Novel non-specific heat-labile nucleases active at low temperature, in a wide pH range and at high concentrations of salt

Abstract

The subject of the invention is a novel thermostable PPR nuclease or enzymatically active fragment thereof, which exhibits high catalyst activity under difficult reaction conditions, in particular high concentrations of salts and other additives commonly used in protein and virus purification processes, low temperatures and wide pH ranges, wherein the nuclease sequence is SEQ ID NO: 2 or a sequence having at least 40% identity thereto. The subject of the invention is also a gene encoding the PPR nuclease or enzymatically active fragment thereof; a particle of nucleic acid encoding the PPR nuclease or enzymatically active fragment thereof; an expression plasmid comprising the sequence of the gene encoding PPR; recombinant strains of Escherichia coli JM109(DE3)pD454-PPR-AmpR and Escherichia coli ArcticExpress(DE3)pD454-PPR-AmpR; a method for the production of PPR nuclease protein; the use of PPR nuclease in the purification process of recombinant proteins with significantly lower DNA content; the use of the reactive reagents and mixtures for the purification of PCR, qPCR, RT-PCR, RT-qPCR, RCA, LAMP and NGS to obtain higher sensitivity and specificity of the relevant genetic analysis; the use of the PPR nuclease in the process of purifying viral vectors, in particular lentivirus [LV], adenovirus [AV, AAV] and retrovirus [RV] used for modulating gene and cell therapy (chimeric antigen receptor [CAR] T cell immunotherapy); the use of the PPR nuclease in the process of exosome purification for therapeutic or diagnostic purposes; the use of the PPR nuclease in the process of purifying recombinant proteins, in particular enzymes, antibodies, vaccination antigens, products for cell therapy and other therapeutic proteins.

Description

[0001] The subject of this invention is a novel thermally unstable nonspecific PPR nuclease or its enzyme activity fragment, or a sequence having at least 40% identity with it, that is active at low temperatures, over a wide pH range, and at high concentrations of salt (e.g., NaCl, KCl, MgCl2, MgSO4, (NH4)2SO4). The subject of this invention is also a gene encoding a PPR nuclease or a fragment of its enzymatic activity; a particle encoding a nucleic acid of a PPR nuclease or a fragment of its enzymatic activity; an expression plasmid containing a sequence encoding a gene for PPR; recombinant strains of *Escherichia coli* JM109(DE3) pD454-PPR-AmpR and *Escherichia coli* ArcticExpress (DE3) pD454-PPR-AmpR; a method for producing PPR nuclease protein; the use of the PPR nuclease in purification processes for recombinant proteins with significantly low DNA content; and the use of reagents and mixtures for purifying PCR, qPCR, RT-PCR, RT-qPCR, and NGS to obtain higher sensitivity and specificity for related gene analyses; and the use of the PPR nuclease in purifying viral vectors (particularly for modern gene and cell therapy (chimeric antigen receptor [CAR]). Use of the PPR nuclease in processes for lentiviruses [LV], adenoviruses [AV, AAV], and retroviruses [RV] for T-cell immunotherapy; use of the PPR nuclease in processes for purifying exogenous proteins for therapeutic or diagnostic purposes; use of the PPR nuclease in processes for purifying recombinant proteins, particularly enzymes, antibodies, vaccine antigens, products for cell therapy, and other therapeutic proteins.

[0002] Currently, the most popular non-specific nuclease is Benzonase. ® (Merck, USA) Its optimal activity temperature is 37°C, and its main drawbacks include the impossibility of effective inactivation at high temperatures and limited tolerance to increased salt concentrations. Benzonase ®(Related products with general characteristics; such as Denarase, which is produced in a different host, namely Bacillus species (c-LEcta, Germany)) exhibit similar parameters. Another example of an enzyme with similar characteristics is Cyanase™ nuclease, which is derived from another microorganism (RiboSolutions, USA). However, the inventors' primary vision was to obtain nonspecific nucleases derived from psychrophilic and halophilic microorganisms that maintain significant activity below 20°C and even under refrigerated conditions (4°C–8°C), maintain optimal activity over a wide range of salt concentrations and pH, and are characterized by irreversible enzyme inactivation at lower temperatures. Currently, only two nonspecific nucleases on the global market exhibit significant activity at lower temperatures. These two nonspecific nucleases are Cryonase™ (Takara, Japan) and HL-SAN (ArcticZymes, Norway), both derived from psychrophilic organisms. However, compared with the invention of this subject, these enzymes are characterized by: lower tolerance to high salt concentrations, weaker activity at low temperatures (<20°C), and a narrower pH tolerance range (residual activity at pH<7.0).

[0003] DNA contamination (which typically occurs in protein products produced by microorganisms) has caused significant problems during the industrial manufacturing of recombinant proteins and enzymes, especially for diagnostic, therapeutic, and scientific purposes.

[0004] For precise diagnostics based on amplification and / or DNA ligation (as well as PCR, qPCR, RT-qPCR, NGS, RCA, LAMP), enzymes with significantly low nucleic acid contamination (so-called "DNA-free") are ideal, requiring the highest sensitivity, specificity, and the absence of fuzzy or false-positive results. In the aforementioned ultrasensitive techniques, even trace amounts of exogenous DNA can lead to artifacts. The DNA contamination problem escalates when the amount of DNA detected is small. Signals from contaminated DNA can interfere with the detection of low-copy DNA as the metric, significantly impacting the sensitivity and reliability of the test.

