RU2698460C1 - Method of producing recombinant β-1,4-galactosidase bgaa from streptococcus pneumoniae - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and solves the problem of obtaining highly purified enzymatically active recombinant β-1,4-galactosidase BgaA, suitable for use at the remodeling step for producing therapeutically functional preparations of glycoproteins with given properties, to develop tools for high-sensitivity analysis of glycan chains of plant, animal and microbial glycoproteins. Method comprises using metal chelate, pseudo-affine, hydrophobic, anion-exchange, concentrating chromatography followed by polishing gel filtration, wherein protein from soluble fraction of cell lysate is purified by two chromatography steps on same Ni²⁺ metal chelate sorbent under different elution conditions and pseudo-affine chromatography in breakthrough mode on Blue Sepharose.

EFFECT: use of said three steps helps to maximize purity of the preparation from major contaminating impurities.

10 cl, 8 dwg, 3 tbl, 6 ex

Description

The invention relates to the field of biotechnology, and in particular to methods for the production of therapeutic drugs, isolation and purification of recombinant proteins, in particular the production of a highly purified recombinant β-1,4-galactosidase (BgaA) preparation. The main areas of application of the bacterial β-1,4-galactosidase BgaA obtained by the proposed method are its inclusion in the technological process of obtaining a number of medical drugs, as well as biomedical research in the field of glycobiology.

The invention solves the problem of creating a method for producing in industrial volumes of highly purified enzymatically active recombinant β-1,4-galactosidase BgaA, suitable for use at the remodeling stage to obtain therapeutically functional preparations of glycoproteins with desired properties, to develop tools for highly sensitive analysis of glycan chains of plant tissue glycoproteins, animals and microorganisms. The invention also relates to a method for producing a recombinant β-1,4-galactosidase BgaA preparation having a higher degree of purity compared to commercial preparations, homogeneous in the target product, with a lower content of endotoxins, proteins and producer DNA in comparison with commercial preparations.

DESCRIPTION OF DRAWINGS

FIG. 1. The structure of the N-glycosylated human glycoprotein. The names of the sugar residues are located under their respective symbols. The lines are the bonds between sugar residues, the numbers above are the specificity of the bond. Arrows are bonds that are potentially cleaved by glycosidases, indicated at the top of the diagram;

FIG. 2. Plasmid map pMT1620 (expression vector based on pET15b to obtain streptococcal β-galactosidase BgaA)

Designations:

T7 promoter - T7 promoter of RNA polymerase; N-terminal His-tagged BGAA_STRPN is a protein gene of His-tagged (N-terminus) BGAA_STRPN, which consists of sequences of 20 amino acid residues containing a hexahistidine tag and 2053 amino acid residues (without signal peptide, G5 domain and anchor peptide) β- galactosidase (bgaA) Streptococcus pneumoniae; T7 terminator - terminator of T7 RNA polymerase; AmpR promoter - promoter of the ampicillin resistance gene; AmpR - ampicillin resistance gene; ori - origin of replication; lacI gene of a lactose repressor; lacI promoter - lactose repressor gene promoter; NdeI — NdeI restriction endonuclease recognition site; BamHI - BamHI restriction endonuclease recognition site

FIG. 3. The dynamics of biomass accumulation during cultivation of the producer strain E. coli 12746 in a volume of 15 l according to the proposed scheme. The abscissa shows the cultivation hours, the ordinate shows the cultivation parameters.

FIG. 4. The cultivation parameters of the producer strain E. Coli VKPM 12746 in a volume of 15 l according to the proposed scheme. The abscissa shows the cultivation hours, the ordinate shows the cultivation parameters.

FIG. 5. Analysis of the accumulation of protein of β-galactosidase BgaA by electrophoresis in reducing conditions in 8% PAAG during cultivation of the producer strain E. Coli VKPM B-12746 in a volume of 15 l according to the proposed scheme. The abscissa shows the cultivation hours, the ordinate shows the accumulation of protein. Marker - molecular weight markers, Arr. 1 - 2.5 hours of growth, Arr. 2 - 3 hours of growth, Arr. 3-4 hours of growth, Arr. 4 - 6 hours of growth, CO - a standard sample of β-galactosidase BgaA.

FIG. 6. The result of the allocation of BgaA. Analysis by electrophoresis in 4 - 20% gradient PAAG. Comparison of the commercial preparation of β-galactosidase and the resulting preparation BgaA.

Lane 1 — Thermo Scientific molecular weight markers;

Lanes 2-4 — Purified BgaA preparation with varying amounts of drug added to the well.

Lane 5 - commercial β-galactosidase;

FIG. 7. Comparison of the chromatographic profiles of β-galactosidase from Streptococcus pneumoniae, recomb, E. coli, Calbiochem, and β-galactosidase BgaA MBC "Generium" using gel filtration. Line 1 - Placebo; Line 2 - Commercial β-galactosidase from S. Pneumoniae, recomb, E. coli, Calbiochem, 3 - β-galactosidase BgaA MBC "Generium"; Line 4 - Molecular Weight Standards. Column: Superdex200 Increase 10/300 GL, GE Healthcare; Flow rate: 0.75 ml / min; Volume of injection: 100 μl; The time and parameters of the registration of the chromatogram: 40 min, t _count = 30 ° C; t _arr = 5 ° C; λ = 280/214 nm; PF: 0.02 M NaH ₂ PO ₄ 0.3 M NaCl pH = 7.0.

FIG. 8. The result of the analysis of GF HPLC. Analysis of fractions from the intermediate series and the finished form of the industrial series BgaA.

Line 1 - Placebo for the Q Sepharose FF fraction; Line 2 - Eluate fraction with Q Sepharose FF; Line 3 - placebo for GF BgaA; Line 4 - GF BgaA; Line 5 - Molecular Weight Standards. Column: Superdex200 Increase 10/300 GL, GE Healthcare; Flow rate: 0.75 ml / min; Volume of injection: 100 μl; The time and parameters of the registration of the chromatogram: 40 min, t _count = 30 ° C; t _arr = 5 ° C; λ = 280/214 nm; PF: 0.02 M NaH2PO4 0.3 M NaCl pH = 7.0.

The lack of domestic commercially available preparations of the full-sized recombinant β-1,4-galactosidase BgaA from Streptococcus pneumoniae expressed in E. coli, and the extremely high cost of drugs from foreign manufacturers such as QA-Bio, NEB, Calbiochem, makes the use of these drugs commercially unprofitable in the production of drugs. The products of these companies are not manufactured in accordance with the Good Manufacturing Practice (GMP) standards and are intended only for analytical research.

