A method for industrial separation and purification of DNase I

Abstract

The present invention provides an industrial separation and purification method of DNaseⅠ, belonging to the field of biotechnology. The industrial separation and purification method of DNaseⅠ of the present invention comprises the steps of centrifuging a cell fermentation liquid or a cell lysate expressing a recombinant DNaseⅠ gene to obtain a supernatant, concentrating and ultrafiltration to replace the liquid, anion exchange chromatography, affinity chromatography, composite chromatography and post-treatment. The separation and purification method of the present invention has simple steps, high yield, low cost, and is easy to industrially scale up production. The purity of the recombinant DNaseⅠ obtained by separation and purification is higher than 95%, the specific activity of the enzyme is higher than 10KU / mg, the endotoxin residue is less than 1EU / mg, and there is no RNase contamination. It can be well applied to DNA removal, DNA labeling by nick translation method, DNaseⅠ footprint analysis experiment, etc. It is widely used and has good economic value and social significance.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an industrial separation and purification method for DNase I. Background Art

[0002] DNase I (Deoxyribonuclease I) is a phosphodiesterase that can hydrolyze deoxyribonucleic acid (DNA). It has a molecular weight of about 39 kD and was originally isolated from bovine pancreas. Compared with restriction endonucleases that need to recognize specific sequences, DNase I can non-specifically degrade DNA. The optimal working pH range of DNase I is 7-8, and its activity depends on Ca. 2+ , and can be oxidized by divalent metal ions such as Co 2+ , Mn 2+ , Zn 2+ DNase I is widely used in the preparation of DNA-free RNA; removal of template DNA after in vitro transcription; preparation of DNA-free RNA before RT-PCR and RT-qPCR reactions; DNA labeling by nick shift in combination with DNA polymerase I; and construction of DNA fragmentation libraries.

[0003] mRNA vaccine technology is one of the new vaccine technologies that has gradually developed in recent years. mRNA vaccines express antigens in cells and can prevent infectious diseases by stimulating the immune system to produce B and T cell immune responses and other effector mechanisms. The research and development and production of mRNA vaccines require the participation of a series of enzymes: T7 RNA polymerase, inorganic pyrophosphatase, RNase inhibitor, capping enzyme, methyltransferase, Poly (A) polymerase, DNase I and other 7 enzymes and nucleotide substrates. The residual DNA is strictly controlled during the production of mRNA vaccines. The residual DNA can be removed by DNase I, which can reduce the difficulty of downstream purification and increase the purity of the product. However, the production process of mRNA vaccines has high requirements for the residual endotoxin and RNase contamination in the enzyme raw materials. Therefore, it is of great economic value and social significance to study a DNase I purification method with low toxin residues, no RNase contamination and industrial preparation. Summary of the invention

[0004] Based on this, it is necessary to provide an industrial separation and purification method for DNaseⅠ, so that the recombinant DNaseⅠ obtained by separation and purification has high purity, good activity, no RNase contamination, and extremely low endotoxin content.

[0005] An industrial separation and purification method for DNase I comprises the following steps:

[0006] S1: Take the cell fermentation liquid or cell lysate expressing the recombinant DNaseⅠ gene and centrifuge to obtain the supernatant, and obtain the crude enzyme solution of the recombinant DNaseⅠ;

[0007] S2: concentrating the clarified solution of the crude enzyme solution of the recombinant DNaseⅠ, and replacing the solution with ultrafiltration to obtain a one-step loading solution;

[0008] S3: taking the one-step loading solution and using anion exchange chromatography to enrich the recombinant DNaseⅠ, thereby increasing the specific activity and reducing the content of endotoxin, and obtaining an anion chromatography eluate;

[0009] S4: taking the anion chromatography eluate and performing affinity chromatography to further purify the recombinant DNase I to obtain an affinity chromatography eluate;

[0010] S5: taking the affinity chromatography eluate and performing composite chromatography to further purify the recombinant DNase I and remove RNase to obtain a composite chromatography eluate;

[0011] S6: taking the composite chromatography eluate for post-processing to obtain separated and purified recombinant DNase I.

[0012] Preferably, in step S2, a low-salt buffer is used for multiple ultrafiltration replacements to reduce the ionic strength of the clarified solution of the crude enzyme solution of the recombinant DNaseⅠ, which is beneficial for the recombinant DNaseⅠ to bind to the anion exchange medium; wherein the low-salt buffer is: 0.02-0.05M Tris-HCl, 0-10% glycerol, 0-0.2% Tween 20, pH 6.8-8.5.

[0013] Preferably, the anion exchange chromatography in step S3 is specifically:

[0014] Regeneration: Use not less than 0.5M NaOH to flush the column at a flow rate of 80-300cm / h for at least 30min to sterilize and remove pyrogens;

[0015] Balance: Use low salt buffer to balance the column bed at a flow rate of 80-300 cm / h for 10 to 20 column volumes;

[0016] Sampling: taking the one-step loading solution and loading it onto the ion column at a flow rate of 80-300 cm / h;

[0017] Re-balance: Continue to use low-salt buffer at a flow rate of 80-300 cm / h to re-balance the column after loading for 5 to 10 column volumes;

[0018] Washing: using a buffer with a specific salt concentration at a flow rate of 80-300 cm / h to wash the column bed for 5 to 10 times the column volume; wherein the buffer with a specific salt concentration is: 0.05-0.15M NaCl, 0.02-0.05M Tris-HCl, 0-10% glycerol, 0-0.2% Tween 20, pH 6.8-8.5;

[0019] Elution: flushing the column bed with an eluent of a specific salt concentration at a flow rate of 80-300 cm / h for 5 to 10 times the column volume; wherein the eluent of a specific salt concentration is: 0.2-0.4 M NaCl, 0.02-0.05 M Tris-HCl, 0-10% glycerol, 0-0.2% Tween 20, pH 6.8-8.5;

[0020] CIP: The column was cleaned with 2M NaCl and 0.5-1M NaOH.

[0021] Preferably, the anion exchange chromatography column comprises a strong anion exchanger, the anion exchanger comprises a strong anion exchanger Q type and a weak anion exchanger DEAE type, the strong anion exchanger comprises Q Bestarose XL, QBestarose FF, Q Sepharose FF, Q Sepharose HP, Q Seplife FF, UniGel-80Q and Nuvia QAnion Exchange Media; the weak anion exchanger comprises DEAE Sepharose FF, DEAE SepharoseHP, DEAE Seplife.

