RU2693660C1 - METHOD OF PRODUCING RECOMBINANT BETA-N-ACETYLGLUCOSAMINIDASE StrH FROM STREPTOCOCCUS PNEUMONIAE - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention relates to the biotechnology. Disclosed is an industrial method of producing a biologically active preparation of recombinant exo-beta-N-acetylglucosaminidase StrH (β-N-GlcNAcase StrH) produced in accordance with GMP rules. Method includes consecutive stages of metal chelate chromatography for sorption of target protein with hexahistidine tag; gel filtration in breakthrough mode to purify the target protein from contaminating impurities of cell proteins which are coelaxing with the target protein at all steps; anion-exchange chromatography on sorbent, optimized for capture and purification of proteins with high molecular weight; hydrophobic chromatography, where the contaminant impurities of hydrophobic proteins and bacterial endotoxins are purified.

EFFECT: method is suitable for use at the remodeling step in preparing the therapeutically functional preparations of glycoproteins with given properties, as well as for developing tools for high-sensitivity analysis of glycan chains of glycoproteins of plant tissues, animals and eukaryotic microorganisms.

9 cl, 8 dwg, 7 tbl, 8 ex

Description

The invention relates to the field of biotechnology, in particular to methods for producing and producing therapeutic drugs, isolating and purifying recombinant proteins, in particular in obtaining a highly purified preparation of recombinant exo-beta-N-acetylglucosaminidase StrH (β-N-GlcNAcase StrH). The main applications obtained by the proposed method of bacterial β-N-GlcNAcase StrH is its inclusion in the technological process of obtaining a number of medical drugs (B), as well as biomedical research in the field of glycobiology.

The invention solves the problem of creating a method for producing large amounts of highly purified enzymatically active recombinant β-N-GlcNAcase StrH, suitable for use at the remodeling stage to produce therapeutically functional glycoprotein preparations with desired properties, to develop tools for highly sensitive analysis of glycan chains of plant, animal and eukaryotic tissue glycoproteins microorganisms. The invention also relates to a method for producing a recombinant β-N-GlcNAcase StrH preparation having a higher degree of purity as compared to commercial preparations, homogeneous in the target product, with a lower content of endotoxins and producer DNA in comparison with commercial preparations.

DESCRIPTION OF THE DRAWINGS

FIG. 1. Scheme of the structure of N-glycosylated human glycoprotein. The names of the sugar residues are located under the corresponding symbols. Lines - connections between sugar residues, numbers above - specificity of connections. Arrows - links that are potentially broken down by glycosidases, indicated at the top of the scheme;

FIG. 2. Substrate specificity of the β-N-GlcNAcase StrH. Data for two constructs: 1 - corresponds to full-size StrH, 2 - catalytic domain GH20A. The complexity of glycans increases from (a) to (e). Activity on all presented substrates is observed only for a full-size construct. For GH20A, there is a reduced activity on the substrate (c) and its absence on the substrates (d) or (e).

FIG. 3. Map of plasmid pMT1618 (expression vector based on pET15b for producing Streptococcal β-N-GlcNAcase StrH). Legend: T7 promoter - T7 RNA polymerase promoter; N-terminal His-tagged β-N-GlcNAcase StrH is a gene of the His-tagged protein (N-terminus) β-N-GlcNAcase StrH, consisting of sequences of 20 amino acid residues containing the hexahistidine tag, and 1117 amino acid residues (without signal peptide, one of two G5 domains, and an anchor peptide) Streptococcus pneumoniae β-N-GlcNAcase StrH; T7 terminator - T7 RNA polymerase terminator; AmpR promoter - promoter of ampicillin resistance gene; AmpR - ampicillin resistance gene; ori - origin of replication; lacI - lactose repressor gene; lacI promoter - lactose repressor gene promoter; NdeI - restriction endonuclease recognition site NdeI; BamHI - BamHI restriction endonuclease recognition site

FIG. 4. The dynamics of biomass accumulation during the cultivation of E. coli producer strain VKPM B-12747 in a volume of 15 liters. The abscissa axis is the cultivation hours, the ordinate is the accumulation of biomass;

FIG. 5. Elution profile of the target protein β-N-GlcNAcase StrH - gel filtration on Superdex 200.

FIG. 6. The result of the selection of β-N-GlcNAcase StrH. Analysis by electrophoresis in PAG. Comparison of the commercial drug and the resulting recombinant drug β-N-GlcNAcase StrH. Lane 1 — Thermo Scientific molecular weight markers; Lane 2 - recombinant commercial β-N-acetylglucosaminidase; Lane 3 - Purified recombinant β-N-GlcNAcase StrH preparation.

FIG. 7. The result of the analysis of GF HPLC. Industrial Series β-N-GlcNAcase StrH.

FIG. 8. The result of the analysis of GF HPLC. Comparison of a commercial preparation and a recombinant β-N-GlcNAcase StrH preparation with enhanced chromatographic purity.