Detailed characteristics of β-1,4-galactosidase BgaA belonging to the large family of galactosidases (lactases, β-galactoside-galactohydralases) that act on N / O-glycosyl compounds and cleave the terminal unreduced β-D-galactose residue in β-galactosides, including lactose, with the formation of free monosaccharides, or the remainder of β-D-galactose is transferred to a molecule of lactose or other β-D-galactosides with the formation of galactooligosaccharides, given in A.A. Kostenevich, L.I. Sapunova, Bacterial β-galactosidases, biochemical and genetic diversity // Transactions of BSU. 2013 - T. 8. 4.1 - p. 52-63, A.E. Braunstein, Enzyme Nomenclature / Recommendations of the International Biochemical Union on the nomenclature and classification of enzymes, as well as on units of enzymes and symbols of the kinetics of enzymatic reactions, // - 1979 - VINITI. - with. 320, Panesar P. S. et al, Microbial production, immobilization and applications of β-D-galactosidase, // J. Chem. Technol. Biotechnol. - 2006. - V. 81 - p. 530-543, Ah-Reum Park, Deok-Kun Oh, Galacto-oligosaccharide production using microbial β-galactosidase: current state and perspectives, // - 2010 - Appl Microbiol Biotechnol - V. 85 - p. 1279-1286).

These enzymes are widely distributed in nature and are of great interest to the biotechnological and pharmaceutical industries; their hydrolytic activity has been used for decades in the food industry to reduce the lactose content in dairy products in order to bypass the lactose intolerance prevailing in more than half of the world's population (Alejandro Vian, Alfonso V. Carrascosa, Jose L. Garcia, Estrella Cortes, Structure of the b-Galactosidase Gene from Thermus sp.Strain T2: Expression in Escherichia coli and Purification in a Single Step of an Active Fusion Protein, // Applied and Environmental Microbiolology - 1998, V. 64 - N.6 - p. 2187-21 91).

Some of the β-galactosidases have been purified, sequenced and widely characterized. Commercially available β-galactosidase preparations are mainly obtained from the yeast Kluyveromyces fragilis, Kluyveromyces lactis and Candida pseudotropicalis, Aspergillus niger and Aspergillus oryzae and Bacillus stearothermophilus (Parmjit S. Panesar, Reeba Panesar and John Harish Kumar, Review.Microbial production, immobilization and applications of β-D-galactosidase // Journal of Chemical Technology and Biotechnology-2006 - V.81 - p. 530-543).

Known work on the isolation and cloning of genes responsible for the synthesis of β-galactosidase in various bacteria. The most studied β-galactosidase gene of E. coli - lacZ, which encodes an enzyme tetramer protein (540 kDa), consisting of identical subunits (A. Kalnins et al, lacZ Sequence of the gene of Escherichia coli // EMBO J. - 1983. - V. 2, N. 4. - p. 593-597).

Despite the long history of the use of β-galactosidases in various industries and scientific research, not all β-galactosidases can serve as tools for the analysis and remodeling of glycans, as well as for the preparation of oligosaccharides from natural materials or substrates for glycosyl transferases. For example, an enzyme from Escherichia coli is used in many fields of biochemistry, but not for glycoconjugates (Reinhard Zeleny, Friedrich Altmann and Werner Praznik, A Capillary Electrophoretic Study on the Specificity of β-Galactosidases from Aspergillus oryzae, Escherichia coli, Streptococcus pneumonia (Jack Bean) // Analitical Biochemistry - 1997 - V. 246. - p. 96-101).

By Kyoko Kojima, Masao Iwamori, Sei-Ichi Takasaki, Shirou Nozawa, Rihachi Iizuka, Yoshitaka Nagalt, Diplococcal β-Galactosidase with a Specificity Reacting to β 1-4 Linkage but Not to β-1-3 Linkage as a Useful Exoglycosidase for the Structural Elucidation of Glycolipids, // Analytical Biochemistrty - 1987 - V. 165 - p. 465-469, it was shown that the enzyme β-1,4-galactosidase BgaA from Streptococcus pneumoniae (formerly Diplococcus pneumoniae) has a rather strict specificity for β-1-4 bonds and, therefore, can serve to remove or determine the position of the galactose residue, in addition, Dominique N. Limoli, Julie A. Sladek, Lindsey A. Fuller, Anirudh K. Singh and Samantha J. King, BgaA acts as an adhesin to mediate attachment of some pneumococcal strains to human epithelial cells, // Microbiology - 2011 - V.157 - p. 2369-2381 it was reported that, unlike other β-galactosidases, the main role of BgaA is not in lactose metabolism, but in the deglycosylation of human glycoconjugates. Unusually high specificity of the BgaA enzyme for β-1,4-glycosidic bonds and more than 10-fold high specificity for N-acetylgalactosamine (Ga1β1-4GlcNAc) than for lactose (Zeleny, R, F. Altmann, and W. Praznik, A capillary electrophoretic study on the specificity of β-galactosidases from Aspergillus oryzae, Escherichia coli, Streptococcus pneumoniae, and Canavalia ensiformis (jack bean) // Anal. Biochem - 1997 - V.246 - p - 96-101), allow the BgaA enzyme to be considered an ideal candidate for attacking polysaccharides conjugated to the surface components of eukaryotic cells (Yang, Y, and R. Orlando, Simplifying the exoglycoside digestion / MALDI-MS procedure for sequencing N-linked carbohydrate side chains, // Anal. Chem - 19 96 - V. 68 - p. 570-572, Vanessa S. Terra, Karen A. Homer, Susmitha G. Rao, Peter W. Andrew and Hasan Yesilkaya, Characterization of Novel - Galactosidase Activity That Contributes to Glycoprotein Degradation and Virulence in Streptococcus pneumoniae. // Infection and Immunity - 2010 - V. 78 - N. 1 - p. 348-357), which makes the BgaA enzyme extremely attractive for use in glycobiology. Amanda M. Burnaugh, Laura J. Frantz, and Samantha J. King, Growth of Streptococcus pneumoniae on Human Glycoconjugates Is Dependent upon the Sequential Activity of Bacterial Exoglycosidases // Journal of Bactereology - 2008 - V. 230 - N. 1 - p. 221-230, it has been demonstrated that BgaA can, together with NanA neuraminidase and StH β-N-acetylglucosaminidase StH, sequentially remove sugars on N-linked glycans of human glycoconjugates (Fig. 1).