[0022] Preferably, the affinity chromatography in step S4 is specifically as follows:

[0023] Regeneration: Use 0.1M EDTA to wash the column to remove metal ions, then use 0.5M NaOH to wash the column for at least 30 minutes to sterilize and remove pyrogens, then use 2M NaCl to wash the column for 5 column volumes, then use pure water to wash the column to pH below 10.0, then use 0.5M metal ion soluble salt to wash the column for 5 column volumes to achieve metal ion ligand attachment, and then use pure water to wash the column for 10 column volumes;

[0024] Balance: Use high salt, low imidazole buffer to balance the column bed at a flow rate of 80-400 cm / h for 10 to 20 times the column volume; wherein the high salt, low imidazole buffer is: 0.5M NaCl, 0.005-0.03M imidazole, 0.02-0.05M Tris-HCl, 0% to 10% glycerol, 0-0.2% Tween 20, pH 7.0-8.0;

[0025] Loading: Take 0.5M imidazole and 2M NaCl mother solutions and add them to the anion chromatography eluent respectively, so that the final concentration of NaCl in the eluent is 0.3-0.5M, and the final concentration of imidazole is consistent with that in the high-salt, low-imidazole buffer. After fully mixing, perform metal column affinity chromatography loading at a flow rate of 80-400cm / h;

[0026] Re-equilibration: After loading, continue to flush the column bed with high-salt, low-imidazole buffer at a flow rate of 80-400 cm / h for 5-6 column volumes;

[0027] Washing: Use a high-salt washing buffer containing a certain amount of surfactant to wash the column bed at a flow rate of 80-400 cm / h for 5 to 10 times the column volume; wherein the high-salt washing buffer containing a certain amount of surfactant is: 0.8-1.5M NaCl, 2% Triton X 100, 0.02-0.05M Tris-HCl, pH 6.8-8.5;

[0028] Re-balance: After washing, continue to flush the column bed with high-salt, low-imidazole buffer at a flow rate of 80-400 cm / h for 5-6 column volumes;

[0029] Elution: using an elution buffer with a specific imidazole concentration to elute the chromatography column bed at a flow rate of 80-400 cm / h for 5 to 10 times the column volume; wherein the elution buffer with a specific imidazole concentration is 0.05-0.2 M imidazole, 0.2-0.5 M NaCl, 0.02-0.05 M Tris-HCl, 0-10% glycerol, 0-0.2% Tween 20, pH 6.8-8.5;

[0030] CIP: The chromatography column was cleaned with 0.5 M imidazole.

[0031] Preferably, the affinity chromatography is metal ion chelate His chromatography, and the metal ion is a transition state metal ion capable of providing an electron donor coordination site, including Cu 2+ 、Co 2+ 、Ni 2+ 、Zn 2+ , Fe 2+ and Ca 2+ .

[0032] Preferably, the affinity chromatography is Ni affinity chromatography using Ni NTA or Ni TED type, and the types of Ni chromatography columns include Ni Sepharose excel, Ni Sepharose FF, Ni Bestarose FF, UniNTA-80Ni and Ni NTA Beads6FF.

[0033] Preferably, the step S5 composite chromatography is specifically:

[0034] Regeneration: Sterilize and remove pyrogens by flushing the column with 0.5 M NaOH for at least 30 min;

[0035] Balance: Use low-salt buffer to balance the chromatography column bed for 10 to 20 times column volume at a flow rate of 50 to 400 cm / h; wherein the low-salt buffer includes 0.02 to 0.05 M Tris-HCl, 0 to 10% glycerol, 0 to 0.2% Tween 20, pH 6.8 to 8.5;

[0036] Loading: diluting the affinity chromatography eluate with a low-salt buffer, with the dilution end point being a conductivity range of 5 to 10 mS / cm, and performing composite chromatography loading at a flow rate of 50 to 400 cm / h;

[0037] Re-balance: Use low-salt buffer to balance the chromatography column bed for 5 to 6 column volumes at a flow rate of 50-400 cm / h;

[0038] Washing impurities: using a buffer with a specific salt concentration to wash the column bed at a flow rate of 50-400 cm / h for 5 to 10 times the column volume, and collecting the washing impurity peak; wherein the buffer with a specific salt concentration is: 0-0.2M NaCl or KCl, 0.02-0.05M Tris-HCl, 0-10% glycerol, 0-0.2% Tween 20, pH 6.8-8.5;

[0039] Elution: using an eluent with a specific salt concentration to elute the column bed at a flow rate of 50-400 cm / h for 5 to 10 times the column volume, and collecting the elution peak; wherein the eluent with a specific salt concentration is: 0.4-0.5 M NaCl or KCl, 0.02-0.05 M Tris-HCl, 0-10% glycerol, 0-0.2% Tween 20, pH 6.8-8.5;

[0040] CIP: The column was cleaned with 2M NaCl and 0.5-1M NaOH.

[0041] Preferably, the composite chromatography condition is a hydrophobic and ionic composite mode filler, and the composite filler includes MMC HF, MMC FF, MMC Bestarose 6FF, Capto MMC, Diamond MMC and NM90 Agarose.

[0042] Preferably, the post-processing of step S6 is specifically as follows:

[0043] Membrane package pyrogen removal treatment: according to the sample, take an ultrafiltration membrane package of appropriate area, the molecular weight cutoff of the ultrafiltration membrane package is not more than 10KD, use 0.5M NaOH to rinse the ultrafiltration membrane package for at least 30 minutes to disinfect and remove pyrogens, and then rinse the membrane package with ultrapure water until the pH is neutral;

[0044] Membrane pack rinsing: Use replacement buffer to rinse the membrane pack as needed until the filtrate pH is 7.6±0.5, ready to start concentration; wherein, the replacement buffer is: 0.02M Tris-HCl, 4mM CaCl2, pH7.6±0.05;

[0045] Concentration: Place the membrane package inlet and the interception end into the composite chromatography eluent for concentration. During the concentration process, the recombinant DNaseⅠ concentration is measured by nano at any time. The recombinant DNaseⅠ concentration is required to be no more than 20 mg / mL. When the concentration is completed, the sample volume is recorded as V, which includes the membrane package volume.

