WO2023014247A1 - Procédé de production d'endonucléase recombinante serriata marcensces - Google Patents

Procédé de production d'endonucléase recombinante serriata marcensces Download PDF

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WO2023014247A1
WO2023014247A1 PCT/RU2022/050237 RU2022050237W WO2023014247A1 WO 2023014247 A1 WO2023014247 A1 WO 2023014247A1 RU 2022050237 W RU2022050237 W RU 2022050237W WO 2023014247 A1 WO2023014247 A1 WO 2023014247A1
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protein
marcescens
recombinant
endonuclease
buffer
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Александр Викторович ЕРШОВ
Ольга Анатольевна ЕРШОВА
Роман Львович АНИСИМОВ
Сергей Александрович КАТОРКИН
Николай Игоревич БОНДАРЬ
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Акционерное общество "ГЕНЕРИУМ"
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/42—Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)

Definitions

  • 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).
  • S. marcescens recombinant endonuclease Serratia 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.
  • FIG. 1 Map of plasmid pGNR-095-001 - expression vector for the production of S. marcescens endonuclease.
  • 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;
  • 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
  • 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;
  • 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.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: Leten zur Substratitati und Kunststoff Katalyse // Inaguration thesis für Erlangung des Grades Doktor der Naturwissenschaften - Dr. rer. nat. -des aus Schlurgiology, 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.
  • restriction enzymes 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.
  • 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).
  • S. marcescens endonuclease namely the removal of both DNA and RNA from samples
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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, Mandarin Peixoto, Ricardo R.J.S. Silva, Paula M. Alves, Jose P. B. Mota, Manuel J. T.
  • 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.
  • 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).
  • 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.
  • nucleases 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).
  • 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 ⁇ m 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 ⁇ m filters to obtain pure enzyme.
  • 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.
  • 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.
  • EU endotoxin units
  • 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.
  • 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.
  • a plasmid carrying the pSmaNuc nuclease activity gene under the control of the Pb promoter was obtained.
  • 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.
  • cells were harvested by centrifugation. The resulting cells were suspended in lysis buffer -10 mM Tris-HCl, 1 mM EDTA, pH 8.2.
  • 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.
  • a plasmid was obtained containing the S. marcescens nuclease gene with an N-terminal Hisb tag under control of the PL-promoter.
  • strains of E. coli - LK111 and TGE900 were used for transformation.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 are placed at the N-terminus, as in the prototype of the invention, probably in order to increase the yield soluble active protein.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • GEC genetically engineered construct
  • 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.
  • 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.
  • 2-Mercaptoethanol 10-20 mM, at pH 8.2, preferably 20 mM
  • 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.
  • 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.
  • 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.
  • 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
  • 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).
  • a genetically engineered construct (GEC) BL21(DE3) /pGNR-095-001 is obtained.
  • GEC genetically engineered construct
  • 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.
  • 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:
  • 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.
  • 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-
  • 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.
  • 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.
  • 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.
  • the chromatography profile (Fig.4.A.)
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 100 ⁇ l 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.
  • IC50io is the IC50 value for the test sample, calculated using the software.
  • 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.
  • 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.
  • ELISA enzyme-linked immunosorbent assay
  • 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.
  • 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.
  • 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.
  • 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.
  • Panda Plus 2000 GAA Niro Soavi
  • 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.
  • 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.
  • Buffer F 10 mM Tris-HCl, 3 mM imidazole, 20 mM mercaptoethanol, 6 M urea, pH 8.2;
  • 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.
  • 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.
  • Buffer K 20sM Tris -NO; 1 M urea; 10% glycerin; 0.3mM L-cystine; 5shM 2-mercaptoethanol; pH-8.2;
  • 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.
  • 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.
  • 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.
  • 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.
  • Buffer Q 40 mM Tris-HCl, 40 mM NaCl, pH 8.0;
  • 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.
  • FILTERMAX sterilizing system 1000 ml, PES, with a pore size of 0.22 ⁇ m, using a diaphragm vacuum pump.
  • Residual proteins of the producer strain Less than 1.8 ng/mg;

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Abstract

L'invention se rapporte au domaine des biotechnologies, et concerne notamment des procédés de production, d'isolation et de purification de protéines recombinantes, et plus particulièrement la production d'une préparation d'une grande pureté d'endonucléase recombinante correctement inversée S.marcescens. Ce procédé consiste à produire une protéine recombinante de production d'E. Coli sous forme de corps d'inclusion insolubles, à produire et rincer les corps d'inclusion suivi d'une solubilisation des corps d'inclusion, purifier la protéine dénaturée dans des conditions de dénaturation et de réduction par chromatographie de chélates métalliques sur Ni2+ IMAC Sepharose FF suivi d'un repliage afin de produire une forme active de ferment grâce à la formation de liaisons disulfure doubles dans la chaîne, puis d'une purification chromatographique de la protéine à partir du mélange de repliage par chromatographie de chélates métalliques sur Ni2+ IMAC Sepharose FF et d'une chromtographie par échanges d'ions sur une DEAE Sepharose FF ayant une action de polissage et de concentration. L'invention permet de créer un procédé de production dans des volumes industriels d'une endonucléase S.marcescens recombinante active par ferments d'une grande pureté pouvant être utilisée au stade de séparation d'acide nucléique dans la production biopharmaceutique.
PCT/RU2022/050237 2021-08-03 2022-08-02 Procédé de production d'endonucléase recombinante serriata marcensces WO2023014247A1 (fr)

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