RU2672816C2 - METHOD OF PRODUCING NON-METHIONINE RECEPTOR ANTAGONIST INTERLEUKIN-36 STRAIN BACTERIA ESCHERICHIA COLI BL21 [DE3] - PRODUCER NON-METHIONINE RECEPTOR ANTAGONIST INTERLEUKIN-36 (VARIANTS), METHOD OF THEIR PREPARATION, AND PLASMID VECTOR pBAD15A pET15A AND DNA SEQUENCE FOR THEIR PREPARATION - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to the field of biotechnology, in particular to a technology for producing a receptor antagonist of interleukin-36, and is a plasmid expression vector pBAD15A for producing a non-methionine recombinant human interleukin-36 (IL-36PA) receptor antagonist of a human, the map of which is shown at Fig. 2, pET15A plasmid expression vector for producing a human without the use of recombinant IL-36PA, the map of which is shown at FIG. 3, as well as the strain producing the non-methionine recombinant human IL-36PA.EFFECT: invention allows to obtain a more active IL-36RA with a higher yield.5 cl, 12 dwg, 7 ex, 4 tbl

Description

The invention relates to the field of biotechnology, in particular to genetic engineering drugs for medical purposes and the technology for their preparation, more precisely, to the technology for producing the receptor antagonist of interleukin-36.

Interleukins-36 alpha, beta and gamma (IL-36α, IL-36β, IL-36γ), as well as the receptor antagonist of interleukins-36 (IL-36Ra) are a separate group in the interleukin-1 family [Gresnigt MS, van de Veer -donk FL Biology of IL-36 cytokines and their role in disease // Semin. Immunol. 2013. Vol. 25, No. 6. P. 458-465]. Interleukins of the IL-36 group are synthesized mainly in epithelial tissues and in the skin, do not have signal peptides and therefore cannot be secreted by the usual secretion pathway [Vigne S. et al. IL-36R ligands are potent regulators of dendritic and T cells // Blood. 2011. Vol. 118, No. 22. P. 5813-5823.].

IL-36 (α, β, γ) play an important role in the activation of the inflammatory process in the skin and lungs. In particular, a number of experimental data indicate the possible participation of IL-36 in the development of psoriasis and inflammatory diseases of the respiratory tract and joints [Ramadas R.A. et al. IL-36α exerts pro-inflammatory effects in the lungs of mice // PloS One. 2012. Vol. 7, No. 9. P. e45784 .; Vigne S. et al. IL-36R ligands are potent regulators of dendritic and T cells // Blood. 2011. Vol. 118, No. 22. P. 5813-5823 .; Foster A.M. et al. IL-36 promotes myeloid cell infiltration, activation, and inflammatory activity in skin // J. Immunol. Baltim. Md 1950. 2014. Vol. 192, No. 12. P. 6053-6061]. Several studies have shown that some mutations in the interleukin-36 receptor antagonist gene (IL-36RA) can cause generalized pustular psoriasis (GLP), a deadly form of psoriasis that affects large areas of the skin and is characterized by periodic exacerbations, hyperleukocytosis and an increase in C level -reactive protein in the blood [Sugiura K. et al. The Majority of Generalized Pustular Psoriasis without Psoriasis Vulgaris Is Caused by Deficiency of Interleukin-36 Receptor Antagonist // J. Invest. Dermatol. 2013. Vol. 133, No. 11. P. 2514-2521 .; Farooq M. et al. Mutation analysis of the IL36RN gene in 14 Japanese patients with generalized pustular psoriasis // Hum. Mutat. 2013. Vol. 34, No. 1. P. 176-183; Griffiths C.E.M., Barker J.N.W.N. Pathogenesis and clinical features of psoriasis // Lancet Lond. Engl. 2007. Vol. 370, No. 9583. P. 263-271 .; Zelickson B.D., Muller S.A. Generalized pustular psoriasis. A review of 63 cases // Arch. Dermatol. 1991. Vol. 127, No. 9. P. 1339-1345.].

Given that GLP is caused, as a rule, by a lack of activity of IL-36PA [Sugiura K. et al. The Majority of Generalized Pustular Psoriasis without Psoriasis Vulgaris Is Caused by Deficiency of Interleukin-36 Receptor Antagonist // J. Invest. Dermatol. 2013. Vol. 133, No. 11. P. 2514-2521 .; Farooq M. et al. Mutation analysis of the IL36RN gene in 14 Japanese patients with generalized pustular psoriasis // Hum. Mutat. 2013. Vol. 34, No. 1. P. 176-183; Hussain S. et al. IL36RN mutations define a severe autoinflammatory phenotype of generalized pustular psoriasis // J. Allergy Clin. Immunol. 2015. Vol. 135, No. 4. P. 1067-1070.e9], the introduction of exogenous IL-36RA into the body seems to be a promising approach to the treatment of this disease. In addition, therapy aimed at suppressing inflammation mediated by IL-36 could also be used to treat other forms of psoriasis, as well as other inflammatory skin diseases, due to the fact that IL-36Ra is able to activate the anti-inflammatory cascade by interacting with the SIGIRR receptor [ Costelloe C. et al. IL-1F5 mediates anti-inflammatory activity in the brain through induction of IL-4 following interaction with SIGIRR / TIR8 // J. Neurochem. 2008. Vol. 105, No. 5. P. 1960-1969.].

Closest to the present group of inventions was the technology previously developed by the authors for the production of the IL-36 receptor antagonist (IL-36Ra), which includes DNA sequences encoding full-length IL-36Ra; The expression vector pET-IL36Raf, as well as the bacterial strain Escherichia coli BL21Star [DE3] (pET-IL36Raf) containing the vector pET-IL36Raf, is a producer of full-sized human IL-36Ra (RU 2582569, 2016).

The disadvantage of using this group of inventions is to obtain a drug with insufficiently high antagonistic activity.

The technical problem solved by the authors was the development of a technology for the production of recombinant biologically active human IL-36Ra with higher activity.

The basis of the proposed solution was the assumption that the activity of IL-36Ra can be increased by removing from its molecule the N-terminal methionine residue as a result of its treatment with methionine aminopeptidase.