[0005] Commercial suppliers of enzymes and reagents (especially DNA polymerase, PCR premixes, and reagents for NGS) have acknowledged the importance of nucleic acid contamination and offer DNA-free products that differ from conventional reagents in terms of manufacturing technology and quality control. However, due to their strong reliance on the sensitivity of DNA contamination detection methods, the contamination levels of these products often fall far short of expectations (according to literature, most companies offer DNA-free polymerases containing 10 to 1000 copies of the DNA genome per 1 U of enzyme).

[0006] Due to high-quality standards, the production of therapeutic proteins and active substances used in pharmaceutical products also requires the removal of process contaminants, particularly those related to (host and exogenous) DNA. Typically, the amount of residual DNA must be limited to 100 pg per drug dose (e.g., in the case of therapeutic antibodies) and, for some vaccines, to 10 ng per drug dose. These values ​​are determined by guidance from the World Health Organization (WHO), as well as the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMEA).

[0007] The ideal tool for purifying nucleic acid contamination appears to be an appropriate, nonspecific, and universal nuclease characterized by high activity at low temperatures (4°C–22°C), a wide pH range (6.0–10.0), and at high concentrations of salts and other additives commonly used in purification processes (downstream treatments). This nuclease can be inactivated at temperatures safe for the enzymes and biopharmaceuticals being purified (proteins, enzymes, antibodies, antigens, viral vectors for gene therapy, etc.).

[0008] Especially in the efficient purification of nucleic acids from viral vectors (and lentiviruses (LV), adenoviruses (AV, AAV), and retroviruses (RV) used in modern gene and cell therapies (e.g., CAR-T therapy), there is a high expectation for purification conditions at high salt concentrations (250–1000 mM NaCl) and pH 6.0–8.0 (Kramberger et al., Hum Vaccin Immunoother [Human Vaccines and Immunotherapy]. 2015; 11(4): 1010–21. doi: 10.1080 / 21645515.2015.1009817), i.e., optimal conditions for PPR nuclease action. Such conditions significantly promote the digestion of nucleic acids that constitute chromatin in host cells and alter the viscosity of solutions containing viral vectors or proteins to facilitate their purification. Furthermore, such conditions are essential for the efficient binding of purified viral vectors or proteins to the stationary phase. This is crucial for improving the efficiency of the production process and significantly reducing production costs.

[0009] In recent years, interest in enzymes derived from psychrophilic microorganisms (i.e., microorganisms adapted to living at low temperatures) has been growing. The significance of these enzymes lies in their high activity at low temperatures (achieving production savings) and thermal instability, which allows them to be effectively, rapidly, and selectively inactivated (after purification processes) with only a slight increase in temperature (without damaging the product being treated with the enzyme).

[0010] This subject (thermally unstable nonspecific nucleases) can be applied to the production of enzymes that do not contain nucleic acids (e.g., DNA-free polymerases, reverse transcriptases, or ligases). These are very expensive and not widely available enzymes, typically required for molecular biology and in vitro diagnostics specialties. PPR nucleases (the subject we invented) can also be used by pharmaceutical and cosmetic companies to purify naturally derived products from nucleic acids. For this purpose, the pharmaceutical market currently uses mesophilic Benzonase. ® (Merck Corporation), characterized by low tolerance to monovalent and divalent salts in the reaction environment.

[0011] International publication WO 2006095769 describes a polypeptide with endonuclease activity derived from a species of Shewanella, a psychrophilic microorganism exhibiting high activity at low temperatures. It can remove any nucleic acids present in protein solutions and reduce the viscosity of protein extracts. However, its inactivation presents some challenges, as, according to literature reports (Saramiento et al., Front Bioeng Biotechnol. [Bioengineering and Biotechnology Frontiers] 2015; 3: 148.), it requires incubation at 70°C for 30 minutes (at such high temperatures, many recombinant proteins may denature).

[0012] Furthermore, international publication WO 2013 / 121228 discloses a nonspecific endonuclease and its enzymatic active fragment, available under the trademark HL-SAN. This invention relates to endonucleases that are inactivated under mild temperature conditions, exhibiting thermal instability. The invention also includes the removal of polynucleotide contamination from biological products by applying this endonuclease. Furthermore, this invention relates to the prevention of false-positive results in nucleic acid amplification reactions, particularly those using PCR methods, by applying an endonuclease.

[0013] The object of this invention is to obtain a thermally unstable nonspecific nuclease with superior properties, which maintains high activity at temperatures below 20°C, particularly under refrigerated conditions (4°C–8°C), high salt concentrations, and a potentially wide pH range. Furthermore, the PPR nuclease is compatible with most buffers and additives used in bioprocessing. This nucleic acid-hydrolyzing enzyme can be a valuable tool for producing recombinant proteins with low nucleic acid content, enzymes, antibodies, vaccine antigens, exogens, and viral vectors for gene or cell therapy; for preparing products for cell therapy; and for purifying other therapeutic proteins from DNA and RNA contamination (e.g., enzymes for molecular biology and precise in vitro diagnostics, proteins and viral vectors for the biopharmaceutical industry, and biological components for the veterinary and cosmetic industries).

[0014] The subject of this invention is: a PPR nuclease or an enzyme activity fragment thereof, wherein the nuclease sequence is SEQ ID NO: 2 or a sequence having at least 40% identity with it.

[0015] In the presence of 1–5 mM DTT, PPR nuclease or its enzyme-active fragments are irreversibly inactivated after incubation at 52°C for 15 minutes, and the inactivation temperature may be lowered by incubation with DTT for a longer period of time.

[0016] The PPR nuclease or its active fragment 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] A gene encoding a PPR nuclease or a fragment of its enzymatic activity, the sequence of which is shown in SEQ ID NO: 1.