[0046] Replacement: add 3-5 times V replacement buffer for replacement, the volume is concentrated to the original volume V for the first time, the replacement times are 3-5 times, the replacement end point is pH 7.6±0.5, the deviation between conductivity and replacement buffer is no more than 1mS / cm, and the ultrafiltration replacement semi-finished product is obtained;

[0047] Preparation: Accurately measure a certain volume of the ultrafiltration replacement semi-finished product, add glycerol in a volume ratio of 1:1, mix thoroughly and filter through 0.22 μm to obtain the separated and purified recombinant DNase I finished product.

[0048] The industrial separation and purification method of DNaseⅠ of the present invention has simple steps, high yield and low cost, and is easy to be industrially scaled up for production. The purity of the recombinant DNaseⅠ obtained by separation and purification is higher than 95%, the specific activity of the enzyme is higher than 10KU / mg, the endotoxin residue is less than 1EU / mg, and there is no RNase pollution. The method can be well applied to DNA removal (such as removal of gDNA before RNA extraction or reverse transcription, removal of template DNA by in vitro transcription, and rRNA removal: applied to RNA library construction and sequencing), DNA labeling by gap translation method, DNaseⅠ footprint analysis experiment, etc., and is widely applied and has good economic value and social significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 It is a schematic diagram of the process of industrial separation and purification of DNaseⅠ of the present invention;

[0050] Figure 2 This is an SDS-PAGE image of the DNase I purification process of the 500 mL-fermentation supernatant in Example 1 of the present invention;

[0051] Figure 3 This is an SDS-PAGE image of the DNase I purification process of the 10L-fermentation supernatant in Example 2 of the present invention;

[0052] Figure 4The SDS-PAGE images of the finished recombinant DNaseⅠ obtained in Example 1 and Example 2 of the present invention; in the figure, 1 is the SDS-PAGE image of the finished recombinant DNaseⅠ of Example 1, and 2 is the SDS-PAGE image of the finished recombinant DNaseⅠ of Example 2;

[0053] Figure 5 A graph showing the results of RNase residue detection of the recombinant DNaseⅠ finished products obtained in Example 1 and Example 2 and DNaseⅠ products labeled RNasefree from three different manufacturers on the market; in the graph, 1 and 2 represent the results of Example 1, 3 and 4 represent the results of Example 2, 5 and 6 represent market sample 1, 7 and 8 represent market sample 2, 9 and 10 represent market sample 3, and 11 represents a negative control. DETAILED DESCRIPTION

[0054] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation of the present invention is described in detail below in conjunction with specific embodiments and drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

[0055] like Figure 1 As shown, the present invention provides an industrial separation and purification method for DNase I, comprising the following steps:

[0056] S1: Take the cell fermentation liquid or cell lysate expressing the recombinant DNaseⅠ gene and centrifuge to obtain the supernatant, and obtain the crude enzyme solution of the recombinant DNaseⅠ;

[0057] S2: concentrating the clarified solution of the crude enzyme solution of the recombinant DNaseⅠ, and replacing the solution with ultrafiltration to obtain a one-step loading solution;

[0058] S3: taking the one-step loading solution and using anion exchange chromatography to enrich the recombinant DNaseⅠ, thereby increasing the specific activity and reducing the content of endotoxin, and obtaining an anion chromatography eluate;

[0059] S4: taking the anion chromatography eluate and performing affinity chromatography to further purify the recombinant DNase I to obtain an affinity chromatography eluate;

[0060] S5: taking the affinity chromatography eluate and performing composite chromatography to further purify the recombinant DNase I and remove RNase to obtain a composite chromatography eluate;

[0061] S6: taking the composite chromatography eluate for post-processing to obtain separated and purified recombinant DNase I.

[0062] The recombinant plasmid carrying the DNaseⅠ gene fragment is transformed into a host cell for induced expression culture, and a cell strain of recombinant DNaseⅠ is obtained. The expression host includes fungal cells and bacterial cells. The cell culture fluid is a cell fermentation fluid of the target protein secretion type produced by the growth of fungal cells and bacterial cells in the culture medium. The cell lysate is a cell body produced by the growth of fungal cells and bacterial cells in the culture medium, and the target protein is expressed intracellularly. After resuspending in a lysis buffer, the cells are broken by high-pressure homogenization or ultrasonic disruption to release the cell contents. Among them, the buffer includes a lysis buffer, which is a pH 6.8-8.5 buffer system, such as a disodium hydrogen phosphate-citric acid buffer system, a Tris-HCl buffer system, a barbiturate buffer system, and a phosphate buffer system, preferably a Tris-HCl (pH 7.5) buffer system.

[0063] Since the cell fermentation liquid or cell lysate has high ionic strength and complex components, the crude enzyme solution of recombinant DNase I is ultrafiltered and replaced multiple times with a low-salt buffer in step S2 to reduce the ionic strength of the crude enzyme solution of recombinant DNase I, which is beneficial for the recombinant DNase I to bind to the anion exchange medium. The low-salt buffer comprises: 0.02-0.05M Tris-HCl (pH 6.8-8.5), 0-10% glycerol, 0-0.2% Tween 20; preferably 0.02M Tris-HCl (pH 6.8-8.5), 0-10% glycerol, 0-0.2% Tween 20.

