RU2795623C2 - Method for producing serratia marcescens recombinant endonuclease - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to methods of obtaining, isolating and purifying recombinant proteins, in particular to obtaining a highly purified preparation of S. marcescens correctly folded recombinant endonuclease.

EFFECT: method for obtaining Serratia marcescens recombinant endonuclease produced in E. coli including the steps of washing and dissolving inclusion bodies, refolding and protein chromatography, characterized by the following: the refolding is carried out by dilution at a concentration of refolded protein from approximately 0.3 to 0.5 g/l.

9 cl, 2 ex, 7 dwg, 4 tbl

Description

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 obtained by the proposed method of recombinant bacterial endonuclease S. marcescens is its inclusion in the technological process of obtaining biopharmaceuticals, as well as to study 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

Fig. 1 Plasmid map 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;

f1 origin - f1 origin of replication; bla - ampicillin resistance gene; pBR322 ori - pBR322 origin of replication; lacI - lactose repressor gene; NdeI - NdeI restriction endonuclease recognition site;

XhoI 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 - Inclusion body solution; Sample 4 - Inclusion bodies solution, 2XP;

Fig. 3A Chromatography profile with Ni 2+IMAC Sepharose FF 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;

Fig. 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;

Fig. 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 - Breakthrough; 2 - washing; 3 - eluate; 4 - Regeneration;

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

Fig. 7 Decrease in fluorescence 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, 2-3.33 U, 3-1 U, 4-0.33 U, 5-0.1 U, 6-0.033 U, 7-0.01 U, 8-0 U and reaction time (extreme left column).

endonucleases// FEMS Microbiology Reviews. - 2001. - 25. - p. 583-613).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-Liebig-Universitat Gieβen, 2001, pp. 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 (ss) NA) (Qing Song and Xiaobo Zhang, Characterization of a novel non- specific nuclease from thermophilic bacteriophage GBSV1 // BMC Biotechnology - 2008. - 8:43 - pp. 1-9., Li Lia, Shumei Linb, 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

endonucleases// FEMS Microbiology Reviews. - 2001. - 25. - p. 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 - p. 1-9).

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 nucleic acid, biopharmaceutical products such as antibodies or enzymes, polysaccharides, lipids, or small molecule substances such as antibiotics or small molecule 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 P., 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-He 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; 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 rapidly 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 nucleases 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 was also 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. - s - 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//.I. 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 Sun, 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 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 the primary structure of 245 amino acids predicted from the DNA sequence (Kirsten Biederman, Pia Knak Jepsen, Erik Riise, Ib Svendsen, Purification and Characterization of a S. marcescens Nuclease produced by Esherichia Coli // Carlsberg Res. Commun.-1989.-V.54.-p.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, provides a method for producing recombinant nucleases from S. marcescens (S. marcescens) and Staphylococcus aureus (S. aureus). It involves the 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 fusion protein, MBP nuclease.

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. by 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 from 250 endotoxin units (EU) to 1 EU per mega units (1,000,000 IU) of nuclease activity.

Table 1 (US 9 796 994 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.

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

, 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) авторы сообщали о создании генетической конструкции, содержащей гены экстрацеллюлярной нуклеазы из S.marcescens, которые были получены в результате скрининга геномной библиотеки S.marcescens. Затем стандартными методами была получена плазмида, несущая ген нуклеазной активности pSmaNuc под контролем P_L-промотора. Для экспрессии нуклеазы плазмида была трансформирована в штамм E.coli DH5α. Культура рутинно выращивалась при 28°С в среде LB. Индукцию P_L-промотора производили ростом температуры с 28°С до 42°С.The closest in purpose to the proposed method is the technical solution described in the article by Peter Friedhoff et al. .- 1994.- V. 22.- No. 16.-p. 3280-3287). In an earlier work (Peter Friedhoff, Oleg Gimadutdinow, Thomas