[0018] A particle encoding a nucleic acid based on the aforementioned PPR nuclease or its enzyme activity fragment thereof, or encoding a protein containing the aforementioned PPR nuclease or its enzyme activity fragment thereof.

[0019] An expression plasmid pD454-PPR-AmpR contains the sequence of the gene encoding PPR as described above. Additionally, the plasmid includes either a T7 phage promoter or another promoter active in E. coli expression systems. The plasmid has the sequence SEQ ID NO: 5.

[0020] The above plasmids were used to transform recombinant strains of Escherichia coli JM109(DE3) pD454-PPR-AmpR or Escherichia coli ArcticExpress (DE3) pD454-PPR-AmpR.

[0021] A method for producing PPR nuclease protein, wherein recombinant strains of Escherichia coli JM109(DE3) pD454-PPR-AmpR or Escherichia coli ArcticExpress (DE3) pD454-PPR-AmpR are cultured in a culture medium, and then PPR nuclease gene expression is induced by adding IPTG; the protein is then isolated and purified.

[0022] A method for isolating and purifying a PPR nuclease or an enzyme-active fragment thereof as defined above, the method involving expressing the previously described nuclease or a fragment thereof in a relevant host cell and thus isolating the nuclease from the host cell and / or the culture medium of the cultured cell.

[0023] The use of PPR nuclease in the purification process of recombinant proteins with significantly low DNA content, and its use in purifying reagents and reaction mixtures for PCR, qPCR, RT-PCR, RT-qPCR, RCA, LAMP, and NGS to obtain increased sensitivity and specificity for related gene analyses.

[0024] The use of this PPR nuclease in the purification of viral vectors, particularly lentiviruses [LV], adenoviruses [AV, AAV], and retroviruses [RV] for modern gene and cell therapies (e.g., chimeric antigen receptor [CAR] T-cell immunotherapy).

[0025] Use of this PPR nuclease in purification processes for exogenous bodies for therapeutic and diagnostic purposes.

[0026] The PPR nuclease has applications in the purification of recombinant proteins, particularly enzymes, antibodies, vaccine antigens, products for cell therapy, and other therapeutic proteins.

[0027] The PPR nuclease has applications in the pharmaceutical, veterinary, and cosmetic industries.

[0028] This PPR nuclease is used in the pharmaceutical and biotechnology industries to remove DNA contamination from culture media used in mammalian fermentation and microbial processes.

[0029] The terms used in the above specification and patent claims have the following meanings: Nuclease - This term refers to an enzyme that hydrolyzes the phosphodiester bonds in the polynucleotide chains of nucleic acids (DNA or RNA).

[0030] Non-specific nucleases are enzymes that hydrolyze all types of nucleic acids, including ssDNA, dsDNA, circular DNA, ssRNA, and dsRNA.

[0031] Psychrophiles - Organisms that live in low temperatures (below 20°C).

[0032] Psychrotropic organisms - organisms that can tolerate low temperatures (they can live at low temperatures, but it is not necessary for them).

[0033] Halophiles are organisms that tolerate high salt concentrations and live in saline water or soil.

[0034] PPR nuclease - a non-specific nuclease that is the subject of this invention, having the sequence SEQ ID NO:2.

[0035] Description of the attached figures and sequences: Figure 1 - The protocol for the pD454-PPR expression plasmid is shown.

[0036] Figure 2 - The effect of pH on PPR nucleus dissolution activity, depending on NaCl salt concentration, is shown. Measurements were performed using a modified Kunitz test under the following conditions: 20 mM MgCl2 and 22°C.

[0037] Figure 3 - The effects of temperature and high salt concentration (500 mM NaCl + 100 mM MgCl2) on the nucleus dissolution activity of PPR are shown. Measurements were performed using a modified Kunitz test.

[0038] Figure 4 - Showing Mg 2+ The effect of ions on the nucleolysis activity of PPR under selected pH and temperature conditions. Maximum PPR activity was obtained in a buffer at pH 8.0, at 37°C, and at concentrations of 50–150 mM. At ambient temperature (22°C), in a buffer at pH 6.5, significantly lower Mg... 2+ The best activity was obtained at ion concentrations of 20-50 mM.

[0039] Figure 5 - This demonstrates PPR deactivation under different temperature conditions in the presence of 5 mM DTT. Complete PPR deactivation was achieved at 52°C.

[0040] Figure 6 - Showing PPR, HL-SAN and Benzonase ® Comparison of nuclease solubility in buffers with different NaCl concentrations (0, 250, 500 mM) and pH values ​​of 7.0, 8.0, and 9.0. The remaining reaction conditions were: 22°C, 50 mM Tris, and 20 mM MgCl2 (5 mM MgCl2 for Beznonase®). PPR showed the greatest competitive advantage at high NaCl concentrations (250–500 mM) commonly used in purification processes for recombinant proteins and viral vectors. The advantage of PPR over HL-SAN nucleases (these two nucleases are most similar in characteristics) increased correlatedly with decreasing pH (7.0–8.0).

[0041] Figure 7 - Showing PPR, HL-SAN and Benzonase ® Comparison of nuclease nucleolysis activity values ​​in DMEM medium, which is commonly used for in vitro mammalian cell culture to produce recombinant proteins, viral vectors and other biotherapeutic agents.

[0042] Figure 8- Showing PPR, HL-SAN and Benzonase ® Comparison of nuclease nucleolysis activity values ​​in buffers (PBS and TBS) with similar physiological salt concentrations and 500 mM NaCl added, which are commonly used in recombinant protein purification methods.

[0043] Figure 9 - This demonstrates the detection of host DNA contamination (E. coli) in UDG enzyme (UDG) and UDG enzyme purified using PPR nuclease (UDG+PPR) samples using qPCR.