The technical result was achieved by creating a group of inventions, including:

- a technology for the preparation of non-methionine human IL-36Ra based on strains transformed with a plasmid vector containing the methionine aminopeptidase gene, followed by isolation and concentration of the target product;

- variants of strains transformed with a plasmid vector containing the methionine aminopeptidase gene, the bacterial strain Escherichia coli BL21Star [DE3] (pET-IL3 6Raf, pBAD15A) and the bacterial strain Escherichia coli BL21Star [DE3] (pET-IL36Raf, pET15A);

- ways of their construction, which consists in transforming the Escherichia coli strain BL21Star [DE3] with plasmid vectors with induction of expression of the human interleukin-36 receptor antagonist (IL-36RA) and methionine aminopeptidase (MAP) genes.

- plasmid expression vector pBAD15A for transformation of the bacterial strain Escherichia coli BL21Star [DE3] (pET-IL36Raf, pBAD15A)

- plasmid expression vector pET15A strain of bacteria Escherichia coli BL21Star [DE3] (pET-IL36Raf, pET15A)

- a DNA sequence encoding methionine aminopeptidase to obtain plasmid vectors.

The DNA sequence encoding methionine aminopeptidase was amplified using Escherichia coli strain BL21Star [DE3] as a DNA template from a bacterial culture. At the ends, oligonucleotides that form the restriction sites NdeI and XhoI were added to the gene. (The obtained DNA sequence is shown on SEQ ID No 1.)

The gene was digested with the above restriction enzymes and cloned into a similarly prepared pBAD22 vector derived from the pET22b + vector to obtain the intermediate plasmid vector pBAD22 / MAP.

The regulatory sequence methionine aminopeptidase gene was then cloned from the plasmid pBAD22 / MAP into plasmid pACYC184 at the HindIII and SalI restriction sites to obtain the expression plasmid vector pBAD15A, the map of which is shown in FIG. 2.

The plasmid expression vector pBAD15A contains the E. coli methionine aminopeptidase gene (MAP), the pBADara promoter, the rrnB gene transcription terminators (rrnB T1T2), the araBAD operon regulatory sequence, the p15A plasmid replication origin, the chloramphenicol acetyl-transferase gene for Cam combining individual fragments in a vector (SalI, HindIII, XhoI, NdeI).

The plasmid expression vector pET15A was obtained in a similar manner. The E. coli methionine aminopeptidase gene was cloned into the pET302 / NT-His vector at the EcoRI and BamHI restriction sites to give the intermediate plasmid pET302 / MAP. Then, the regulatory sequence methionine aminopeptidase gene was cloned from the pET302 / MAP plasmid into the pACYC184 plasmid at the HindIII and SalI restriction sites to obtain the pET15A expression plasmid vector, the map of which is shown in FIG. 3.

The plasmid expression vector pET15A (Fig. 3) contains the E. coli methionine aminopeptidase gene (MAP) under the control of the regulated T7 promoter, T7 transcriptional terminator, p15A replication origin, chloramphenicol acetyl transferase gene (CamR), restriction sites necessary for association individual fragments in the vector (EcoRI, BamHI, HindIII, SalI).

The obtained strains of Escherichia coli BL21Star [DE3] (pET-IL36Raf, pBAD15A) and Escherichia coli BL21Star [DE3] (pET-IL36Raf, pET15A) are characterized by the same cultural-morphological and physico-biochemical properties:

Cultural and morphological features of the strains. Gram-negative straight sticks, 1.1-1.5 × 2.0-3.0 microns in size, single, do not form spores and capsules. Catalopositive. Oxidase-negative. Optional anaerobes. Cells grow well on simple nutrient media containing and not containing ampicillin and chloamphenicol, for example, on LB medium. On an agar medium, colonies are smooth, round, slightly convex, with a smooth edge. A uniform light-scattering suspension is formed in liquid media; when stored without stirring, they settle to the bottom. Cells grow in the temperature range from 8 ° C to 43 ° C, the interval for cultivation is 28-38 ° C, the optimum growth at 37 ° C. The pH range is 5-8. Catalyze D-glucose and some other carbohydrates with the formation of acid and gas, do not ferment galactose. Voges-Proskauer reaction is negative, do not form H ₂ S, hydrolyze urea.

Characteristics of a useful substance synthesized by strains.

Recombinant protein is a human interleukin-36 receptor antagonist with a cleaved N-terminal methionine residue (36 people are a non-methionine interleukin receptor antagonist).

The activity of the strains. The productivity of the strains - recombinant IL-36Ra is at least 3% of the mass of the proteins of the cell lysate during cultivation under conditions of induction.

Cryopreservation. In sealed ampoules, the strains are freeze-dried in a medium containing 0.8-1.2% sucrose and 0.8-1.2% mannitol, stored at room temperature for 20 years.

The cultivation of strains. Cultivation at a temperature of 28-38 ° C in a thermostat or rocking chair, in agar (2% agar) or liquid LB-medium, respectively. Selective conditions - the addition of 50-125 μg / ml ampicillin and 20-40 μg / ml chloramphenicol.

Fermentation. For fermentation of the obtained strains, 1-2 times LB medium in 0.8-1.2 times M9 medium with the addition of 50-125 μg / ml ampicillin and 20-40 μg / ml chloramphenicol are used. The cultures are grown in flasks with a volume of 0.5-2.5 L (preferably 2 L) at a temperature of 35-38 ° C (preferably + 37 ° C) to a density of 2-5 OU / ml (preferably 3 OU / ml). At this optical density, an IPTG inductor of 0.1-1 mM (preferably 0.5 mM) is introduced. In the case of the Escherichia coli strain BL21Star [DE3] (pET-IL36Raf, pBAD15A), arabinose 0.1-1% (preferably 0.2%) is also introduced. Induction is continued for 2-4 hours (preferably 3 hours) at a temperature of 27-37 ° C (preferably + 30 ° C).