[0064] In some embodiments, the anion exchange chromatography step S3 is specifically:

[0065] Regeneration: Use not less than 0.5M NaOH to flush the column at a flow rate of 80-300cm / h for at least 30min to sterilize and remove pyrogens;

[0066] Balance: Use low salt buffer to balance the column bed at a flow rate of 80-300 cm / h for 10 to 20 times the column volume; wherein the low salt buffer includes 0.02-0.05M Tris-HCl (pH 6.8-8.5), 0% to 10% glycerol, 0-0.2% Tween 20, and the preferred equilibrium flow rate is 140 cm / h;

[0067] Loading: taking the one-step loading solution and loading it on the ion column at a flow rate of 80-300 cm / h, preferably the loading flow rate is 100 cm / h;

[0068] Re-equilibration: Continue to use low-salt buffer to re-equilibrate the chromatography column after loading for 5 to 10 column volumes at a flow rate of 80-300 cm / h, preferably the re-equilibration flow rate is 100 cm / h;

[0069] Washing: using a buffer with a specific salt concentration at a flow rate of 80-300 cm / h to wash the column bed for 5 to 10 times the column volume; wherein the buffer with a specific salt concentration is: 0.05-0.15M NaCl, 0.02-0.05M Tris-HCl (pH6.8-8.5), 0-10% glycerol, 0-0.2% Tween 20; preferably, the washing flow rate is 140 cm / h;

[0070] Elution: using an eluent of a specific salt concentration to flush the column bed at a flow rate of 80-300 cm / h for 5 to 10 times the column volume; wherein the eluent of the specific salt concentration is: 0.2-0.4 M NaCl, 0.02-0.05 M Tris-HCl (pH 6.8-8.5), 0-10% glycerol, 0-0.2% Tween 20; preferably, the elution flow rate is 140 cm / h;

[0071] CIP: The column was cleaned with 2M NaCl and 0.5-1M NaOH.

[0072] Among them, the anion exchange chromatography column includes Q strong anion exchangers and DEAE weak anion exchangers with different resolutions, such as Q Bestarose XL, Q Bestarose FF, Q Sepharose FF, Q Sepharose HP, Q SeplifeFF, DEAE Sepharose HP, DEAE Seplife, UniGel-80Q, Nuvia Q Anion Exchange Media, etc., preferably Q Sepharose FF.

[0073] In some embodiments, the affinity chromatography step S4 is specifically:

[0074] Regeneration: Use 0.1M EDTA to wash the column to remove metal ions, then use 0.5M NaOH to wash the column for at least 30 minutes to sterilize and remove pyrogens, then use 2M NaCl to wash the column for 5 column volumes, then use pure water to wash the column until the pH is below 10.0, then use 0.5M metal ion soluble salt (such as NiSO4) to wash the column for 5 column volumes to achieve metal ion ligand attachment, and after completion, use pure water to wash the column for 10 column volumes;

[0075] Balance: Use high salt, low imidazole buffer to balance the column bed at a flow rate of 80-400 cm / h for 10 to 20 times the column volume; wherein the high salt, low imidazole buffer includes: 0.5M NaCl, 0.005-0.03M imidazole, 0.02-0.05M Tris-HCl (pH7.0-8.0), 0%-10% glycerol, 0-0.2% Tween 20; preferably, the equilibrium flow rate is 180 cm / h;

[0076] Loading: Take 0.5M imidazole and 2M NaCl mother solutions and add them to the anion chromatography eluent respectively, so that the final concentration of NaCl in the eluent is 0.3-0.5M, and the final concentration of imidazole is consistent with that in the high-salt, low-imidazole buffer. After fully mixing, perform metal column affinity chromatography loading at a flow rate of 80-400cm / h, preferably at a loading flow rate of 150cm / h;

[0077] Re-equilibration: After loading, continue to flush the column bed with high-salt, low-imidazole buffer at a flow rate of 80-400 cm / h for 5-6 column volumes;

[0078] Washing: Use a high-salt washing buffer containing a certain amount of surfactant to wash the column bed at a flow rate of 80-400 cm / h for 5 to 10 times the column volume; wherein the high-salt washing buffer containing a certain amount of surfactant is: 0.8-1.5M NaCl, 2% Triton X 100, 0.02-0.05M Tris-HCl (pH6.8-8.5); the preferred washing flow rate is 180 cm / h;

[0079] Re-balance: After washing, continue to flush the column bed with high-salt, low-imidazole buffer at a flow rate of 80-400 cm / h for 5-6 column volumes;

[0080] Elution: using an elution buffer with a specific imidazole concentration to elute the chromatography column bed at a flow rate of 80-400 cm / h for 5 to 10 times the column volume; wherein the elution buffer with a specific imidazole concentration is 0.05-0.2 M imidazole, 0.2-0.5 M NaCl, 0.02-0.05 M Tris-HCl (pH 6.8-8.5), 0-10% glycerol, 0-0.2% Tween 20; preferably, the elution flow rate is 180 cm / h;

[0081] CIP: The chromatography column was cleaned with 0.5 M imidazole.

[0082] The affinity chromatography is metal ion chelating His chromatography, the purification tag of the fusion protein is His, and the metal ion is a transition state metal ion that can provide an electron donor coordination site, including Cu 2+ 、Co 2+ 、Ni 2+ 、Zn 2+ , Fe2+ and Ca 2+ etc.; preferably Ni 2+ Ni affinity chromatography, using Ni NTA and Ni TED types with different resolutions, such as Ni Sepharose excel, Ni Sepharose FF, Ni Bestarose FF, UniNTA-80Ni and Ni NTA Beads 6FF, etc., preferably Ni Sepharose FF.

[0083] In some embodiments, the step S5 composite chromatography is specifically:

[0084] Regeneration: Sterilize and remove pyrogens by flushing the column with 0.5 M NaOH for at least 30 min;

[0085] Balance: Use low-salt buffer to balance the chromatography column bed for 10 to 20 times the column volume at a flow rate of 50-400 cm / h; wherein the low-salt buffer includes 0.02-0.05M Tris-HCl (pH 6.8-8.5), 0-10% glycerol, and 0-0.2% Tween 20; the preferred balance flow rate is 130 cm / h;

[0086] Loading: diluting the affinity chromatography eluate with a low-salt buffer, the dilution end point is a conductivity range of 5 to 10 mS / cm, and performing composite chromatography loading at a flow rate of 50 to 400 cm / h, preferably a loading flow rate of 120 cm / h;

[0087] Re-balance: Use low-salt buffer to balance the chromatography column bed for 5 to 6 column volumes at a flow rate of 50-400 cm / h;

[0088] Washing impurities: using a buffer with a specific salt concentration to wash the column bed at a flow rate of 50-400 cm / h for 5 to 10 times the column volume, and collecting the washing impurity peak; wherein the buffer with a specific salt concentration: 0 to 0.2M NaCl or KCl, 0.02-0.05M Tris-HCl (pH 6.8 to 8.5), 0 to 10% glycerol, 0 to 0.2% Tween 20; preferably, the washing impurity flow rate is 130 cm / h;

[0089] Elution: using an eluent with a specific salt concentration to elute the column bed at a flow rate of 50-400 cm / h for 5 to 10 times the column volume, and collecting the elution peak; wherein the eluent with a specific salt concentration is: 0.4-0.5 M NaCl or KCl, 0.02-0.05 M Tris-HCl (pH 6.8-8.5), 0-10% glycerol, 0-0.2% Tween 20; preferably, the elution flow rate is 130 cm / h;

[0090] CIP: The column was cleaned with 2M NaCl and 0.5-1M NaOH.