[0044] Figure 10 - This demonstrates the removal of genomic DNA contamination from the post-culture medium of CHO cells that produce cetuximab and bevacizumab monoclonal antibodies.

[0045] Sequence description: SEQ ID NO: 1- shows the nucleotide sequence of the PPR nuclease.

[0046] SEQ ID NO: 2- shows the amino acid sequence of the PPR nuclease protein.

[0047] SEQ ID NO: 3- shows the amino acid sequence of the PelB signal peptide.

[0048] SEQ ID NO: 4- shows the His6 tag that is permitted for purification.

[0049] SEQ ID NO: 5- shows the sequence of the recombinant pD454-PPR-AmpR expression plasmid.

[0050] The invention is illustrated by the following examples of its performance, but there are no limitations on its application.

[0051] Example 1 The expression plasmid pD454-PPR-AmpR was obtained.

[0052] To obtain the expression plasmid pD454-PPR-AmpR (purified by ethanol precipitation), the DNA fragment in the pattern of SEQ ID NO: 1 was digested with the restriction enzyme SapI and subsequently ligated with DNA from the pD454-SR plasmid vector (ATUM Corporation, Newark, CA, USA, 94560) digested with the same restriction enzyme.

[0053] The ligation mixture was used to transform competent TOP10F *E. coli* cells placed in Piper dishes containing LA medium (1% peptone; 0.5% yeast extract; 1% NaCl; 1.5% agar) and ampicillin (100 μg / ml). As a result of plasmid DNA isolation, the expression plasmid pD454-PPR-AmpR with the sequence SEQ ID NO: 5 was obtained from the developing bacterial colonies. The map of the pD454-PPR-AmpR plasmid is shown below. Figure 1 As shown.

[0054] Example 2 Recombinant strains of Escherichia coli JM109(DE3) pD454-PPR-AmpR or Escherichia coli ArcticExpress (DE3) pD454-PPR-AmpR were obtained.

[0055] To obtain recombinant strains of *Escherichia coli* JM109(DE3) pD454-PPR-AmpR or *Escherichia coli* ArcticExpress(DE3) pD454-PPR-AmpR, circular DNA (SEQ ID NO: 5) of the pD454-PPR-AmpR expression plasmid obtained as described in Example 1 was used to transform *Escherichia coli* JM109(DE3) or *Escherichia coli* ArcticExpress(DE3) cells. Bacterial cells were placed on LB medium (1% peptone; 0.5% yeast extract; 1% NaCl) containing ampicillin (100 μg / ml), and colonies of the obtained recombinant strains of *Escherichia coli* JM109(DE3) pD454-PPR-AmpR or *Escherichia coli* ArcticExpress(DE3) pD454-PPR-AmpR were then used for the biosynthesis of PPR nucleases.

[0056] Example 3 PPR nuclease was obtained from cells containing recombinant strains of Escherichia coli JM109(DE3) pD454-PPR-AmpR or Escherichia coli ArcticExpress (DE3) pD454-PPR-AmpR.

[0057] Recombinant strains of *Escherichia coli* JM109(DE3) pD454-PPR-AmpR or *Escherichia coli* ArcticExpress (DE3) pD454-PPR-AmpR obtained according to Example 2 were cultured at 37°C in LB medium (1% peptone; 0.5% yeast extract; 1% NaCl) containing ampicillin (50 μg / ml) for 16–18 h. Subsequently, overnight cultures were inoculated at a 1:50 ratio with the same medium contents. Culture was continued at 30°C until OD was obtained. 600 (Optical density) = 0.4-0.5, and then PPR nuclease gene expression was induced by adding IPTG to a final concentration of 0.2 mM. The cells were cultured at 18°C ​​for 20-22 h, and then separated from the culture medium by centrifugation. The cell pellet suspended in buffer contained 20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 10 mM imidazole, and 5 mM MgCl2; each 1 g of cell pellet contained at least 5 ml of buffer solution.

[0058] Subsequently, the cell suspension was disintegrated using sonication (3 sonication cycles; energy intensity 100 J / ml suspension). The obtained cell lysates were centrifuged at 16000 RCF to remove insoluble proteins and cell fragments, and then filtered through a 0.2 μM membrane. The PPR nuclease protein was separated from the remaining bacterial proteins using immobilized metal affinity chromatography (IMAC, using a stationary phase with immobilized divalent nickel ions). The PPR nuclease bound to the stationary phase was then eluted with a buffer containing 20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 300 mM imidazole, and 5 mM MgCl2. Under refrigeration, the fraction containing the PPR nuclease was dialyzed against a buffer containing 20 mM Tris-HCl (pH 8.0), 500 mM NaCl, and 5 mM MgCl2 (at least 100 ml per 1 ml of enzyme) for at least 18 hours. The obtained enzyme formulation was mixed with glycerol at a 1:1 ratio and frozen at -20°C. Protein concentration was measured spectrophotometrically at a wavelength of 280 nm.

[0059] Example 4 Test of the enzymatic properties of PPR recombinant nuclease protein.

[0060] For the recombinant PPR nuclease obtained according to Example 3, the specific nucleolysis activity of the relevant nucleic acid necessary to define the optimal conditions for enzyme activity and inactivation was determined.