, 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 on the creation of a genetic construct containing extracellular nuclease genes from S. marcescens, which were obtained by screening the S. marcescens genomic library. Then, using standard methods, a plasmid carrying the pSmaNuc nuclease activity gene under the control of the P _L promoter was obtained. To express the nuclease, the plasmid was transformed into the E. coli DH5α strain. The culture was routinely grown at 28°C in LB medium. The P _L 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 disruptor. The precipitate was collected by centrifugation and dissolved in buffer 10 mm Tris-HCl, 6 M urea, pH 8.2, overnight. The lysate was clarified by centrifugation, the precipitate was discarded, and the supernatant was dialyzed against a buffer of 50 mM sodium acetate, pH 5.2. The insoluble precipitate was removed by centrifugation, the clarified lysate was subjected to phosphocellulose P11 chromatography, the target protein was eluted from the carrier with a linear molarity gradient and pH: from 50 mM sodium acetate, pH 5.2 to 250 mM 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 His6 tag under the control of the P _l promoter. For transformation were used strains of E. coli - LK111 and TGE900. The induction was also started by raising the temperature to 42°C for 15 min. 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 target protein was isolated as follows, 500 mg of induced cells were thawed and resuspended in about 20 ml of 10 mM Tris-HCl, 1 mM EDTA, pH 8.2. Cells were destroyed by sonication at 4°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 loaded onto a Ni-NTA resin column equilibrated with 10 mM Tris-HCl, 6 M urea, 10 mM imidazole buffer. The fraction not adsorbed on the column was again applied to the Ni-NTA resin column and this procedure was repeated twice. Protein was eluted using buffer 10 mM Tris-HCl, 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 8.2, containing imidazole, but about three times less concentration, namely 3 mM, and not 10 mM, as a denaturing agent, and using for purification of denatured protein by affinity chromatography on Ni-NTA sorbent under denaturing conditions.

To solve the problem of obtaining a highly purified and active preparation 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 a plasmid vector encoding a chimeric protein with C-terminal chimerization, 255 amino acid residues long 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 inclusion bodies, followed by solubilization of 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 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.

, Susanne van den Berg,

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). Благодаря предлагаемому способу достигается высокий выход целевого продукта, что делает процесс рентабельным. На Фиг. 1. представлена плазмидная карта pGNR-095-001 (экспрессионный вектор для получения эндонуклеазы S.marcescens).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 of the S.marcescens 6His-tag endonuclease molecule, the tag is placed at the N-terminus, as in the prototype of the invention, probably in order to increase the yield of soluble active protein. We propose a variant with a 6His 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 data (Esmeralda A. Woestenenk, Martin

, Susanne van den Berg,

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. On FIG. 1. shows the plasmid map of pGNR-095-001 (expression vector for the production of S. marcescens endonuclease).

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 molecules 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 0.3 mM L-cystine / 5 mM 2-mercaptoethanol, however, in its absence, refolding is also possible. When refolding, the best results were obtained in 20 mM Tris-HCl buffer, pH 8.2, with 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, in conjunction with the use of some standard protein purification techniques (affinity chromatography on Ni-IMAC 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 target protein, cellular protein impurities, endotoxins, with commercially attractive product yields capable of providing preparations of recombinant S. marcescens endonuclease of high purity and activity and obtaining a stable finished form of a highly purified preparation of recombinant S. marcescens endonuclease, at least 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 the 6His-tag 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. On FIG. 2 shows the electrophoregram of cell lysate samples, washing solutions, and inclusion body solution obtained using washing solutions. Sample 1 - 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 impurity proteins in the washing 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 involved when working with industrial scale protein production, significant time and resources can be saved by eliminating or simplifying one or more process steps, such as increasing the concentration of protein 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 1.0 M urea denaturant, 10% aggregation inhibitor glycerol, redox components 0.3 mM L-cystine / 5 mM 2-mercaptoethanol in 20 mM buffer 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 by series. 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. - p. 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.

Labahn, Frank

, Immobilized-metal affinity chromatography (IMAC): a review. In: Richard, B.R. and Deutscher, M.R, eds. // Methods Enzymol. - 2009. -463. -p. - 439-473). На этой стадии происходит разделение правильно свернутого белка, от неправильно свернутого белка, у которого сайты связывания с металлом расположены таким образом, что смещают равновесие ассоциации / диссоциации больше в сторону ассоциации, приводящую к более сильному связыванию, чем у белка, имеющего правильную трехмерную структуру.For the first stage of purification, based on the presence of 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, retaining the Ni2+ ion with four valences, leaves two valencies of the metal ion available for interaction with the imidazole rings of histidine residues (Helena Block, Barbara Maertens, Anne Spriestersbach, Nicole Brinker, Jan Kubicek, Roland Fabis,

Labahn, Frank

, Immobilized-metal affinity chromatography (IMAC): a review. In: Richard, BR and Deutscher, MR, eds. // Methods Enzymol. - 2009. -463. -p. - 439-473). This step separates the correctly folded protein from the misfolded protein, which has its metal binding sites arranged in such a way that it shifts the association/dissociation balance more towards association, resulting in stronger binding than a properly 3D protein.