The absence of domestic commercially available recombinant β-N-GlcNAcase StrH preparations from Streptococcus pneumoniae expressed in E. coli, and the extremely high cost of preparations from foreign manufacturers such as QA-Bio, Amsbio, ProZyme, Sigma makes commercially unprofitable to use these preparations in the production drugs. The preparations of these companies are not made in accordance with the standards of "Good Manufacturing Practice" / "Good Manufacturing Practice" (GMP) and are intended only for analytical studies.

Detailed characteristics of the β-N-GlcNAcase StrH, belonging to the large family of 20 glycosidase hydrolases, which hydrolyze the terminal sugar residue from the non-reducing end of oligosaccharides and the carbohydrate chain of glycoconjugates, such as glycoproteins and glycolipids (Fig. 1.), are given in Kob publications Use of Endo- and Exoglycosidases for Structural Studies of Glycoconiugates, // Analytical biochemistry - 1979. - V.100. - P. 1-14; Intra J at al., Phylogenetics analyzes for changes in glycosil hydrolase 20 family, // BMC Evol. Biol. - 2008. - P. 8214; .Lowrie R. Glascow at al, Systematic Purification of Five Glycosidases from Streptococcus (Diplococcus) pneumoniae, // The Journal of Biological Chemistry - 1977. - V. 252. - N. 23. - Issue of December 10. - p. 8615-8623. A large number of exo-acetylglucosaminidases isolated from a variety of organisms, such as bacteria, viruses, plant and animal tissues, which are widely used as a tool for analyzing the structure and functions of glycoconjugates, oligosaccharides associated with glycoproteins and cell membranes, and are in great demand for glycobiological research and industrial processes. However, they all have different substrate-specificity and many of the glycosidases described are of limited importance, as they are active only at low pH, or only on low molecular weight substrates, and, as a rule, there are difficulties in obtaining pure preparations from natural sources. They are usually contaminated with impurities of other glycosidases, proteases, and bacterial endotoxins. In addition, a limited number of exo-acetylglucosaminidases, which are isolated and produced from natural sources, are commercially available. The list is presented in Table 1. (4. US Patent 7094563 B2, Isolation and composition of novel glycosidases, // 2006.).

For the first time β-N-GlcNAcase StrH Diplococcus pneumonia, isolated from a natural source, described in the publication R. Colin Hughes at al., The Extracellular Glycosidases of Diplococcus pneumoniae. Purification and Properties of β-N-Acetylglucosaminidase. Action on a Derivative of the α-Acid Glycoprotein of Human Plasma, // Biochemistry - 1964. - V. 3. - N. 10. - P. 1543-1548. It was found that the β-N-GlcNAcase StrH is active both at neutral pH and on low and high molecular weight substrates.

In the publication Valerie A. Clarke at al., Cloning and Expression of the β-N-Acetylglucosaminidase Gene from Streptococcus pneumoniae. Generation of truncated enzymes with modified aglycon specificity, // The Journal of Biological Chemistry - 1995. - V. 270. - N. 15. - P. 8805-8814), the structure of the β-N-GlcNAcase StrH gene from Streptococcus pneumoniae was first identified . The expression of an almost complete DNA clone in Escherichia coli was shown to produce functional and authentic β-N-GlcNAcase StrH, which has aglycon specificity identical to that of the wild type. It was also shown that the β-N-GlcNAcase StrH completely hydrolyzes the terminal GlcNAc residues from N-glycans regardless of which branch in glycans (1-3) or (1-6), (Fig. 2.), (Samantha J. King at al., Deglycosylation of human glycoconjugates expressed by Streptococcus pneumoniae, // Molecular Microbiology - 2005. - V. 59. - N. 3. - P. 961-974).

The production of β-N-GlcNAcase StrH in Escherichia coli as a homologous variant of the enzyme was previously carried out for research purposes and is disclosed in Clarke V.A. At al. // int. J. Biochrom. - 1994. - V. 1. - p. 151-158. The authors obtain a β-N-GlcNAcase StrH in the form of a fusion protein with glutathione S-transferase using affinity chromatography on Glutathione Sepharose and the subsequent cleavage of the fusion protein by a protease factor Xa. This isolation method has its obvious drawbacks for the production of an enzyme on an industrial scale:

1. The commercially available glutathione (γ-glutamylcysteinylglycine) Sepharose sorbent can be affected by γ-glutamyl transpeptidase, which is found in unpurified cell lysates. Consequently, the glutathione sorbent has a finite service life and can be regenerated and reused 4-20 times, unlike, for example, IMAC sorbents, which can be used almost infinitely (Michelle E. Kimple at al, HHS Public Access Author manuscript, // Curr Protoc Protein Sci. - 2015 - V.73. P. 1-26).

2. Chaperonin - 70 kDa E. coli (heat shock protein) is also adsorbed on the sorbent and co-eluted with the target GST-fusion protein.

