A novel nonspecific heat-labile nuclease active at low temperature, a wide pH range, and at high salt concentrations.

Abstract

The subject of the present invention is a novel heat-labile PPR nuclease or an enzymatically active fragment thereof that exhibits high catalytic activity under difficult reaction conditions (especially high concentrations of salt and other additives commonly used in protein and virus purification processes, low temperatures, and a wide range of pH), wherein the nuclease sequence is SEQ ID NO: 2 or a sequence sharing at least 40% identity thereto. The subject of the present invention also includes a gene encoding a PPR nuclease or an enzymatically active fragment thereof; a nucleic acid particle encoding a PPR nuclease or an enzymatically active fragment thereof; an expression plasmid containing the sequence of the PPR-encoding gene; recombinant strains of Escherichia coli JM109(DE3)pD454-PPR-AmpR and E. coli ArcticExpress(DE3)pD454-PPR-AmpR; a method for producing the PPR nuclease protein; and a process for purifying the recombinant protein with a significantly lower DNA content. and the application of PPR nuclease for decontamination of reaction reagents and mixtures of PCR, qPCR, RT-PCR, RT-qPCR, RCA, LAMP, and NGS to achieve higher sensitivity and specificity of related genetic analyses; application of PPR nuclease in the process of viral vector purification (especially lentiviruses [LV], adenoviruses [AV, AAV], and retroviruses [RV] used in modern gene therapy and cell therapy (chimeric antigen receptor [CAR] T-cell immunotherapy)); application of PPR nuclease in the process of exosome purification for therapeutic or diagnostic purposes; application of PPR nuclease in the process of recombinant protein purification, in particular enzymes, antibodies, vaccination antigens, products used in cell therapy, and other therapeutic proteins.

Description

[Technical field]

[0001] The subject of the present invention is a novel heat-labile non-specific PPR nuclease or an enzymatically active fragment thereof or a sequence sharing at least 40% identity therewith, which is active at low temperature, over a wide pH range, and at high salt concentrations (e.g. NaCl, KCl, MgCl2, MgSO4, (NH4)2SO4). The subject of the present invention is also a gene encoding a PPR nuclease or an enzymatically active fragment thereof; a particle of nucleic acid encoding a PPR nuclease or an enzymatically active fragment thereof; an expression plasmid comprising the sequence of a PPR-encoding gene; a recombinant strain of Escherichia coli JM109(DE3)pD454-PPR-AmpR and an expression vector of Escherichia coli JM109(DE3)pD454-PPR-AmpR. coli ArcticExpress(DE3)pD454-PPR-AmpR strain; a method for PPR nuclease protein production, application of PPR nuclease in the process of purification of recombinant proteins with a fairly low DNA content and for the decontamination of PCR, qPCR, RT-PCR, RT-qPCR and NGS reagents and mixtures to achieve higher sensitivity and specificity of the associated genetic analyses; application of PPR nuclease in the process of viral vectors purification (in particular lentiviruses [LV], adenoviruses [AV, AAV] and retroviruses [RV] used in modern gene therapy and cell therapy (chimeric antigen receptor [CAR] T cell immunotherapy); application of PPR nuclease in the process of exosome purification for therapeutic or diagnostic purposes; application of PPR nuclease in the process of recombinant protein purification, in particular enzymes, antibodies, vaccination antigens, products used in cell therapy and other therapeutic proteins. [Background technology]

[0002] Currently, the most common non-specific nuclease is Benzonase® (Merck, USA), whose activity optimum is 37°C, but its main disadvantages include the inability to be effectively inactivated by high temperatures and limited tolerance to high salt concentrations. Similar parameters are exhibited by non-branded products related to Benzonase®, such as, for example, Denarase (c-LEcta, Germany), which is produced in another host - Bacillus species. Another example of an enzyme with similar characteristics is Cyanase™ nuclease, which is derived from another microorganism (RiboSolutions, USA). However, the inventors mainly envisage obtaining a non-specific nuclease derived from a psychrophilic and halophilic microorganism, which retains considerable activity below 20°C and even in frozen conditions (4-8°C), maintains optimal activity in a wide range of salt concentrations and pH, and is characterized by irreversible enzyme inactivation by low temperatures. Currently, there are only two non-specific nucleases on the world market that exhibit considerable activity at low temperatures. These are Cryonase™ (Takara, Japan) and HL-SAN (ArcticZymes, Norway) derived from psychrophilic organisms. However, compared to the present invention, these enzymes are characterized by low tolerance to high salt concentrations, weak activity at low temperatures (<20° C.), and a narrow pH tolerance range (residual activity at pH<7.0).