For the first time, the BgaA enzyme was purified from the filtrate of Streptococcus 6646 culture and characterized by Takao Kiyohara, Tadao Terao, Kohei Shioiri-Nakano and Toshiaki Osawa, Purification and Characterization of β-N-Acetylhexosaminidases and β-Galactosidase from Streptococidus 6646 Streptococidus Biochem - 1976 - V. 80 - p. 9-17, and Lowrie R. Glasgows, S. James, C. Paulson and Robert L. Hill, Systematic Purification of Five Glycosidases from Streptococcus (Diplococcus) pneumoniae ", // The Journal of Biological Chemistry-1977 - V. 252 - N. 23 - p. 8615-3623. In a study by Dorothea Zahner and Regine Hakenbeck, The Streptococcus pneumoniae Beta-Galactosidase Is a Surface Protein // Journal of Bacteriology - 2000, - V. 182 - N. 20 - p. 5919-5921 , a gene encoding BgaA β-galactosidase from S. Pneumoniae has been identified. Unlike typical galactosidases, which are usually cytoplasmic proteins, S. Pneumoniae BgaA gene encodes a surface linked β-1,4-galactosidase consisting of 2,235 amino acid residues with a molecular weight of more than 200 k A. The complete sequence of the S. Pneumoniae genome is presented by Herve Tettelin et al, Complete Genome Sequence of a Virulent Isolate of Streptococcus pneumonia // - Science - 2001 - V. 293 - p. 498-506. In the study by Anirudh K. Singh et al, Unravelling the Multiple Functions of the, Architecturally Intricate Streptococcus pneumoniae β-galactosidase BgaA, // PLOS Pathogens / www.plospathogens.org - 2014 - V. 10 - I.9 - e1004364, due to detailed structural and functional analysis, a complex BgaA architecture and variety of features.

The complete genomic sequences of seven S. Pneumoniae strains have been studied: S. Pneumoniae ATCC 700669, S. Pneumoniae G54, S. Pneumoniae CGSP14, S. Pneumoniae Hungary 19A-6, S. PneumoniaeD39, S. Pneumoniae R6 and S. Pneumoniae TIGR4 , available in public databases (Lanie, JA, WL Ng, KM Kazmierczak, T.M. Andrzejewski, T.M. Davidsen, KJ Wayne, N. Tettelin, JI Glass, and ME Winkler. Genome sequence of Avery's virulent serotype 2 strain D 39 of Streptococcus pneumoniae and comparison with that of unencapsulated laboratory strain R6. // - J. Bacteriol. - 2007. - V. 189. - p. 38-51).

Jae Car Jeong, Ohsuk Kwon, Yun Mi Lee, 1 Doo-Byoung Oh, Jung Mi Lee, Seonghun Kim, Eun-Hye Kim, Tu Nhat Le, Dong-Kwon Rhee, and Hyun Ah Kang, Characterization of the Streptococcus pneumoniae BgaC Protein as a Novel Surface - Galactosidase with Specific Hydrolysis Activity for the Gal1-3GlcNAc Moiety of Oligosaccharides. // -Journal of Bacteriology. - 2009. - V. 191. - N. 9.- p. 3011-3023 another BgaC Streptococcus pneumoniae β-galactosidase is reported, which is also a surface-associated β-galactosidase with high specific hydrolysis activity for the Gal1-3GlcNAc fragment of oligosaccharides, for a β-1,3-glycosidic bond without any or modifications, consisting of 595 amino acid residues with a molecular weight of about 70 kDa. In the patent Kang et al. Recombinant beta-Galactosidase derived from Streptococcus Pneumoniae, // US 0287463 A1., - 2011, discloses the preparation of a recombinant B-Galactosidase BgaC with a molecular weight of 69 kDa expressed in a soluble fraction of E. Coli cells with further destruction of the cells by ultrasound and subsequent centrifugation and obtaining target protein by purification of the supernatant by affinity chromatography on a column of Ni-NTA agarose. This β-1-3 galactosidase has an extremely narrow substrate specificity and is not used for remodeling of glycoconjugates. The preparation of β-1-4 galactosidase from Bacteroides fragilis is disclosed in Sharon T. Wong - Madden, Ellen P. Guthrie et al, Isolation and compositionof novel glycosidases, // US 7094563 B2, - 2006, the enzyme was isolated from a coarse cell lysate of Bacteroides fragilis chromatographic purification on DEAE Sepharose FF in slip mode followed by chromatography on Phenyl Sepharose FF, dialysis of active fractions, subsequent chromatography on Heparin-Sepharose and dialysis of active fractions, chromatography on Mono Q with final chromatography on Heparin TSK.

Although some commercial β-1,4 galactosidase preparations, both from natural sources and recombinant ones expressed in E. Coli, are currently available, there is a need for new, much cleaner β-1,4 galactosidases with a strictly defined substrate specificity and reproducible enzymatic activity.

In S. Campuzano, B. Serra, D. Llull, J.L. Garcia, E. Garcia, P. Garcia, Cloning, Expression, and Characterization of a Peculiar Choline-Binding-Galactosidase from Streptococcus mitis, // Applied and Environmental Microbiology, - 2009. - V.75, - N.18. - p. 5972-5980, describes the closest to the claimed invention a method for producing orthologous β-1,4 galactosidase from Streptococcus mitis. A Streptococcus mitis genomic DNA fragment carrying the SMT1224 gene encoding β-galactosidase was identified, cloned and expressed in Escherichia coli. This gene encodes a 2411 amino acid protein with a predicted molecular weight of 268 kDa. The recombinant protein contains an N-terminal signal peptide and a C-terminal choline binding domain. The choline binding capacity of the protein facilitates its purification using affinity chromatography on DEAE cellulose, although complete purification of the target protein was achieved by constructing a fusion protein with a polyhistidine sequence.

A variant of the recombinant protein with a choline-binding domain was purified using DEAE-cellulose affinity chromatography, the active fractions obtained after DEAE-affinity chromatography were combined, dialyzed and concentrated 10 times in a dialysis bag with polyethylene glycol 6000. The concentrated enzyme solution was further purified by gel filtration chromatography. Sephacryl S-300. In this way received 0.36 mg of the target protein with 1 l of culture fluid.