The method of purification of recombinant IL-36PA from the biomass of producer strains involves lysing cells by adding a Tris buffer solution, after which the resulting lysate is clarified and then purified from impurity proteins by chromatography successively with Q FF sorbent (GE) and Butyl-650 sorbent ( Tosoh) and concentration by ultrafiltration using a Millipore Amicon Stirred Cell stirred cell using a Sartorius Ultracel PLC10 membrane followed by dialysis.

Lysis, as a rule, is carried out using a buffer solution containing from 30 to 70 mm Tris-HCl (optimally 50 mm), pH from 6.8 to 7.2 (optimally 7.0).

The lysate is clarified by centrifugation at 5-12 thousand rpm./min, after which a calcium chloride solution is added to the resulting supernatant to a final concentration of 20 to 40 mM (optimally 30) and a trisodium phosphate solution to a final concentration of 10 to 30 mM (optimally 20 ), mix, and then clarify again by centrifugation at 5-12 thousand rpm.

Chromatography on Q FF (GE) is carried out on a sorbent with a balanced buffer solution containing 30-70 mM Tris-HCl (optimally 50 mM), pH 6.8-7.2 (optimally 7.0), followed by washing with a buffer solution containing 30-70 mM Tris-HCl (optimally 50 mM), 0-0.3 M NaCl (optimally 0.15), pH 6.8-7.2 (optimally 7.0). The column is regenerated with a buffer solution containing 30-70 mM Tris-HCl (optimally 50 mM), 1.5-2 M NaCl (optimally 2 M), pH 6.8-7.2 (optimally 7.0).

Purification of the target protein from impurity proteins on the sorbent Butyl-650 (Tosoh) is carried out as follows. Ammonium sulfate is added to 0.15-0.5 M (optimally 0.3). To the unbound fraction after chromatography on Q FF (GE) containing IL-36PA, ammonium sulfate was added to 0.15-0.5 M (optimally 0.3), then applied to a Butyl-650 sorbent (Tosoh), balanced 0, 3 ± 0.05 L buffer solution containing 30-70 mM Na ₂ HPO ₄ (optimally 50 mM), 0.15-0.5 M (NH ₄ ) ₂ SO ₄ (optimally 0.3), pH 5.8 -6.2 (optimally 6.0). Then, IL-36RA was eluted with a buffer solution containing 30-70 mM Na2HPO4 (optimally 50 mM), pH 5.8-6.2 (optimally 6.0). The column regenerate 0.5 ± 0.1 l of distilled water.

The concentration of IL-36RA is carried out by ultrafiltration using a Millipore Amicon Stirred Cell with a Sartorius Ultracel PLC 10 membrane with a cut-off limit of 10 kDa, and then the solution is changed by protein dialysis against a solution containing 30-70 mm sodium citrate (optimally 50 mm ), pH 5.8-6.2 (optimally 6.0).

After that, the protein solution can be sterilized if necessary using Millipore Millex syringe filters with a pore diameter of 0.22 μm.

After all stages of purification, the obtained IL-36RA has a purity of> 95% by RP-HPLC, has <5% impurities of an unprocessed form with an uncleaved initiator methionine residue and <5% of a deamidated form.

If necessary, for better storage, the production technology of IL-36RA can be supplemented by introducing into the target product 20-100 mM nitrate buffer solution (optimally 50 mM) with a pH of 5.5-6.5 (optimally 6.0), tween-80 in quantity 0.01-1% and EDTA in an amount of 0.1-10 mm.

The resulting preparation was tested on a model of psoriasis-like dermatitis caused by cutaneous application of an agonist of Toll-like receptors 7 and 8 of imichimode [van der Fits L. et al. Imiquimod-induced psoriasislike skin inflammation in mice is mediated via the IL-23 / IL-17 axis // J. Immunol. Baltim. Md 1950. 2009. Vol. 182, No. 9. P. 5836-5845.]. It was shown that daily subcutaneous administration of the developed preparation of recombinant human IL-36RA in doses of 50 ± 1 and 10 ± 1 μg / mouse significantly reduces the severity of experimental dermatitis caused by imichimod. Moreover, the effect appears within 3-4 days after the first injection of the drug and exceeds the effect of the control drug - methotrexate.

The invention is illustrated by sequences and figures.

FIG. 1. The nucleotide sequence of the methionine aminopeptidase gene of E. coli.

FIG. 2. Map of plasmid vector pBAD15A carrying the sequence

the methionine aminopeptidase gene, where:

MAP - E. coli methionine aminopeptidase gene

pBADara promoter is a prokaryotic promoter necessary to initiate gene transcription

rrnB T1T2 terminators - terminators of transcription of the rrnB gene of E. coli

araBAD operon is the regulatory sequence of the araBAD operon necessary for regulating the expression of genes controlled by the pBADara promoter

p15A origin - plasmid replication origin site

CamR - chloramphenicol acetyltransferase gene sequence ensuring bacterial resistance to chloramphenicol

SalI, HindIII, XhoI, NdeI - restriction sites necessary for combining individual fragments in the vector

FIG. 3. Map of the plasmid vector pET15A carrying the sequence of the methionine aminopeptidase gene, where

MAP - methionine aminopeptidase gene sequence

T7 promoter - T7 phage promoter needed to initiate gene transcription

T7 terminator - phage T7 gene transcription terminator

lacI is the lacI gene sequence that regulates the expression of genes under the control of the T7 promoter.

p15A origin - plasmid replication origin site

CamR - chloramphenicol acetyltransferase gene sequence ensuring bacterial resistance to chloramphenicol EcoRI, BamHI, HindIII, SalI - restriction sites necessary for combining individual fragments in a vector

FIG. 4. Electrophoregram of biomass lysates of various producer strains of IL-36RA after cultivation under the conditions of adding inductors and without adding them. The thick blue arrow marks the strip corresponding to IL-36RA; a thin white arrow marks the band corresponding to the MAP.