[0091] The conditions for composite chromatography include composite fillers with different resolutions, such as MMC HF, MMC FF, MMCBestarose 6FF, Capto MMC, Diamond MMC and NM90 Agarose, etc., preferably MMC Bestarose 6FF.

[0092] In some embodiments, the post-processing of step S6 is specifically as follows:

[0093] Membrane package pyrogen removal treatment: according to the sample, take an ultrafiltration membrane package of appropriate area, the molecular weight cutoff of the ultrafiltration membrane package is not more than 10KD, use 0.5M NaOH to rinse the ultrafiltration membrane package for at least 30 minutes to disinfect and remove pyrogens, and then rinse the membrane package with ultrapure water until the pH is neutral;

[0094] Membrane package washing: Use replacement buffer to wash the membrane package as needed until the filtrate pH is 7.6±0.5, ready to start concentration; wherein, the preferred replacement buffer is: 0.02M Tris-HCl (pH7.6±0.05), 4mM CaCl2;

[0095] Concentration: Place the membrane package inlet and the interception end into the composite chromatography eluent for concentration. During the concentration process, the recombinant DNaseⅠ concentration is measured by nano at any time. The recombinant DNaseⅠ concentration is required to be no more than 20 mg / mL. When the concentration is completed, the sample volume is recorded as V, which includes the membrane package volume.

[0096] Replacement: add 3-5 times V replacement buffer for replacement, the volume is concentrated to the original volume V for the first time, the replacement times are 3-5 times, the replacement end point is pH 7.6±0.5, the deviation between conductivity and replacement buffer is no more than 1mS / cm, and the ultrafiltration replacement semi-finished product is obtained;

[0097] Preparation: Accurately measure a certain volume of the ultrafiltration replacement semi-finished product, add glycerol in a volume ratio of 1:1, mix thoroughly and filter through 0.22 μm to obtain the separated and purified recombinant DNase I finished product.

[0098] The industrial separation and purification method of DNaseⅠ of the present invention has simple steps, high yield and low cost, and is easy to be industrially scaled up for production. The purity of the recombinant DNaseⅠ obtained by separation and purification is higher than 95%, the specific activity of the enzyme is higher than 10KU / mg, the endotoxin residue is less than 1EU / mg, and there is no RNase pollution. The method can be well applied to DNA removal (such as removal of gDNA before RNA extraction or reverse transcription, removal of template DNA by in vitro transcription, and rRNA removal: applied to RNA library construction and sequencing), DNA labeling by gap translation method, DNaseⅠ footprint analysis experiment, etc., and is widely applied and has good economic value and social significance.

[0099] Example 1

[0100] An industrial separation and purification method for DNase I specifically comprises the following steps:

[0101] 1. Obtaining yeast fermentation supernatant of recombinant DNase I

[0102] Referring to the method described in patent document CN106520724B, a wild-type gene was used to construct a plasmid, and expression was induced in a Pichia pastoris host to obtain a yeast fermentation broth of recombinant DNase I.

[0103] 500 mL of yeast fermentation broth expressing recombinant DNaseⅠ was taken and centrifuged at 7000 rpm for 20 min to collect the supernatant of the fermentation broth.

[0104] 2. Ultrafiltration Fluid Replacement

[0105] The fermentation supernatant was ultrafiltered and concentrated using a 10kDa ultrafiltration membrane. When the concentration reached 1 / 3 of the original volume, 3 times the volume of replacement Buffer A0 was added and ultrafiltration was continued until the concentration reached 1 / 3 of the original volume. This process was repeated 3 times until the conductivity of the concentrated solution reached 1.1 mS / cm. The top membrane was then replaced with replacement Buffer A0 to obtain a one-step loading solution. Buffer A0: 0.05M Tris-HCl (pH 7.50), 5% glycerol, 0.1% Tween 20.

[0106] As shown in Table 1, when the processing volume of the fermentation supernatant was amplified by 20 times and the ultrafiltration membrane area was linearly amplified, the protein and enzyme activity recovery rates after ultrafiltration were stable, indicating that the process can be stably amplified.

[0107] Table 1 Summary and analysis results of ultrafiltration section parameters during process amplification

[0108]

[0109] 3. Anion exchange chromatography (one-step chromatography)

[0110] Regeneration: Use 0.5M NaOH to flush the column at a flow rate of 140cm / h for 30min to sterilize and remove pyrogens.

[0111] Equilibration: Equilibrate a Q Sepharose FF column (column height 10 cm, column volume 50 mL) with Buffer A1 at a flow rate of 140 cm / h until the pH of the effluent is 7.50 and the conductivity is within 1 mS / cm of the conductivity of the equilibration buffer, then prepare for sample loading. Buffer A1: 0.02 M Tris-HCl (pH 7.50), 5% glycerol, 0.1% Tween 20.

[0112] Sample loading: The one-step loading solution obtained by ultrafiltration is loaded onto the ion chromatography column at a flow rate of 100 cm / h. The one-step loading volume is controlled to not exceed 30 mL of loading solution per mL of filler.

[0113] Re-equilibration: Continue to equilibrate the column bed with Buffer A1 buffer for 5 column volumes at a flow rate of 140 cm / h.

[0114] Wash: Wash with Buffer B1 for 5 column volumes at a flow rate of 140 cm / h. Buffer B1: 0.15 M NaCl, 0.02 M Tris-HCl (pH 7.5), 5% glycerol, 0.1% Tween 20.