[0003] DNA contamination occurs commonly in protein products produced in microorganisms and poses significant problems especially during the industrial production of recombinant proteins and enzymes for diagnostic, therapeutic and scientific purposes.

[0004] Enzymes with very low nucleic acid contamination (so-called "DNA-free") are ideal for accurate diagnostics based on amplification and / or DNA ligation (among others: PCR, qPCR, RT-qPCR, NGS, RCA, LAMP), where the highest sensitivity, specificity and absence of ambiguous or false positive results are required. Even traces of exogenous DNA may lead to artifacts in the ultrasensitive techniques mentioned above. The problem of DNA contamination escalates when the amount of DNA to be detected is low. Signals from contaminating DNA may interfere with low copy DNA detection and be the subject of measurement, significantly affecting the sensitivity and reliability of the test.

[0005] Commercial suppliers of enzymes and reagents (especially DNA polymerases, PCR master mixes, and reagents for NGS) recognize the importance of nucleic acid contamination and offer DNA-free products, which are distinguished from conventional reagents by their production technology and quality control. However, the level of contamination in these products is very often far from expectations, as it strongly depends on the sensitivity of the DNA contamination detection method (a survey of the literature shows that most companies offer DNA-free polymerases, which contain 10 to 1000 copies of DNA genome per U of enzyme).

[0006] The production of therapeutic proteins and active pharmaceutical ingredients also requires the removal of process contaminants, especially those related to DNA (host and foreign), for high quality standards. The amount of residual DNA must usually be limited to 100 pg per drug dose (e.g. for therapeutic antibodies) and for some vaccines to 10 ng per drug dose. These values ​​are determined by the World Health Organization (WHO) as well as the Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) guidelines.

[0007] The ideal tool for purification of contaminating nucleic acids appears to be the application of suitable non-specific and versatile nucleases characterized by high activity at low temperatures (4-22°C), a wide range of pH (6.0-10.0), and high salt concentrations, which can be inactivated at temperatures that are safe for the enzymes and biopharmaceuticals being purified (proteins, enzymes, antibodies, antigens, viral vectors for gene therapy, etc.), as well as other additives commonly used in the purification process (downstream processing).

[0008] In particular, in terms of the effective purification of nucleic acids of viral vectors used in modern gene and cell therapies (such as CAR-T therapy) (among others lentiviruses (LV), adenoviruses (AV, AAV), retroviruses (RV)), it is highly desirable to apply purification conditions at high salt concentrations (250-1000 mM NaCl) and a pH within 6.0-8.0 (Kramberger et al., Hum Vaccin Immunother. 2015;11(4):1010-21.doi:10.1080 / 21645515.2015.1009817), i.e. optimal conditions for the function of PPR nucleases. Such conditions considerably promote the digestion of the host cell nucleic acids constituting the chromatin as well as change the viscosity of the solution containing the viral vectors or proteins, facilitating their purification. In addition, such conditions are essential for the effective binding of the purified viral vectors or proteins to the stationary phase. This is a key to increasing the efficiency of the production process and significantly reducing production costs.

[0009] In recent years, there has been an increasing interest in enzymes derived from psychrophilic microorganisms (i.e. microorganisms adapted to live at low temperatures). The great significance of these enzymes is related to their high activity at low temperatures (generating process savings) and their thermolability, which allows their effective, fast and selective inactivation (after the purification process) by a small increase in temperature (without damaging the product under enzymatic treatment).

[0010] The subject heat-labile non-specific nucleases may be applied for the production of nucleic acid-free enzymes, such as DNA-free polymerases, reverse transcriptases, or ligases. These are very expensive and not commonly available enzymes, but they are often desirable for specialized techniques such as molecular biology and in vitro diagnostics. The subject PPR nucleases of the present invention may also be used by pharmaceutical and cosmetic companies for the purification of natural products from nucleic acids. For this purpose, the pharmaceutical market currently uses the mesophilic Benzonase® (Merck), which is characterized by a low tolerance to monovalent and divalent salts in the reaction environment.

[0011] WO2006095769 describes a polypeptide with endonuclease activity from the psychrophilic microorganism Shewanella sp., which exhibits high activity at low temperatures. This polypeptide may remove any nucleic acids present in the protein solution and may reduce the viscosity of the protein extract. However, its inactivation, as reported in the literature, causes certain problems (Saramiento et al., Front Bioeng Biotechnol. 2015; 3: 148.), and inactivation requires incubation at 70°C for 30 minutes (at such high temperatures many recombinant proteins may be denatured).

