WO2023014247A1 - Method for producing recombinant serratia marcescens endonuclease - Google Patents

Abstract

The invention relates to the field of biotechnology, and more particularly to methods for producing, isolating and purifying recombinant proteins, and even more particularly to the production of a high-purity preparation of correctly folded recombinant S.marcescens endonuclease. The claimed method envisages obtaining, by production in E. coli, a recombinant protein in the form of insoluble inclusion bodies, obtaining and washing the inclusion bodies, then solubilizing same and purifying the denatured protein under denaturing and reducing conditions by metal chelate affinity chromatography on Ni2+ IMAC Sepharose FF, with subsequent refolding to produce an active form of the enzyme by the formation of two intrachain disulphide bonds, and then chromatographically purifying the protein from the refolded mixture by metal chelate affinity chromatography on Ni2+ IMAC Sepharose FF, followed by polishing and concentrating anion-exchange chromatography on DEAE Sepharose FF. The invention solves the problem of developing a method for producing industrial volumes of a high-purity enzymatically active recombinant S.marcescens endonuclease suitable for use in the removal of nucleic acids in biopharmaceutical manufacturing.

Description

METHOD FOR PRODUCING RECOMBINANT ENDONUCLEASE SERRATIA MARCESCENS

The invention relates to the field of biotechnology, and in particular to methods for obtaining, isolating and purifying recombinant proteins, in particular to obtaining a highly purified preparation of recombinant endonuclease Serratia marcescens (hereinafter S. marcescens). The main areas of application of the recombinant bacterial endonuclease S. marcescens obtained by the proposed method is its inclusion in the technological process for obtaining biopharmaceuticals, as well as for studying the structure and properties of polynucleotides.

The invention solves the problem of creating a method for producing in industrial volumes a highly purified enzymatically active recombinant S. marcescens endonuclease suitable for use at the stage of removing nucleic acids in biopharmaceutical production.

The invention also relates to a method for obtaining a preparation of recombinant S. marcescens endonuclease, having a high degree of purity, homogeneous in the target product, with a low content of bacterial endotoxins and proteins - the host producer.

DESCRIPTION OF THE DRAWINGS

Figure 1 Map of plasmid pGNR-095-001 - expression vector for the production of S. marcescens endonuclease.

Designations: T7 promoter - promoter of T7 RNA polymerase;

Endonuclease S. marcescens - endonuclease gene, consisting of 768 nucleotide residues; 6His-Tag - hexahistidine tag;

T7 terminator - terminator of T7 RNA polymerase; fl origin - fl origin of replication; N-gene of resistance to ampicillin; pBR322 ori - pBK322 - origin of replication; lacl - lactose repressor gene;

Ndel - Ndel restriction endonuclease recognition site;

Xhol is the recognition site for the Xhol restriction endonuclease.

Fig.2 Electrophoregram of samples of lysate, washing solution and solution of inclusion bodies.

Sample 1 - Cell Lysate; Sample 2 - Wash 1; Sample 3 - Wash 2; Sample 3 - A solution of inclusion bodies; Sample 4 - Inclusion bodies solution, 2XP;

FIG. 3A Ni 2+ IMAC Sepharose FF Chromatography Profile under Denaturing and Reducing Conditions

Fraction 1 - Slip; Fraction 2 - Laundering; Fraction 3 - Eluate 1; Fraction 4 - Eluate 2;

FIG. 3B Electropherogram of fractions from Ni 2+IMAC Sepharose FF chromatography under denaturing and reducing conditions.

Sample 1 - Cell Lysate; Sample 2 - Inclusion body solution; Sample 3 - Breakthrough with Ni 2 + IMAC Sepharose FF; Sample 4 - Wash 1 with Ni 2+IMAC Sepharose FF; Sample 5 - eluate 1 with Ni 2+IMAC Sepharose FF, fraction 1; Sample 6 - eluate 1 with Ni 2+IMAC Sepharose FF, fraction 2; Sample 7 - eluate 2 with Ni 2+IMAC Sepharose FF;

4A Chromatography profile of the refolded mixture with Ni 2 + IMAC Sepharose FF

1 - slip; 2 - Wash 1; 3 - Wash 2; 4 - Eluate 1; 5- Eluate 2; 6- Eluate 3;

4B Electropherogram of Ni 2+IMAC Sepharose FF chromatography fractions

Sample 1 - Refolded mixture; Sample 2 - Eluate 1 with Ni 2+IMAC Sepharose FF, fraction 3; Sample 3 - Eluate 1 with Ni 2+IMAC Sepharose FF, fraction 4; Sample 4 - Eluate 1 with Ni 2+IMAC Sepharose FF, fraction 5; Sample 5 - Eluate 1 with Ni 2 + IMAC Sepharose FF, fraction 6; Sample 6 - Eluate 1 with Ni 2+IMAC Sepharose FF, fraction 7, 10XP; Sample 7 - Eluate 1 with Ni 2+IMAC Sepharose FF, fraction 8, 10XP; Sample 8 - Eluate 2 with Ni 2+IMAC Sepharose FF; Sample 9 - Eluate 3 with Ni 2+IMAC Sepharose FF;

Fig. 5 Chromatography profile with DEAE Sepharose FF

1 - slip; 2 - washing; 3 - eluate; 4 - Regeneration;

Fig.6 Typical electropherogram of the drug recombinant endonuclease S. marcescens.

Fig. 7 Fluorescence drop of ethidium bromide upon destruction of plasmid DNA by alpha-DNase 1, rNucSm (commercial Serratia m. nuclease), rNucSm series 1 (recombinant Serratia m. endonuclease), and rNucSm series 2 (recombinant S. marcescens endonuclease). Dilutions where lane 1 corresponds to 10 U, 2 to 3.33 U, 3 to 1 U, 4 to 0.33 U, 5 to 0.1 U, 6 to 0.033 U, 7 to 0.01 U, 8 to 0 U and reaction time (extreme left column).

Nucleases are hydrolytic enzymes that are capable of hydrolyzing phosphodiester bonds in nucleic acids. Nucleases are found ubiquitously in all organisms and are of great scientific and economic importance (Bettina Haberland, Die extrazellulare Endonuklease aus S.marcescens: Untersuchungen zur Substratbindung und zur Katalyse // Inaguration dissertation zur Erlangung des Grades Doktor der Naturwissenschaften - Dr. rer. nat. -des Fachbereichs Biologie, Chemie und Geowissenschaften, FB 08 der Justus-Eiebig-Universitat GieBen.-2001.-p.1-119). Nucleases are classified depending on the nature of the hydrolysable substrate (DNA, RNA - DNases and RNAases), exonucleases (cleaves bonds inside DNA) and / or endonucleases (acts on the free ends of DNA), specific (structurally dependent (action only on double-stranded (ds) NA (nucleic acid) or single-stranded (ss) NA) or non-specific (structurally independent (action on both double-stranded (ds) NA and single-stranded (88) NA) (Qing Song and Xiaobo Zhang, Characterization of a novel nonspecific nuclease from thermophilic bacteriophage GBSV1// BMC Biotechnology.- 2008.- 8:43.- p.1-9., Fi Fia, Shumei Finb, Feng Yangaa, Functional identification of the non-specific nuclease from white spot syndrome virus // Virology .- 2005. -337.-p. 399 - 406, E. Srinivasan Rangarajan, Vepatu Shankar , Sugar non-speci0c endonucleases// FEMS Microbiology Reviews .- 2001.- 25.- pp. 583-613).

Specific nucleases, such as RNases and DNases, are commonly known as restriction enzymes and are indispensable tools in molecular biology for DNA characterization, mapping, and the creation of new genetic constructs (Richard J. Roberts, How restriction enzymes became the workhorses of molecular biology // PNAS.- 2005.- V. 102.- N. 17.- p. 5905-5908, Francesca Di Felice, Gioacchino Micheli and Giorgio Camilloni, Restriction enzymes and their use in molecular biology: An overview// J. Biosci .-2019.-p.44:38.). Non-specific nucleases, characterized by the ability to hydrolyze both DNA and RNA without a clear base preference, have been found in a wide variety of sources such as viruses, bacteria, fungi and mammals. Nonspecific nucleases play an important role in various aspects of basic genetic mechanisms, including mutation prevention, DNA repair, replication and recombination, nucleotide and phosphate removal for growth and metabolism, host defense against foreign nucleic acid molecules, apoptosis and infection. Given their important role in nucleic acid metabolism, non-specific nucleases are widely used in molecular biology research, such as nucleic acid structure determination, rapid RNA sequencing, nucleic acid removal for protein purification, and as antiviral agents (Qing Song and Xiaobo Zhang, Characterization of a novel non-specific nuclease from thermophilic bacteriophage GBSV1// BMC Biotechnology.- 2008.- 8:43.-pl-9).