3. High expression of GST fusion proteins in E. coli can lead to accumulation of aggregated protein in inclusion bodies. Chromatography on Glutathione Sepharose depends on proper GST refolding. Therefore, a refolding must be carried out before cleaning, however, the large size of GST of 26 kDa and its dimerization in solution may affect the properties of the fusion protein.

4. The high cost of factor Xa, additional purification of factor Xa and hydrolysis products, as well as additional control of the presence of factor Xa in the finished product significantly increase the cost of the drug.

The closest to the claimed invention can be considered the purification method β-N-GlcNAcase StrH, disclosed in Benjamin Pluvinage at al, Conformational Analysis of StrH, the Surface-Attached exo-β-dN-Acetylglucosaminidase from Streptococcus pneumoniae, // Journal of Molecular Biology - 2013. - V. 425. - N. 23. - P. 334-349. This method involves the purification of a homologous variant of the enzyme and its variants in the form of a fusion protein with a polyhistidine sequence, in analytical quantities. The target proteins were purified using metal-chelate chromatography on IMAC Sepharose, followed by hydrolysis with thrombin and anion-exchange chromatography on UNOsphere Q (Bio-Rad Laboratories), gel-filtered on Sephacryl S200 (GE Biosciences), followed by concentration by ultrafiltration using a 10 cm membrane using 10 cm. This purification scheme using thrombin is unacceptable for large-scale production of the drug due to the reasons already mentioned for hydrolysis by factor Xa. In addition, the authors do not report the level of bacterial endotoxins in the preparation. The removal of bacterial endotoxins is a fundamental and necessary requirement, both in terms of Russian and international standards (GF XIII, Document Q AS / 11.452 FINAL July 2012). However, it often remains technically difficult task, therefore, the content of bacterial endotoxins in intermediate products used in the manufacture of drugs is a critical parameter. When assessing the quality of such intermediate products as glycosidases, in addition to the correspondence of the substrate specificity and activity, such potentially dangerous contaminants as bacterial endotoxins and cellular DNA, including potentially oncogenic, as well as the homogeneity of the drug, should be specifically evaluated.

To solve the problem of obtaining highly purified β-N-GlcNAcase StrH in industrial volumes, the inventors propose a new method of obtaining recombinant β-N-GlcNAcase StrH from Streptococcus pneumoniae, including the synthesis of β-N-GlcNAcase protein optimized for translation into E. coli DNA StrH, the nucleotide sequence of which is shown in SEQ ID NO 1, constructs an expression plasmid vector encoding a chimeric protein with N-terminal chimerization, composed of six histidine peptide sequences, site recognized thrombin and 1117 amino acid residues (without signal peptide, one of two G5 domains and anchor peptide) β-N-GlcNAcase StrH from Streptococcus pneumoniae (amino acid sequence represented by SEQ ID NO 2), preparation of recombinant protein production in E. coli, purification protein from the soluble fraction of cell lysate using metal chelate chromatography on IMAC Sepharose, chromatography in skip mode on Blue Sepharose FF, anion-exchange chromatography on Toyopearl Giga Cap Q-650M, chromatography in skip mode on Butyl Capto, concentrating anion exchange chromatograph at Q Sepharose FF, with the release of the highly purified active drug pGlcNAcase StrH, suitable for use in the manufacture of drugs and glycobiological studies, not less than 0.04 g per liter of bacterial culture.

In order to obtain the most chromatographically homogeneous preparation (more than 98% by gel filtration HPLC) used for accurate glycobiological studies, concentration by ultrafiltration using a 50 kD membrane and gel filtration on Superdex 200 may be additionally included.

The main technical result of the invention is to obtain on an industrial scale highly purified active drug β-N-GlcNAcase StrH, suitable for use in the manufacture of drugs and glycobiological studies.

For the invention was created a strain genetically modified microorganism E. coli VKPM B-12747: strain obtained by transformation of the recipient host strain BL21 (DE3) Plasmids pLysS, carrying the T7 lysozyme gene, an inhibitor of T7 RNA polymerase, rMT1618 (pET15b-STRH_STRPN ), carrying the β-N-GlcNAcase StrH protein gene, which consists of sequences of 20 amino acid residues containing a hexahistidine tag, a thrombin recognition site and 1117 amino acid residues (without a signal peptide, one of two G5 domains and an anchor peptide Teed) β-N-GlcNAcase StrH (Fig. 3.).

Product refolding is not required, since it is synthesized in the bacterial cytoplasm mainly in a dissolved state during the cultivation of E. coli VKPM B-12747 with a level of biosynthesis of variants of the target protein of at least 40 mg / l of culture fluid.

Highly efficient extraction of the target protein β-N-GlcNAcase StrH from cells is achieved by the procedure of lysis of bacterial cells using a high-pressure homogenizer.