[0012] Furthermore, WO 2013 / 121228 discloses a non-specific endonuclease and its enzymatically active fragments available under the trade name HL-SAN. The present invention relates to an endonuclease that is inactivated by mild temperature conditions and exhibits heat-labile properties. The present invention also includes the application of this endonuclease to remove contaminating polynucleotides from biological preparations. The present invention also relates to the prevention of false positive results in nucleic acid amplification reactions, particularly in amplification reactions by PCR, by the application of the endonuclease. Summary of the Invention [Problem to be solved by the invention]

[0013] The aim of the present invention was to obtain a better characterized heat-labile non-specific nuclease that maintains high activity at temperatures below 20°C, especially at refrigeration conditions (4-8°C), at high salt concentrations and possibly over a wide pH range. Furthermore, PPR nucleases are compatible with most buffers and additives used in bioprocessing. This enzyme, which hydrolyzes nucleic acids, may become a very valuable tool used for the production of recombinant proteins, enzymes, antibodies, vaccination antigens, exosomes, viral vectors for gene therapy or cell therapy with low nucleic acid content, for the preparation of products used in cell therapy, as well as for the purification of other therapeutic proteins from DNA and RNA contaminants (e.g. enzymes for molecular biology and accurate in vitro diagnostics, proteins and viral vectors for the biopharmaceutical industry, and biological components for the livestock and cosmetics industries). [Means for solving the problem]

[0014] A subject of the present invention is a PPR nuclease or an enzymatically active fragment thereof, wherein the nuclease sequence is SEQ ID NO: 2 or a sequence sharing at least 40% identity therewith.

[0015] PPR nuclease or an enzymatically active fragment thereof is irreversibly inactivated in the presence of 1-5 mM DTT after incubation at 52° C. for 15 minutes, and the inactivation temperature can be lowered by longer incubation with DTT.

[0016] PPR nuclease or an enzymatically active fragment thereof is typically active at the following salt concentrations: NaCl: 0-1400 mM, MgCl2 5-200 mM, urea: 0-6000 mM, ammonium sulfate: 0-200 mM, imidazole: 0-400 mM.

[0017] The gene encoding the PPR nuclease or an enzymatically active fragment thereof has the sequence shown as SEQ ID NO:1.

[0018] A nucleic acid particle encoding the PPR nuclease or an enzymatically active fragment thereof according to claims 1 to 3, or encoding a protein containing the PPR nuclease or an enzymatically active fragment thereof.

[0019] An expression plasmid, pD454-PPR-AmpR, containing the sequence of the PPR-encoding gene according to claim 4. In addition, the plasmid contains: a T7 phage promoter or another promoter active in an E. coli expression system. The plasmid has the sequence of SEQ ID NO:4.

[0020] The recombinant strains of Escherichia coli JM109(DE3)pD454-PPR-AmpR or Escherichia coli ArcticExpress(DE3)pD454-PPR-AmpR have been transformed with the plasmids described above.

[0021] A method for PPR nuclease protein production, in which a recombinant strain of Escherichia coli JM109(DE3)pD454-PPR-AmpR or Escherichia coli ArcticExpress(DE3)pD454-PPR-AmpR is cultivated in a medium, then induction of PPR nuclease gene expression is performed by adding IPTG; the protein is isolated and purified.

[0022] A method for the isolation and purification of a PPR nuclease or an enzymatically active fragment thereof as defined above, comprising the expression of the nuclease or a fragment thereof as described above in a suitable host cell and, consequently, the separation of the nuclease from the host cell and / or the medium in which the cell is cultured.

[0023] Application of PPR nuclease in the process of purification of recombinant proteins with significantly lower DNA content as well as for decontamination of reagents and reaction mixtures of PCR, qPCR, RT-PCR, RT-qPCR, RCA, LAMP and NGS to achieve increased sensitivity and specificity of related genetic analyses.

[0024] Application of PPR nucleases in the process of viral vector purification (especially lentiviruses [LV], adenoviruses [AV, AAV], and retroviruses [RV]) used in modern gene and cell therapies (e.g. chimeric antigen receptor [CAR] T-cell immunotherapy).

[0025] Application of PPR nuclease in a process for purifying exosomes to be used for therapeutic and diagnostic purposes.

[0026] Application of PPR nucleases in the process of recombinant protein purification, especially enzymes, antibodies, vaccination antigens, products used in cell therapy, and other therapeutic proteins.

[0027] Applications of PPR nucleases in pharmaceutical, livestock, and cosmetic industries.

[0028] Application of PPR nucleases in the pharmaceutical and biotechnology industries to remove DNA contamination in culture media for mammalian fermentation and microbiological processes.