1 - Biomass lysate of Escherichia coli strain BL21Star [DE3]

2 - Biomass lysate of Escherichia coli strain BL21Star [DE3] (pET-IL36Raf) without induction

3 - Biomass lysate of Escherichia coli strain BL21Star [DE3] (pET-IL36Raf) after induction

4 - Biomass lysate of Escherichia coli strain BL21Star [DE3] (pET-IL36Raf) without induction, another method of lysis

5 - Biomass lysate of Escherichia coli strain BL21Star [DE3] (pET-IL36Raf) after induction, another lysis method

6 - Biomass lysate of Escherichia coli strain BL21Star [DE3] (pET-IL36Raf, pET15A) without induction

7 - Biomass lysate of Escherichia coli strain BL21Star [DE3] (pET-IL36Raf, pET15A) after induction

8 - Biomass lysate of Escherichia coli strain BL21Star [DE3] (pET-IL36Raf, pBAD15A) without induction

9 - Biomass lysate of Escherichia coli strain BL21Star [DE3] (pET-IL36Raf, pBAD15A) after induction

FIG. 5. Comparison of the production of IL-36RA with different producer strains using solid-phase ELISA using monoclonal antibodies to IL-36RA. The designations correspond to the plasmid with the MAP gene present in the strain:

"PBAD15A" - strain Escherichia coli BL21Star [DE3] (pET-IL36Raf, pBAD15A)

"PET15A" - Escherichia coli strain BL21 Star [DE3] (pET-IL36Raf, pET15A) "without co-expression with MAP" - Escherichia coli strain BL21Star [DE3] (pET-IL36Raf)

FIG. 6. Comparison of the impurity of the unprocessed form of IL-36RA containing the methionine initiator residue in IL-36RA samples obtained from various producer strains. The designations correspond to the plasmid with the MAP gene present in the strain:

"PBAD15A" - strain Escherichia coli BL21Star [DE3] (pET-IL36Raf, pBAD15A)

"PET15A" - Escherichia coli strain BL21Star [DE3] (pET-IL36Raf, pET15A) "without co-expression with MAP" - Escherichia coli strain BL21Star [DE3] (pET-IL36Raf)

FIG. 7. Electrophoregram fractions obtained by chromatographic purification of IL-36RA. The arrow marks the band corresponding to IL-36RA.

1 - clarified biomass lysate of the producer strain IL-36PA Escherichia coli BL21Star [DE3] (pET-IL36Raf, pBAD15A)

2 - fraction not associated with Q

3 - fraction not associated with Butyl-650

4 - fraction eluted with Butyl-650

5 - passing fraction after ultrafiltration concentration

6 - molecular weight marker

FIG. 8. Chromatogram OF-HPLC separation of the IL-36RA sample after chromatographic purification.

FIG. 9. Overlaying OF-HPLC chromatograms of the separation of IL-36RA samples obtained by co-expression with MAP and without co-expression with MAP.

Figure 10. Electrophoregram of separation of native (1) and deamidated (2) forms of IL-36RA in KZEF.

FIG. 11. Change in the PASI index in the experimental and control groups (* p <0.01 compared with the corresponding values of the control group). On the ordinate axis - the severity of signs of psoriasis-like dermatitis in points.

FIG. 12. Change in the thickness of the ear at the site of application of imichimod in the experimental and control groups (* p <0.05 compared with the corresponding values of the control group). The ordinate axis is the index of the change in the thickness of the ear at the site of application of imichimode.

The essence and industrial applicability of the claimed group of inventions is illustrated by the following examples

Example 1. Construction of expression plasmid vectors.

Construction of the intermediate plasmid pBAD22. The plasmid pBAD22 contains the beta-lactamase gene, which provides resistance to antibiotics of the beta-lactam group, the pBR322 replication site, as well as the regulatory elements and the promoter of the araBAD operon, which allow inducing and regulating the expression of the target protein by changing the concentration of arabinose in the medium.

At the first stage, the pBAD plasmid was modified (ThermoFisher Scientific, USA): the plasmid region containing the NdeI restriction site was deleted, and the original polylinker was replaced by the polylinker of plasmid pET22b (+) (Novagen, USA). For this purpose, the fragment of plasmid pBAD was amplified using a pair of 5'-phosphorylated primers (product length 3885 bp):

5'-GCTGCATGTGTCAGAGGTT-3 '

5'-GCACAGATGCGTAAGGAGAA-3 '

It is acceptable to use primers in the concentration range from 5 to 25 μM, however, the best result is achieved when using primers at a concentration of 10 μM. The amplified fragment was treated with ligase, which led to its closure in the ring. The amount of ligase added can vary from 0.5 to 5 units, however, the optimal amount is 1 unit. to the reaction. Then, the obtained plasmid was used as a template in the second PCR with the following pair of primers (product length 3793 bp):

5'-ATGCATATGTATATCTCCTTCTTAAAGTT-3 '

5'-TAAGACGTCTCCAGCTTGGCTGT-3 '

It is acceptable to use primers in the concentration range from 5 to 25 μM, however, the best result is achieved when using primers at a concentration of 10 μM. In parallel, PCR of the polylinker site was carried out on a pET22b (+) template using the following pair of primers (product length 256 bp):

5'-TAATACGACTCACTATAGGG-3 '

5'-AGCGACGTCTTATCAGTGGTGGTGGTGGT-3 '

It is acceptable to use primers in the concentration range from 5 to 25 μM, however, the best result is achieved when using primers at a concentration of 10 μM. The ends of the amplified DNA fragments were opened using restriction enzymes NdeI and AatII and directionally ligated with each other. The amount of restriction enzymes added can vary from 0.1 to 1 units, however, the optimal amount is 0.25 units. each restrictase for 1 reaction.

As a result, the plasmid pBAD22 was obtained. The correct assembly of the modified plasmid region was confirmed by sequencing.

Construction of intermediate plasmids pET302 / MAP and pBAD22 / MAP To obtain intermediate expression vectors used commercial plasmid pET302 / NT-His (Thermo Fisher Scientific, USA) and plasmid pBAD22.

The plasmid pET302 / NT-His contains regulatory elements and the T7lac promoter, which induces and regulates the expression of the target protein by changing the concentration of IPTG in the medium, as well as the beta-lactamase gene, which provides resistance to beta-lactam antibiotics, and the pBR322 replication site .