Obtaining active, chromatographically homogeneous with a low content of bacterial endotoxins and host DNA of the drug β-N-GlcNAcase StrH is achieved through chromatographic purification scheme, including: metal chelate chromatography on IMAC Sepharose 6 FF, chromatography in the overshoot mode on Blue Sepharose FF, anion exchange chromatography on Toyopearl Giga Cap Q-650M, slippage chromatography on Butyl Capto, concentrating anion exchange chromatography on Q Sepharose FF. To obtain the most chromatographically homogeneous preparation, concentration by ultrafiltration using a membrane with a cut-off of 50 K and gel filtration on Superdex 200 can be additionally included.

Introduction to the purification scheme of the stage of chromatography on Blue Sepharose in the overshoot mode allows to significantly purify the target protein from contaminating impurities of cellular proteins that co-elute with the target protein at all stages.

Application of TOYOPEARL GigaCap Q-650M, a high molecular weight, high resolution anion exchange sorbent optimized for capturing and purifying high molecular weight proteins. The conditions of planting and removal of the target protein were selected, allowing maximum purification of the target protein from contaminating impurities of cellular proteins and lipopolysaccharides. In addition, the method differs from the previously known as the use of chromatography on Butyl Capto in the mode of breakthrough, which allows to remove contaminating impurities of hydrophobic proteins and lipopolysaccharides.

Materials and methods

For the expression of β-N-GlcNAcase StrH, genetically engineered constructs (GICs) pMT1618 are obtained. In order to obtain GICs encoding the β-N-GlcNAcase StrH, a cloning scheme based on the pET-15b vector (Novagen) was developed, which implies obtaining a full version with 6 × His-tag at the N-terminus. To generate the appropriate GIC, TOP Gene Technologies ordered a synthetic gene encoding a β-N-GlcNAcase StrH (the genomic sequence of Streptococcus pneumoniae, encoding 1117 amino acid residues (without a signal peptide, one of two G5 domains and an anchor peptide) β-N- acetylglucosaminidase (strH), fused at the 5'-end with part of the vector sequence of the parent plasmid pET15b (+), encoding 20 amino acid residues containing hexa-histidine tag), flanked by the restriction sites NdeI, BamHI, containing the internal restriction sites NheI to obtain the "catalytic" domain and stop codons at the 3'-end of the sequence (TGATAA). Top Gene Technologies carried out a codon-sequence optimization for protein expression in E. coli, taking into account the requirement that the sequence contains no “internal” sites for restriction endonucleases NdeI, BamHI, XhoI and NheI. The codon optimization of the obtained β-N-GlcNAcase StrH sequence was verified at http://gcua.schoedl.de/sequential_v2.html. Compliance of the translated amino acid sequence ordered confirmed by alignment in the program MEGA 6.0. The absence of undesirable restriction sites has been verified using the CloneManager 9.0 software. In order to obtain the pMT1618 GIC (pET15b-STRH_STRPN), the accumulation and restriction mapping of the synthetic gene pAPG110-β-N-GlcNAcase StrH was carried out. According to the developed scheme, cloning of the fragment encoding Streptococcal β-N-GlcNAcase StrH from plasmid pAPG110-STRH_STRPN into the vector plasmid pET15b was carried out at the restriction sites NdeI, BamHI. The restriction fragments were purified and used for ligation. The ligase mixture was used to transform chemically competent cells of E. coli XL10-GOLD. Cells were sown to obtain transformed clones.

Then plasmid DNA was isolated and restriction mapping was performed using the restriction endonuclease PstI. A sample of plasmid DNA was transferred for sequencing to Evrogen. The resulting sequence was aligned with the “theoretical” sequence of pMT1618.

To conduct analytical expression, the plasmid DNA of pMT1618 GIC was transformed into strains BL21 (DE3) pLysS. Based on the analytical expression, clones superproducers β-N-GlcNAcase StrH were selected. Then received the strain superproducer β-N-GlcNAcase StrH. The strain was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) under accession number - VKPM B-12747.

To obtain the target protein β-N-GlcNAcase StrH, preparative expression was performed by culturing E. coli VKPM B-12747 in a complete nutrient medium with a relative content of dissolved oxygen of at least 10% at a temperature of 37 ° C, pH value - 6.5-7, 6 The duration of the cultivation process was 5-6 hours. Thanks to the enriched culture medium, active cell growth took place during the first stage. The presence of glucose in the medium suppresses the expression of the recombinant protein, and, consequently, reduces the load on the cell of the producer strain, which leads to an increase in the amount of accumulated biomass.

From 2.5-3 hours of cultivation, the stage of hybrid protein biosynthesis begins. Expression of the β-N-GlcNAcase StrH protein is under the control of a promoter, which is de-repressed by introducing the IPTG solution to a final concentration of 0.0476 wt. % The level of biosynthesis of variants of the target protein β-N-GlcNAcase StrH is at least 15 mg / l of culture fluid.