[0029] The terms used in the above description and claims have the following meanings: Nuclease - This term refers to an enzyme that hydrolyzes phosphodiester bonds in a polynucleotide chain of nucleic acid (DNA or RNA). Nonspecific nuclease - an enzyme that hydrolyzes all types of nucleic acids, including ssDNA, dsDNA, circular DNA, ssRNA, and dsRNA. Psychrophiles - Organisms that live at low temperatures (below 20°C). Psychrotolerant - Organisms that can tolerate low temperatures (they may live at low temperatures but do not require low temperatures). Halophile - an organism that lives in salt water or soil and can tolerate high salt concentrations. PPR nuclease - a non-specific nuclease which is the subject of the present invention according to SEQ ID NO:2. [Brief description of the drawings]

[0030] [Figure 1] FIG. 1 shows the construction of the pD454-PPR expression plasmid. [Diagram 2] Figure 2 shows the effect of pH on PPR nucleolytic activity depending on NaCl salt concentration, measured using a modified Kunitz test, conditions: 20 mM MgCl2, temperature 22°C. [Diagram 3] Figure 3 shows the effect of temperature and high salt (500 mM NaCl + 100 mM MgCl2) on PPR nucleolytic activity, measured using a modified version of the Kunitz test. [Figure 4] Figure 4 shows the effect of Mg2+ ions on PPR nucleolytic activity at selected pH and temperature conditions. Maximal PPR activity is obtained at concentrations of 50-150 mM in a pH 8.0 buffer at 37°C. At ambient temperature (22°C) in a pH 6.5 buffer, optimal activity is obtained at much lower Mg2+ ion concentrations (20-50 mM). [Diagram 5] Figure 5 shows PPR inactivation at different temperatures in the presence of 5 mM DTT. Complete PPR inactivation is obtained at 52°C. [Figure 6]FIG. 6 shows a comparison of the nucleolytic activity values ​​of PPR, HL-SAN, and Benzonase® nuclease in buffers with various NaCl concentrations (0, 250, 500 mM) at pH 7.0, 8.0, and 9.0, respectively. The remaining reaction conditions are as follows: temperature 22°C, 50 mM Tris, 20 mM MgCl2 (5 mM MgCl2 was used for Benzonase®). PPR shows the greatest competitive advantage at high NaCl concentrations (250-500 mM), which are commonly used in the process of recombinant protein and viral vector purification. The advantage of PPR over HL-SAN nuclease, whose characteristics are most similar, increases in correlation with the decrease in pH (7.0-8.0). [Figure 7] FIG. 7 shows a comparison of nucleolytic activity values ​​of PPR, HL-SAN, and Benzonase® nuclease in DMEM medium commonly used for mammalian in vitro cell culture in which recombinant proteins, viral vectors, and other biological therapeutics are produced. [Figure 8] FIG. 8 shows a comparison of nucleolytic activity values ​​of PPR, HL-SAN, and Benzonase® nuclease in buffers with similar physiological salt concentrations (PBS and TBS) with the addition of 500 mM NaCl, commonly used in recombinant protein purification procedures. [Figure 9] FIG. 9 shows detection of contaminating host DNA (E. coli) in samples of UDGase (UDG) and UDGase purified using PPR nuclease (UDG+PPR) using qPCR methodology. [Figure 10]Figure 10 shows the removal of genomic DNA contamination from the post-culture medium of CHO cells producing the cetuximab and bevacizumab monoclonal antibodies. SEQ ID NO:1 - shows the PPR nuclease nucleotide sequence. SEQ ID NO:2 - shows the amino acid sequence of the PPR nuclease protein. SEQ ID NO:3A - shows the amino acid sequence of the PelB signal peptide. SEQ ID NO:3B - shows the His6-tag allowing purification. SEQ ID NO:4 - shows the sequence of the recombinant pD454-PPR-AmpR expression plasmid. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] The present invention is illustrated by the following examples of its practice, without any limitation on its application. EXAMPLES

[0032] Example 1 Obtain the expression plasmid pD454-PPR-AmpR. To obtain the expression plasmid pD454-PPR-AmpR, purified by ethanol precipitation, the DNA fragment of the type of SEQ ID NO:1 is digested with SapI restriction enzyme, which is then ligated with the DNA of the pD454-SR plasmid vector (ATUM, Newark, CA94560, USA) digested with the same restriction enzyme.

[0033] The ligation reaction mixture is transformed into competent TOP10F Escherichia coli cells placed on Petri dishes with LA medium (1% peptone; 0.5% yeast extract; 1% NaCl; 1.5% agar) containing 100 μg / ml ampicillin. As a result of plasmid DNA isolation, pD454-PPR-AmpR and the expression plasmid of the sequence of SEQ ID NO: 4 are obtained from the grown bacterial colonies. The map of the pD454-PPR-AmpR plasmid is shown in FIG. 1.