One aspect of the use of S. marcescens endonuclease, namely the removal of both DNA and RNA from samples, is of significant economic interest. This function is attractive in the production of a wide range of molecules by cellular or cell-free biological systems, in which the target product is not a nucleic acid, but biopharmaceutical products such as antibodies or enzymes, polysaccharides, lipids, or small molecular weight substances such as antibiotics or small molecular weight chemicals. The need to remove nucleic acids becomes especially important if the production of molecules occurs intracellularly or if a part of the produced cells is lysed during production. As a result, large amounts of nucleic acids are also released or contained in the preparation during the preparation of the target products. The presence of residual host nucleic acids in the final product raises concerns about potential transfer and integration into the patient's genetic material, contributes to the viscosity of the process fluid and contamination of chromatographic resins, reduces usable capacity, causes codeposition and aggregation (Gousseinov E., Kools W., Pattnaik R., Nucleic acid impurity reduction in viral vaccine manufacturing// Bioprocess Int.- 2014.- 12.-p.-59-68, John O. Konz, Ann L. Lee, John A. Lewis and Sangeetha L. Sagar, Development of a Purification Process for Adenovirus: Controlling Virus Aggregation to Improve the Clearance of Host Cell DNA//Biotechnol.Prog.-2005.-21,-p.-466-472).

Viral vectors and viral vaccines are playing an increasingly important role in modern medical approaches. Gene vectors such as adenoviruses, adeno-associated viruses or retroviruses are being developed as a means of delivering genetic material for target cells in gene therapy (R. Morenweiser, Downstream processing of viral vectors and vaccines. -Gene Therapy.-2005.- 12.p. 103-110). Host cell DNA clearance is critical for vaccine production and for the purification of adenoviral and lentiviral vectors. (Si-Ming Li, Fu-Liang Bai, Wen-Juan Xu, Yong-Bi Yang, Ying An, Tian-Not Li, Yin-Hang Yu, De-Shan Li, Wen-Fei Wang, Removing residual DNA from Vero- cell culture-derived human rabies vaccine by using nuclease,// Biologicals.- 2014.-xxx.- p.1-6). Two types of DNA activity that are potential risk factors: the first is oncogenic activity, the second is the risk of infectivity.

All viral vaccines are known to contain residual contaminating DNA. The infectivity of contaminant DNA can be reduced to below detectable levels either by reducing the average size of cellular DNA to 200-350 base pairs or by treatment with chemicals. The most common method for reducing the size of residual cell substrate DNA in vaccines is by non-specific nuclease digestion (Li Sheng-Fowler, Andrew M. Lewis Jr., Keith Peden, Quantitative determination of the infectivity of the proviral DNA of a retrovirus in vitro: Evaluation of methods for DNA inactivation,// Biologicals.- 2009.- 37.-p.259-269, Si-Ming Li et al., Removing residual DNA from Vero-cell culture-derived human rabies vaccine by using nuclease,// Biologicals. - 2014. -xxx.- p.1-6).

Non-specific nuclease treatment is used not only to reduce the size of the DNA, but also to reduce the bulk viscosity, since nucleic acids increase the viscosity of preparations to such an extent that subsequent steps such as filtration or chromatography are not possible. In addition, nuclease treatment prevents the formation of complexes with viral particles, since the strong negative charge of DNA facilitates interaction with viral particles. Prevention of such aggregation increases the yield of the final product (John O. Konz, Ann L. Lee, John A. Lewis, and Sangeetha L. Sagar, Development of a Purification Process for Adenovirus: Controlling Virus Aggregation to Improve the Clearance of Host Cell DNA/ / Biotechnol. Prog.- 2005.- 21.- p. 466-472, A. J. Hagen, R. A. Aboud, P. A. De Phillips, C. N. Oliver, C. J. Orella, R. D. Sitrin, Use of nuclease enzyme in the purification of VAQTA, a hepatitis A vaccine // Biotechnol. Appl. Biochem. -1996. -23.-3.-p.209-215).

Most publications refer to the use of a nuclease from S. marcescens, an enzyme marketed by Merck under the brand name Benzonase, (WO 2016/156613 Al, Schlegl R. et al., Aseptic purification process for viruses; US 2009/0017523 Al, Weggeman et al. ., Virus purification methods ; Piergiuseppe Nestola, Cristina Peixoto, Ricardo R.J.S. Silva, Paula M. Alves, Jose P. B. Mota, Manuel J. T. Carrondo, Improved Virus Purification Processes for Vaccines and Gene Therapy// Bioseparations and Downstream Processing Biotechnology and Bioengineering; Gousseinov E , Kools W, Pattnaik P. Nucleic acid impurity reduction in viral vaccine manufacturing.// Bioprocess Int.-2014.-12.-p.59-68).

It is a nuclease of the gram-negative bacterium S.marcescens, which has high activity and stability. In Marion Nestle, D W. K. Roberts, Purification and some properties of the enzyme,// The Journal of Biological Chemistry. -1969. -V. 244.- N. 19.- p.5213-5218, describes the isolation of an extracellular nuclease from S. marcescens, which hydrolyzed DNA and RNA at the same rate. In another work, the authors (Marion Nestle, D W. K. Roberts, Specificity of the enzyme,// The Journal of Biological Chemistry.- 1969.- V. 244.- N. 19.- p. 5219-5225) investigated the specificity of the isolated extracellular nuclease from S. marcescens and concluded that the enzyme hydrolyzes both double-stranded and single-stranded DNA, as well as RNA to 5'-phosphorylated tetra, tri-, di-, and also in a small number of mononucleotides. The enzyme requires Mg2+ as a cofactor at concentrations of 5 to 10 mM, which can be replaced by other divalent metal ions such as Mn2+. It is active over a wide pH range from 7 to 10. Kensuke Yonemura et al. (Yonemura et al., Isolation and Characterization of Nucleases from a Clinical Isolate of S.marcescens kums 3958, //Biochem.- 1983. -93. -p. 1287-1295) found that the enzyme has the highest activity at pH 8.0 and is also highly resistant to certain denaturing agents such as urea or mercaptoethanol.

Filimonova M.N. with co-authors in their work report some physicochemical properties of S. marcescens endonuclease. They determined the amino acid composition of the enzyme. It has also been shown that the protein molecule contains 1 SH-group and 1 S-S-bond. It has been established that the N-terminal amino acid is threonine (Filimonova M.N., Baratova L.A., Vospelnikova N.D., Zheltova A.O., Leschinskaya I.B., S. marcescens endonuclease. Characterization of the enzyme.// Biochemistry.- 1981. - T. 46.- Issue 9.- p.- 1660-1665).

S.marcescens, a pathogenic gram-negative intestinal bacterium, extracellularly secretes several other proteins in addition to the nuclease: two lipases, two chitinases, and two proteases (Mitchell D. Miller, Michael J. Benedik, Merry C. Sullivan, Nancy S. Shipley and Kurt L. Krause , Crystallization and Preliminary Crystallographic Analysis of a Novel Nuclease from S. marcescens //.!. Mol. Biol. -1991.-222.-p.27-30). S.marcescens uses various secretory systems by which it exports proteins into the culture medium. The nuclease export mechanism remains unclear and has features suggesting that it may be unique. The efficiency and kinetics of Serratia nuclease secretion is regulated by many factors such as cell physiology, nutrient composition, growth conditions, and host cell mutations (Yousin Suh, Shida Jin, Timothy K. Ball, Michael J. Benedik, Two-Step Secretion of the S. marcescens Extracellular Nuclease // Journal of Bacteriology.- 1996.- 7.- pp. 3771-3778.; Michael J. Benedik, Ulrich Strych, S. marcescens and its extracellular nuclease// FEMS Microbiology Letters.- 1998. - 165.-p.-1-13). In addition, in addition to the main isoform of the secreted nuclease, other isoforms are also present in the medium, identified by capillary electrophoresis (Jytte Pedersen and Mikael Pedersen, Separation of isoforms of S. marcescens nuclease by capillary electrophoresis// Journal of Chromatography.-1993.-645.-p. 353-361). All of these above aspects make it difficult to use the bacterium S. marcescens as a culture for the economical production of nuclease in sufficient quantities and with the required purity.