[0061] To determine the nucleolysis activity of PPR nuclease, serially diluted enzyme was incubated for 10 min in reaction buffer containing 20 mM Tris-HCl (pH 8.0), 20 mM MgCl2, and 1 μg pUC19 plasmid DNA (20 μl volume). The reaction was stopped by adding 5 μl of 25 mM DTT solution to a final concentration of 5 mM, and the sample was heated to 55°C for 10 min. As a control, 1 μg of pUC19 plasmid DNA was incubated without nuclease. Subsequently, the sample was loaded onto a 1% agarose gel, and DNA separation was performed in the gel at 130°C for 40 min. The remaining undegraded DNA in the gel was stained with ethidium bromide and recorded by gel photography. 1 U of activity was determined as the amount of enzyme required to completely degrade 1 μg of pUC19 plasmid DNA within 10 min at 37°C.

[0062] To determine the specific activity of PPR nuclease, serially diluted enzyme was incubated for 30 min at 37°C in a reaction buffer containing 20 mM Tris-HCl (pH 8.0), 20 mM MgCl2, and 100 μg of herring sperm genomic DNA (300 μl volume). The reaction was stopped by adding 300 μl of 4% perchloric acid solution to a final concentration of 2%, and the sample was incubated on ice for 60 min. As a control, 100 μg of DNA was incubated without the addition of nuclease. The sample was then centrifuged for 10 min until the undegraded DNA precipitate was separated. The content of free nucleotides and oligonucleotide fragments smaller than 10 bp in the supernatant was determined by measuring the absorbance at 260 nm. 1 U of nuclease activity was defined as the amount of enzyme that increased the absorbance of the studied sample by 1.0 at 260 nm within 30 min of reaction at 37°C.

[0063] Example 4A Determination of the optimal pH for nuclear dissolution PPR nuclease activity.

[0064] To determine the optimal pH for enzyme nucleolysis activity, the reaction was performed as described in Example 4. 1 U of enzyme solution (pH 6.0–10.0) was added to reaction mixtures containing 0, 250, and 500 mM NaCl, respectively. The PPR nuclease exhibited the highest nucleolysis activity in a buffer solution at pH 8.0 with 500 mM NaCl concentration, as shown in Example 4. Figure 2 As shown.

[0065] Example 4B Determination of the optimal temperature for PPR nuclease nucleolysis activity.

[0066] As described in Example 4, the optimal temperature for nucleolysis activity was determined at different temperatures (from 6°C to 45°C), wherein 1 U of enzyme was contained in a reaction mixture containing 50 mM Tris (pH 8.0) and different concentrations of MgCl2 (5 mM and 100 mM). The PPR nuclease maintained nucleolysis activity throughout the tested temperature range. Figure 3 However, it exhibited the highest activity at 37°C and with high concentrations of NaCl (500 mM) and MgCl2 (100 mM). It should be emphasized that under these conditions, by applying the relevant salt concentrations of NaCl (500 mM) and MgCl2 (100 mM), 100% standard activity can be obtained under refrigeration at 6°C.

[0067] Example 4C Regarding the nucleolysis activity of PPR nuclease, Mg 2+ Determining the optimal concentration of ions.

[0068] To determine Mg 2+ The effect of ion concentration on the nucleolysis activity of PPR, as described in Example 4, is that at different Mg concentrations... 2+ The reaction is carried out in solutions with ion concentrations ranging from 5 mM to 200 mM, wherein the reaction mixture contains 1 U of enzyme. In a slightly alkaline environment (pH 8.0), the PPR nuclease is reacted in a solution containing 150 mM (optimal 50-150 mM) of Mg. 2+ The highest nucleolysis activity was observed in the ion buffer, such as Figure 4 As shown. In a low pH environment (pH 6.5), PPR in the presence of 20 mM Mg (optimal 20-50 mM) 2+ The highest nucleolysis activity was observed in the ion buffer. PPR concentrations showed high specific activity across all observed MgCl2 ranges (5–200 mM).

[0069] Example 4D Confirmation of the effect of potential inhibitors on PPR enzyme activity.

[0070] To determine the enzyme's tolerance to common ionic components present in the reaction buffer used for recombinant protein preparation, nucleolysis reactions were performed in solutions with varying amounts of individual potential inhibitors (NaCl, urea, ammonium sulfate, imidazole), as described in Example 4. The PPR nuclease maintained high nucleolysis activity in the presence of increasing concentrations of the tested substances, as shown in Table 1.

[0071] Table 1. Effects of inhibitors on PPR nucleolysis activity

[0072] Example 4E Heat inactivation of PPR enzyme activity To determine the parameters for PPR nuclease inactivation, a 0.2 ml PCR probe series containing 100 U PPR in 50 μl reaction buffer (as described in Example 4A, except for pUC10 plasmid DNA) containing 5 mM DTT was prepared. The probes were then incubated at the appropriate temperature for 15 minutes, followed by incubation on ice for 5 minutes. As a control, 100 U PPR nuclease in the same buffer was stored on ice. Subsequently, after inactivation as described in Example 4A, a nucleolysis reaction was performed using 5 μl of PPR solution from the previous stage. As controls, plasmid DNA-only (negative control) and plasmid DNA containing 100 U PPR stored on ice (positive control) were incubated under the same conditions as the reaction assay. The extent of plasmid DNA degradation during the assay was analyzed on an agarose gel. Figure 5 As shown, PPR is completely deactivated in the presence of DTT at 52°C or higher.

[0073] Example 4F PPR, HL-SAN, Benzonase ® Determination of nuclease nucleolysis activity in buffer solutions with different salt concentrations and pH values.