A variant of the recombinant protein with a polyhistidine sequence expressed in E. coli MC4100 (pHGM02) was purified in one step by affinity chromatography on IMAC cartridges (Bio-Rad) with a yield of 0.004 g per 1 liter of culture fluid.

These purification schemes are unacceptable for large-scale production of the drug, since the authors received the drug with a low yield and only for analytical purposes. In addition, the authors did not report the level of bacterial endotoxins in the preparation. The removal of bacterial endotoxins is a fundamental and necessary requirement, both from the point of view of Russian and international standards (GF XIII, Document Q AS / 11.452 FINAL July 2012). However, it often remains a technically difficult task, therefore, the content of bacterial endotoxins in the intermediate products used in the manufacture of drugs is a critical parameter. When assessing the quality of intermediates such as glycosidases, in addition to substrate specificity and activity, potentially hazardous contaminants such as bacterial endotoxins, cellular DNA, including potentially oncogenic, the amount of host proteins of the producer strain and the homogeneity of the drug should be specially evaluated. And, as a rule, in the production of drugs from natural sources, there are difficulties in obtaining pure drugs. Usually they are contaminated with impurities of other glycosidases, proteases.

To solve the problem of obtaining highly purified BgaA in industrial volumes, the inventors propose a new method for producing recombinant β-1,4 galactosidase BgaA from Streptococcus pneumoniae, including the synthesis of a DNA sequence optimized for translation into E. coli that encodes a BgaA protein, the nucleotide sequence of which is shown in SEQ ID NO1, construction of an expression plasmid vector encoding a chimeric protein with N-terminal chimerization composed of peptide sequences of six histidines, recognition site t rhombin and 2053 amino acid residues (without signal peptide, G5 domain and anchor peptide) β-1,4-galactosidase (BgaA) Streptococcus pneumoniae with a molecular weight of 231 kDa (amino acid sequence represented by SEQ ID NO 2), obtaining a recombinant protein with products in E .coli, and then chromatographic purification of the protein from the soluble fraction of the cell lysate, where the first two stages are carried out on the same Ni + metal chelate sorbent under different different elution conditions, pseudo-affinity chromatography in slip mode on Blue Sepharose anion exchange chromat graphs on Toyopearl Giga Cap Q-650M, hydrophobic slip chromatography on Butyl Capto, concentration anion exchange chromatography on Q Sepharose FF followed by gel filtration on Superdex 200 with the release of a highly purified active drug BgaA, suitable for use in the manufacture of drugs and glycobiological studies, not less than 0.01 g per liter of bacterial culture.

The main technical result of the invention is the production on an industrial scale of a highly purified active preparation of an enzymatically active recombinant β-1,4-galactosidase BgaA containing minimal residual concentrations of proteins and DNA of the host cell, which makes it suitable for use at the remodeling stage in the production of therapeutically functional glycoprotein preparations with desired properties, as well as for the development of tools for highly sensitive analysis of glycan chains of tissue glycoproteins second plants, animals and microorganisms.

To carry out the invention, a strain of a genetically modified microorganism E.coli VKPM B-12746 was created: the recipient host strain BL21 (DE3) with plasmids pLysS carrying the T7 lysozyme gene, which is an inhibitor of T7 RNA polymerase, pMT1620 (pET15b-BGAA_STRPN), BGAA_STRPN protein gene, of which a sequence of 20 amino acid residues containing a hexahistidine tag and 2053 amino acid residues (without signal peptide, G5 domain and anchor peptide) of β-galactosidase BgaA Streptococcus pneumoniae with an estimated molecular weight of 231 kD but. (Fig. 2).

Refolding of the product is not required, since it is synthesized in the bacterial cytoplasm mainly in the dissolved state during the cultivation of E. coli VKPM B-12746 with the level of biosynthesis of variants of the target protein of at least 10 mg / l of culture fluid.

Highly efficient extraction of the target BgaA protein from cells is achieved as a result of bacterial cell lysis using a high pressure homogenizer.

The preparation of an active, chromatographically homogeneous with a low content of bacterial endotoxins, DNA and host proteins BgaA preparation is achieved through a chromatographic purification scheme, which, in combination with some standard methods of protein purification (anion exchange chromatography on high molecular weight anion exchange sorbent, hydrophobic chromatography in slip mode, concentrating anion exchange chromatography and polishing gel - filtration), includes two stages of chromatography on the same Ni + met an allhelate sorbent under different elution conditions and then pseudo-affinity chromatography in slip mode using a sorbent matrix to which Cibacron Blue 3G, an anion anthraquinone dye, is covalently attached. Typically, such a sorbent is used to purify albumin and interferon both on a laboratory and technological scale. It is also used to isolate enzyme groups and remove albumin, as the blue dye binds many proteins, such as albumin, interferon, lipoproteins and blood coagulation factors. It also binds several enzymes, including kinases, dehydrogenases, and most enzymes requiring adenyl-containing cofactors, such as NAD +. In the claimed invention, this type of sorbent was used to remove some cellular proteins of E. coli, due to the unexpected discovery by the authors of the fact that, when purified, β-galactosidase BgaA passes through the sorbent without adsorption, while the cellular proteins are retained. Usually this sorbent is not used for such purposes. Thus, in contrast to previously known methods, the technical result is achieved by introducing into the scheme of purification from cellular proteins the pseudo-affinity chromatography stage on sorbents with covalently bound Cibacron Blue 3G.

The example presented in the invention illustrates the use of two Ni ²⁺ metal chelate chromatographs on IMAC Sepharose 6 FF with different elution conditions of the target protein, which made it possible to maximally separate the target protein from major contaminating impurities of cell proteins having an affinity for IMAC Sepharose. This technical result is one of the decisive factors for the quality of further purification by reducing the load of contaminating proteins on all subsequent sorbents. Other sorbents are available on the market that can also be used for metal chelate chromatography in the proposed method, for example, IMAC Sepharose High Performance can be used. Chelating Sepharose Fast Flow sorbent is less preferred as it has a lower capacity and less affinity for the protein of interest. For both stages of chromatography, a single equilibration buffer is acceptable, the pH of which varies in the range of 7.5 - 7.8. Elution in the first stage is carried out with NaCl-containing sodium acetate buffer having a pH in the range of 4.2–4.3 units; in the second stage, the target protein is eluted with 150 mM – 200 mM imidazole. The preferred pH of the wash buffer used in the second stage of metal chelate chromatography is in the range of 6.5-6.6.