With the use of recombinant technologies, the desired product is obtained using standard expression organisms by heterologous expression, i.e. the genetic information for the desired protein is incorporated into the expressing organism, which then carries out the expression, ie. e. foreign protein synthesis. This often has the advantage that the productivity of these expression organisms can be greatly increased compared to the parent organism, and established processes have been developed for culturing the expression organisms and further purifying them for the manufacture of the final product.

The peculiarity of nucleases is that they can have a high toxic potential on the host organism, if the nuclease is already converted into an active form in the cytosol, it cleaves the nucleic acids of the host cell and causes their death or inhibits their growth, which makes it difficult for the expression of the nuclease in other cells - hosts (Michael J. Benedik, Ulrich Strych, S. marcescens and its extracellular nuclease// FEMS Microbiology Letters.- 1998.- 165. -p.- 1-13).

Timothy K. Ball et al reported the cloning of the secreted nuclease gene from S. marcescens and its complete nucleotide sequence. It turned out that extracellular secretion of nuclease in E. coli does not require additional genes, from which it was concluded that E. coli is able to secrete certain proteins extracellularly (Timothy K. Ball, Peter N. Saurugger and Michael J. Benedik, The extracellular nuclease gene of S. marcescens and its secretion from Escherichia coli // Gene.- 1987.-57.- pp. 183-192). Kirsten Biederman et al reported the determination of the primary structure and physicochemical properties of the nuclease expressed and secreted by Escherichia coli. The plasmid carried a DNA sequence isolated from S. marcescens encoding the enzyme. During the cultivation of E. coli cells, 85% of the enzyme was released into the culture fluid. The enzyme was purified and showed a single band with a molecular weight of about 30,600 daltons on SDS-PAGE and was similar to the nuclease isolated from S. marcescens. The amino acid composition and amino acid sequence of the resulting enzyme, determined by the authors, confirmed a 245 amino acid primary structure predicted from a DNA sequence (Kirsten Biederman, Pia Knak Jepsen, Erik Riise, lb Svendsen, Purification and Characterization of a S.marcescens Nuclease produced by Esherichia Coli // Carlsberg Res. Commun.- 1989.-V 54, - pp. 17-27).

EP O 229 866 Al Bacterial enzymes and method for their production, US 5173418, Molin et al., Production in Esherichia Coli of Extracellular Serratia SPP Hydrolases, describes a method for obtaining a recombinant nuclease from S. marcescens, free from other bacterial proteins, when this portion of the enzyme is secreted by E. coli into the culture medium and collected from the culture medium. After 16-20 hours in stationary growth phase, fermentation medium from 25 liters of E. coli culture containing expressed recombinant S. marcescens nuclease was collected by filtration through a 0.45 µm filter followed by concentration by ultrafiltration through a 10,000 dalton membrane. After dialysis against 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, the preparation was filtered through a glass filter and 0.45 and 0.22 µm filters to obtain pure enzyme.

US 2008/0118949 Al, Pei-Chung Hsieh, Intracellular Production of Nuclease, presents a method for producing recombinant nucleases from S. marcescens (S. marcescens) and Staphylococcus aureus (S. aureus). It involves overexpression of MBP (maltose-binding protein), which is part of the hybrid sequence of the recombinant MBP protein and a truncated nuclease (without signal peptide) in a soluble form in the cytoplasm of the host cell, from which it is easily extracted by affinity chromatography methods. A feature of the modified nuclease is that more than 80% of the expressed nuclease fusion protein is dispersed in the cytoplasm of the host cell. With 100 ml of E. coli cell culture, the authors received 3.2 mg of the recombinant MBR nuclease fusion protein.

US 9 796 994 B2, Thomas Greiner, Stefan Schoenert, Method for Producing S. marcescens Nuclease using a Bacillus expression host, describes a method for producing recombinant nuclease from Serratia marscens in Bacillus sp. It has been shown that nucleases from gram-negative bacteria can be obtained with high yields and high purity in gram-positive bacteria by heterologous expression. In particular, it has been found that S. marcescens nuclease can be efficiently expressed in Bacillus sp. secretion into the culture medium. By the claimed method, the authors received up to 100,000 units of activity per 1 ml of culture medium. The nuclease preparation of the invention preferably has 250 endotoxin units (EU) to 1 EU per mega units (1,000,000 IU) of nuclease activity.

Table 1 (US 9796 994 B2, Thomas Greiner; Stefan Schoenert Method for Producing S. marcescens Nuclease using a Bacillus expression host) compares the yields of expressed nuclease obtained in different hosts. Column (1) shows the best nuclease expression yield from a Gram negative S. marcescens host in a Gram negative Escherichia coli host and (2) shows a nuclease expression yield from a Gram negative S. marcescens host in a Gram positive Bacillus subtilis host.

Table 1.

Yields of Expressed Nuclease Obtained in Different Hosts

The above production methods have obvious disadvantages for obtaining the enzyme on an industrial scale, the most significant of which is the low yield of the target protein even when expressed in Bacillus subtilis (see table 1).

Other disadvantages of these methods for the production of non-specific nuclease include the fact that the microorganisms or bacterial sources used for production are either human pathogens, or these microorganisms and sources require relatively complex growth media and conditions, or require special methods and technical solutions to obtain high-quality purified product.

Thus, there is an obvious demand for an economical, high-quality domestic preparation of non-specific nuclease, which meets the strict requirements for drugs used for biopharmaceutical production.

The goal of the present invention is to provide a method for producing a S. marcescens endonuclease enzyme recombinantly expressed in Escherichia coli and accumulated predominantly as an insoluble fraction.

The closest in purpose to the proposed method is technical the solution outlined in the article by Peter Friedhoff et al. .- No.16.-p. 3280-3287). In his earlier work (Peter Friedhoff, Oleg Gimadutdinow, Thomas Riiter, Wolfgang Wende, Claus Urbanke, Hubert Thole and Alfred Pingoud, A procedure for Renaturation and Purification of the Extracellular S. marcescens Nuclease from Genetically Engineered Escherichia coli // Protein Expression and purification.-1994.-5.-p.37-43), the authors reported the creation of a genetic construct containing extracellular nuclease genes from S. marcescens, which were obtained by screening the genomic library of S. marcescens. Then, using standard methods, a plasmid carrying the pSmaNuc nuclease activity gene under the control of the Pb promoter was obtained. To express the nuclease, the plasmid was transformed into the E. coli DH5a strain. The culture was routinely grown at 28°C in LB medium. The Pb promoter was induced by increasing the temperature from 28°C to 42°C. The expressed protein accumulated mainly in the cell in the form of an insoluble fraction - inclusion bodies. After 2.5 hours of fermentation, cells were harvested by centrifugation. The resulting cells were suspended in lysis buffer -10 mM Tris-HCl, 1 mM EDTA, pH 8.2. Cells were disrupted at 4°C using an ultrasonic disintegrator. The precipitate was collected by centrifugation and dissolved in 10 mM Tris-HCl, 6 M urea, pH 8.2 buffer overnight. The lysate was clarified by centrifugation, the precipitate was discarded, and the supernatant was dialyzed against 50 nM sodium acetate buffer, pH 5.2. The insoluble precipitate was removed by centrifugation, the clarified lysate was subjected to chromatography on phosphocellulose RP, the target protein was eluted from the carrier with a linear molarity gradient and pH: from 50 nM sodium acetate, pH 5.2 to 250 nM sodium acetate, pH 7.5. The protein enriched fractions were collected, dialyzed against 10 mM Tris-HCl, pH 8.2, and the protein was concentrated on DE 52, the protein was eluted with 10 mM Tris-HCl, 0.5 M NaCl, pH 8.2. Received with 0.5 l of culture medium 10 mg of the target protein, with a total activity of 80,000,000 units (protein activity was determined by the modified Kunitz method for DNase I). In their next work, the authors (Peter Friedhoff, Oleg Gimadutdinow and Alfred Pingoud, Identification of catalytically relevant amino acids of the extracellular S. marcescens endonuclease by alignment-guided mutagenesis// Nucleic Acids Research.- 1994.- V. 22.- No. 16.-p. 3280-3287) describe the creation of a genetic construct closest to the present invention. A plasmid was obtained containing the S. marcescens nuclease gene with an N-terminal Hisb tag under control of the PL-promoter. For transformation were used strains of E. coli - LK111 and TGE900. Induction was also started by raising the temperature to 42 ⁰ C for 15 minutes. After 1 - 1.5 h of fermentation, cells were harvested by centrifugation, washed with STE buffer (8% (w/v) sucrose, 50 mM Tris-HCl, pH 8.0, 5 mM EDTA) and centrifuged again. The induced cells were stored at -20°C or used directly for protein isolation. The cyle protein was isolated as follows, 500 mg of induced cells were thawed and resuspended in about 20 ml of 10 mM Tris-HC1, 1 mM EDTA, pH 8.2. The cells were destroyed by sonication at ⁴⁰ C. The insoluble fraction was precipitated by centrifugation. The pellet was resuspended in 20 ml of buffer 10 mM Tris-HCl, 6 M urea, 10 mM imidazole, pH 8.2 overnight. Insoluble debris was removed by centrifugation. The supernatant was applied to a Ni-NTA resin column equilibrated with 10 mM Tris-HCl buffer, 6 M urea, 10 mM imidazole. The fraction not adsorbed on the column was again applied to the Ni-NTA resin column and this procedure was repeated twice. The protein was eluted using 10 mM Tris-HCl buffer, 6 M urea, 200 mM imidazole, pH 8.2 by fractions. Fractions containing nuclease were pooled and extensively dialyzed against 10 mM Tris-HCl, pH