[0034] Example 2 A recombinant strain of E. coli JM109(DE3)pD454-PPR-AmpR or E. coli ArcticExpress(DE3)pD454-PPR-AmpR is obtained. To obtain recombinant strains of E. coli JM109(DE3)pD454-PPR-AmpR or E. coli ArcticExpress(DE3)pD454-PPR-AmpR, transformation of E. coli JM109(DE3) cells or E. coli ArcticExpress(DE3) cells is carried out with the circular DNA of the pD454-PPR-AmpR expression plasmid (SEQ ID NO: 4) obtained as described in Example 1. The bacterial cells are plated on LB medium (1% peptone; 0.5% yeast extract; 1% NaCl) containing 100 μg / ml ampicillin, and the colonies obtained for the recombinant strains of E. coli JM109(DE3)pD454-PPR-AmpR or E. coli ArcticExpress(DE3)pD454-PPR-AmpR are then used for the biosynthesis of PPR nuclease.

[0035] Example 3 Obtaining PPR nuclease using cells of recombinant strains of E. coli JM109(DE3)pD454-PPR-AmpR or E. coli ArcticExpress(DE3)pD454-PPR-AmpR. The recombinant strains of E. coli JM109(DE3)pD454-PPR-AmpR or E. coli ArcticExpress(DE3)pD454-PPR-AmpR obtained according to Example 2 are cultivated in LB medium (1% peptone; 0.5% yeast extract; 1% NaCl) containing 50 μg / ml of ampicillin at 37 °C for 16-18 hours. The overnight culture is then used to inoculate a medium of the same content in a ratio of 1:50. OD 600The culture is continued at 30°C until an optical density of 0.4-0.5 is obtained, then induction of the PPR nuclease gene expression is carried out by adding IPTG to a final concentration of 0.2 mM. The culture is maintained at 18°C ​​for 20-22 hours, then the bacterial cells are separated from the medium by centrifugation. The cell sediment is suspended in a buffer containing 20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 10 mM imidazole, 5 mM MgCl2; at least 5 ml of buffer solution per 1 g of cell sediment.

[0036] The cell suspension is then disrupted using ultrasound, performing 3 cycles of sonication; energy intensity 100 J / ml of suspension. The resulting cell lysate is centrifuged at 16000 RCF to remove non-soluble proteins and cell fragments, and then it is filtered through a 0.2 μm membrane. The PPR nuclease protein is separated from the remaining bacterial proteins by applying an immobilized metal affinity chromatography method (IMAC) using a stationary phase with immobilized divalent ions of nickel. The PPR nuclease bound to the stationary phase is then washed with a buffer containing 20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 300 mM imidazole, 5 mM MgCl2. The fraction containing the PPR nuclease is dialyzed for at least 18 hours in refrigerated conditions against a buffer solution containing 20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 5 mM MgCl2; at least 100 ml per ml of enzyme. The resulting enzyme preparation is mixed 1:1 with glycerol and frozen at −20° C. Protein concentration determination is performed using spectrophotometry at a wavelength of 280 nm.

[0037] Example 4 Examination of the enzymatic properties of the PPR nuclease recombinant protein. The specific nucleolytic activity towards nucleic acids, necessary to define the optimal conditions of enzymatic activity and inactivation, was determined for the recombinant PPR nuclease obtained according to Example 3.

[0038] To determine the PPR nuclease nucleolytic activity, serial dilutions of the enzyme are incubated for 10 min in a 20 μl volume of reaction buffer containing 20 mM Tris-HCl (pH 8.0), 20 mM MgCl2, and 1 μg pUC19 plasmid DNA. The reaction is stopped by adding 5 μl 25 mM DTT solution to a final concentration of 5 mM, and the samples are heated to 55° C. for 10 min. As a control, 1 μg pUC19 plasmid DNA is incubated without adding nuclease. Subsequently, the samples are loaded onto a 1% agarose gel, and DNA separation is carried out in the gel for 40 min at a voltage of 130. The remaining undegraded DNA in the gel is recorded by staining with ethidium bromide and taking a photograph of the gel. 1 U activity is determined as the amount of enzyme necessary for the complete degradation of 1 μg pUC19 plasmid DNA at 37° C. for 10 min.

[0039] To determine the PPR nuclease specific activity, serial dilutions of the enzyme are incubated for 30 min at 37° C. in a volume of 300 μl of a reaction buffer containing 20 mM Tris-HCl (pH 8.0), 20 mM MgCl2, and 100 μg of herring sperm genomic DNA. The reaction is stopped by adding 300 μl of a 4% solution of perchloric acid to a final concentration of 2%, and the samples are incubated on ice for 60 min. As a control, 100 μg of DNA is incubated without adding nuclease. The samples are then centrifuged for 10 min until the precipitate of non-degraded DNA is separated. The content of free nucleotides and oligonucleotide fragments smaller than 10 bp is determined in the supernatant by measuring the absorbance at a wavelength of 260 nm. 1 U nuclease activity is determined as the amount of enzyme that causes an increase in absorbance in the studied sample at a wavelength of 260 nm of 1.0 within 30 min of reaction at 37° C.