The introduction of the pseudo-affinity chromatography stage of the sorbent with covalently bound Cibacron Blue 3G in the slip mode is another key stage, which allows one to significantly purify the target protein from contaminants that coelute with the target protein in all previous stages. In addition to the Blue Sepharose 6 Fastp Flow used by the inventors, a number of sorbents suitable for use in the claimed invention are available on the market: Capto Blue (high sub), Capto Blue and others with similar properties. The preferred equilibration buffer composition at this stage is 20 mM Tris-HCl, 80 mM NaCl, 5% isopropanol, pH 8.2-8.3.

After two metal chelate chromatography and pseudo-affinity chromatography in slip mode, protein purification can be carried out using various types of chromatography routinely used in protein purification: hydrophobic, anion exchange, concentration chromatography and polishing gel filtration. The choice of sorbents and conditions can be carried out by a specialist on the basis of general knowledge of the prior art and the recommendations of sorbent manufacturers. In the claimed invention, the authors gave examples of the use of specific brands of sorbents in specific application conditions, however, with the change of sorbent, the conditions can be changed in accordance with the manufacturer's recommendations and general rules. The most preferred sorbents for subsequent anion exchange chromatography are TOYOPEARL GigaCap Q-650M (Toson) and MacroCap Q (GE) sorbents, optimized for sorption and purification of high molecular weight proteins. The preferred pH of the equilibration buffer for this step is in the range of 7.5-7.7. The salt composition of the elution buffer is a gradient from 5 mM to 260 mM NaCl in the start buffer. The proposed conditions for planting and removal of the target protein allow maximum purification of the target protein from contaminating impurities of cellular proteins and endotoxins. The use of hydrophobic chromatography in slip mode allows you to remove contaminating impurities of hydrophobic proteins and lipopolysaccharides. As a sorbent for a pre-column, one can use a number of sorbents on the market: Butyl Capto, Butyl Sepharose High Performance Capto Butyl ImpRes, SuperButyl-550C, etc. As a concentrating sorbent, in addition to Q Sepharose FF, which is used in an example embodiment of the present invention, Capto Q, Capto Q ImpRes, Capto Q XP. These sorbents have a larger capacity, which should be considered when choosing the concentration of salt in the elution buffer. The preferred pH of the equilibration buffer is in the range of 7.5-7.7. The composition of the preferred elution buffer for this step is 350 mM — 360 mM NaCl in 20 mM Tris — HCl, pH 7.5-7.7. The final stage of gel filtration chromatography is polishing, allowing you to get a homogeneous preparation in the buffer of the finished form. It is preferable to use the pre-packed column HiLoad Superdex 200 26/600 (GE), it is also possible to use Superdex 200, HiPrep 26/60 Sephacryl S-400 HR, and the sorbent Sephacryl S-400 HR. The preferred formulation buffer is 20 mM Tris HCl, 0.05 M NaCl, 1 mM EDTA pH 7.5-7.6.

Materials and methods

For the expression of BgaA, genetic engineering constructs (HECs) of pMT1620 are obtained. To obtain GICs encoding BgaA, a cloning scheme based on the pET-15b vector (Novagen) was developed, which implies the receipt of the full version with 6 × His-tag at the N-terminus. To obtain the corresponding GIC, TOP Gene Technologies ordered a synthetic gene encoding BgaA (the genomic sequence of the bacterium Streptococcus pneumoniae encoding 2053 amino acid residues (without a signal peptide, one of two G5 domains and an anchor peptide) β-1,4-galactosidase (BgaA ) Streptococcus pneumoniae fused at the 5'-end to a part of the vector sequence of the mother plasmid pET15b (+) encoding 20 amino acid residues containing a hexahistidine tag) flanked by NdeI restriction sites, BamHI, containing internal Nhe restriction sites I to obtain a "catalytic" domain and stop codons at the 3'-end of the sequence (TGATAA). TOP Gene Technologies performed codon optimization for protein expression in E. coli, taking into account the requirement that there are no NdeI, BamHI, XhoI, and NheI restriction endonuclease sites in the sequence. The codon optimization of the obtained BgaA sequence was verified at http://gcua.schoedl.de/sequential_v2.html. The correspondence of the translated amino acid sequence ordered is confirmed by alignment in the MEGA 6.0 program. The absence of unwanted restriction sites was confirmed using CloneManager 9.0 software. To obtain the GIC pMT1620 (pET15b-BGAA_STRPN), the run time and restriction mapping of the synthetic gene pAPG110-BgaA_STRPN were run and the restriction mapping of the synthetic gene pAPG110-BgaA_STRPN was performed. According to the developed scheme, the fragment encoding streptococcal BgaA from plasmid pAPG110-BgaA_STRPN was cloned into the vector plasmid pET15b at the NdeI, BamHI restriction sites. The fragments obtained during restriction were purified and used for ligation. Chemically competent E. coli XL10-GOLD cells were transformed with a ligase mixture. Cells were seeded to obtain transformed clones.

Plasmid DNA was then isolated and restriction mapping was performed using BglII restriction endonuclease. A plasmid DNA sample was transferred for sequencing to Eurogen. The resulting sequence was aligned with the "theoretical" sequence pMT1620.

For analytical expression, plasmid DNA GIC pMT1620 was transformed into strains BL21 (DE3) pLysS. BgaA super-producing clones were selected based on analytical expression. Then received a strain of superproducer BgaA. The strain was deposited in the All-Russian collection of industrial microorganisms (VKPM) under inventory number - VKPM V-12746.

To obtain the target BgaA protein, preparative expression was carried out by culturing E.Coli VKPM B-12746 in a complete nutrient medium with a relative dissolved oxygen content of at least 10% at a temperature of 37 ° C and a pH value of 6.5-7.5. The duration of the cultivation process is 5-7 hours.

Induction of the expression of the target product at the stage of biosynthesis was provided by introducing a solution of Isopropyl-β-D-1-thiogalactopyranoside to a final concentration of 1 mM in a nutrient medium for 2.5-3.0 hours of growth to achieve an optical density of the culture of 2.5-5.0 about .е

During the first stage, cells grow actively due to the enriched culture medium. The presence of glucose in the medium inhibits the expression of the recombinant protein, and, therefore, reduces the load on the cell of the producer strain, which leads to an increase in the amount of accumulated biomass.