8.2. After dialysis, any precipitate was removed by centrifugation. The supernatant contained pure nuclease and was stored at 4°C. The yield of wild-type nuclease (wt HisNuc) per liter of culture was 25 mg (0.025 mg/ml) (45,000,000 U; protein activity was determined by the Kunitz method).

The disadvantages of the prototype are:

- underestimated yield of recombinant nuclease S.marcescens compared to the method proposed in the claimed invention;

- difficulties of a technical nature when scaling a laboratory technique into industrial production;

- The resulting product has not been characterized for the content of impurities of host cellular proteins and endotoxins for use in biopharmaceutical production;

The known method was used in the claimed invention in terms of using 6M urea in 10 mM Tris buffer, pH as a denaturing agent.

8.2 containing imidazole, but about three times less concentration, namely 3 mm, not 10 mm, and the use of affinity chromatography on a Ni-NTA sorbent for purification of denatured protein under denaturing conditions.

To solve the problem of obtaining a highly purified and active drug of recombinant S. marcescens endonuclease in industrial volumes, the claimed invention proposes an original method for obtaining recombinant S. marcescens endonuclease, including the synthesis of a DNA sequence optimized for translation in E. coli, encoding the S. marcescens endonuclease protein, construction of an expression plasmid vector encoding a chimeric protein with C - terminal chimerization, 255 amino acid residues in length and an estimated molecular weight of 29.9 kDa, obtaining a recombinant protein by production in E. coli in the form of insoluble inclusion bodies, obtaining and washing the inclusion bodies, followed by solubilization of the inclusion bodies and purification of the denatured reduced protein on Ni-IMAC Sepharose under denaturing and reducing conditions, refolding to obtain the active form of the protein due to the formation of 2 disulfide bonds within the molecule, and then chromatographic purification of the protein from the refolded mixture, where the first stage is carried out using a metal chelate chromatography on Ni-IMAC Sepharose. Next, concentrating and polishing anion-exchange chromatography is carried out to obtain a finished form of a highly purified active preparation of recombinant S.marcescens endonucleases, suitable for removing nucleic acid impurities in biopharmaceutical production, at least 700 mg per liter of bacterial culture.

The main technical result of the invention is the production on an industrial scale of a highly purified and active preparation of recombinant S. marcescens endonuclease suitable for use in the manufacture of drugs and for studying the structure and properties of polynucleotides.

The implementation of the invention is disclosed in one of its possible embodiments, specialists understand that different strains of E. Coli can be used, nucleotide and amino acid sequences can be modified within certain limits, purification parameters by chromatography at each individual stage can also vary. In one of the embodiments, a strain of a genetically modified microorganism E. coli BL21(DE3)/pGNR-095-001 was created: the strain was obtained by transforming the recipient host strain BL21(DE3) with the plasmid pGNR095-001 carrying the S. marcescens endonuclease gene, a composite part of which are the sequences of 249 amino acid residues of S. marcescens endonuclease and 6 amino acid residues containing a hexahistidine tag at the C-terminus. In the existing his-tagged variants, the S. marcescens 6His-Tar endonuclease molecule is placed at the N-terminus, as in the prototype of the invention, probably in order to increase the yield soluble active protein. We propose a variant with an eHis tag at the C-terminus with an increase in the yield of overexpressed insoluble protein, which is true for many proteins, according to the literature (Esmeralda A. Woestenenk, Martin Hammarstrom, Susanne van den Berg, Torleif Hard and Helena Berglund, His tag effect on solubility of human proteins produced in Escherichia coli: a comparison between four expression vectors // Journal of Structural and Functional Genomics.- 2004.-5.-p. 217-229). Thanks to the proposed method, a high yield of the target product is achieved, which makes the process cost-effective. In Fig.1. plasmid map pGNR-095-001 (expression vector for S. marcescens endonuclease production) is presented.

In the claimed invention, the refolding scheme is of critical importance. Refolding is required, since the protein is synthesized in the bacterial cytoplasm mainly in the undissolved state in the form of inclusion bodies during cultivation of E. coli BL21(DE3)/pGNR-095-001 with a biosynthesis level of at least 1300 mg/l of culture fluid. In the proposed scalable purification method, the refolding of the recombinant Serratia marcescens endonuclease is carried out by the dilution method at the concentration of the refolded protein from 0.3 to 0.5 g/L. This is a significant difference from the prototype, which uses the method of dialysis, which is usually technically difficult to scale and transfer to production. Typical refolding concentrations for complex molecules, such as those containing two or more disulfides, are less than 0.05 g/l and more typically 0.001-0.01 g/l (Biomolecules.- 2014.- 4.- p.- 235 -251; Biotechnol. J. -2012.-7.-p.1-15), thus, this modification is not obvious, while allowing a significant improvement in the method.

Using the presented description, it will be clear to the specialist that in various embodiments of the invention, various special cases of its implementation are possible, without affecting the scalability of the proposed purification method. While the best results were obtained using a refolding buffer containing the denaturing agent urea at a concentration of 1 M, the aggregation inhibitor glycerol at a concentration of 10%, these values may vary within the limits known to the person skilled in the art. The best yields of the target protein were obtained using the redox pair 0.3mM L-cystine / 5mM 2-mercaptoethanol, however, in its absence, refolding is also possible. During refolding, the best results were obtained in 20 mM Tris-HCl buffer, pH-8.2, at constant stirring at 4 °C under aerobic conditions for 60 hours.

The proposed refolding variant demonstrated stable scalability from 1.5 L to 10.5 L with approximately 50% yield of the desired product on both scales. This technical result is necessary to obtain an active target protein and carry out further purification, since it is at this stage that the structural assembly of the target protein molecule occurs due to the formation of disulfide bonds within the molecule. A properly folded active protein must have two intrachain disulfide bonds. Further purification can be carried out as indicated in the prototype, or, to obtain a highly purified protein, apply additional purification steps.

One particular embodiment is also the dissolution of the washed inclusion bodies in a solution of 6 M urea in the presence of the reducing agent 20 mM 2-Mercaptoethanol at pH 8.2. In contrast to the known methods, when only a chaotropic agent, namely 6M urea, is used in the solubilization of the insoluble fraction, the reducing agent 2-Mercaptoethanol is also used in the claimed method.

One particular embodiment is the chromatographic purification of denatured and reduced protein from a solution of inclusion bodies on an IMAC-Ni sorbent under reducing and denaturing conditions. The compatibility of the IMAC matrix with a wide range of chemicals such as chaotropes, salts, organic solvents, detergents, reducing agents makes it indispensable for the purification of His-tagged recombinant proteins that need to be purified in denatured form.

Highly efficient extraction of inclusion bodies from cells became possible as a result of the bacterial cell lysis procedure using a high-pressure homogenizer and the use of the recombinant S. marcescens endonuclease enzyme to reduce the solution viscosity and increase the yield of the active target product.

Obtaining the finished form of a highly purified S.marcescens endonuclease preparation can be achieved due to the developed scheme for obtaining cellular proteins of inclusion bodies purified from impurities as a result of washings and purification of the denatured reduced protein by chromatography on Ni-IMAC Sepharose under denaturing and reducing conditions, optimal refolding and subsequent chromatographic purification, which, together with some standard protein purification techniques (Ni-IMAC affinity chromatography) Sepharose, anion exchange chromatography on a weak anion exchange sorbent DEAE Sepharose FF and subsequent transfer to the buffer of the finished form by adding stabilizing additives directly to the eluate), is highly effective for separating correctly folded active protein of recombinant S. marcescens endonuclease and misfolded recombinant S. marcescens endonuclease and contaminants such as degradation products, misfolds, aggregates and other forms of the target protein, cellular protein contaminants, endotoxins, with commercially attractive yields of the product, capable of providing preparations of recombinant S.marcescens endonuclease of high purity and activity and obtaining a stable finished form of a highly purified recombinant preparation endonuclease S. marcescens, not less than 700 mg per liter of bacterial culture.