[0040] Example 4A Determination of the optimal pH for the activity of nucleic acid degrading PPR nuclease. To determine the optimal pH for the enzyme nucleolytic activity, reactions are carried out as described in Example 4. Solutions of pH 6.0 to 10.0 with 1 U of enzyme in reaction mixtures containing 0, 250, and 500 mM NaCl, respectively. PPR nuclease exhibits the highest nucleolytic activity in a pH 8.0 buffer with a NaCl concentration of 500 mM, as shown in Figure 2.

[0041] Example 4B Determination of the optimum temperature for PPR nuclease nucleolytic activity. The determination of the optimum temperature of the enzymatic nucleolytic activity reaction is carried out as described in Example 4, at various temperatures from 6°C to 45°C, with 1U enzyme, in a reaction mixture containing 50 mM Tris, pH 8.0 and various concentrations of MgCl2 (5 mM and 100 mM). PPR nuclease maintains nucleolytic activity in the entire range of temperatures tested (Figure 3). However, the highest activity is exhibited at 37°C, at high concentrations of NaCl (500 mM) and MgCl2 (100 mM). It should be emphasized that in these conditions, 100% standard activity may be obtained by applying the appropriate salt concentrations: NaCl (500 mM) and MgCl2 (100 mM) in refrigerated conditions at 6°C.

[0042] Example 4C Nucleolytic PPR nuclease activity Mg 2+ Determination of optimal concentrations of ions. Mg on PPR nucleolytic activity 2+ To determine the effect of ion concentration, reactions were run with 1 U enzyme and 100% Mg in the reaction mixture. 2+ Solutions with various contents of ions; 5 mM to 200 mM, carried out as described in Example 4. In a weakly alkaline environment (pH 8.0), PPR nuclease exhibits a high Mg content at 150 mM concentration (optimal condition 50 to 150 mM), as shown in FIG. 2+ The highest nucleolytic activity is observed in buffers containing Mg ions. In a low pH environment (pH 6.5), PPR exhibits high nucleolytic activity at a concentration of 20 mM (optimum condition: 20-50 mM). 2+The highest nucleolytic activity is observed in buffers containing ions. Over the entire observed range of MgCl2 (5-200 mM), the PPR concentrate shows high specific activity.

[0043] Example 4D Identification of the effect of potential inhibitors on PPR enzyme activity. To determine the range of enzyme tolerance to common ionic constituents used in recombinant protein preparations present in reaction buffers, nucleolytic reactions are carried out in solutions of varying amounts of each potential inhibitor: NaCl, urea, ammonium sulfate, imidazole, as described in Example 4. PPR nucleases retain high nucleolytic activity in the presence of high concentrations of test substances, as shown in Table 1.

[0044] [Table 1]

[0045] Example 4E Heat inactivation of PPR enzyme activity To determine the parameters of PPR nuclease inactivation, a series of 0.2 ml test probes for PCR are prepared containing 100U PPR in 50 μl reaction buffer containing 5 mM DTT as described in Example 4A, except for pUC10 plasmid DNA. The probes are then incubated at the appropriate temperature for 15 minutes and then on ice for 5 minutes. As a control, 100U PPR nuclease in the same buffer is kept on ice. Then, a nucleolytic reaction is carried out after inactivation using 5 μl PPR solution from the previous step as described in Example 4A. As controls, only plasmid DNA in reaction buffer (negative control) and 100U PPR and plasmid DNA kept on ice (positive control) are incubated under the same conditions as the reaction test. The degree of plasmid DNA degradation in the test is analyzed on an agarose gel. As shown in Figure 5, PPR is completely inactivated in the presence of DDT at 52°C or higher.

[0046] Example 4F Determination of nucleolytic activity of PPR, HL-SAN, and Benzonase® nuclease in buffers of various salt concentrations and pH. The determination of the nucleolytic activity of PPR, HL-SAN, and Benzonase® nucleases is carried out at various temperatures; 6°C, 22°C, and 37°C, with various additions of NaCl to final concentrations; 0, 250, 500 mM, at pH 7.0, 8.0, 9.0, respectively, in the presence of 50 mM Tris, 20 mM MgCl2 (5 mM MgCl2 for Benzonase®). As shown in Figure 6, PPR nuclease exhibits the highest nucleolytic activity of all tested nucleases in buffers with high salt content (250 and 500 mM NaCl) at pH 7.0, 8.0, and 9.0. (Only HL-SAN shows slightly higher activity at 500 mM NaCl and pH 9.0). Benzonase® nuclease is virtually nonfunctional at any condition of high salt (250, 500 mM NaCl), regardless of pH value. At lower pH (7.0, 8.0) and ambient temperature (22° C.), PPR nuclease is significantly more active than HL-SAN and Benzonase®. Similar correlations were observed at 6° C. and 37° C. (data not shown).