Immediately before introducing the inducer, glycerin was added to the medium, which serves as a source of carbon and energy, without inhibiting the expression of the recombinant protein. This leads to an increase in the amount of accumulated recombinant β-galactosidase protein.

From 3-3.5 hours of cultivation, upon reaching optical density, the stage of hybrid protein biosynthesis begins. The expression of the BgaA protein is under the control of a promoter that is depressed by introducing an IPTG solution to a final concentration of 0.0238 wt. %

Cultivation was carried out for 5-7 hours

The level of biosynthesis of the variants of the target BgaA protein is at least 5 mg / l of culture fluid. Bacterial biomass was collected by centrifugation and stored at - 70 ° C. Cell biomass was lysed using a Panda Plus 2000 high pressure homogenizer (GEA Niro Soavi), the lysate was clarified by centrifugation, and BgaA protein was isolated from the lysate by purification of the target protein from impurity proteins by two metal chelate chromatography with different elution conditions on columns with Ni ²⁺ IMAC Sepharose FF . The enzyme was further purified by sequential chromatography on Blue Sepharose FF in slip mode, anion exchange Toyopearl Giga Cap Q-650M, Butyl Capto in slip mode, chromatography on Q Sepharose FF, concentration and gel filtration. At each stage, the degree of purification was monitored by SDS page electrophoresis.

The protein yield is at least 0.01 mg per liter of bacterial culture.

Measurements of the parameters of various batches of the obtained target BgaA protein showed that as a result of purification it has the following characteristics:

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

- the content of bacterial endotoxins (HF XIII, gel thrombus test with LAL reagent OFS.1.2.4.0006.15) not more than 2 EU / mg;

- Chromatographic purity (HPLC) not less than 94%;

- Enzymatic activity (colorimetric method, synthetic substrate ONPG (ortho-nitrophenyl-β-D-galactopyranoside), "β-Gal Assay Kit" (Invitrogen)) not less than 13000 U / mg;

- Residual DNA of the producer strain (Real-time quantitative PCR) not more than 0.015 pg / mg;

- Residual proteins of the producer strain not more than 4 ng / mg;

Thus, it is shown that using the purification method proposed in the claimed invention, a BgaA preparation is obtained with a chromatographic purity determined by gel filtration HPLC — more than 94% homogeneity, specific activity is 13,000 units / mg or more of protein, and bacterial content endotoxins is not more than 2 EU / mg of protein, the residual DNA of the producer strain is not more than 0.015 pg / mg of protein, the residual protein content of the producer strain is not more than 4 ng / mg of protein.

Obtaining BgaA 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 hexahistidine tag, which simplifies the cleaning and removes the use of expensive tag removal agents from the purification scheme. The amino acid sequence of BgaA is represented by SEQ ID NO 2. The experiments showed that, despite the presence of a hexahistidine tag, having a high molecular weight (231 kDa), the target BgaA protein penetrates poorly into the pores of the sorbent, being adsorbed on its surface. At the same time, the deep layers of the sorbent are occupied by impurities that have different, including higher affinity for the sorbent than the target protein. To remove these impurities, without which efficient protein purification is impossible, the claimed invention proposed an unexpected and original approach, consisting in two stages of metal chelate chromatography with different elution conditions, allowing to remove impurities that interfere with further purification. Thus, the inventors consider the introduction of these two stages of purification the most essential for obtaining a technical result.

At the first stage of chromatography on a Ni + metal chelate sorbent, the inventors use elution of the target protein with a low pH and high ionic strength buffer, at the second stage of chromatography at the same sorbent, the authors use washings with a buffer with a slightly acidic pH, preferably pH 6.5, and high ionic strength, and then elution with a buffer containing imidazole in a low concentration. The use of Blue Sepharose pseudo-affinity chromatography in slip mode allows purification of the target protein from impurities of cellular proteins coeluting with the target protein at all stages. Anion exchange chromatography is preferably carried out on a high molecular weight, high resolution anion exchange sorbent optimized for the capture and purification of high molecular weight proteins. Further, anion exchange chromatography allows practically without loss to concentrate the target protein without the process of membrane concentration and diafiltration. After carrying out the final polishing gel filtration, a highly purified active preparation BgaA is obtained, suitable for use in the manufacture of drugs and glycobiological studies, with a yield of at least 0.01 g per liter of bacterial culture.

The claimed method allows to obtain a recombinant BgaA preparation having a higher degree of purity compared to commercial analogues, the comparison results are presented in Table 1.

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

Example 1. Microbiological synthesis of protein BgaA

The inventive method is implemented for the producer strain E. Coli VKPM B-12746 when cultured in a fermenter (Biotron LiFlus SP / SL-20L, South Korea) in a volume of nutrient medium 15 l. pH is maintained at a level of 6.9-7.1. Cultivation is carried out at a temperature of 37 ° C. The relative content of dissolved oxygen is maintained at a level of 5-100% by stirring at a speed of 800 rpm and air supply with a volumetric flow rate of 15 l / min. Before the start of the process, a 50% glucose solution is aseptically introduced into the cooled nutrient medium to a final concentration of 0.5 wt. % The inoculum in a volume of 0.75 l, grown to an optical density measured at a wavelength of 600 nm (OD600) equal to 1.8 p.u. (optical units) are introduced into the fermenter with a sterile nutrient medium containing casein hydrolyzate, yeast extract, potassium dihydrogen phosphate, ammonium sulfate, sodium hydrogen phosphate.

During the cultivation process, the main parameters of culture growth are monitored: OD600, pH, cell morphology, relative dissolved oxygen content. At 2.5 hours of cultivation, when the optical density of the culture reaches 3.0 p.u., a sterile 50% glycerol solution is added successively to a final concentration of 0.5 wt. % and inducer - 23.8% sterile solution-propyl-β-D-1-thiogalactopyranoside - to a final concentration of 0.0238 wt. % (1 mmol). Cultivation is carried out for 6 hours. The final optical density is -20.3 p.u., the biomass yield is 28 g / l. The productivity of the target product of the strain E.Coli VKPM B-12746 is 10.0 mg / l of culture fluid.

In FIG. 4. presents the dynamics of biomass accumulation during cultivation of the producer strain E. coli VKPM B-12746.

