

The invention is as follows. Recombinant plasmid DNA p3PTEIL3 was constructed. For this, the vector fragment of the plasmid pTE2IL3 [6] is first obtained by its cleavage with the endonucleases BamHI and XbaI. After purification by electrophoresis on a 1% agarose gel, the vector fragment is ligated with the BamHI / EcoRI fragment of plasmid pKK223-3 containing the Ptac promoter, a synthetic lac operator [7] and a synthetic fragment containing the Pt7 promoter. The competent E. coli BL21 (DE3) cells are transformed with the ligase mixture and plated on YT agar containing 50 μg / ml ampicillin or another penicillin antibiotic. The obtained clones are analyzed by hybridization with ³² P-labeled oligonucleotides that make up the synthetic fragments, and plasmid DNAs are isolated from hybridizing with probe clones, which are subjected to restriction analysis using restriction enzymes HindIII, EcoRI, BamHI and BglII, as well as nucleotide sequence determination between BamHI and EcoRI sites . The producer strain E / coli BL21 (DE3) / p3PTEIL3 is grown in a rich medium (YT-, LB-broth, etc.), isopropylthio-bD-galactoside is induced, and grown again to reach the maximum culture density.

Isolation of rhIL3 protein from producer cells involves the following steps:
the destruction of the grown cells by one of the commonly used methods;
washing with buffer solutions of inclusion bodies from soluble components of the cell;
solubilization of the denatured target protein from inclusion bodies in either a 5 8 M urea solution, or a 5 M guanidine hydrochloride solution, or in another suitable solvent;
purification of the target protein as a result of stepwise dilution of solutions in urea or guanidine hydrochloride, or other methods;
renaturation of rhIL3 by treating the denatured protein with a mixture of the reduced and oxidized forms of glutathione;
final purification of rhIL3 by HPLC.

 In fig. 1 shows the partial structure of plasmid p3PTEIL3 in the promoter region of the rhIL3 gene and the corresponding amino acid sequence of rhIL3.

 In fig. Figure 2 shows the structures of synthetic fragments with the lac operator (A) and the Pt7 promoter (B).

 In fig. 3 shows the construction scheme of plasmid p3PTEIL3.

 Example 1. Chemical synthesis of oligonucleotides.

The oligonucleotides are synthesized using the solid-state phosphoamidite method on an ASM-102U DNA synthesizer (NPO Biosan, Novosibirsk) with the oligonucleotide chain being extended from the 3'-end to the 5'-end using 5'-dimethoxytrityl-N-acyl-protected phosphamidites 2'-deoxynucleoside-3'-O- (b-cyanethyl diisopropylamino) phosphites activated with tetrazole. The synthesis is carried out on a scale of 0.5 to 0.7 μmol, using porous glass (pore size 500 A) as a support, to which the first nucleoside unit (load 20 30 μmol / g) is connected via a 3'-succinate bond. Using the synthetic cycle described in [9]
Example 2. Construction of recombinant plasmid DNA p3PTEIL3.

To prepare the plasmid DNA vector, pTE2IL3 (3 μg) is treated with 40 μl KGB buffer (100 mM K-glutamate, pH 8.8, 25 mM Tris-acetate, pH 7.6, 10 mM Mg-acetate, 1 mM mercaptoethanol) restrictase BamHI (10 units of act.) And XbaI (10 units of act.) For 1 h at 37 ^o C. Vector fragment 3.34 kbp. after electrophoresis in a 1% agarose gel, 0.5% LGT agarose is electrophoretically transferred to the gel layer and then eluted by freezing the agarose and subsequently precipitating the DNA from the solution with ethanol.

 Under the same conditions, from 3 μg of plasmid pKK223-3 using restriction enzymes BamHI (10 units act) and EcoRI (10 units act), a fragment containing a Ptac promoter 0.26 kb in length was obtained.

 Synthetic fragments (A) and (B) are obtained by annealing the corresponding complementary 5'-phosphorylated oligonucleotides.

5 μl of solutions of synthetic fragments (A) and (B), each containing 20 pmol, are added to a solution of 1 μg of the above vector fragment and 0.1 μg of the fragment containing the Ptac promoter in 10 μl of L buffer (20 mM Tris-HCl , pH 7.56, 10 mm MgCl ₂ , 0.2 mm rATP, 10 mm dithiothreitis) and are ligated with 20 units Act. T4 DNA ligase for 6 hours at 10 ^o C.

An aliquot of the reaction mixture is used to transform competent E. coli BL21 (DE3) cells. Transformants are plated on a YT agar plate containing 50 μg / ml ampicillin. Recombinant screening is performed by in situ hybridization of colonies with ³² P-labeled oligonucleotides comprising fragments (A) and (B). DNA is isolated from the hybridizing clones and analyzed using endonucleases HindIII, BamHI, EcoRI and BglII and determining the nucleotide sequence of the regulatory region and the structural gene.

 Example 3. Obtaining a strain of E. coli BL21 (DE3) / p3PTEIL3 (VKPM B6511) - producer of rhIL3 protein and determination of its productivity.

 E. coli BL21 (DE3) cells carrying the plasmid p3PTEIL3, the structure of which is confirmed by the analysis data (see Example 2), are the producer of the rhIL3 protein.