[0115] Elution: Elution was performed by column chromatography at a flow rate of 140 cm / h using Buffer B2. The eluted peak was collected in a pyrogen-free container. Buffer B2: 0.4 M NaCl, 0.02 M Tris-HCl (pH 7.5), 5% glycerol, 0.1% Tween 20.

[0116] CIP: The column was cleaned with 2 M NaCl and 0.5 M NaOH for 5 column volumes.

[0117] As shown in Table 2, after the anion exchange chromatography was linearly scaled up 20 times, the protein and enzyme activity recovery rates of the eluate were stable, indicating that the process scale-up was stable and feasible. The salt concentration would affect the enzyme activity, so when testing salty samples, sampling and desalting were required before the activity could be detected.

[0118] Table 2Q FF chromatography purification table

[0119]

[0120] 4. Affinity chromatography (two-step chromatography)

[0121] Regeneration of nickel column: Use 0.1M EDTA to wash the Ni Sepharose FF (NTA type) column at a pump speed of 180cm / h to denickelize the column, then wash the column with 0.5M NaOH for at least 30min to sterilize and remove pyrogens, then use 2M NaCl to wash the column for 5 column volumes, continue to wash the column with pure water to below pH 10.0, then use 0.5M NiSO4 to wash the column for 5 column volumes at a pump speed of 80cm / h to load nickel, after the nickel loading is completed, use pure water to wash the column for 10 column volumes.

[0122] Balance: Use Buffer A2 to balance Ni FF column (column height 12 cm, column volume 25 mL) for 20 column volumes at a pump speed of 180 cm / h, and measure the pH of the effluent to 7.51 for sample loading. Buffer A2: 0.5 M NaCl, 0.03 M imidazole, 0.02 M Tris-HCl (pH 7.50), 5% glycerol, 0.1% Tween 20.

[0123] Loading: Slowly add 2M NaCl solution to the Q FF eluate to make the final concentration of NaCl to 0.5M, add 2M imidazole solution to make the final concentration of imidazole to 30mM, mix thoroughly and load the sample at a flow rate of 150cm / h. The two-step loading capacity is controlled so that the sample amount per mL of filler does not exceed 2mL of sample solution.

[0124] Re-equilibration: Continue to equilibrate the column with Buffer A2 for 5 column volumes at a flow rate of 180 cm / h.

[0125] Washing: Use Buffer B3 at a flow rate of 180 cm / h to wash the column bed for 10 times the column volume. Buffer B3: 0.8-1.5 M NaCl, 2% Triton X 100, 0.02 M Tris-HCl (pH 7.50).

[0126] Re-balance: After washing, continue to balance the column bed with Buffer A2 at a flow rate of 180 cm / h for 5 times the column volume.

[0127] Elution: Use Buffer B4 for elution at a flow rate of 180 cm / h. Buffer B4 includes 0.2 M imidazole, 0.2 M NaCl, 0.02 M Tris-HCl (pH 7.50), 5% glycerol, and 0.1% Tween 20. Collect the elution peak in a pyrogen-free container.

[0128] CIP: The chromatography column was cleaned with 0.5 M imidazole.

[0129] As shown in Table 3, after linear amplification of affinity chromatography, the protein and enzyme activity recovery rates of the eluate were stable, indicating that the process amplification was stable and feasible. The salt concentration will affect the determination of enzyme activity, so when testing salt-containing samples, the sample must be desalted before the activity can be detected.

[0130] Table 3 Ni FF chromatography purification table

[0131]

[0132] 5. Composite chromatography (three-step chromatography)

[0133] Regeneration: Sterilize and depyrogenate the column by flushing with 0.5 M NaOH at a linear flow rate of 130 cm / h for 30 min.

[0134] Balance: Use Buffer A3 to balance an MMC Bestarose 6FF chromatography column (column height 10 cm, column volume 5 mL) at a flow rate of 130 cm / h, balance for 20 times the column volume, measure the pH of the effluent to be 7.54 and prepare for sample loading, Buffer A3: 0.02 M Tris-HCl (pH 7.50), 5% glycerol, 0.1% Tween 20.

[0135] Loading: Dilute the Ni FF eluted sample with Buffer A3, and the dilution end point is 8mS / cm. After thorough mixing, load the sample onto the MMC Bestarose 6FF column at a flow rate of 120cm / h. The second step loading volume is controlled to not exceed 5mL of sample solution per mL of filler.

[0136] Re-equilibration: Continue to equilibrate the column bed with Buffer A3 for 5 column volumes at a flow rate of 130 cm / h.

[0137] Washing: Continue to wash the column with Buffer B5 for 5 column volumes at a flow rate of 130 cm / h. Buffer B5 includes 0.1 M NaCl, 0.02 M Tris-HCl (pH 7.5), 5% glycerol, and 0.1% Tween 20.

[0138] Elution: Use Buffer B6 to elute the column at a flow rate of 130 cm / h, and collect the elution peak in a pyrogen-free container. Buffer B6: 0.4 M NaCl, 0.02 M Tris-HCl (pH 7.5), 5% glycerol, 0.1% Tween 20.

[0139] CIP: The column was cleaned with 2M NaCl and 0.5-1M NaOH.

[0140] As shown in Table 4, after the composite chromatography was linearly amplified 20 times, the protein and enzyme activity recovery rates of the eluate were stable, indicating that the process amplification was stable and feasible. The salt concentration will affect the determination of enzyme activity, so when testing salt-containing samples, the sample must be desalted before the activity can be detected.

[0141] Table 4MMC Bestarose 6FF chromatography purification table

[0142]

[0143] 6. Post-processing

[0144] Depyrogenation treatment of membrane package: according to the sample, take an ultrafiltration membrane package of appropriate area, the retention molecular weight of the ultrafiltration membrane package is 10KD, and use 0.5M NaOH to rinse the ultrafiltration membrane package for at least 30 minutes to sterilize and remove pyrogens, and then rinse the membrane package with ultrapure water to pH 7.0.

[0145] Membrane pack rinsing: Then use replacement Buffer C to rinse the membrane pack until the filtrate pH is 7.6±0.5, and then start concentration. Buffer C: 0.02M Tris-HCl (pH7.62), 4mM CaCl2.