Bacterial biomass was collected by centrifugation and stored at –70 ° C. Cell biomass was lysed, the lysate was clarified by centrifugation, and β-N-GlcNAcase StrH protein was isolated from the lysate by binding the target protein by metal chelate chromatography on a Ni ²⁺ IMAC Sepharose FF column. Finished the enzyme by successive chromatography on Blue Sepharose FF in skip mode, Toyopearl Giga Cap Q-650M anion-exchange, Butyl Capto in skip mode, HP HP chromatography, concentration and gel filtration. At each stage, the control of the degree of purification is carried out by PAGE.

The protein yield is at least 0.04 mg per liter of bacterial culture.

The final product obtained by the method of the invention has the following parameters:

- protein content (UV spectrophotometry) - not less than 0.5 mg / ml;

- the content of bacterial endotoxins (GF XIII, gel-thrombus test with LAL-reagent OFS.1.2.4.0006.15) is not more than 100 EU / mg;

- Chromatographic purity (GF HPLC) of at least 85%;

- Enzymatic activity (Colorimetric method, β-N-Acetylglucosaminidase Assay Kit "(Sigma)) not less than 150 U / mg;

- Residual DNA of the producer strain (Quantitative real-time NDP) not more than 20 pg / mg;

Using the purification methods of the present invention, it was possible to obtain the β-N-GlcNAcase StrH preparation with chromatographic purity determined by gel filtration HPLC, 90% homogeneity and more purified - 98.8% homogeneity.

The specific activity of the β-N-GlcNAcase StrH preparations of the present invention is 230 units / mg protein and higher.

The content of bacterial endotoxins in the β-N-GlcNAcase StrH preparation of the present invention is not more than 4 U / mg protein.

The content of residual DNA of the producer strain in the β-N-GlcNAcase StrH preparation of the present invention is preferably not more than 4.0 pg / mg protein.

Production of the β-N-GlcNAcase StrH based on an optimized synthetic DNA sequence with a short sequence of six histidines at the N-terminus of the recombinant protein occurs without removing the hexa-histidine tag, which simplifies the purification and removes from the purification scheme the use of expensive drugs to remove the tag. The amino acid sequence of the β-N-GlcNAcase StrH is represented by SEQ ID NO 2. As a first step, the inventors used metal chelate chromatography on IMAC Sepharose 6 FF for complete sorption of the target protein with a hexa-histidine tag. The use of washings with an acidic buffer with high ionic strength at this stage makes it possible to substantially remove impurities of cellular proteins from the target product. The use of chromatography on Blue Sepharose in the breakthrough mode allows the target protein to be purified from contaminating impurities of cellular proteins that co-elute with the target protein at all stages. Anion-exchange chromatography is performed on Toyopearl Giga Cap Q-650M, a high-resolution, high-molecular-weight, anion-exchange sorbent optimized for capturing and purifying high molecular weight proteins. Conditions of planting and removal of the target protein are selected in such a way as to maximally purify the target protein from contaminating impurities of cellular proteins and bacterial endotoxins. Chromatography in the mode of breakthrough on Butyl Capto allows you to remove contaminating impurities of hydrophobic proteins and bacterial endotoxins. The final concentrating anion-exchange chromatography on Q Sepharose FF allows to obtain a highly purified active drug β-N-GlcNAcase StrH, suitable for use in the production of drugs and glycobiological studies, with a yield of at least 0.04 g per liter of bacterial culture.

Using the purification methods of the present invention, it was possible to obtain a β-N-GlcNAcase StrH preparation with chromatographic purity determined using gel filtration HPLC and 90.0% homogeneity (Fig. 7.).

To obtain the most chromatographically homogeneous preparation (more than 98% according to the results of gel filtration HPLC, Fig. 8) used for accurate glycobiological studies, the scheme can be additionally included concentration by ultrafiltration using a 50 kDa cut-off membrane and gel filtration on Superdex 200 .

The specific activity of the β-N-GlcNAcase StrH preparations obtained by the method disclosed in the invention is 240 units / mg protein; the content of bacterial endotoxins ranges from 2 EE / mg to 4 EE / mg protein; the content of residual DNA of the producer strain is not more than 4.0 pg / mg protein.

The claimed method allows to obtain a recombinant drug βN-GlcNAcase StrH, having a higher degree of purity compared with commercial analogues, the comparison results are presented in Table 2.

For a better understanding of the invention, the following are examples of its specific implementation:

Example 1. Microbiological protein synthesis β-N-GlcNAcase StrH

The claimed method is implemented for the producer strain E. coli VKPM B-12747 when cultured in a fermenter (Biotron LiFlus SP / SL-20L, South Korea) in a nutrient medium volume of 15 liters. The pH is maintained at 7.0-7.6. Cultivation is carried out at a temperature of 37 ° C. The relative content of dissolved oxygen is maintained at a level of 20-100% by mixing at a speed of 800 rpm and air supply with a volume flow rate of 15 l / min. Inoculum in a volume of 0.75 l, grown to an optical density measured at a wavelength of 600 nm (OD600), equal to 1.5 p.u. (optical units), bring in a fermenter with a sterile nutrient medium.