At the first stage, the methionine aminopeptidase gene sequence (Fig. 1) was amplified with the genomic DNA of E. coli BL21 cells [DE3] using the following pair of primers:

5'-GAACATATGGCTATCTCAATCAAGACCCCACA-3 '

5'-GAACTCGAGTTATTATTCGTCGTGCGAGATTA-3 '

It is acceptable to use primers in the concentration range from 5 to 25 μM, however, the best result is achieved when using primers at a concentration of 10 μM. The amplified DNA fragment of 800 bp, the ends of which were opened using restriction enzymes NdeI and XhoI, were cloned into the plasmid pBAD22 linearized using the same restriction enzymes. The amount of restriction enzymes added can vary from 0.1 to 1 units, however, the optimal amount is 0.25 units. each restrictase for 1 reaction.

The following pair of primers was used to clone the methionine aminopeptidase gene into the pET302 / NT-His plasmid:

5'-GAATTCAGCTATCTCAATCAAGACCCCACA-3 '

5'-GGATCCTTATTATTCGTCGTGCGAGATTA-3 '

It is acceptable to use primers in the concentration range from 5 to 25 μM, however, the best result is achieved when using primers at a concentration of 10 μM. The amplified 800 bp DNA fragment, the ends of which were opened using restriction enzymes EcoRI and BamHI, was cloned into the plasmid pET302 / NT-His linearized using the same restriction enzymes. The amount of restriction enzymes added can vary from 0.1 to 1 units, however, the optimal amount is 0.25 units. each restrictase for 1 reaction.

Cloning resulted in plasmids pBAD22 / MAP and pET302 / MAP, respectively. The correct assembly of plasmids was confirmed by sequencing.

At the second stage, in the obtained plasmids, the pBR322 replication site and the beta-lactamase gene were replaced by the p15A replication site and the chloramphenicol resistance gene from pACYC184 plasmid (NEB). This modification is necessary for the effective coexpression of two proteins from different plasmids within the same cell.

Construction of the expression vector pBAD15A

A DNA sequence fragment containing the methionine aminopeptidase gene and the sequences regulating its expression was amplified from the plasmid pBAD22 / MAP using the following pair of primers:

5'-GATAAGCTTCTGTGCGCTGCATGTGTCAGAG-3 '

5'-GAAGTCGACGTCCTACTCAGGAGAGCGTTCAC-3 '

It is acceptable to use primers in the concentration range from 5 to 25 μM, however, the best result is achieved when using primers at a concentration of 10 μM. The amplified DNA fragment of 2529 bp, the ends of which were opened using restriction enzymes HindIII and SalI, were cloned into the plasmid pACYC184, linearized using the same restriction enzymes. The amount of restriction enzymes added can vary from 0.1 to 1 units, however, the optimal amount is 0.25 units. each restrictase for 1 reaction.

Cloning resulted in the plasmid pBAD15A (Fig. 2) containing the methionine aminopeptidase gene under the control of the regulated arabinose promoter, the p15A replication site and the chloramphenicol resistance gene for selection. The correct assembly of the plasmid was confirmed by sequencing.

Primers

pBAD-HindIII-F 5'-GATAAGCTTCTGTGCGCTGCATGTGTCAGAG-3 'FW Location - 3536-3557 bp plasmids pBAD22 / MAP. pBAD-SalI-R 5'-GAAGTCGACGTCCTACTCAGGAGAGCGTTCAC-3 'RV Location - 1446-1468 bp plasmids pBAD22 / MAP.

Construction of the expression vector pET15A

A DNA sequence fragment containing the methionine aminopeptidase gene and the sequences regulating its expression was amplified from plasmid pET302 / MAP using the following pair of primers:

5'-GTTAAGCTTCGGGTTACTGATGATGAACATG-3 '

5'-GGAGTCGACATCCGGATATAGTTCCTCCTTTC-3 '

It is acceptable to use primers in the concentration range from 5 to 25 μM, however, the best result is achieved when using primers at a concentration of 10 μM. The amplified 3755 bp DNA fragment, the ends of which were opened with the restriction enzymes HindIII and SalI, was cloned into the pACYC 184 plasmid linearized with the same restriction enzymes. The amount of restriction enzymes added can vary from 0.1 to 1 units, however, the optimal amount is 0.25 units. each restrictase for 1 reaction.

Cloning yielded the plasmid pET15A (Fig. 3) containing the methionine aminopeptidase gene under the control of the regulated T7 promoter, the p15A replication site and the chloramphenicol resistance gene for selection. The correct assembly of the plasmid was confirmed by sequencing.

Primers

pET-HindIII-F 5'-GTTAAGCTCCGGGTTACTGATGATGAACATG-3 'FW Location - 3788-3809 bp plasmids pET302 / MAP.

pRh15A-SalI-R 5'-GGAGTCGACATCCGGATATAGTTCCTCCTTTC RV

Location - 1048-1070 bp plasmids pET302 / MAP.

Example 2. Obtaining strains of cultured cells - producers of methionine-free human IL-36RA

To express the IL-36RA gene, we used the previously created plasmid expression vector pET-IL36Raf (RU 2582569, 2016). Electrocompetent cells of E. coli BL21 [DE3] were transformed with a mixture of plasmids pET-IL36Raf and pET15A in an equimolar ratio. Transformation was performed using a Bio-Rad MicroPulser apparatus (Bio-Rad, USA) according to the manufacturer's instructions. Positive transformants were selected on an agar medium containing a mixture of two antibiotics (ampicillin at a final concentration of 50 to 125 μg / ml, preferably 100 μg / ml; and chloramphenicol at a final concentration of 20 to 40 μg / ml, preferably 25 μg / ml), which guaranteed the content of both plasmids in the selected colonies. The presence of both plasmids in transformants was also confirmed by PCR using two pairs of primers specific for the IL-36RA gene and the MAP gene, respectively. The resulting strain was called Escherichia coli BL21Star [DE3] (pET-IL36Raf, pET15A).