The example presented in the invention illustrates the production of a genetically engineered construct (GEC) pGNR-095-001, in which 6His-Tar is located at the C-terminus of the molecule and the expression of the target protein by culturing E. Coli BL21(DE3) /pGNR-095-001 in reactor Biostat Bplus Twin 5L. The process of culturing the BL21(DE3)/pGNR-095-001 cell bank was focused on obtaining a high amount of biomass with targeted localization of the S.marcescens recombinant endonuclease protein in the inclusion bodies.

The example presented in the invention illustrates the lysis of cell biomass and the production of inclusion bodies using a recombinant S. marcescens endonuclease enzyme and Mg2+ up to 2 mM. Removal of the E. Coli genomic DNA impurity released during lysis with the help of nuclease not only eliminates bulk viscosity and facilitates lysis on a high-pressure flow disintegrator, but also increases the yield of the target protein during refolding, eliminating the substrate on which the adsorbed target protein is not refolded. The use of washings of inclusion bodies to remove contaminating impurities of cellular components already at the first stage with a buffer solution: 10 mM Tris, 0.2% Triton X-100, pH 8.2 makes it possible to remove some contaminating impurities of pigments, cellular proteins and endotoxins already at the first stage. Figure 2 shows the electrophoregram of samples of cell lysate, wash solutions and a solution of inclusion bodies obtained using wash solutions. Sample 1 is a cell lysate, on the electropherogram we can observe the absence of a soluble form of the target protein, samples 2 and 3 demonstrate the presence of impurities proteins in the wash solutions and the absence of the target protein, samples 4 and 5 - the solution of inclusion bodies shows the presence of a denatured chimeric protein and the presence of some remaining cellular proteins. To more completely remove the remaining impurities of cellular proteins, the inventors use chromatography under denaturing and reducing conditions of the denatured and reduced chimeric protein.

The example presented in the invention illustrates an example of the dissolution of the resultant inclusion bodies in an appropriate urea solution, typically at a level equivalent to 6 M urea. The protein was reduced with 2-Mercaptoethanol, 10-20 mM, at pH 8.2, preferably 20 mM, and incubated at 4° C. for approximately 16-18 hours. Unlike the prototype (Peter Friedhoff, Oleg Gimadutdinow and Alfred Pingoud , Identification of catalytically relevant amino acids of the extracellular S. marcescens endonuclease by alignment-guided mutagenesis// Nucleic Acids Research.- 1994.- V. 22.- No.16.-p. 3280-3287), when solubilized insoluble fraction, only a chaotropic agent is used, namely 6M urea, the authors of the present invention also use the reducing agent 2-Mercaptoethanol at a concentration of 20 mm.

The example presented in the invention illustrates the chromatographic purification of a denatured and reduced protein from a solution of inclusion bodies on an IMAC-Ni sorbent under reducing and denaturing conditions. The selection of optimal conditions for planting, washing and elution allows the maximum purification of the denatured target protein from contaminating impurities of cellular proteins.

The example of refolding presented in the invention illustrates the identified and evaluated refolding conditions that optimize the yield of the target protein and minimize the formation of aggregates and misfolds. First of all, the concentration of the refolded protein was selected. Typical refolding concentrations for complex molecules, such as those containing two or more disulfides, are less than 0.05 g/L and more typically 0.001-0.01 g/L (Hiroshi Yamaguchi, and Masaya Miyazaki, Refolding Techniques for Recovering Biologically Active Recombinant Proteins from Inclusion Bodies// Biomolecules.- 2014.- 4.- p.- 235-251; Satoshi Yamaguchi, Etsushi Yamamoto, Teruhisa Mannen and Teruyuki Nagamune, Protein refolding using chemical refolding additives// Biotechnol. J. -2012. - 7.-p.1-15). Due to the large volumes of material and pool sizes used when working with industrial scale protein production, significant time and resources can be saved by eliminating or simplifying one or more stages of the process, for example, by increasing the protein concentration in the refolded mixture.

In the claimed purification method, we propose to carry out refolding of recombinant S.marcescens endonuclease by dilution at a concentration of refoldable protein from 0.3 to 0.5 g/l, which is the difference between the method and the prototype. Optimal values of stabilizing concentrations of urea, glycerol, pH, redox couple, temperature and refolding time are proposed. The best results were obtained using a refolding buffer containing the denaturing agent urea at a concentration of 1.0 M, the aggregation inhibitor glycerol at a concentration of 10%, the redox components 0.3mM L-cystine / 5mM 2-mer captoethanol in 20mM Tris- HCl, pH-8.2, with constant stirring at 4 °C under aerobic conditions for 60 hours. The described process demonstrated stable scalability from 1.5 L to 10.5 L with a yield of the desired product of approximately 50% on both scales.

Timothy K. Ball et al reported that the presence of two disulfide bonds is a clear requirement for S. marcescens nuclease activity and stability. The study used site-directed mutagenesis when cysteine residues were replaced with serine. All mutants showed low specific activity in the absence of one or more disulfides and were less stable than the wild type (Timothy K. Ball, Yousin Suh and Michael J. Benedik, Disulfide bonds are required for S. marcescens nuclease activity//Nucleic Acids Research. -1992.- V. 20.- N. 19.- pp. 4971-4974).

The example presented in the invention illustrates the chromatographic purification of a refoldable mixture in order to obtain a highly purified finished form of recombinant S. marcescens endonuclease.

For the first stage of purification, based on the presence of the His-tag in the chimeric protein, the authors used metal chelate chromatography on IMAC Shepharose FF, a specially designed affinity sorbent based on an agarose matrix and a nitrilotriacetic acid (NTA) ligand, which, holding the Ni2+ ion with four valences, leaves available two metal ion valences for interaction with imidazole rings of histidine residues (Helena Block, Barbara Maertens, Anne Spriestersbach, Nicole Brinker, Jan Kubicek, Roland Fabis, Jorg Labahn, Frank Schafer, Immobilized-metal affinity chromatography (IMAC): a review. In: Richard, BR and Deutscher, MR, eds // Methods Enzymol.-2009.-463.-p.-439-473). At this stage, the correctly folded protein is separated from the misfolded protein, in which the sites The metal bindings are arranged in such a way that they shift the association/dissociation balance more towards association, resulting in stronger binding than for a protein that has a regular three-dimensional structure.

The main purification of the target protein from bacterial endotoxins, impurity proteins, remaining aggregates and misfolds, and at the same time, concentration, is carried out by anion exchange chromatography on a weak DEAE Sepharose FF anion exchanger. The conditions for sorption and elution of the target protein were chosen in such a way that for the preparation of the finished form of the drug it was enough to add stabilizing additives to the resulting eluate, such as glycerol up to 50% and Mg2+ up to 2 mM Use of strong anion exchangers based on quaternary amines, such as Q Sepharose FF , YMC Q30, Source 30 Q, does not allow you to achieve such results.

As a result of purification by the claimed method, it was possible to obtain the target protein recombinant S. marcescens endonuclease - a homogeneous preparation that actually corresponds to the calculated molecular weight (on the electrophoregram of the sample there is one main band corresponding to a molecular weight of about 30 kDa (Fig. 6)), with a content of bacterial endotoxins less than 0.3 EU/mg, the content of residual proteins of the E.Coli producer strain is less than 1.8 ng/mg and has a specific biological activity from 900,000 to 1,000,000 U/mg (the activity is determined by a spectrophotometric method using a DNA-methyl substrate green).

Materials and methods

For the expression of recombinant S. marcescens endonuclease, a genetically engineered construct (GEC) BL21(DE3) /pGNR-095-001 is obtained. To obtain an expression vector, a codon-optimized for expression in E. coli, encoding S. marcescens endonucleases, a nucleotide sequence flanked at the 5' and 3' ends with Ndel and Xhol restriction sites, respectively, was synthesized (SEQ ID NO 1). The synthesized sequence was cloned at the above sites into a vector in which protein expression from the cloned sequence is under the control of the T7 promoter. The resulting cloning frame encodes an endonuclease with a 6His C-terminal tag (SEQ ID NO 2). The resulting vector is hereinafter referred to as pGNR095-001.