During the cultivation process, the main parameters of culture growth are monitored: OD600, pH, cell morphology, and the relative content of dissolved oxygen. At 2.5 hours of cultivation, when the optical density of the culture reaches 3.2 OE, an inducer-sterile solution of isopropyl-β-D-1-thiogalactopyranoside is applied to a final concentration of 0.0476 wt. % (2 mM). Cultivation is carried out for 5-6 hours. The final optical density is 28.2 pu, the biomass yield is 44 g / l. The productivity of the target product of E. coli strain VKPM B-12747 is 40 mg / l of culture fluid. FIG. 4. shows the dynamics of biomass accumulation during the cultivation of E. coli producer strain VKPM B-12747.

Example 2. Isolation and purification of protein β-N-GlcNAcase StrH from bacterial biomass. Obtaining an industrial series of the drug β-N-GlcNAcase StrH.

2.1 Biomass disintegration.

Buffer A - 20 mM Tris-HCl, 0.5 M NaCl, pH 7.8;

180 g of frozen cell biomass, 60 g of 3 XP are suspended in 350 ml of buffer A with PMSF up to 0.003% for 45 minutes on ice and destroyed in a Panda Plus 2000 high-pressure flow homogenizer (GEA Niro Soavi). The homogenizer is washed with 250 ml of cooled to 0 ° С purified water, adjusted by buffer A cooled to 0 ° С, setting II of the stage is 140 bar, setting I of the stage is 800 bar, then lyse the cell suspension in this mode, collecting the lysate in a container placed in an ice bath, wash the homogenizer with 30 ml of buffer A. Lysis is repeated 2 more times in the same mode. A 1500 ml lysate was clarified by centrifuging 3,250 × g (JLA-16.250) at 4 ° C for 60 minutes.

Chromatography was performed at 4 ° C, Akta Purifer 100 chromatography system (GE).

2.2. Chromatography on Ni ²⁺ IMAC Sepharose FF.

1500 ml of supernatant, diluted with buffer A to 1800 ml, adjusted to pH 7.8 with 2M NaOH, filtered through a Sartopor 2 150 depth filter and applied to an IMAC Sepharose Ni ²⁺ column, equilibrated with buffer A. After application, the column is washed with buffer A and sodium - acetate buffer with 1 M NaCl, pH 5.0. The target protein is eluted with 250 mM imidazole.

2.3 Chromatography on Blue Sepharose 6 FF 30 ml in slip mode.

The Ni ²⁺ IMAC Sepharose eluate is diluted with a starting buffer (50 mM MES, pH 6.5) to a conductivity of 5.7 mSm / sm, the pH is adjusted to 6.5, and applied to a column with Blue Sepharose 6 FF 30 ml, balanced with starting a buffer. The target protein is collected in the mode of breakthrough.

2.4 Chromatography on Toyopearl Giga Cap Q-650M.

The breakthrough with Blue Sepharose 6 FF is diluted with cooled to 4 ° C purified water to a conductivity of 1.8 mSm / sm, the pH is adjusted to 6.5 and applied to the column with Toyopearl Giga Cap Q-650M balanced with a buffer of 50 mM MES, pH 6.5 . The target protein was eluted with a gradient from 3 mM to 0.3 M NaCl in the starting buffer.

2.5 Concentration on Q Sepharose FF with a 5 ml HiTrap Butyl Capto precolumn.

The eluate from Toyopearl Giga Cap Q-650M is diluted with purified water to a conductivity of 2.04 mSm / sm, the pH is adjusted to 7.5 and applied to Q Sepharose FF columns with a HiTrap Butyl Capto pre-column, equilibrated with 20 mM Tris-HCl buffer, pH 7.5 . Wash off with starting buffer until the eluate of the non sorbed components exits, remove the HiTrap Butyl Capto column, wash with buffer B (20 mM Tris-HCl, ImM EDTA, pH 7.5) and elute with 250 mM NaCl in buffer B.

2.6 Breeding, sterilizing filtration and packaging.

The eluate with Q Sepharose FF is diluted with buffer B until the finished form is obtained and sterile filtered through FILTER-MAX, "rapid" - 150 ml, 0.22 μm, aliquoted and stored at minus 20 ° C.

Received:

73.6 mg, 118.0 ml, ε280 = 1.07, C = 0.624 mg / ml,

The content of bacterial endotoxins: From 9.6 to 19.2 EE / ml.

Synthetic substrate activity: 234 U / mg.

The purity of the sample by GF HPLC is: 90.0%.

Residual DNA of producer strain (E. colli): ≤ 4.0 pg / mg.

Example 3. Obtaining protein β-N-GlcNAcase StrH with improved quality characteristics.

Gel filtration is performed at RT, Akta Pure, GE chromatography system.