[0074] PPR, HL-SAN, and Benzonase were performed under the following conditions. ® Determination of nuclease nucleolysis activity: Different temperatures (6°C, 22°C, and 37°C), different amounts of NaCl added (final concentrations of 0, 250, and 500 mM), pH values ​​of 7.0, 8.0, and 9.0, with the presence of 50 mM Tris and 20 mM MgCl2 (used for Benzonase). ® (5 mM MgCl2). Figure 6 As shown, in buffers with increased salt concentrations (250 and 500 mM NaCl), at pH 7.0, 8.0, and 9.0, PPR nuclease exhibited the highest nucleolysis activity among all tested nucleases (only HL-SAN showed slightly higher activity at 500 mM NaCl and pH 9.0). Regardless of pH, Benzonase... ® Nucleases are practically inactive under any conditions with increased salt concentrations (250, 500 mM NaCl). They also function under lower pH (7.0, 8.0) and ambient temperature (22°C) conditions, in contrast to HL-SAN and Benzonase. ®In comparison, the PPR nuclease exhibited significantly higher activity. Similar correlations were observed at 6°C and 37°C (data not shown).

[0075] Example 4G PPR, HL-SAN, Benzonase ® Determination of nuclease nucleolysis activity in DMEM medium.

[0076] As described in Example 4, PPR, HL-SAN, and Benzonase were performed at different temperatures (6°C, 22°C, and 37°C) but using DMEM medium (commonly used for mammalian cell cultures such as CHO and HEK for the production of recombinant proteins and viral vectors) instead of reaction buffer. ® Determination of the nuclease lysis activity. For example... Figure 7 As shown, PPR nuclease, added directly to DMEM medium at all tested temperatures, exhibited the highest nucleolysis activity among all tested enzymes. At 37°C, PPR's activity was six times that of Benzonase® nuclease. Under refrigerated conditions (6°C) and ambient temperature (22°C), PPR's activity was approximately three times that of the other tested nucleases (i.e., HL-SAN and Benzonase®). Figure 7 Under the conditions recommended by the manufacturer, the activity of each enzyme is assumed to be 100%.

[0077] Example 4H PPR, HL-SAN, Benzonase ® Determination of nuclease nucleolysis activity in PBS and TBS buffers.

[0078] As described in Example 4, PPR, HL-SAN, and Benzonase were performed at 6°C, 22°C, and 37°C using the following buffers commonly used in recombinant protein purification processes: 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), instead of reaction buffers. ® The nuclease nucleolysis activity was determined. Furthermore, the activities of the nucleases in TBS supplemented with 500 mM NaCl were compared. Figure 8As presented, PPR nucleases exhibited the highest nucleolysis activity of all tested enzymes in all tested buffers—PBS, TBS, and TBS with a high salt content (500 mM NaCl)—and at all tested temperatures. Under the manufacturer's recommended conditions, the activity of each enzyme is assumed to be 100% activity. Figure 8 ).

[0079] Example 5 Applications of recombinant PPR nuclease.

[0080] Example 5A Use of PPR nuclease in the production of recombinant UDG enzymes with low host DNA content.

[0081] Following Example 3, the obtained PPR nuclease was used in the purification processes of other recombinant enzymes commonly used in scientific research and molecular diagnostics, particularly in the purification processes of polymerases, ligases, and UDG enzymes containing significantly lower levels of host DNA contamination. The standard protocol for UDG enzyme purification from *E. coli* bacteria was modified to add PPR nuclease to prepared bacterial lysates containing overproduced UDG enzyme as follows: 40 U per 1 ml of lysate, followed by incubation at 20°C–25°C for 1 hour using a magnetic mixer set to 200 rpm. Thus, the lysates were processed according to the standard UDG enzyme procedure. The host DNA contamination level was measured using qPCR (using 16S bacterial-specific primers). Compared to enzymes purified using PPR nuclease, UDG enzymes purified using the additional step (using PPR nuclease) contained 100-fold less host DNA contamination, such as... Figure 9 As shown. The UDG enzyme purified using this method, which is used in scientific research or molecular diagnostics, improves the sensitivity of the method and significantly reduces the risk of potential false positive results.

[0082] Example 5B The use of PPR nuclease in the purification process of monoclonal antibodies in mammalian cells to remove DNA contamination.