[0047] Example 4G Determination of nucleolytic activity of PPR, HL-SAN, and Benzonase® nuclease in DMEM medium. The determination of the nucleolytic activity of PPR, HL-SAN, and Benzonase® nucleases is carried out at different temperatures: 6°C, 22°C, and 37°C, as described in Example 4, but using DMEM medium (commonly used in the culture of mammalian cells, e.g., CHO, HEK, for the production of recombinant proteins and viral vectors) instead of the reaction buffer. As shown in Figure 7, PPR nuclease added directly to DMEM medium shows the highest nucleolytic activity of all tested enzymes at all tested temperatures. At 37°C, PPR is 6 times more active than Benzonase® nuclease. At refrigeration conditions (6°C) and ambient temperature (22°C), PPR is approximately 3 times more active than the other tested nucleases, i.e., HL-SAN and Benzonase® (Figure 7). The activity at the conditions recommended by the manufacturer was assumed as 100% activity for all enzymes.

[0048] Example 4H Determination of nucleolytic activity of PPR, HL-SAN, and Benzonase® nuclease in PBS and TBS buffers. The determination of the nucleolytic activity of PPR, HL-SAN and Benzonase® nucleases is carried out at 6°C, 22°C and 37°C as described in Example 4, but instead of the reaction buffer, the following buffers are used: PBS (phosphate buffered saline) pH 7.4 (10 mM Na2HPO4, 1.8 mM KH2PO4; 2.7 mM KCl; 137 mM NaCl) and TBS (Tris buffered saline) (50 mM Tris-Cl, pH 7.6; 150 mM NaCl), which are commonly used in the process of recombinant protein purification. In addition, the activity of the nucleases in TBS supplemented with 500 mM NaCl was compared. As shown in Figure 8, PPR nuclease shows the highest nucleolytic activity of all tested enzymes at all tested temperatures in all tested buffers, i.e. PBS, TBS and TBS with a high content of salt (500 mM NaCl). The activity under the conditions recommended by the manufacturer was assumed as 100% activity for all enzymes (Figure 8).

[0049] Example 5 Application of recombinant PPR nuclease. Example 5A Application of PPR nuclease in the production of recombinant UDGase with low host DNA content. Following Example 3, the obtained PPR nuclease is used in the process of purification of other recombinant enzymes commonly used in scientific research and molecular diagnostics, in particular polymerases, ligases, and UDGase, which contain significantly less host DNA contaminants. The standard protocol of E. coli bacterial UDGase purification was modified in order to add PPR nuclease to the prepared bacterial lysate containing overproduced UDGase as follows: 40 U per ml of lysate and subsequent incubation at 20-25°C for 1 hour using a magnetic mixer set at 200 revolutions / min. The resulting lysate is processed according to standard procedures for UDGase. The measurement of the content of host DNA contamination is carried out using 16S bacteria-specific primers and qPCR methods. The UDGase purified using the additional step with PPR nuclease contains 100 times less host DNA contaminants compared to the enzyme purified without PPR nuclease, which is shown in Figure 9. Such purified UDGase enzyme used in scientific research or molecular diagnostics increases the sensitivity of qPCR methods and significantly reduces the risk of potential false positive results.

[0050] Example 5B Application of PPR nuclease for the removal of DNA contamination in the process of mammalian cell monoclonal antibody purification. The PPR nuclease enzyme obtained as described in Example 3 is used for the removal of DNA contaminants in the process of purification of recombinant monoclonal antibodies isolated from Chinese Hamster Ovary (CHO) cells. Mammalian cells are cultured in an appropriate medium for 5 days. The cells are then separated by centrifugation and the supernatant is used to purify the antibody by standard chromatographic methods. In the medium, besides the antibody and medium components, there is a large amount of genomic DNA from the host cells that is degraded during the culture. The implementation of a preliminary step, such as incubation of the post-culture medium with PPR nuclease, significantly reduces the content of DNA contamination in the medium and therefore increases the efficiency of the antibody binding to the stationary phase. A step of post-culture medium incubation with the addition of up to 50 U / ml of PPR nuclease for 60 minutes at 20-22°C was introduced into the standard process of antibody purification. After this, DNA is isolated from 1 ml of medium treated and not treated with PPR nuclease using a genomic DNA isolation kit. The resulting total DNA is loaded onto a 1% agarose gel and ethidium bromide is added to visualize the nucleic acids. The medium then undergoes standard procedures for antibody purification. As a control, the post-culture medium is incubated under the same conditions without adding PPR nuclease. As shown in Figure 10, the addition of PPR nuclease significantly reduces the contamination of genomic DNA in the post-culture medium, which accumulates during the growth of cells that secrete cetuximab and bevacizumab antibodies into the medium.