The producer strain E. coli BL21 (DE3) / p3PTEIL3 was grown at 37 ^° C in 100 ml of YT broth (pH 7.0) with 50 μg / ml ampicillin for 2 hours on a shaker at a speed of 190 rpm until turbidity A ₅₅₀ 0.7 0.8, add isopropylthio-bD-galactoside to a concentration of 0.2 mM and continue the process for another 6 hours. Each hour, a 2 ml sample is taken, A ₅₅₀ is determined and the amount of culture corresponding to 1 ml with A ₅₅₀ 1, 0, centrifuged for 5 minutes at 6000 rpm. Precipitated cells in 100 μl of lysing buffer with dye bromphenol blue are treated with ultrasound for 20 minutes, heated for 3 min at 100 ^° C and 1 μl samples are used for electrophoresis in 15% SDS-PAGE. The gel was stained with Coomassie R-250 according to standard procedures and scanned to determine the relative amount of protein in the band corresponding to the rhIL3 protein.

Example 4. Isolation and primary purification of rhIL3 protein. Wet cells (100 g) are suspended in 200 ml of buffer (50 mm Na-phosphate, 1 mm EDTA, pH 7.5), lysozyme (100 μg / ml) is added, incubated for 30 min at 20 ^° C, MgCl ₂ solution is added ( up to 10 mM) and DNase (10 μg / ml), incubated at 20 ^o C until the viscosity is lost (30 min), diluted with 2 L of buffer (50 mM Na-phosphate, 10 mM EDTA, 4 M urea, 1% Triton X- 100, pH 7.5) and centrifuged for 20 min at 10,000 g. The precipitate was suspended in 2 L of buffer (50 mM Na-phosphate, 10 mM EDTA, 4 M urea, 1% Triton X-100, pH 7.5) using a Sonifier 240 ultrasonic disintegrator (Branson) and centrifuged for 20 min at 10,000 g. Buffer washing is repeated once more, then similarly washed with buffer (50 mM Na-phosphate, 1 mM EDTA, pH 7.5).

 Example 5. Renaturation and final purification of recombinant interleukin-3 (rhIL3).

The precipitate of denatured rhIL3 after the initial purification described in Example 4 was resuspended in 2 L of buffer (50 mM Na-phosphate, 1 mM EDTA, 5 M guanidine hydrochloride, pH 7.5) and incubated at 20 ^° C. The resulting solution was diluted with stirring 8 l of buffer (50 mm Na-phosphate, 1 mm EDTA, pH 7.5), adjust the pH of the suspension to 8.5 and add a mixture of oxidized and reduced glutathione to a concentration of 1.5 mm. The mixture was stirred at 20 ^° C, monitoring the progress of the oxidation of thiol groups in rhIL3 using the Ellman method [10] and the renaturation was considered to be complete with a negative reaction. After oxidation is complete, the solution is concentrated in an ultrafiltration cell (Amicon) on a PM-10 membrane, trifluoroacetic acid is added to 0.1% and centrifuged for 20 min at 10,000 g. The clarified solution is chromatographed on a Nucleosil 300-7C ₄ preparative column (Masherey-Nagel) in a linear gradient of 0 60% acetonitrile in 0.1% trifluoroacetic acid. The fraction of purified rhIL3 (leaves at 40 43% acetonitrile) was lyophilized. The purity of the drug is more than 98% (according to analytical HPLC). The yield of pure rhIL3 is 20 25% of the protein determined in the producer's wet cells.

 Thus, the claimed group of inventions allows to obtain recombinant interleukin-3 by microbiological synthesis according to simple technology and with high yield.

Sources of information
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 3. Wright A. Fundamentals of immunology. 1991. M. Mir, p. 130 134.

 4. Kimmenade A. Bond M.W. Shumacher J.H. Laquoi C. Eur. J. Biochem. 1988. V. 173. No. 1, p. 109 114.

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 6. Gurevich A.I. Skaptsova N.V. Lutsenko S.V. Smirnov V.A. Kurkin A.R. Azhayev A.V. Bioorgan. chemistry. 1991.V. 17.No. 5, p. 647 -652.

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Claims (3)

 1. The method of producing recombinant human interleukin-3, comprising cultivating a producer of the species Escherichia coli in a rich medium with induction of protein biosynthesis with isopropylthio-β-D-galactoside, solubilization, renaturation and purification of the target product, characterized in that Escherichia bacteria strain is used as a producer coli VKPM B-6968.  2. Recombinant plasmid DNA p 3PTEIL3 encoding recombinant human interleukin-3, molecular weight 2.34 MDa, containing the tandem of the P8 promoters of phage fd and Ptac, two lac operators, the Pt7 promoter, translational amplification gene 10 of the phage T7, interleukin-3 gene and phage fd transcription terminator, β-lactamase gene determining penicillin antibiotic resistance, unique restriction endonuclease recognition sites located at the following distance to the right of the BamHL site: XPL 325 bp, ECORV 361 bp, NdeL 365 and 1292 bp, HpaL 411 bp, Hind III 545 bp, ECOPL 774 bp, Bg III 1128 bp, PstI-2527 bp.  3. The bacterial strain Escherichia coli VKPM B-6969 producer of recombinant human interleukin-3.

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