For cultivation of the obtained strain used 1-2x (preferably 1.5x) LB medium (10-20 g / l (preferably 15 g / l) tryptone, 5-10 g / l (preferably 7.5 g / l) yeast extract, 4-6 (preferably 5 g / l) sodium chloride) in 0.8-1.2x (preferably 1x) M9 medium (Na ₂ HPO ₄ 3.84-5.76 mm (preferably 4.8 mmol), KH ₂ PO ₄ 1.76-2.64 mm (preferably 2.2 MM); NaCl 0.68-1.02 mm (preferably 0.85 mm), NH ₄ Cl 1.5-2.24 mm (preferably 1, 87 mM), MgSO ₄ 0.8-1.2 mM (preferably 1 mM), CaCl ₂ 0.08-0.12 mM (preferably 0.1 mM) supplemented with antibiotics at the above concentrations. to a density of 0.05-0.5 OE (preferably 0.1 OE), grew 12-18 hours (preferably 16 hours) at a temperature of 35-38 ° C (preferably + 37 ° C) and a stirring speed of 150-300 rpm min (preferably 250 rpm). Then these cultures were inoculated in 1-2x LB (preferably 1.5x LB) + 0.8-1.2x (preferably 1x) M9 with a volume of 50-2500 ml (preferably 200 ml) to a density 0.1-1 OE (preferably 0.3 OE) and grown in flasks with a volume of 0.5-2.5 L (preferably 2 L) at a temperature of 35-38 ° C (preferably + 37 ° C) with a stirring speed of 150- 300 rpm (preferably 250 rpm) to a density of 2-5 OU / ml (preferably 3 OU / ml). At this optical density, an IPTG inductor of 0.1-1 mM (preferably 0.5 mM) was added.

Induction was continued for 2-4 hours (preferably 3 hours) at a temperature of 27-37 ° C (preferably + 30 ° C) at a stirring speed of 150-300 rpm (preferably 250 rpm). Then the bacteria biomass was collected by centrifugation at a speed of 2000-12000 g (preferably 8000 g) at a temperature of 3-18 ° C (preferably + 4 ° C) for 15-60 minutes (preferably 30 minutes). Initially, the expression of IL-36PA protein was confirmed by electrophoresis of total cell lysates (Fig. 4). Then, the level of production of IL-36RA by various producer strains was evaluated using solid-phase ELISA using monoclonal antibodies to IL-36RA, obtained in the Federal State Unitary Enterprise State Research Institute of General Medicine (Fig. 5). An admixture of the unprocessed form of IL-36RA containing the methionine initiator residue was evaluated by RP-HPLC (Fig. 6, 9).

Example 3. Obtaining a strain of Escherichia coli BL21Star [DE3] (pET-IL36Raf, pBAD15A).

Obtaining Escherichia coli strain BL21Star [DE3] (pET-IL36Raf, pBAD15A) and all work with it was carried out according to the technology described in example 2 with two differences: when it was created, plasmid pBAD15A was used, and not pET15A, and arabinose was also introduced during induction 0 , 2%).

Example 4. Isolation of the receptor antagonist of IL-36.

After thawing 150 ± 15 g of bacterial biomass from the freezer (-70 ° C) to room temperature, 1400 ± 50 ml of buffer containing 30 to 70 mM Tris-HCl (optimally 50 mM), pH 6.0 to 7.2 (optimally 7.0), and mixed on an electric stirrer for 45-75 minutes (optimally 60) until a homogeneous solution is obtained. The volume of the obtained lysate was 1500 ± 100 ml. The lysate was then poured into Beckman centrifugation tubes, placed in a JA-10 rotor and centrifuged at a speed of 5000 to 12000 rpm (optimally 10000) for 45-75 minutes (optimally 60). The precipitate was removed, and a calcium chloride solution was added to the supernatant to a final concentration of 20 to 40 mM (optimally 30) and a trisodium phosphate solution to a final concentration of 10 to 30 mM (optimally 20). The resulting solution was stirred on an electric stirrer overnight. Next, the resulting solution was poured into Beckman centrifugation tubes, placed in a JA-10 rotor and centrifuged at a speed of 5000 to 12000 rpm (optimally 10000) for 45-75 minutes (optimally 60). The precipitate was removed, and the supernatant was applied to a chromatographic sorbent. This stage of preparation of the raw material allowed to remove most of the mechanical impurities that irreversibly clog the chromatographic sorbent.

Chromatographic purification on a Q FF sorbent (300 ± 10 ml) allows the IL-36RA to be purified from a portion of impurity proteins, as well as lipopolysaccharides (LPS) and DNA. The chromatography conditions were selected so that the IL-36 receptor antagonist, unlike impurities, did not bind to the sorbent and remained in the fraction that did not bind to the sorbent. The fraction was collected in a volume of 2200 ± 200 ml, starting from the moment of increasing the UV absorption of the passing fraction at 280 nm by 30 mAU compared to the equilibration buffer.

Sorbent volume: 300 ± 10 ml

Balancing buffer: 30-70 mM Tris-HCl (optimally 50 mM), pH 6.0-7.2 (optimally 7.0) (0.5 ± 0.025 L)

Washing buffer: 30-70 mM Tris-HCl (optimally 50 mM), 0-0.3 M NaCl (optimally 0.15), pH 6.0-7.2 (optimally 7.0) (0.7 ± 0.035 L ) Regeneration buffer: 30-70 mM Tris-HCl (optimally 50 mM), 1.5-2 M NaCl (optimally 2 M), pH 6.0-7.2 (optimally 7.0) (0.5 ± 0.025 L) .

Chromatographic purification using Butyl-650 sorbent (100 ± 5 ml) is intended for purification of the target protein from impurity proteins. The fraction that did not bind to the sorbent Q FF was added to the sorbent with the addition of ammonium sulfate up to 0.15-0.5 M (optimally 0.3). Chromatography conditions were selected so that the IL-36 receptor antagonist, unlike most impurities, binds to the sorbent and then elutes under certain conditions.