[0083] The PPR nuclease obtained as described in Example 3 was used to remove DNA contaminants from the purification process of recombinant monoclonal antibodies isolated from Chinese hamster ovary (CHO) cells. Mammalian cells were cultured in the appropriate medium for 5 days. The cells were then separated by centrifugation, and the supernatant was used to purify the antibody using standard chromatography. In addition to the antibody and medium components, the medium contained a significant amount of genomic DNA derived from host cells degraded during culture. The initial stage of incubation in the post-culture medium containing the PPR nuclease significantly reduced the amount of DNA contamination in the medium, thereby improving the efficiency of antibody binding to fixation. A stage of post-culture medium incubation at 20°C–22°C for 60 minutes by adding PPR nuclease to 50 U / ml of medium was introduced into the standard antibody purification process. After this time, DNA was isolated from 1 ml of medium treated with and untreated with PPR nuclease using a genomic DNA isolation kit. All the obtained DNA was loaded onto a 1% agarose gel, and ethidium bromide was added to visualize the nucleic acids. The medium was then subjected to the standard antibody purification procedure. As a control, the culture medium was incubated under the same conditions (but without the addition of PPR nuclease). Figure 10 As shown, the addition of PPR nuclease significantly reduced genomic DNA contamination in the culture medium after culture, which accumulates during the growth of cells to which cetuximab and bevacizumab antibodies are secreted into the culture medium. sequence list <110> Britt Corporation <120> Novel nonspecific, heat-labile nucleases active at low temperatures, over a wide pH range, and at high salt concentrations. <130> 4PCT / MM / 2020 <140> PCT / PL2020 / 000076 <141> 2020-09-14 <150> P.431144 <151> 2019-09-13 <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 846 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence <400> 1 caggcgctct tctatgaaat atctgctgcc gaccgcagca gcgggtctgc tgctgctggc agcacagcct gcaatggcag catccatgga gcatcaccat catcatcatg gtagcgaaaa tctgtatttt cagtccgcac cgcctagcag ctttagcaaa gcaaaacgtc tggcagttga aatctatcag gatcatccga ccagcttttta ttgtggttgt gcaattacct ggcagggtaa aaaaggtctg ccggatctgg caagctgtgg ttatgaagtt cgtaaacaag aaaaaacgtgc caaccgtatt gaatgggaac atgttgttcc ggcagaaaat tttggtcgtg catttgttga atggcgtgaa ggtcatccgc agtgtgttaa tagcaaaggt aaagcatata aaggtcgtag ctgtgccacc aaaatgaatc cgacctatcg tagcatgcag gccgatctgc ataatctgac accggcagtt ggtgaagtta atggtgatcg tagcaattat gcatttctgc cgtggaatgg fathercggt gcattttatg gtcagtgcga fathercggtc gacttcaaaa atcgtcgtgc agatcctccg gaacagagcc gtggtgcaat tgcccgtacc tatctgtata tgaatcaaga gtataaaatg gccctgagca gccagcagcg tcagctgatg gaagcatgga atcgtcagta 720 tccgattagc acctgggaat gtgaacgtga tcgtcgtatt gcaaatatcc aggataacca 780 taacagcttt gttctgcagg catgtaaaaa agccggttat tgagtcgact ggtagaagag 840 cgctgc 846 <210> 2 <211> 269 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 2 Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala Met Ala Ala Ser Met Glu His His His His His His 20 25 30 Gly Ser Glu Asn Leu Tyr Phe Gln Ser Ala Pro Pro Ser Ser Phe Ser 35 40 45 Lys Ala Lys Arg Leu Ala Val Glu Ile Tyr Gln Asp His Pro Thr Ser 50 55 60 Phe Tyr Cys Gly Cys Ala Ile Thr Trp Gln Gly Lys Lys Gly Leu Pro 65 70 75 80 Asp Leu Ala Ser Cys Gly Tyr Glu Val Arg Lys Gln Glu Lys Arg Ala 85 90 95 Asn Arg Ile Glu Trp Glu His Val Val Pro Ala Glu Asn Phe Gly Arg 100 105 110 Ala Phe Val Glu Trp Arg Glu Gly His Pro Gln Cys Val Asn Ser Lys 115 120 125 Gly Lys Ala Tyr Lys Gly Arg Ser Cys Ala Thr Lys Met Asn Pro Thr 130 135 140 Tyr Arg Ser Met Gln Ala Asp Leu His Asn Leu Thr Pro Ala Val Gly 145 150 155 160 Glu Val Asn Gly Asp Arg Ser Asn Tyr Ala Phe Leu Pro Trp Asn Gly 165 170 175 Asn Asn Gly Ala Phe Tyr Gly Gln Cys Asp Met Lys Ile Asp Phe Lys 180 185 190 Asn Arg Arg Ala Asp Pro Pro Glu Gln Ser Arg Gly Ala Ile Ala Arg 195 200 205 Thr Tyr Leu Tyr Met Asn Gln Glu Tyr Lys Met Ala Leu Ser Ser Gln 210 215 220 Gln Arg Gln Leu Met Glu Ala Trp Asn Arg Gln Tyr Pro Ile Ser Thr 225 230 235 240 Trp Glu Cys Glu Arg Asp Arg Arg Ile Ala Asn Ile Gln Asp Asn His 245 250 255 Asn Ser Phe Val Leu Gln Ala Cys Lys Lys Ala Gly Tyr 260 265 <210> 3 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 3 Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala Met Ala 20 <210> 4 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 4 Ala Ser Met Glu His His His His His Gly Ser Glu Asn Leu Tyr 1 5 10 15 Phe Gln Ser <210> 5 <211> 4824 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence <400> 5 tctagaaata attttgttta actttttgag accttaagga ggtaaaaaat gaaatatctg 60 ctgccgaccg cagcagcggg tctgctgctg ctggcagcac agcctgcaat ggcagcatcc 120 atggagcatc accatcatca tcatggtagc gaaaatctgt attttcagtc cgcaccgcct 180 agcagcttta gcaaagcaaa acgtctggca gttgaaatct atcaggatca tccgaccagc 240 ttttattgtg gttgtgcaat tacctggcag ggtaaaaaag gtctgccgga tctggcaagc 300 tgtggttatg aagttcgtaa acaagaaaaa cgtgccaacc gtattgaatg ggaacatgtt 360 gttccggcag aaaattttgg tcgtgcattt gttgaatggc gtgaaggtca tccgcagtgt 420 gttaatagca aaggtaaagc atataaaggt cgtagctgtg ccaccaaaat gaatccgacc 480 tatcgtagca tgcaggccga tctgcataat ctgacaccgg cagttggtga agttaatggt 540 gatcgtagca attatgcatt tctgccgtgg aatggtaata acggtgcatt ttatggtcag 600 tgcgatatga aaatcgactt caaaaatcgt cgtgcagatc ctccggaaca gagccgtggt 660 gcaattgccc gtacctatct gtatatgaat caagagtata aaatggccct gagcagccag 720 cagcgtcagc tgatggaagc atggaatcgt cagattccga ttagcacctg ggaatgtgaa 780 cgtgatcgtc gtattgcaaa tatccaggat aaccataaca gctttgttct gcaggcatgt 840 aaaaaagccg gttatgagt cgactggttg aggtctcacc ccctagcata accccttggg 900 gcccttaaac gggtcttgag gggttttttg cccctgagac gcgtcaatcg agttcgtacc 960 taagggcgac acccccctaat tagcccgggc gaaaggccca gtctttcgac tgagcctttc 1020 gttttatttg atgcctggca gttccctact ctcgcatggg gagtccccac actaccatcg 1080 gcgctacggc gtttcacttc tgagttcggc atggggtcag gtgggaccac cgcgctactg 1140 ccgccaggca aacaaggggt gttatgagcc atattcaggt ataaatgggc tcgcgataat 1200 gttcagaatt ggttaattgg ttgtaacact gacccctatt tgtttatttt tctaaataca 1260 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa 1320 aaagaat atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt 1380 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca 1440 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag 1500 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc 1560 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca 1620 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt 1680 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct 1740 gacaacgatc ggaggaccga aggagctaac cgctttttg cacaacatgg gggatcatgt 1800 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga 1860 caccacgatg cctgtagcga tggcaacaac gttgcgcaaa ctattaactg gcgaactact 1920 tactctagct tcccggcaac attaataga ctggatggag gcggataaag ttgcaggacc 1980 acttctgcgc tcggcccttc cggctggctg gttattgct gataaatccg gagccggtga 2040 gcgtggttct cgcggtatca tcgcagcgct ggggccagat ggtaagccct cccgtatcgt 2100 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaatagac agatcgctga 2160 gataggtgcc tcactgatta agcattggta agcggcgcgc catcgaatgg cgcaaaacct 2220 ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatatgaaac 2280 cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc tttcagacc gtttcccgcg 2340 tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg 2400 cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc 2460 tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga 2520 ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg 2580 gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga 2640 tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg 2700 ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt attttctccc 2760 atgaggacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg 2820 cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata 2880 aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca 2940 tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc 3000 tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc 3060 gcgttggtgc ggatatctcg gtagtgggat acgacgatac cgaagatagc tcatgttata 3120 tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc 3180 gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg ccagtctcac 3240 tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg 3300 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgactca 3360 tgaccaaaat cccttaacgt gagttacgcg cgcgtcgttc cactgagcgt cagaccccgt 3420 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 3480 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 3540 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta 3600 gccgtagtta gcccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 3660 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 3720 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 3780 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 3840 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 3900 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 3960 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 4020 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 4080 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt 4140 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga 4200 ggaagcggaa ggcgagagta gggaactgcc aggcatcaaa ctaagcagaa ggcccctgac 4260 ggatggcctt tttgcgtttc tacaaactct ttctgtgttg taaaacgacg gccagtctta 4320 agctcgggcc ccctgggcgg ttctgataac gagtaatcgt taatccgcaa ataacgtaaa 4380 aacccgcttc ggcgggtttt tttatggggg gagtttaggg aaagagcatt tgtcagaata 4440 tttaagggcg cctgtcactt tgcttgatat atgagaatta tttaacctta taaatgagaa 4500 aaaagcaacg cactttaaat aagatacgtt gctttttcga ttgatgaaca cctataatta 4560 aactattcat ctattattta tgatttttg tatatacaat atttctagtt tgttaaagag 4620 attaagaaa ataaatctcg aaaataataa agggaaaatc agttttgat atcaaaatta 4680 tacatgtcaa cgataataca aaatataata caaactataa gatgttatca gtatttatta 4740 tcatttagaa taaatttgt gtcgcccttc cgcgaaatta atacgactca ctatagggga 4800 attgtgagcg gataacaatt cccc 4824