Example 2. Isolation and purification of BgaA protein from bacterial biomass. Obtaining an industrial series of the drug BgaA.

2.1 Disintegration of biomass.

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

Frozen cell biomass 288 g, suspended in 1400 ml of buffer A with PMSF up to 0.003% for 45 min on ice and destroyed in a high-pressure flow homogenizer Panda Plus 2000 (GEANiro Soavi). The homogenizer is washed with purified water cooled to 0 ° C, adjusted to buffer A cooled to 0 ° C, setting of stage II is 130 bar, setting of stage I is 750 bar, then the cell suspension is lysed in this mode, collecting the lysate in a container placed in ice the bath, the homogenizer is washed with buffer A. The lysis is repeated 2 more times in the same mode. A 2200 ml lysate is clarified by centrifugation at 33250 × g (JLA-16.250) at 4 ° C for 90 minutes.

Purifer 100 (GE).Chromatography is carried out at 4 ° C, chromatographic system

Purifer 100 (GE).

2.2. Chromatography on Ni ²⁺ IMAC Sepharose FF - stage 1.

The supernatant of 2200 ml was adjusted to pH 7.8 with 2M NaOH, filtered through a Sartopor 2,300 depth filter, diluted with buffer A to 3,000 L and applied to a 300 ml volume column equilibrated with buffer A ² Ni IMAC Sepharose, equilibrated with buffer A. After application, the column was washed with buffer A, the target protein is eluted with sodium acetate buffer with 1 M NaCl, pH 4.3.

2.3. Chromatography on Ni ²⁺ IMAC Sepharose FF - stage 2.

The eluate with Ni ²⁺ IMAC Sepharose FF - 1 was diluted with 20 mM Tris-HCl buffer cooled to 4 ° C, pH 7.8, adjusted to pH 7.8 with 2M NaOH and applied to a 150 Ni ²⁺ IMAC Sepharose FF column, 0 ml equilibrated with buffer A. After application, the column is washed with 20 mM sodium phosphate buffer with 1 M NaCl, pH 6.5. The target protein is eluted with 150 mM imidazole.

2.4. Pseudo-affinity chromatography

The eluate with Ni ²⁺ IMAC Sepharose was diluted with start buffer (20 mM Tris-HCl, 80 mM NaCl, 5% isopropanol, pH 8.3) to a conductivity of 2.3 mSm / sm, adjusted to pH 8.3, and applied to a Blue column Sepharose 6 FF 50 ml, balanced with start buffer. The target protein is collected in slip mode.

2.5. Anion exchange chromatography

The breakthrough with Blue Sepharose 6 FF is diluted with purified water to 4 ° C, purified to a conductivity of less than 1.0 mSm / sm, adjusted to pH 7.5 and applied to a 50 ml Toyopearl Giga Cap Q-650M column equilibrated with 20 mM buffer Tris-HCl, pH 7.5. The target protein is eluted with a gradient of 5 to 0.25 M NaCl in the start buffer, collecting in fractions.

2.6. Concentration with a pre-column containing a hydrophobic sorbent. The target protein eluate fractions with Toyopearl Giga Cap Q-650M are diluted with purified water to a conductivity of 2.3 mSm / sm, adjusted to pH 7.5 and applied to Q Sepharose FF columns with Butyl Capto pre-column equilibrated with 20 mM Tris buffer - HCl, pH 7.5. Washed with starting buffer until the eluate of the adsorbed components exited, the Butyl Capto column was disconnected, washed with starting buffer and eluted with 350 mM NaCl in the starting buffer

2.7. Polishing gel - filtration.

The eluate with Q Sepharose FF is applied to a Superdex 200 26/600 column, equilibrated with buffer B - 20 mM Tris HCl, 0.05 M NaCl, 1 mM EDTA pH 7.5. Elute from the column in the same buffer.

2.8. Dilution, sterilizing filtration and packaging.

The eluate with Superdex 200 is diluted with buffer B to obtain the finished form and sterilely filtered through FILTER-MAX, "rapid" - 150 ml, 0.22 μm, aliquoted and stored at minus 20 ° C.

Received:

122.46 mg, 157.0 ml, ε ₂₈₀ = 1.17, C = 0.78 mg / ml,

The content of bacterial endotoxins: less than 0.6 EE / ml

Activity on a synthetic substrate: 17061 U / mg.

The purity of the sample by GF HPLC is: 99.5%.

Residual DNA of producer strain (E. colli): ≤ 0.01385 pg / mg.

Residual proteins of the producer strain (E. colli): ≤ 4.0 ng / mg.

Example 3. Analysis of the activity of recombinant BgaA

Determination of the activity of the recombinant BgaA β-galactosidase is carried out by setting the test on a synthetic substrate ONPG (ortho-nitrophenyl-β-D-galactopyranoside) using the β-Gal Assay Kit (Invitrogen). As a result of the hydrolysis of the ONPG substrate by the β-galactosidase enzyme, the product ortho-nitrophenol (ONP) is formed, the content of which is measured colorimetrically at a wavelength of 420 nm.

For the test, prepare single PBS and Cleavage Buffer solutions by diluting the stock solutions with purified water (10 ml of 10xPBS solution is added to 90 ml of purified water and 10 ml of 10xCleavage Buffer solution is added to 90 ml of purified water). Before use, 270 μl of β-mercaptoethanol are added to 100 ml of a single Cleavage Buffer solution. Samples of test solutions are used in the reaction with preliminary dilution in a single PBS solution 10, 20, 40, 80 times. The resulting dilutions of the samples in a volume of 2 μl are added to the wells of a 96-well plate containing 50 μl of a single Cleavage Buffer solution with β-mercaptoethanol added and 17 μl of ONPG substrate. The tablet is covered with a film and incubated at a temperature of 37 ° C for 30 minutes. Add 125 μl Stop Buffer solution to the wells and measure the optical density of the samples at a wavelength of 420 nm. The final volume of the reaction mixture is 192 μl.

The amount of hydrolyzed ONPG substrate (nmol) was calculated by the formula:

(OD420) × (1.92 × 105nl) / (4500nl / nmol-cm) × (1cm), where

OD420 - optical density of the sample solution at a wavelength of 420 nm

1.92 × 105 nl - the volume of the reaction mixture, nl

4500 - substrate extinction coefficient

To determine the specific activity of β-Galactosidase, expressed in units of nmol / min / mg, the amount of nmol hydrolyzed ONPG calculated by the above formula is divided by the incubation time (30 minutes) and the amount of β-galactosidase protein contained in the reaction mixture.