Concentration anion-exchange chromatography fractions containing the target protein (preparation according to Example 2) are combined and concentrated on Amicon Ultrac centricons, 50 K. Then the concentrate is applied to a column with Superdex 200, using 20 mM Tris HCl buffer, 0.05 M NaCl buffer as the mobile phase, 1 mM EDTA pH 7.5. The elution of the target protein is carried out in the same buffer. The elution profile is shown in FIG. 5. Then the eluted target protein β-N-GlcNAcase StrH is diluted with buffer of the mobile phase to a protein concentration of not more than 1 mg / ml and, observing aseptic conditions, is sterile filtered through a 0.22 μm filter, packed in aliquots and stored at –20 ° C .

Example 4. Analysis of the activity of recombinant β-N-GlcNAcase StrH.

Determination of recombinant β-N-GlcNAcase StrH activity is carried out by testing a synthetic substrate NAG (4-Nitrophenyl N-acetyl-β-D-glucosaminide) using the β-N-Acetylglucosaminidase Assay Kit (Sigma). Hydrolysis of the substrate NAG (4-Nitrophenyl N-acetyl-β-D-glucosaminide) with the enzyme β-N-Acetylglucosaminidase produces the product p-nitrophenol, the content of which is measured colorimetrically at 405 nm. One unit of activity (1 unit) should hydrolyze 1.0 μmole of the substrate 4-Nitrophenyl N-acetyl-β-D-glucoside to form p-nitrophenol and N-acetyl-β-D-glucosaminide products in 1 minute at pH 4.7 and temperature 37 ° C For the test, a solution of the substrate "4-Nitrophenyl N-acetyl-β-D-glucosaminide" with a concentration of 1 mg / ml is prepared. To do this, dissolve 10 mg of the substrate in 10 ml of 0.09M solution "Citrate Buffer Solution". As a positive control, a solution of β-N-Acetylglucosaminidase from Jack beans is prepared by diluting 100 times in a solution of Dilution Buffer. Prepare a solution of "Stop Solution" by diluting the contents of the bottle in 118 ml of purified water. As a control, dilutions of β-N-Acetylglucosaminidase (NAG) (Sigma, cat # A6805) are prepared 50, 100,200, 400 times in a Dilution Buffer solution.

Dilutions of purified recombinant β-N-GlcNAcase StrH are prepared 50, 100, 200, 400 times in a Dilution Buffer solution. Before adding 5 μl of Standard Solution (10 mM p-Nitrophenol) to a well of a 96-well plate, dilute it with 995 μl of Stop Solution.

98 μl of the substrate solution are introduced into the wells of the plate, 2 μl of the test samples, the NAG solution and the positive control are added in each. Diluted standard solution is poured into 300 μl per well. As “Blank”, wells are used in which 100 μl of the substrate solution are added. The scheme of the reaction is presented in Table 3.

Incubate the plate at 37 ° C for 11 minutes.

Add 200 μl of Stop Solution solution to the wells, except for the wells with the standard solution.

Measure the optical density of the solutions at a wavelength of 405 nm.

The enzyme activity is calculated by the formula:

Units / ml = (Asample-Ablank) * 0.05 * 0.3 * DF / Astandard / time / V, where

Asample - optical density of the studied samples, 405 nm

Ablank - optical density of the solution “Blank”, 405 nm

0.05 - μmole / ml p-Nitrophenol in standard solution

0.3 - the final volume of the reaction mixture in the well after adding the Stop Solution solution, ml

DF - dilution factor

A standard - the optical density of the standard solution, 405 nm

time - incubation time, min

V - sample volume, ml

The test results of the β-N-GlcNAcase StrH are presented in Table 4.

Example 5. Electrophoretic separation of proteins

Electrophoresis is carried out in 4-20% gradient PAG in reducing conditions, stained with silver salts (Thermo Scientific).

Sample preparation is carried out by diluting the samples to a concentration of ~ 50 μg / ml by adding sample loading buffer (S.b.) with the addition of 2 M dithiothreitol solution (DTT). Heat up for 5 minutes at (100 ± 2) 0С. Centrifuged for 1 min., 13000 rpm and put samples (solutions obtained by dilution of samples) 10 µl per well of gel, molecular weight markers - 5 µl per well of gel.

Electrophoresis is carried out at a voltage of 400 V, a current of 30 mA, and the time until the dye is released. Then it is stained with silver salts according to the instructions for the Thermo Scientific kit. The results are presented in FIG. 6

Example 6. Testing of purified preparations of recombinant β-N-GIcNAcase StrH in comparison with the commercial preparation β-N-GlcNAcase for the content of bacterial endotoxins.