[0146] Concentration: Place the membrane package inlet and the interception end into the eluted protein solution of the composite chromatography in step (5) for concentration. Concentrate to 1 / 4 of the original volume. Use nano to measure the protein concentration at any time during the concentration process. The protein concentration is required to be no more than 20 mg / mL. When the concentration is completed, record the sample volume as V1.

[0147] Replacement: Add 3 to 5 times the volume of V1 replacement Buffer C for replacement. The first pass is when the volume is concentrated to V1. The replacement times are 4 times, and the replacement endpoint is pH 7.63.

[0148] Preparation: Accurately measure a certain volume of the ultrafiltration displacement semi-finished product, add glycerol in a volume ratio of 1:1, mix thoroughly and filter through 0.22μm to obtain the finished product.

[0149] The SDS-PAGE diagram of the purification process in this example is as follows Figure 2 shown.

[0150] Example 2

[0151] The difference from Example 1 is that the amount of yeast fermentation broth expressing recombinant DNaseⅠ is different. In this example, 10 L of yeast fermentation broth expressing recombinant DNaseⅠ is taken. The SDS-PAGE image of the purification process is as follows: Figure 3 shown.

[0152] Example 3

[0153] Purity test of the products obtained in Examples 1 and 2

[0154] The recombinant DNaseⅠ was separated and purified from 500mL and 10L of yeast fermentation broth expressing recombinant DNaseⅠ, and then analyzed by SDS-PAGE. Figure 4 As shown, the grayscale was scanned by gel imager and the peak area normalization method was used, and the purity reached more than 99.0%.

[0155] Example 4

[0156] The products obtained in Examples 1 and 2 were tested for endotoxin content.

[0157] The samples of the recombinant DNase I products obtained in Example 1 and Example 2 before preparation were taken, and the endotoxin content was determined using the Zhanjiang Andus horseshoe crab kit. The measurement results showed that the endotoxin content in the recombinant DNase I product obtained in Example 1 ranged from 0.21 to 0.53 EU / mg, and the endotoxin content in the recombinant DNase I product obtained in Example 2 ranged from 0.30 to 0.80 EU / mg, both of which were far below the general standard limit of 10 EU / mg for mRNA vaccine enzyme raw materials.

[0158] Example 5

[0159] The products obtained in Examples 1 and 2 were subjected to RNase contamination assay

[0160] Take 1 μL of DNase I sample and 1.6 μg of MS2 RNA, incubate at 37℃ for 4 hours, and detect contamination. Figure 5 As shown, the recombinant DNaseⅠ products obtained in Examples 1 and 2 were free of RNase contamination. The DNaseⅠ products marked as RNase free from three different manufacturers in the market were purchased for RNase detection. Among them, one manufacturer had no RNase contamination, and the other two manufacturers had varying degrees of contamination. Therefore, the DNaseⅠ product prepared by the separation and purification method of the present invention has obvious advantages over the same type of samples in the market.

[0161] Example 6

[0162] The products obtained in Examples 1 and 2 were tested for activity

[0163] The definition of DNaseⅠ unit enzyme activity is as follows: using calf thymus DNA at a final concentration of 33.33 μg / mL as substrate, at 37°C and pH 5.0, the amount of enzyme required to increase the absorbance at 260 nm per milliliter of reaction system by 0.01 within 1 minute is defined as 1 enzyme activity unit (U).

[0164] Specific enzyme activity: Specific enzyme activity is defined as the unit of activity per milligram of enzyme protein, expressed in U / mg.

[0165] The specific activity determination operation method is described in patent document CN106520724B. After measurement, the specific activity of the recombinant DNase I product obtained in Example 1 is 13655 U / mg, and the specific activity of the recombinant DNase I product obtained in Example 1 is 13782 U / mg, both of which can meet market requirements.