Example 4. Electrophoretic separation of proteins

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

Sample preparation of samples is carried out by diluting the samples to a concentration of ~ 50 μg / ml by adding a sample buffer (S.b) with the addition of a 2 M solution of dithiothreitol (DTT). Warm up for 5 minutes at (100 ± 2) 0С. Centrifuged for 1 min, 13000 rpm and apply samples (solutions obtained by diluting the samples) of 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, current strength of 30 mA, the time until the dye is released. Then stained with silver salts according to the instructions for the Thermo Scientific kit. The results are presented in FIG. 6.

Example 5. Testing purified preparations of recombinant BgaA in comparison with the commercial preparation of β-galactosidase 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 GF XIII, gel-thrombus test with LAL-reagent OFS.1.2.4.0006.15. Samples are diluted with water for lal test (Pirotest, Russia). Used for work: LAL-reagent Endosafe (Charles River, USA) with a sensitivity of 0.03 EU / ml; Control standard sample of endotoxin (Charles River, USA) with a content of 20 EU / ml 100 μl of samples and 100 μl of LAL reagent are placed in assay tubes and incubated for 60 min at 37 ° C. Positive result: the formation of a dense clot of gel. When the tube is turned 180 degrees, the gel does not break. Negative result: the gel does not form or breaks when the tube is turned over.

The endotoxin content at the endpoint is expressed in EE / ml and is calculated by the formula: “C” = (dilution at the endpoint) × (sensitivity of the LAL reagent). The results obtained are presented in Table 2.

Notes to the table:

"+" - a positive result; "-" - negative result.

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

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

Example 6. Testing the chromatographic purity of purified preparations of recombinant BgaA in comparison with the commercial preparation of β-galactosidase.

Testing is performed by HPLC gel filtration on a column with Superdex200 Increase 10/300 GL (GE) on an Alliance 2695 HPLC chromatography system, UV / Visible Detector 2487 (Waters). A buffer of 0.02 M NaH2PO4 is used as the mobile phase; 0.3 M NaCl; pH = 7.0. The preparation BgaA MBC "Generium" and the commercial preparation β1,4-Galactosidase, Streptococcus pneumoniae, Recombinant, E. Coli, Calbiochem Cat. 345806-50MIU Lot 2625690, 3 U / ml, 11.8 U / mg, 0.254 mg / ml, M.w. 350,000 before entering the system is diluted 2 times with the mobile phase. A comparison of the chromatographic profiles of the test samples shows (Fig. 7) that the BgaA MBC “Generium” is significantly more uniform in composition, while the commercial β-galactosidase is more heterogeneous, which causes difficulties in determining the main peak in the chromatogram of a commercial sample. Testing of industrial batches of the recombinant BgaA preparation is carried out in the same way. In FIG. Figure 8 shows a typical chromatographic profile of the tested BgaA industrial series.

Example 7. Testing of recombinant BgaA preparations in comparison with the commercial preparation β-galactosidase for residual DNA of a producer strain.

To determine the residual DNA of the producer strain, a real-time quantitative PCR method is used. For the design of the quantitative polymerase chain reaction (QPCR), the Taqman system was chosen, in which, upon propagation of a specific DNA sequence, the probe is degraded by DNA polymerase. A set of primers and a 16SER2 probe was used to detect the DNA of a producer strain of E. coli.

Sample Preparation: Samples were purified using the QIAamp DNA Blood Mini Kit (Qiagen). The sample volume for isolation was 0.5 ml. The final volume during elution is 0.1 ml. 0.005 ml of the sample was added to the qPCR reaction (sample). The number of repetitions for each sample (sample) is 3.

Analysis Results:

The concentration of DNA in the samples is calculated by the formula:

X is the concentration of DNA relative to the protein in the test sample (pg / mg protein);

C (protein) is the concentration of protein in the test sample (mg / ml); A is the amount of DNA in the sample, pg; V sample - sample volume for isolation taken for isolation, ml (0.5 ml); Vпср is the volume of the eluate introduced into the PCR tube, ml (0.005 ml); Values - volume of elution, ml (0.1 ml).

The analysis of each sample was carried out in three steps. The results obtained (A) are averaged.

* If the obtained values are below 0.27 pg in the sample, then the amount of DNA is below the FFP (Quantification Limit) and a value of less than 0.27 pg (≤0.27 pg) is accepted. The results of the analysis are presented in Table 3.

Claims (10)

1. The method of purification in industrial volumes of recombinant β-1,4-galactosidase BgaA in E. coli using metal chelate, pseudo-affinity, hydrophobic, anion exchange, concentration chromatography and polishing gel filtration, characterized in that the protein from the soluble fraction of the cell lysate is purified with using two chromatography steps on the same Ni ²⁺ metal chelate sorbent, wherein the first elution step is carried NaCl containing sodium acetate buffer having a pH in the range from 4.2 to 4.3, in a second step the target protein is eluted with buffer, with ERZHAN imidazole, and the eluate from the second stage is used for pseudoaffine chromatography slip mode sorbent with covalently bound Cibacron Blue 3G. 2. The method according to p. 1, where the preferred pH of the wash buffer in the second stage of metal chelate chromatography is in the range from 6.5 to 6.6. 3. The method according to p. 1, where both stages of metal chelate chromatography are carried out on IMAC Sepharose 6 FF. 4. The method according to p. 1, where both stages of metal chelate chromatography are carried out on IMAC Sepharose High Performance. 5. The method according to p. 1, where in the first stage of metal chelate chromatography, the preferred pH of the elution buffer is 4.3. 6. The method according to any one of paragraphs. 1-5, where the washing in the second stage of metal chelate chromatography is carried out with a buffer with a pH of 6.5 and high ionic strength. 7. The method according to p. 1, where in the second stage of metal chelate chromatography, the elution buffer contains from 150 mm to 200 mm imidazole. 8. The method of claim 1, wherein the pseudo-affinity chromatography is performed on Blue Sepharose. 9. The method of claim 1, wherein 5% isopropanol and 80 mM NaCl, pH 8.3, are used on equilibration pseudo-affinity chromatography. 10. The method according to p. 1, where the amino acid sequence of the recombinant β-1,4-galactosidase BgaA with hexahistidine tag is identical to the sequence of SEQ ID NO 2.

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