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 to prepare 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 green substrate).

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 NdeI and XhoI 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 to express the recombinant S. marcescens endonuclease.

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 phase of the fermentation 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 phase of fermentation was at least 30 o.u. The yield of biomass 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. 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. Inclusion bodies were obtained by centrifugation from a disintegrated cell suspension at 30350 x g at 4°C for 35 min.

The resulting inclusion bodies were washed twice with 10 mM Tris-HCl, 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 mM Tris-HCl, 5 mM 2-Mercaptoethanol, 6M urea, pH 7.7. Contaminating cellular proteins were removed by washing with a 20 mM sodium acetate buffer containing 1 M NaCl, 5 mM 2-Mercaptoethanol, and 6 M urea, pH 5.8, and a 10 mM Tris-HC1 buffer solution containing 0.03 M imidazole, 5 mM 2-Mercaptoethanol. , 6M urea, pH 7.7. The target protein was eluted from the carrier with a 10 mM Tris-HCl buffer solution containing 0.2 M imidazole, 5 mM 2-Mercaptoethanol, 6 M urea, pH 7.7.

On FIG. 3A. and Fig. 3B. shows the chromatography profile and electrophoregram 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 sorbed to the carrier (Fraction 1, Breakthrough, Fig. 3A.; Sample 3, Fig. 3.B), the peak corresponding to the elution of the buffer 20 mM sodium acetate, 1 M NaCl, 5 mM 2-Mercaptoethanol 6M 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. 3.B) 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.

Refolding of the target protein of the recombinant S.marcescens endonuclease was carried out by dilution in 20 mM Tris-HCl buffer; 1 M urea; 10% glycerin; 0.3 mM L-cystine; 5mM 2-mercaptoethanol; 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 Ni2 + IMAC Sepharose FF, under washing conditions with a 10 mM Tris-HCl buffer solution containing 0.03 M imidazole, pH-7, 7 by eluting the target correctly folded protein with 10 mM Tris-HCl buffer containing 0.2 M imidazole, pH 7.7. Misfolds and aggregates were eluted with 10 mM Tris-HCl buffers containing 0.5 M imidazole, pH 7.7 and 10 mM Tris-HCl containing 0.5 M imidazole, 0.5 M NaCl, 6 M urea, pH 7.7.

On 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 Fig. 4.B). A peak in absorbance can also be observed corresponding to elution with 10 mM 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 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.

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 mM 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 mM Tris-HCl buffer, pH 8.0 until the conductivity of the buffer was indicated. Eluted with 40 mM Tris-HCl, 40 mM NaCl, pH 8.0. On FIG. 5 shows a chromatography profile where the peak absorbance can be observed corresponding to the eluate of the target protein (3 Eluate). 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.

To prepare the finished form of the S. marcescens recombinant endonuclease preparation, the protein content in the eluate was measured, diluted to the desired 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. 6). On FIG. 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 the example of hydrolytic cleavage of plasmid DNA. 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. On FIG. Figure 7 shows a 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 the 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. Dilutions of S.marcescens recombinant endonuclease CO and dilutions of the test sample of S.marcescens recombinant endonuclease were added to the wells of a 96-well non-sorbent plate. Then, 100 µl of DNA substrate with methyl green was added to each well, and mixed gently. After that, the tablet 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 IC ₅₀ values for recombinant S. marcescens CO endonuclease solution and test sample was performed automatically using the option “constrain-shared value for all data set” for the upper (top) and lower (bottom) asymptotes and 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:

And _CO - specific specific activity

recombinant S.marcescens endonuclease according to the manufacturer's certificate, U/mg;

IC50 _CO - IC ₅₀ value for a solution of CO endonuclease S. marcescens recombinant

calculated using software;