Claims

1. A PPR nuclease, characterized in that, The amino acid sequence of the PPR nuclease is SEQ ID NO:

2.

2. The PPR nuclease according to claim 1, wherein the PPR nuclease is irreversibly inactivated after incubation, the incubation comprising: Incubate at 52°C or above for 15 minutes in the presence of 1-5 mM DTT.

3. The PPR nuclease according to claim 1, characterized in that, The PPR nuclease is active at salt concentrations within the following ranges: NaCl: 0-1400 mM MgCl2: 5-200 mM Urea: 0-6000 mM Ammonium sulfate: 0-200 mM Imidazole: 0-400 mM.

4. A compound, characterized in that, Contains the PPR nuclease according to claim 1 and a salt in one or more concentrations selected from the following: NaCl: 0-1400 mM MgCl2: 5-200 mM Urea: 0-6000 mM Ammonium sulfate: 0-200 mM, and Imidazole: 0-400 mM.

5. A nucleic acid sequence encoding the PPR nuclease according to claim 1.

6. An expression plasmid having the nucleic acid sequence shown in SEQ ID NO: 5, comprising the nucleic acid sequence according to claim 5, wherein the expression plasmid further comprises: a T7 phage promoter.

7. A recombinant Escherichia coli strain, wherein the recombinant Escherichia coli strain is transformed with Escherichia coli JM109 (DE3) or Escherichia coli ArcticExpress (DE3) using the expression plasmid according to claim 6, thereby causing the recombinant Escherichia coli strain to contain the expression plasmid.

8. A method for producing PPR nuclease according to claim 1, characterized in that, The recombinant Escherichia coli strain of claim 7 was cultured in a culture medium, and then PPR nuclease gene expression was induced by adding IPTG, and the protein was isolated and purified.

9. A method for isolating and purifying the PPR nuclease according to claim 1, characterized in that, The nuclease is expressed in a suitable host cell and isolated from the host cell and / or the culture medium of the cultured cell.

10. Use of the PPR nuclease according to claim 1 in the removal of DNA contaminants in a monoclonal antibody purification process for mammalian cells.

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