E. coli strain BL21(DE3) was transformed with the pGNR-095-001 vector. The resulting BL21(DE3)/pGNR-095-001 producer strain was used for expression of recombinant endonuclease S. marcescens.

The cultivation process of the BL21(DE3)/pGNR-095-001 cell bank was focused on obtaining a high amount of biomass and proceeded in two stages. At the first stage, BL21(DE3) /pGNR-095-001 cells were cultivated in Erlenmeyr flasks, which were then used as seed culture at the second stage of production. One cryovial with a working bank of BL21(DE3) /pGNR-095-001 cells was inoculated into 250 ml of sterile 2YT medium and grown at 37°C for 16 hours at 180 rpm.

The second stage of fermentation included the following steps:

(1) 250 ml of the BL21(DE3)/pGNR-095-001 starter culture grown in the first step was loaded into a Biostat Bplus Tween 5L reactor with 4 L of 2YT medium;

(2) pH in the fermenter was maintained at 7.0 with solutions of 12.5% ammonia and 10% glacial acetic acid;

(3) DO in the reactor is set to 40% saturation, which is controlled by the stirrer speed and changing the percentage of dissolved oxygen;

(4) the fermentation temperature is set to 37°C;

Expression of the recombinant S. marcescens endonuclease is induced by adding IPTG to a final concentration of 1.0 mM, when the optical density of the BL21(DE3) /pGNR-095-001 culture is 10-20 optical units. The post-induction fermentation phase continues for another 3 hours with hourly sampling to control the optical density of the culture. The final optical density of the culture 3 hours after the post-induction fermentation phase was at least 30 o.u. The biomass yield ranged from 30-40 g/l of culture. Recombinant endonuclease S.marcescens is expressed at the level of 20-30% of the total protein and is found exclusively in the form of inclusion bodies. The bacterial biomass was collected by centrifugation and stored at -70°C.

Frozen biomass of E. coli cells was suspended in disintegration buffer 10 mM Tris HC1, 300 mM NaCl, pH 8.2 with the addition of a protease inhibitor to inhibit proteolysis of phenyl methylsulfonyl fluoride (PMSF), 500 U/ml recombinant endonuclease S. marcescens and Mg 2+ up to 2 mM. The destruction of cells was carried out on a high-pressure flow-through disintegrator in the following mode: pressure of stage II - 80 bar, stage I - 450-800 bar, collecting the lysate in a container placed in an ice bath. The inclusion bodies were obtained by centrifugation from a disintegrated cell suspension at 30350 xg at 4°C for 35 min.

The resulting inclusion bodies were washed twice with 10 sM Tris-HC1, pH 8.2, with a buffer solution containing 0.2% Triton X-100. The washed inclusion bodies were dissolved in a buffer solution of 10 mM Tris-HC1, 3 mM imidazole, 20 mM 2-Mercaptoethanol, 6 M urea, pH 8.2 at 4°C for 16–18 h. ) at 4°C for 40 min. Then, in order to increase the yield of correctly folded protein in refolding, the denatured protein was purified by metal chelate chromatography under denaturing and reducing conditions on a column with an IMAC Sepharose FF sorbent, preliminarily charged with Ni 2+ and equilibrated with a buffer of 10 gpM Tris-HC1, 5 mM 2-

Mercaptoethanol, 6M urea, pH 7.7. To remove contaminating cellular proteins, washing with a 20 gpM sodium acetate buffer containing 1 M NaCl, 5 mM 2-Mercaptoethanol and 6 M urea, pH 5.8 and a 10 gpM Tris-HC1 buffer solution containing 0.03 M imidazole, 5 mM 2-Mer captoethanol, 6M urea, pH 7.7. The target protein was eluted from the carrier with a 10 gpM Tris-HC1 buffer solution containing 0.2 M imidazole, 5 mM 2-Mer captoethanol, 6 M urea, pH 7.7.

On FIG. BEHIND. and Fig. ZV. shows the chromatography profile and electropherogram of fractions with Ni 2 + IMAC Sepharose FF, carried out under denaturing and reducing conditions, where a peak in optical density can be observed corresponding to the fraction not adsorbed onto the carrier (Fraction 1, Breakthrough, Fig. 3 .; Sample 3, Fig. 3.C), the peak corresponding to the elution of the buffer 20 gpM sodium acetate, 1 M NaCl, 5 mM 2-Mercaptoethanol 6 M urea, pH 5.8, in which contaminant proteins and trace amounts of the target protein are eluted (Fraction 2, Washing, Fig. 3A. ; Sample 4, Fig.3.B), a high peak in optical density corresponding to the elution of the target protein (Fraction 3, Eluate 1, Fig. 3A.; Sample 5 and Sample 6, Fig.Z.V) with a small amount of contaminating cellular proteins and Fraction 4, Eluate 2 (FIG. 3A.), in which no optical density peaks are observed (small amounts of the target protein are present in the sample on the electropherogram (Sample 7, Fig. 3B.), which indicates the completeness of the elution.

A summary table of the results of chromatography on with Ni 2 + IMAC Sepharose FF, carried out under denaturing and reducing conditions, is presented in table 2. Table 2 Chromatography on Ni 2+IMAC Sepharose FF under Denaturing and Reducing Conditions

Refolding of the target protein of recombinant S.marcescens endonuclease was performed by dilution in 20mM Tris-HC1 buffer; 1 M urea; 10% glycerin; 0.3mM L- cystine; 5pM 2-mer captoethanol; pH-8.2, at a protein concentration (from 0.3 to 0.5 g/l) for 60 hours at 4°C with constant stirring. The yield of the refolded target protein was maintained when the process was scaled from 1.5 L to 10.5 L of the refolded mixture and was at least 50% of the active recombinant S. marcescens endonuclease. Purification of the target protein was carried out by separating correctly folded and misfolded conformations of this protein, separating contaminating impurities from cellular proteins by affinity metal chelate chromatography on N12+ IMAC Sepharose FF, under washing conditions with a 10 nM Tris-HCl buffer solution containing 0.03 M imidazole, pH 7.7 , elution of the target correctly folded protein with a 10 nM Tris-HCl buffer solution containing 0.2 M imidazole, pH-7.7. Misfolds and aggregates were eluted with 10 nM Tris-HCl buffers containing 0.5 M imidazole, pH 7.7 and 10 nM Tris-HCl containing 0.5 M imidazole, 0.5 M NaCl, 6 M urea, pH 7.7.

In Fig.4.A and Fig.4.B. the chromatographic profile of the refolded mixture and the electropherogram of the fractions with Ni 2+IMAC Sepharose FF are presented. According to the chromatography profile (Fig.4.A.), one can observe a slight absorption of the optical density, corresponding to the fraction not sorbed to the carrier (1 Breakthrough), a high peak of optical density, corresponding to the elution of a correctly folded target protein (Eluate 2, Fig.4.A. , Samples 3 to 7 of Fig. 4.B). A peak in absorbance can also be observed corresponding to the elution with 10 nM Tris-HCl buffer containing 0.5 M imidazole, pH-7.7, (Eluate 2, Fig. 4.A, Sample 8, Fig. 4.B), which shows the elution compounds with a higher concentration of imidazole - 0.5 M compared with the correctly folded enzyme, which indicates that they were more strongly bound to the carrier. These molecules are misfolded enzyme molecules, most likely soluble aggregates and misfolds. The absorbance peak corresponding to the elution with a buffer containing 0.5 M imidazole, 0.5 M NaCl and 6 M urea, pH-7.7, (Eluate 3, Fig.4.A, Sample 9, Fig.4.B) shows, that the eluting compounds are so strongly bound to the carrier that they are removed only by a high molarity solution of imidazole and salt in the presence of 6M urea, indicating that these compounds are most likely insoluble aggregates and unrefolded protein.

A summary table of the results of chromatography on with Ni 2 + IMAC Sepharose FF is presented in table 3.

Table 3 Chromatography on Ni 2 + IMAC Sepharose FF

To carry out the next stage of purification on the sorbent DEAE Sepharose FF, the eluate from Ni 2 + IMAC Sepharose FF chromatography was diluted 10–14 times with purified water cooled at 4°C to an electrical conductivity of 0.5 to 0.8 mS/cm, the pH of the solution was adjusted to a value of 8 .0+0.05 and applied to a DEAE Sepharose FF column equilibrated with 10 pM Tris-HCl buffer, pH 8.2. After application, the carrier was washed with equilibration buffer to remove non-specific adsorbed impurities, then the carrier was washed with 40 nM Tris-HCl buffer, pH 8.0 until the conductivity of the buffer was indicated. Eluted with buffer 40 nM Tris-HCl, 40 nM NaCl, pH 8.0. On

Fig. 5 shows a chromatography profile where the absorbance peak corresponding to the eluate of the target protein (3 eluate) can be observed. A small double peak is also observed, corresponding to the peak of regeneration (4 Regeneration), at this stage the unrefolded target protein, insoluble aggregates, impurities of cellular proteins, and residual endotoxins are removed.