IC50 _IE - IC ₅₀ value for the subject

sample calculated by 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 recombinant S. marcescens endonuclease preparation 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 Global Fund of the Russian Federation, GPM.1.2.4.0006.15 "Bacterial endotoxins" and was 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 S.marcescens endonuclease was determined by the enzyme-linked immunosorbent assay (ELISA), in accordance with the requirements of the State 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 the level of 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 IPPT to a final concentration of 1.0 mM. The post-induction fermentation phase then 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. The bacterial biomass is collected by centrifugation at 7000 rpm for 20 min and stored 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 mM Tris, 0.3 M NaCl, 0.001% PMSF, pH 8.2;

A frozen cell biomass of 110 g is suspended in 1100 ml of buffer A for 15 min on ice. 2.2 ml of a 1 M MgCl ₂ solution and 1,000,000 U of S. marcescens recombinant endonuclease enzyme are added to the suspension, then the cells are destroyed 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 the 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 the 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 the rotor (JLA-16.250) of a centrifuge (Avanti J-20XP) and centrifuged 30350 x g at 4°C for 30 min. 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 sediment 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 sediment. 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 complete, the supernatant is carefully removed.

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

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

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

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

Buffer J: 10 mM 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 loaded onto a Ni ²⁺ IMAC Sepharose column preloaded with 0.2 M NiCl ₂ and equilibrated with buffer G. After loading, the column was washed with buffer G, buffer J, wash 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 closed inlet and outlet at a temperature of 2-8°C.

2.5. Refolding.

Buffer K: 20 mM Tris-HCl; 1 M urea; 10% glycerin; 0.3mM L-cystine; 5 mM 2-mercaptoethanol; pH-8.2;

The eluate from metal chelate chromatography -1 with a volume of 500 ± 10 ml is slowly added dropwise with constant stirring into buffer K cooled to 4 ± 1 ° C with a volume of 10000 ml, the pH of the solution is measured, if the pH deviates from 8.2, it is corrected 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 L: 10 mM Tris-HCl, 1 M urea, pH 7.7;

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

Buffer N: 10 mM 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.

In the refolded mixture, the pH 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 NiCl ₂ solution and equilibrated with buffer L. At the end of the application, column 3 was successively washed column volumes of 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 closed inlet and outlet at a temperature of 2-8°C.

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

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

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

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

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

Pure 150 при температуре окружающей среды 4°С.Anion exchange chromatography is carried out on an XK-50/40 chromatographic column filled with 200 ml DEAE Sepharose FF sorbent using a chromatographic system

Pure 150 at 4°C ambient.

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 8.0 ± 0.05 with concentrated hydrochloric acid or a 5 M 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 the application is completed, the column is washed with 3 column volumes of solution O, 3 column volumes (or more) of solution P until the conductivity reaches 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: 1M MgCl ₂

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 IU, less than 0.3 EU/mg; Specific activity: 913000 U/mg; Residual proteins of the producer strain: Less than 1.8 ng/mg.

Claims (10)

1. A method for obtaining recombinant Serratia marcescens endonuclease produced in E. coli, including the steps of washing and dissolving inclusion bodies, refolding and protein chromatography, characterized in that refolding is carried out by dilution at a concentration of refolded protein from approximately 0.3 to 0.5 g / l. 2. The method according to p. 1, where the dissolution of the washed inclusion bodies is carried out in a solution of 6 M urea in the presence of a reducing agent 20 mm 2-mercaptoethanol at pH 8.2. 3. The method according to claim 1, wherein the refolding buffer contains the denaturing agent urea at a concentration of 1 M and the aggregation inhibitor glycerol at a concentration of 10%. 4. The method according to p. 1, where the refolding is carried out using a redox pair of 0.3 mm L-cystine/5 mm 2-mercaptoethanol. 5. The method according to p. 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. 6. 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. 7. 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. 8. The method of claim 6 wherein the protein polishing after metal chelate chromatography on Ni2+IMAC Sepharose FF is performed by anion exchange chromatography on DEAE Sepharose FF. 9. The method according to p. 8, where after the chromatographic purification on DEAE Sepharose FF, the conversion into the buffer of the finished form is carried out by adding stabilizing additives directly to the eluate. 10. 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.

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