Claims

1. 1. An isolated recombinant protein comprising a nuclease or an enzymatically active fragment thereof, the amino acid sequence of said isolated recombinant protein being: (b) as set forth in residues 23-269 of SEQ ID NO:2; Recombinant proteins.

2. The nuclease or an enzymatically active fragment thereof comprises: (a) for 15 minutes at 52°C in the presence of 1-5 mM DTT, or (b) for more than 15 minutes at a temperature of 52° C. or less in the presence of 1-5 mM DTT 2. An isolated recombinant protein comprising the nuclease of claim 1 or an enzymatically active fragment thereof, which is irreversibly inactivated after incubation.

3. 2. An isolated recombinant protein comprising the nuclease of claim 1 or an enzymatically active fragment thereof, which is active at the following salt concentrations: NaCl: 0-1400 mM, MgCl2: 5-200 mM, urea: 0-6000 mM, ammonium sulfate: 0-200 mM, imidazole: 0-400 mM.

4. 10. A composition comprising an isolated recombinant protein comprising the nuclease of claim 1 or an enzymatically active fragment thereof, and salts at the following concentrations: NaCl: 0-1400 mM, MgCl2: 5-200 mM, urea: 0-6000 mM, ammonium sulfate: 0-200 mM, imidazole: 0-400 mM.

5. A nucleic acid encoding a nuclease or an enzymatically active fragment thereof according to any one of claims 1 to 3, or a nucleic acid encoding an isolated recombinant protein containing a nuclease or an enzymatically active fragment thereof according to any one of claims 1 to 3.

6. 1. An expression plasmid pD454-PPR-AmpR (SEQ ID NO:4) containing the sequence shown as SEQ ID NO:1 encoding the isolated recombinant protein of claim 1 and the T7 phage promoter or alternatively another promoter active in an E. coli expression system.

7. 7. A recombinant Escherichia coli strain selected from JM109(DE3)pD454-PPR-AmpR and Escherichia coli ArcticExpress(DE3)pD454-PPR-AmpR, wherein said recombinant Escherichia coli strain has been transformed with the plasmid of claim 6, whereby said Escherichia coli strain contains the plasmid of claim 6.

8. 13. A method for producing a recombinant protein comprising the nuclease protein of claim 1, wherein a recombinant strain of Escherichia coli JM109(DE3)pD454-PPR-AmpR or Escherichia coli ArcticExpress(DE3)pD454-PPR-AmpR is cultivated in a medium, then induction of nuclease gene expression is performed by adding IPTG, and the protein is isolated and purified.

9. 4. A method for the isolation and purification of a recombinant protein according to any one of claims 1 to 3, wherein said recombinant protein is expressed in a suitable host cell, such that said recombinant protein is isolated from said host cell and / or the medium in which said cells are cultured.

10. 2. Use of the isolated recombinant protein of claim 1, (a) the process of purification of recombinant proteins; (b) a process for purification of the recombinant protein to reduce the DNA content; (c) processes for decontamination of reagents and reaction mixtures for PCR, qPCR, RT-PCR, RT-qPCR, RCA, LAMP, and NGS; (d) processes for decontamination of PCR, qPCR, RT-PCR, RT-qPCR, RCA, LAMP, and NGS reagents and reaction mixtures to achieve increased sensitivity and specificity of the associated genetic analyses; (e) the process of viral vector purification; (f) lentivirus (LV), adenovirus (AV, AAV), and retrovirus (RV) viral vector purification processes; (g) Processes for purifying viral vectors for gene and cell therapy; (h) A process for purifying viral vectors for chimeric antigen receptor [CAR] T cell immunotherapy; (i) processes for purifying exosomes for therapeutic and diagnostic purposes; (j) recombinant protein purification processes; (k) processes for recombinant protein purification of enzymes, antibodies, vaccination antigens, products used in cell therapy, and other therapeutic proteins; or (l) Processes in the pharmaceutical, livestock, and cosmetics industries Use in.

11. 2. Use of an isolated recombinant protein comprising the nuclease of claim 1 in processes within the pharmaceutical and biotechnology industries to remove DNA contamination in culture media for mammalian fermentation processes and microbiological processes.

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