A summary table of the results of chromatography on DEAE Sepharose FF is presented in table 4.

Table 4 Chromatography on DEAE Sepharose FF

To prepare the finished form of the S.marcescens recombinant endonuclease preparation, the protein content in the eluate was measured, diluted to the required concentration with a buffer of 40 mM Tris-HCl, 40 mM NaCl, pH 8.0, glycerol was added to 50% and Mg 2+ to a concentration of 2 mM in the final volume. The finished product was sterile filtered. The protein yield was at least 700 mg per liter of bacterial culture.

Measurements of the parameters of various batches of the finished form of the preparation of recombinant S. marcescens endonuclease showed that as a result of purification by the claimed method, a homogeneous preparation is obtained, in fact, corresponding to the calculated molecular weight of about 30 kDa (Fig.b).

Figure 7 shows the results of measuring the relative biological activity of different batches of preparations of the finished form of recombinant S. marcescens endonuclease in comparison with DNase 1 and a commercial preparation of recombinant S. marcescens endonuclease using hydrolytic cleavage of plasmid DNA as an example. The analysis is based on the ability of the dye ethidium bromide (2,7 - diamino - 10 - ethyl - 9 - phenylphenanthrene bromide) to intercalate between two base pairs of the DNA helix and sharply increase the fluorescence intensity. When DNA is treated with nucleases, the complex is destroyed and the fluorescence intensity decreases. Figure 7 shows the drop in fluorescence upon destruction of plasmid DNA by alpha - DNase 1, rNucSm (commercial S. marcescens nuclease) and rNucSm series 1 (recombinant S. marcescens endonuclease) and rNucSm series 2 (recombinant S. marcescens endonuclease) of various dilutions , where lane number 1 corresponds to 10 units of activity, 2- 3.33, 3-1, 4- 0.33, 5- 0.1, 6-0.033, 7-0.01 and 8-0, respectively, and reaction time (leftmost column). The assay was performed on ice to slow down the reaction rate. Analyzing the presented data, we can conclude that the preparations obtained in the claimed manner have biological activity even at high dilutions and at 4°C.

The specific biological activity of the drug was determined by the spectrophotometric method using the DNA methyl green substrate. The wells of a 96-well non-sorbent plate were loaded with dilutions of S. marcescens recombinant endonuclease CO and dilutions of the test sample of S. marcescens recombinant endonuclease. Then, 100 μl of DNA substrate was added to each well. methyl green, stir gently. After that, the plate was sealed with a film and incubated without stirring in a thermoshaker for 2 h ± 5 min at a temperature of (37+1)°C.

The optical density of the contents of the wells was measured on a spectrophotometer at wavelengths of 630 nm and 492 nm (correction wavelength). GraphPad Prism 6.0 software was used to plot absorbance vs. decimal logarithm of S. marcescens recombinant endonuclease content in dilutions of S. marcescens recombinant CO endonuclease and test sample dilutions. For this, a 4-parameter logistic function of inhibition was used. Calculation of IC50 values for the recombinant S.marcescens CO endonuclease solution and the test sample was performed automatically using the option “constrain-shared value for all data set” for the upper (top) and lower (bottom) asymptotes and the slope (HillSlope) of the curves.

The specific activity of S. marcescens recombinant endonuclease in the test sample (A), in U/mg, was calculated by the formula:

Where:

Aso - specific specific activity of S.marcescens endonuclease recombinant according to the manufacturer's certificate, IU/mg;

IC50co - IC50 value for a solution of CO endonuclease S. marcescens recombinant calculated using software;

IC50io is the IC50 value for the test sample, calculated using the software.

The specific biological activity of different batches of the finished form of the preparation of recombinant S.marcescens endonuclease averaged 950,000 +50,000 U/mg.

The finished form of the preparation of recombinant S.marcescens endonuclease may contain various impurities of a biological nature associated with the producer: (endotoxins, host cell proteins). The content of these impurities may affect the safety of the drug, and therefore should be evaluated in the purified product.

Quantitative determination of bacterial endotoxins in various batches of the finished form of recombinant S. marcescens endonuclease was determined by the gel thrombus test, in accordance with the requirements of the RF GF, GPM.1.2.4.0006.15 "Bacterial endotoxins" and amounted to less than 0.0004 EU per 1000 units.

The quantitative determination of the proteins of the producer strain (E. coli) in various batches of the finished form of the recombinant endonuclease S. marcescens was determined by the method of enzyme-linked immunosorbent assay (ELISA), in accordance with the requirements of the Global Fund of the Russian Federation, OFS.1.7.2.0033.15 "Method of enzyme immunoassay" and amounted to less than 2 ng/mg.

For a better understanding of the essence of the invention, examples of its implementation follow. It will be understood by those skilled in the art that the parameters indicated in the examples may vary:

Example 1 Microbiological Synthesis of S.marcescens Recombinant Endonuclease Protein

The claimed method is implemented for the producer strain BL21(DE3) pGNR-095-001 when cultivated in a fermenter (Sartorius Biostat B Plus Twin 5L, Germany) in a medium volume of 4 liters. The entire cultivation process is carried out at a temperature of 37°C. The pH of the medium is maintained at 7.0-7.1 by titration with 10% acetic acid solution and 12.5% ammonia solution. An aeration level of 4 l/min and a dissolved oxygen concentration of 40%, which is controlled by a cascade of agitator speeds.

The grown inoculum in a volume of 250 ml is introduced into a bioreactor with a sterile nutrient medium 2YT. Glucose in the amount of 10 g/l and glycerol in the amount of 30 g/l are introduced into the medium as a starting substrate.

During the pre-induction phase of growth, cells utilize glucose from the medium. The pH level will drop. After 3-4 hours, the cells reach an optical density of 10-20 optical units. Expression of the recombinant S. marcescens endonuclease is induced by adding IPTG to a final concentration of 1.0 mM. Then the post-induction phase of fermentation continues for another 3 hours with hourly sampling to control the optical density of the culture. After 3 hours, the final optical density of the culture reaches 30-50 p.u. The fermentation process is stopped. bacterial biomass collected by centrifugation at 7000 rpm for 20 min and placed in storage at - 70°C.

Example 2 Isolation and Purification of S.marcescens Recombinant Endonuclease Protein from Bacterial Biomass. Obtaining an industrial batch of the preparation of recombinant S.marcescens endonuclease.

2.1 Disintegration of biomass.

Buffer A - 10 sM Tris, 0.3 M NaCl, 0.001% PMSF, pH 8.2;

Frozen cell biomass PO g is suspended in 1100 ml of buffer A for 15 min on ice. 2.2 ml of a 1 M MgCh solution and 1,000,000 U of S. marcescens recombinant endonuclease enzyme were added to the suspension, followed by cell disruption in a Panda Plus 2000 (GEA Niro Soavi) high pressure flow homogenizer. The homogenizer is washed with 250 ml of purified water cooled to 0°C, adjusted to buffer A cooled to 0°C, stage II setting - 80 bar, stage I setting - 450-800 bar, then the cell suspension is lysed in this mode, collecting the lysate in a container placed in an ice bath, wash the homogenizer with 30 ml of buffer A. The lysate is clarified by centrifugation 30350 x g at 4°C for 40 min. The supernatant is discarded, the sediment of inclusion bodies is collected.

2.2 Washing out the inclusion bodies

Buffer B: 10 mM Tris, 0.2% Triton X-100, pH 8.2;

Buffer E: 10 mM Tris HC1, pH 8.2;

The precipitate of inclusion bodies is placed in a 1-liter container, 1000+10 ml of buffer solution B is added to the precipitate. A dispersant nozzle is placed in a beaker and dispersed for 3-5 minutes. The suspension is poured into 0.25 l centrifuge beakers, balanced in pairs on a balance, placed in a rotor (JLA-16.250) of a centrifuge (Avanti J-20XP) and centrifuged 30350 x g at 4°C for 30 minutes. After centrifugation is complete, the supernatant is discarded.

The above procedure is sequentially repeated one more time with buffer B, then with buffer E.