Sorbent volume: 100 ± 5 ml

Balancing buffer: 30-70 mM Na ₂ HPO ₄ (optimally 50 mM), 0.15-0.5 M (NH ₄ ) ₂ SO ₄ (optimally 0.3), pH 5.8-6.2 (optimally 6 , 0) (1.5 ± 0.2 L) Elution buffer: 30-70 mM Na ₂ HPO ₄ (optimally 50 mM), pH 5.8-6.2 (optimally 6.0) (0.4 ± 0 , 05 l) Regeneration buffer: distilled water

It is fundamentally possible to isolate IL-36 solely by hydrophobic chromatography. In this case, however, the service life of the hydrophobic sorbent will be significantly reduced, therefore, the use of two-stage chromatographic purification appears to be more economical.

Protein concentration was carried out by ultrafiltration using a Millipore Amicon Stirred Cell with a Sartorius Ultracel PLC 10 membrane with a cutoff limit of 10 kDa. The sample was concentrated to 10 ± 0.3 mg / ml. Then the protein was dialyzed against a solution containing 30-70 mM sodium citrate (optimally 50 mM), pH 5.8-6.2 (optimally 6.0), in a MFPI CelluSep RC T2 dialysis bag with a cutoff limit of 6-8 kDa. After this, the protein solution was sterilized using Millipore Millex syringe filters with a pore diameter of 0.22 μm. The total loss of IL-36RA at all stages of treatment amounted to 37.5 ± 9.3%.

The electrophoregram of samples from various stages of purification of IL-36RA is presented in FIG. 7.

Example 5. Analysis of the purity and homogeneity of the obtained IL-36RA

To determine the amount of protein impurities in the obtained IL-36RA, IL-36RA samples were separated by reverse phase HPLC on a Phenomenex Jupiter C18 column. 10 μg (± 2 μg) of IL-36PA in 20 μl (± 4 μl) of the solution was applied to the column in 50 mM (± 5 mM) phosphate buffer, pH 6.0 (pH range 5.8-6.2). Elution was carried out with a gradient of acetonitrile from 30% to 70% in 0.1% TFA, pH 2.5 (± 0.2), for 20 minutes (± 5 min) at a flow rate of 1.5 ml / min and a temperature of + 35 ° C (± 2 ° C). Detection was carried out on a UV detector at a wavelength of 220 nm. A typical RP-HPLC chromatogram of the IL-36RA separation by this technique is shown in FIG. 8. To determine the impurity of the IL-36RA form containing the uncleaved N-terminal methionine residue, RP-HPLC was performed according to the method described above. In this case, elution was carried out with a gradient of acetonitrile from 39.6% to 40.8%), otherwise the conditions were the same. A typical RP-HPLC chromatogram of the IL-36RA separation by this technique is shown in FIG. 9. To determine the impurity of the deamidated form of IL-36RA, capillary zone electrophoresis (KZEF) was performed on a Kapel-105M instrument (Lumex, Russia). For analysis, uncoated quartz capillaries with an inner diameter of 50 μm and a total length of 50 cm (± 5 cm) (effective length 40 cm (± 5 cm) (Lumex, Russia) were used. The capillary was thermostatically controlled at 25 ° С (± 2 ° С), detection was carried out at a wavelength of 214 nm. A 0.12 M (± 20 mM) solution of potassium dihydrogen phosphate (pH 3 ((± 0.2)) was used as the background electrolyte. Before the analysis, the capillary was washed with 0.1 M (± 20 mm) a solution of sodium hydroxide, deionized water and a background electrolyte for 3 min at a pressure of 2000 mbar ((± 100 mbar). Sample for capillary zone electrophoresis was an aqueous solution of a recombinant receptor antagonist of human interleukin-36 with a concentration of 0.5 mg / ml (allowable concentration range of 0.4 mg / ml - 0.6 mg / ml) containing 2 μg (± 0.2 μg) of the internal standard - the synthesized peptide Trp-Glu-His-Arg. The sample was injected hydrodynamically at a pressure of 40 mbar for 5 s. The analysis was carried out at a voltage of 10 kV (± 2 kV) for 35 minutes (± 5 min). Instrumental control, collection and registration of electrophoretic data was carried out using the Elforan system (Lumex, Russia). To identify the peaks of the intact protein and its deamidated form, the relative migration time of each of these peaks (RMT) was calculated, equal to the ratio of the migration time of the protein peak to the migration time of the internal standard. The content of the deamidated form of the protein was calculated by the relative peak area (in percent) equal to the ratio of the peak area of the deamidated form to the sum of the peak areas corresponding to the recombinant human interleukin-36 receptor antagonist. In FIG. 10 shows the electrophoregram of separation of native (A) and deamidated (B) forms of IL-36RA in KZEF.

After all stages of purification according to the results of the analyzes, the obtained IL-36RA has a purity of> 95% by HPLC, has <5% impurities of an unprocessed form with an uncleaved initiator methionine residue, and <5% of a precipitated form.

Example 6. The creation of a composition of recombinant human IL-36RA, stable during storage. When testing the conditions of chromatographic purification of IL-36RA, it was found that when the target protein was exposed to pH> 7.0, a significant impurity of the deamidated form of the protein was observed (Fig. 10). Therefore, to obtain a homogeneous product, the purification conditions were modified so that the pH was always not higher than 7.0. In addition, during storage of the protein in phosphate buffer with a pH of 7.4, an accumulation of a deamidated form was also recorded. However, at pH <5.5, IL-36RA aggregation was observed, because these values are too close to the isoelectric point of the protein - 5.28. To stabilize the IL-36RA solution, a composition was developed that included 1-100 mM citrate or phosphate buffer (optimally 5 mM) with a pH of 5.5-6.5 (optimally 6.0), in which protein deamidation stability analysis does not occur . Also, the selected concentration of sodium citrate or sodium phosphate is suitable for injectable administration to humans. In addition, to prevent the aggregation of IL-36RA, tween-80 in the amount of 0.01-1% was introduced into the composition. At concentrations <0.01% tween-80 did not reduce the ability of the drug to aggregate, concentrations of tween-80> 1% did not show further improvement in the properties of the drug. To protect the drug from possible proteolysis, an EDTA metalloprotease inhibitor in an amount of 0.1-10 mM was introduced into the composition. The described effects are summarized in table 4.