[0166] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An industrial separation and purification method for DNase I, characterized in that: The steps include: S1: Take the cell fermentation liquid or cell lysate expressing the recombinant DNaseⅠ gene and centrifuge to obtain the supernatant, and obtain the crude enzyme solution of the recombinant DNaseⅠ; S2: concentrating the clarified solution of the crude enzyme solution of the recombinant DNaseⅠ, and replacing the solution with ultrafiltration to obtain a one-step loading solution; S3: taking the one-step loading solution and using anion exchange chromatography to enrich the recombinant DNase I, thereby increasing the specific activity and reducing the content of endotoxin, and obtaining an anion chromatography eluate; S4: taking the anion chromatography eluate and performing affinity chromatography to further purify the recombinant DNase I to obtain an affinity chromatography eluate; S5: taking the affinity chromatography eluate and performing composite chromatography to further purify the recombinant DNase I and remove RNase to obtain a composite chromatography eluate; S6: taking the composite chromatography eluate and performing post-treatment to obtain separated and purified recombinant DNase I; In step S2, a low-salt buffer is used for multiple ultrafiltration replacement to reduce the ionic strength of the clarified solution of the crude enzyme solution of the recombinant DNase I, which is conducive to the binding of the recombinant DNase I to the anion exchange medium; wherein the low-salt buffer includes: 0.05M Tris-HCl, 5% glycerol, 0.1% Tween 20, and Tris-HCl pH is 7.5; The anion exchange chromatography step S3 is specifically as follows: Regeneration: Use not less than 0.5M NaOH to flush the column at a flow rate of 80-300cm / h for at least 30min to sterilize and remove pyrogens; Balance: Use low-salt buffer to balance the column bed for 10 to 20 column volumes at a flow rate of 80-300 cm / h; the low-salt buffer includes: 0.02M Tris-HCl, 5% glycerol, 0.1% Tween 20, Tris-HCl pH 7.5; Sampling: taking the one-step loading solution and loading it onto the ion column at a flow rate of 80-300 cm / h; Rebalancing: continue to use low-salt buffer to rebalance and wash the column after loading for 5 to 10 times of column volume at a flow rate of 80-300 cm / h; low-salt buffer includes: 0.02M Tris-HCl, 5% glycerol, 0.1% Tween 20, Tris-HCl pH is 7.5; washing: use a buffer with a specific salt concentration to wash the column bed for 5 to 10 times of column volume at a flow rate of 80-300 cm / h; wherein, the buffer with a specific salt concentration includes: 0.15M NaCl, 0.02MTris-HCl, 5% glycerol, 0.1% Tween 20, Tris-HCl pH is 7.5; Elution: using an eluent of a specific salt concentration to flush the column bed at a flow rate of 80-300 cm / h for 5 to 10 times the column volume; wherein the eluent of the specific salt concentration is: 0.4 M NaCl, 0.02 M Tris-HCl, 5% glycerol, 0.1% Tween 20, and the pH of Tris-HCl is 7.5; CIP: The chromatography column was cleaned by CIP using 2 M NaCl and 0.5 M NaOH; The anion exchange chromatography column is Q Sepharose FF; the affinity chromatography in step S4 is specifically as follows: Regeneration: Use 0.1M EDTA to wash the column to remove metal ions, then use 0.5M NaOH to wash the column for at least 30 minutes to sterilize and remove pyrogens, then use 2M NaCl to wash the column for 5 column volumes, then use pure water to wash the column to pH below 10.0, then use 0.5M metal ion soluble salt to wash the column for 5 column volumes to achieve metal ion ligand attachment, and then use pure water to wash the column for 10 column volumes; Balance: Use high salt, low imidazole buffer to balance the column bed at a flow rate of 80-400 cm / h for 10 to 20 times the column volume; wherein, high salt, low imidazole buffer: 0.5 M NaCl, 0.03 M imidazole, 0.02 M Tris-HCl, 5% glycerol, 0.1% Tween 20, Tris-HCl pH 7.5; Loading: Take 0.5M imidazole and 2M NaCl mother solutions and add them to the anion chromatography eluent respectively, so that the final concentration of NaCl in the eluent is 0.3-0.5M, and the final concentration of imidazole is consistent with that in the high-salt, low-imidazole buffer. After fully mixing, perform metal column affinity chromatography loading at a flow rate of 80-400cm / h; Re-equilibration: After loading, continue to flush the column bed with high-salt, low-imidazole buffer at a flow rate of 80-400 cm / h for 5-6 column volumes; Washing: Use a high-salt washing buffer containing a certain amount of surfactant to wash the column bed at a flow rate of 80-400cm / h for 5 to 10 times the column volume; High salt washing buffer containing a certain amount of surfactant: 0.8-1.5 M NaCl, 2% Triton X100, 0.02 M Tris-HCl, Tris-HCl pH 7.5; Re-balance: After washing, continue to flush the column bed with high-salt, low-imidazole buffer at a flow rate of 80-400 cm / h for 5-6 times the column volume; Elution: elution of the chromatography column bed by 5 to 10 column volumes with an elution buffer having a specific imidazole concentration at a flow rate of 80-400 cm / h; wherein, The elution buffer for a specific imidazole concentration was 0.2 M imidazole, 0.2 M NaCl, 0.02 M Tris-HCl, 5% glycerol, 0.1% Tween 20, Tris-HCl pH 7.5; CIP: The chromatography column was cleaned with 0.5 M imidazole; The affinity chromatography is Ni affinity chromatography, and the type of Ni chromatography column is Ni Sepharose FF; The step S5 composite chromatography is specifically as follows: Regeneration: Sterilize and remove pyrogens by flushing the column with 0.5 M NaOH for at least 30 min; Balance: Use low-salt buffer to balance the chromatography column bed for 10 to 20 column volumes at a flow rate of 50-400 cm / h; wherein the low-salt buffer includes 0.02 M Tris-HCl, 5% glycerol, 0.1% Tween 20, and the pH of Tris-HCl is 7.5; Loading: diluting the affinity chromatography eluate with a low-salt buffer, with the dilution end point being a conductivity range of 5 to 10 mS / cm, and performing composite chromatography loading at a flow rate of 50 to 400 cm / h; Re-balance: Use low-salt buffer to balance the chromatography column bed for 5 to 6 column volumes at a flow rate of 50-400 cm / h; Washing impurities: Use a buffer with a specific salt concentration to wash the column bed for 5 to 10 times the column volume at a flow rate of 50-400 cm / h, and collect the washing impurity peak; wherein the buffer with a specific salt concentration is: 0.1M NaCl, 0.02M Tris-HCl, 5% glycerol, 0.1% Tween 20, and the pH of Tris-HCl is 7.5; Elution: using an eluent with a specific salt concentration to elute the column bed at a flow rate of 50-400 cm / h for 5 to 10 times the column volume, and collecting the elution peak; wherein the eluent with a specific salt concentration is: 0.4 M NaCl, 0.02 M Tris-HCl, 5% glycerol, 0.1% Tween 20, and the pH of Tris-HCl is 7.5; CIP: Use 2M NaCl and 0.5-1M NaOH to clean the column. The conditions of the composite chromatography are ionic and hydrophobic composite mode filler, and the composite filler is MMC Bestarose 6FF; The post-processing of step S6 is specifically as follows: Membrane package pyrogen removal treatment: according to the sample, take an ultrafiltration membrane package of appropriate area, the molecular weight cutoff of the ultrafiltration membrane package is not more than 10KD, use 0.5M NaOH to rinse the ultrafiltration membrane package for at least 30 minutes to disinfect and remove pyrogens, and then rinse the membrane package with ultrapure water until the pH is neutral; Membrane pack rinsing: Use replacement buffer to rinse the membrane pack as needed until the filtrate pH is 7.6±0.5, ready to start concentration; wherein, the replacement buffer is: 0.02M Tris-HCl, 4mM CaCl2, pH7.6±0.05; Concentration: Place the membrane package inlet and the interception end into the composite chromatography eluent for concentration. During the concentration process, the recombinant DNaseⅠ concentration is measured by nano at any time. The recombinant DNaseⅠ concentration is required to be no more than 20 mg / mL. When the concentration is completed, the sample volume is recorded as V, which includes the membrane package volume. Replacement: add 3-5 times V replacement buffer for replacement, the volume is concentrated to the original volume V for the first time, the replacement times are 3-5 times, the replacement end point is pH 7.6±0.5, the deviation between conductivity and replacement buffer is no more than 1mS / cm, and the ultrafiltration replacement semi-finished product is obtained; Preparation: Accurately measure a certain volume of the ultrafiltration replacement semi-finished product, add glycerol in a volume ratio of 1:1, mix thoroughly and filter through 0.22 μm to obtain the separated and purified recombinant DNase I finished product.

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