As a result, at least - 45 +2 g of washed inclusion bodies are obtained. The resulting product is stored at a temperature of minus 80°C.

2.3. Dissolution of inclusion bodies

Buffer F: 10 mM Tris-HCl, 3 mM imidazole, 20 mM mercaptoethanol, 6 M urea, pH 8.2;

The precipitate of inclusion bodies (m = 45+2 g) from the refrigerator is placed in a 1.0-liter plastic beaker, 600+0.1 ml of freshly prepared solution F is added to the precipitate. Dispersed for 5 minutes. The beaker is placed on a magnetic stirrer and stirred at 4°C for 16-18 hours. The suspension is centrifuged 40,000 x g at 4°C for 90 minutes. After centrifugation is completed, the sedimentary fluid is carefully removed from the supra.

2.4. Chromatography on Ni ²⁺ IMAC Sepharose FF under reducing and denaturing conditions. Metal chelate chromatography -1.

Buffer G: 10 sM Tris, 5 mM mercaptoethanol, 6 M urea, pH 7.7;

Buffer H: 10 sM Tris, 0.5 M imidazole, 5 mM mercaptoethanol, 6 M urea, pH 7.7;

Buffer I: 20 gpM sodium acetate, 1 M NaCl, 5 mM mercaptoethanol, 6 M urea, pH 5.8;

Buffer J: 10 gpM Tris, 6 M urea, pH 7.8;

The stage of purification of the denatured and reduced target protein is carried out on a column filled with 350 ml IMAC Sepharose FF sorbent using an Akta Pure 150 chromatographic system at a temperature of 4°C.

The supernatant was diluted 3-fold with buffer J, adjusted to pH 7.8 with 2M NaOH, and applied to a Ni ²⁺ IMAC Sepharose column preloaded with 0.2 M NiCh and equilibrated with buffer G. After loading, the column was washed with buffer G, buffer J , washed with 6% buffer H in buffer G (containing 30 mm) imidazole. The target protein is eluted with 40% buffer H in buffer G (containing 200 mM imidazole). The completeness of the elution is checked with 100% buffer H.

For regeneration, sanitation and storage, the sorbent is sequentially washed with 3 column volumes of 0.5 M EDTA solution, 3 column volumes of 0.5 M sodium hydroxide solution, 3 column volumes of 0.1 M Tris-HCl solution, pH 7.5, 3 column volumes 20% ethyl alcohol. Until the next use, store the column with the inlet and outlet closed at 2-8°C.

2.5. Refolding.

Buffer K: 20sM Tris -NO; 1 M urea; 10% glycerin; 0.3mM L-cystine; 5shM 2-mercaptoethanol; pH-8.2;

Eluate from metal chelate chromatography -1 with a volume of 500 + 10 ml slowly drop by drop with constant stirring, add to buffer K cooled to 4 ± 1 °C with a volume of 10,000 ml, measure the pH of the solution, if the pH deviates from 8.2, adjust with hydrochloric acid or sodium hydroxide solution. The container is placed on a stirrer and the process of protein renaturation is continued at a temperature of 4 ± 1 °C with constant stirring for 60 hours.

2.6. Chromatography on Ni2+ IMAC Sepharose FF. Metal chelate chromatography - 2.

Buffer E: 10 nM Tris-HCl, 1 M urea, pH 7.7;

Buffer M: 10 nM Tris-HCl, pH 7.7;

Buffer N: 10 gpM Tris-HCl, 0.5 M imidazole, pH 7.7;

Metal chelate chromatography-2 is carried out on a chromatographic column filled with 350 ml IMAC Sepharose FF sorbent using an Akta Pure 150 chromatographic system at an ambient temperature of 4 °C.

The pH of the mixture to be refolded was adjusted to 7.7+0.05 with 5 M hydrochloric acid and applied to a Ni2+ IMAC Sepharose FF column preliminarily charged with 0.2 M NiCh and equilibrated with buffer E. After loading, the column was successively washed with 3 column volumes solution L, 3 column volumes of solution M, 1-2 column volumes of 6% buffer solution N in solution M until the optical density reaches a plateau.

Elute the target protein from the carrier with a 40% solution of N in solution M.

For regeneration, sanitation and storage, the sorbent is sequentially washed with 3 column volumes of 0.5 M EDTA solution, 3 column volumes of 0.5 M sodium hydroxide solution, 3 column volumes of 0.1 M Tris-HCl solution, pH 7.5, 3 column volumes 20% ethyl alcohol. Until the next use, store the column with the inlet and outlet closed at 2-8°C.

2.7 Chromatographic purification on DEAE Sepharose FF sorbent. Anion exchange chromatography.

Buffer O: 10 nM Tris-HCl, pH 8.2;

Buffer P: 40 gpM Tris-HCl, pH 8.0;

Buffer Q: 40 sM Tris-HCl, 40 sM NaCl, pH 8.0;

Buffer R: 5 nM Tris-HCl, pH 8.0;

Anion exchange chromatography is carried out on an XK-50/40 chromatographic column filled with 200 ml DEAE Sepharose FF sorbent using chromatographic system Akta Pure 150 at an ambient temperature of 4 °C.

The eluate after chromatography on Ni2+ IMAC Sepharose FF is diluted 10-14 times with buffer R, the pH of the solution is adjusted to a value of 8.0 + 0.05 with concentrated hydrochloric acid or a 5M sodium hydroxide solution (if necessary), the electrical conductivity of the solution is adjusted from 0.5 to 0.8 mS/cm with purified water cooled to 4°C and applied to a DEAE Sepharose FF column equilibrated with buffer O. After completion of the application, wash the column with 3 column volumes of solution O, 3 column volumes (or more) of solution P until the conductivity reaches a plateau . Elute the target protein from the carrier with buffer Q.

2.8 Making the finished form.

Buffer Q: 40 mM Tris-HCl, 40 mM NaCl, pH 8.0;

Solution R: IM MgCh

The eluate with DEAE Sepharose FF is diluted with buffer Q to a protein concentration of 2.0 -2.1 mg/ml, glycerol is added to a concentration of 50% and solution R to a concentration of 2 mm in the final solution. The solution of the finished form is filtered in a laminar air flow. For sterilizing filtration, use a FILTERMAX sterilizing system, 1000 ml, PES, with a pore size of 0.22 µm, using a diaphragm vacuum pump.

After filtration, a sample is taken for process control. The resulting solution is a finished form of recombinant S.marcescens endonuclease.

Received:

2419.5 ml; C= 1.09 mg/ml; 2637.2 mg; 2 407 763 600 units;

Content of bacterial endotoxins:

Less than 0.0003 EU per 1000 U, less than 0.3 EU/mg;

Specific activity: 913,000 U/mg;

Residual proteins of the producer strain: Less than 1.8 ng/mg;

Claims

32

A method for producing recombinant Serratia marcescens endonuclease produced in E. coli, including the steps of washing and dissolving inclusion bodies, refolding and protein chromatography, characterized in that the refolding is carried out by the dilution method at a concentration of refolded protein from approximately 0.3 to 0.5 g / l. The method according to claim 1, where the dissolution of the washed inclusion bodies is carried out in a solution of 6M urea in the presence of a reducing agent 20mM 2-mercaptoethanol at pH 8.2. The method according to claim 1, wherein the refolding buffer contains the denaturing agent urea at a concentration of 1 M and the aggregation suppressor glycerol at a concentration of 10%. The method according to claim 1, where the refolding is carried out using the redox pair 0.3mM b-cystine/5mM 2-mercaptoethanol. The method according to claim 1, where the chromatographic purification of denatured and reduced protein from a solution of inclusion bodies is carried out on an IMAC-Ni sorbent under reducing and denaturing conditions. The method according to claim 1, where refolding to obtain the active form of the enzyme due to the formation of 2 intrachain disulfide bonds is carried out after metal chelate chromatography on Ni2+ IMAC Sepharose FF. The method according to claim 6, wherein the chromatographic purification of the protein from the refolded mixture is carried out by metal chelate chromatography on Ni2+ IMAC Sepharose FF. The method according to claim 6, wherein the protein polishing after metal chelate chromatography on Ni2+ IMAC Sepharose FF is carried out by anion exchange chromatography on DEAE Sepharose FF. The method according to claim 8, where after chromatographic purification on DEAE Sepharose FF, conversion into a buffer of the finished form is carried out by adding stabilizing additives directly to the eluate. The method of claim 1, wherein the E. coli expression construct encodes a full-length S. Marcescens endonuclease protein fused to a C-terminal histag.