Example 7. Evaluation of the biological activity of the composition of IL-36PA on a model of psoriasis-like dermatitis in mice

To determine the biological activity of the obtained IL-36RA, a model of psoriasis-like dermatitis in mice induced by treatment with an imichimod agonist of Toll-like receptors 7 and 8 [van derFits L. et al. Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23 / IL-17 axis // J. Immunol. Baltim. Md 1950. 2009. Vol. 182, No. 9. P. 5836-5845.]. An imihimod was applied cutaneously in the form of Aldar cream (5% imihimod). This model of psoriasis is widely used in scientific practice and has been repeatedly described in the literature [Stjernholm T. et al. Leptin-deficiency in mice counteracts imiquimod (IMQ) -induced psoriasis-like skin inflammation while leptin stimulation induces inflammation in human keratinocytes // Exp. Dermatol. 2016 .; Li R. et al. Increased βTrCP are associated with imiquimod-induced psoriasis-like skin inflammation in mice via NF-κB signaling pathway // Gene. 2016 .; Huang S.-W. et al. Azithromycin impairs TLR7 signaling in dendritic cells and improves the severity of imiquimod-induced psoriasis-like skin inflammation in mice // J. Dermatol. Sci. 2016.].

To induce psoriasis-like dermatitis, Aldar cream was applied to the skin of the right ear of mice of three groups once a day at a dose of 55-68 μg (optimally 62.5 μg), daily, for 7 days. Animals were divided into 4 groups. The first group received an injection of 200 ± 5 μl of saline subcutaneously into the withers daily 1 time per day; the second and third groups received 50 ± 1 or 10 ± 1 μg / mouse IL-36RA, respectively, in a volume of 200 ± 5 μl subcutaneously to the withers daily once a day, the fourth group received a solution of methotrexate at a dose of 20 ± 1 μg / mouse daily intragastrically through a tube. Each group included 10 mice. A daily morphometric study of the thickness of the ear and visual assessment of skin lesions at the site of application of the drug were performed, followed by the calculation of the PASI index [van der Fits L. et al. Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23 / IL-17 axis // J. Immunol. Baltim. Md 1950. 2009. Vol. 182, No. 9. P. 5836-5845.].

It was shown that daily application of the drug Aldara on the skin of the ear of mice receiving injections of physiological saline had a visible effect already 24 hours after the first application. Pronounced erythema and peeling were recorded, reaching a maximum on the 5th day of the experiment. A morphometric study of the thickness of the ear showed a significant increase in this parameter compared with intact animals, starting from 24 hours after the first application of Aldar cream. Analysis of changes in the PASI indices calculated for all groups of animals during the experiment showed that the severity of psoriasis-like dermatitis was significantly reduced in groups who received the studied preparation of recombinant human IL-36RA in doses of 50 ± 1 and 10 ± 1 μg / mouse, as well as methotrexate in a dose of 20 ± 1 μg / mouse per day, compared with that in control group, starting with the third day and throughout the experiment (Fig. 11). On the 6th day of the experiment, the effectiveness of the drug IL-36RA at a dose of 50 ± 1 μg / mouse exceeded the effectiveness of methotrexate. A morphometric study of the thickness of the ear showed that in the group receiving the study drug at a dose of 50 ± 1 μg / mouse, the index of change in the thickness of the ear was significantly lower compared to the control group, starting from the 3rd day until the end of the experiment. In the group of animals treated with methotrexate, this indicator significantly decreased only on the 6th and 7th days of the experiment (Fig. 12).

Thus, it was shown that daily subcutaneous administration of the developed preparation of recombinant human IL-36PA at doses of 50 ± 1 and 10 ± 1 μg / mouse significantly reduces the severity of experimental dermatitis caused by imichimod. The effect appears within 3-4 days after the first injection of the drug and exceeds the effect of methotrexate.

Claims (19)

1. Plasmid expression vector pBAD15A for producing a methionine-free recombinant human interleukin-36 (IL-36PA) receptor antagonist by culturing Escherichia coli strain BL21Star [DE3] containing the plasmid expression vector pET-IL36Raf and the aforementioned vector, and subsequent isolation of IL-36PA : - E. coli methionine aminopeptidase gene, - pBADara promoter, - rrnB gene transcription terminators (rrnB T1T2), - regulatory sequence of araBAD operon, - origin of replication of plasmids p15A, - gene chloramphenicol acetyltransferase (CamR) and - restriction sites necessary for combining individual fragments in a vector, a map of which is shown in FIG. 2. 2. Plasmid expression vector pET15A for the production of recombinant human IL-36PA without methionine by culturing a strain of Escherichia coli BL21Star [DE3] containing the plasmid expression vector pET-IL36Raf and the aforementioned vector, and subsequent isolation of IL-36PA containing: - E. coli methionine aminopeptidase gene, - promoter of phage T7, - phage T7 gene transcription terminator, - regulatory sequence of the lacI gene, - origin of replication of plasmids p15A, - gene chloramphenicol acetyltransferase (CamR) and - restriction sites necessary for combining individual fragments in a vector, a map of which is shown in FIG. 3. 3. The strain is a producer of non-methionine recombinant human IL-36RA, which is a strain of Escherichia coli BL21Star [DE3], containing the plasmid expression vector pET-IL36Raf and the vector according to claim 1 or 2. 4. The method of obtaining the strain according to claim 3, which consists in transforming the Escherichia coli strain BL21Star [DE3] with the plasmid expression vector pET-IL36Raf and the vector according to claim 1 or 2, followed by induction of expression of the human interleukin-36 receptor antagonist genes (IL-36RA) and methionine aminopeptidases of E. coli. 5. The use of a preparation of a non-methionine recombinant human interleukin-36 receptor antagonist obtained using the strain of claim 3 for the treatment of dermatitis.

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