Analysis of the content of bacterial endotoxins is carried out by the method of gel-thrombus test in accordance with the GF XIII, gel-thrombus test with LAL-reagent OFS.1.2.4.0006.15. Samples diluted with water for lal-test ("Pyrotest", Russia). Used for work: Endosafe LAL-reagent (Charles River, USA) with a sensitivity of 0.03 U / ml; Control standard sample of endotoxin (Charles River, USA) with a content of 20 EE / ml. 100 μl of samples and 100 μl of LAL-reagent are placed in test tubes for analysis and incubated for 60 minutes at 37 ° C. Positive result: the formation of a dense clot of the gel. When turning the tube 180 degrees, the gel does not break. Negative result: the gel does not form or breaks when turning the tube.

The endotoxin content at the end point is expressed in EE / ml and is calculated by the formula: “C” = (dilution at the end point) × (sensitivity of the LAL reagent). The results are presented in Table 5.

Notes to the table:

“+” - positive result; "-" - negative result.

Positive result: the formation of a dense clot of the gel. When turning the tube 180 degrees, the gel does not break. Negative result: the gel does not form or breaks when turning the tube.

The endotoxin content at the end point is expressed in EE / ml and is calculated by the formula: “C” = (dilution at the end point) × (sensitivity of the LAL reagent).

Example 7. Testing the chromatographic purity of purified recombinant β-N-GlcNAcase StrH preparations in comparison with the commercial preparation β-N-GlcNAcase.

Testing is performed by HPLC gel filtration on a Superdex200 Increase 10/300 GL (GE) column on an Alliance 2695, UV / Visible Detector 2487 (Waters) chromatography system. A buffer of 0.02 M NaH2PO4 is used as the mobile phase; 0.3 M NaCl; pH = 7.0. The purified β-N-GlcNAcase StrH preparation is applied without dilution, the commercial one is diluted 2 times with a mobile phase before being introduced into the system. The results of the analysis are presented in FIG. 7. Testing the purified recombinant β-N-GlcNAcase preparation StrH in comparison with the commercial preparation β-N-GlcNAcase is carried out in the same way. Comparison of the chromatographic profiles of the test samples shows (Fig. 8.) that the β-N-GlcNAcase StrH StrH from the IBC "Generium" is significantly more uniform in composition, whereas the commercial β-N-GlcNAcase is more heterogeneous, which causes difficulties in determining the main peak on the chromatogram of a commercial sample.

Example 8. Testing purified preparations of recombinant β-N-GlcNAcase StrH in comparison with the commercial preparation β-N-GlcNAcase for residual DNA of the producer strain.

Testing for residual DNA of the producer strain is carried out by quantitative real-time PCR, not more than 20 pg / mg. For the design of a quantitative polymerase chain reaction (The quantitative polymerase chain reaction - QPCR), the Taqman system was chosen, in which, when a specific DNA sequence is propagated, the probe is degraded by DNA polymerase. The primer set and probe for detecting the DNA of the E. colli producer strain are presented in Table 6. Before testing, samples were purified using the QIAamp DNA Blood Mini Kit (Qiagen). The final volume upon elution was 50 μl. In a qPCR reaction (sample), 10 μl of sample was added. The number of repetitions for each sample is 3.

The amount of DNA in the sample is less than 0.5 pg below the limit of reliable detection. The results of the analysis are presented in Table 7.

Claims (10)

1. Industrial purification method of recombinant exo-beta-N-acetylglucosaminidase StrH from a soluble fraction of a producer cell lysate, characterized in that chromatographic purification is carried out using a hexa-histidine tag, using successively: the stage of metal-chelate chromatography for sorption of the target protein with hexa-histidine by sortizing the target protein with hexa-histidine breakthrough gel filtration to purify the target protein from contaminating impurities of cellular proteins that co-elute with the target protein at all stages; anion exchange chromatography on a sorbent optimized for capturing and purifying high molecular weight proteins; hydrophobic chromatography, where they carry out purification from contaminating impurities of hydrophobic proteins and bacterial endotoxins. 2. The method of claim 1, wherein the metal chelate chromatography is performed on IMAC Sepharose 6 FF. 3. A method according to claim 1, where the washing at the stage of metal chelate chromatography is carried out with an acidic buffer with high ionic strength. 4. The method according to claim 1, where the gel filtration is carried out on Blue Sepharose in the overshoot mode. 5. A method according to claim 1, where anion-exchange chromatography is carried out on Toyopearl Giga Cap Q-650M. 6. The method according to claim 1, where hydrophobic chromatography is carried out on a sorbent of Capto Butyl. 7. A method according to claim 1, where, after hydrophobic chromatography, the preparation is concentrated on an anion exchange sorbent. 8. The method according to claim 7, where the concentration of the drug is carried out on Q Sepharose FF. 9. The method according to claim 1, wherein the protein is further concentrated by ultrafiltration using a membrane with 50 kDa cut-off and gel filtration on Superdex 200. 9. The method according to claim 1, wherein the amino acid sequence of the recombinant exo-beta-N-acetylglucosaminidase StrH with the hexahistidine tag is identical to the sequence of SEQ ID NO 2.

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