CN115433726A - Industrial separation and purification method of DNase I - Google Patents
Industrial separation and purification method of DNase I Download PDFInfo
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
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Abstract
The invention provides an industrial separation and purification method of DNase I, belonging to the technical field of biology. The industrial separation and purification method of DNase I comprises the steps of centrifuging cell fermentation liquor or cell lysate expressing recombinant DNase I genes to obtain supernatant, concentrating, performing ultrafiltration liquid exchange, performing anion exchange chromatography, performing affinity chromatography, performing composite chromatography and performing post-treatment.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an industrial separation and purification method of DNase I.
Background
DNase I (Deoxyribonuclease I) is a hydrolysable deoxyribonucleic acid (DNA) phosphodiesterase with a molecular weight of about 39kD, originally isolated from bovine pancreas. DNase I can non-specifically degrade DNA in contrast to restriction enzymes that need to recognize specific sequences. The optimum working pH range for DNase I is 7-8, the activity of which is Ca-dependent 2+ And may be substituted with divalent metal ions such as Co 2+ ,Mn 2+ , Zn 2+ And so on. DNase I is widely applied to preparation of RNA without DNA; removing the template DNA after in vitro transcription; preparing RNA without DNA before RT-PCR and RT-qPCR reaction; DNA labeling by nick translocation in combination with DNA polymerase I; and constructing a DNA fragmentation library.
The mRNA vaccine technology is one of new vaccine technologies which are gradually developed in recent years. The mRNA vaccine expresses antigen in cells, and can achieve the effect of preventing infectious diseases by stimulating an immune system to generate various effect mechanisms such as B and T cell immune response and the like. The development and production of mRNA vaccines requires the involvement 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 control of DNA in the production process of mRNA vaccine is very strict, and the residual DNA can be removed by DNase I, thereby reducing the downstream purification difficulty and increasing the purity of the product. And has high requirements on endotoxin residue in enzyme raw materials and RNase pollution in the production process of mRNA vaccines. Therefore, the research on the purification method of DNase I which has low toxin residue and no RNase pollution and can be industrially prepared has good economic value and social significance.
Disclosure of Invention
Therefore, it is necessary to provide an industrial separation and purification method of DNase I, so that the recombinant DNase I obtained by separation and purification has high purity, good activity, no RNase pollution and extremely low endotoxin content.
An industrial separation and purification method of DNase I comprises the following steps:
s1: centrifuging cell fermentation liquor or cell lysate expressing recombinant DNase I gene to obtain supernatant, and obtaining crude DNase I enzyme supernatant;
s2: concentrating the crude DNase I enzyme solution clarified liquid, and performing ultrafiltration liquid exchange to obtain a one-step sample solution;
s3: enriching and recombining DNase I by the one-step sample loading solution by adopting anion exchange chromatography, improving specific activity, and reducing endotoxin content to obtain anion chromatography eluent;
s4: carrying out affinity chromatography on the anion chromatography eluent, and further purifying the recombinant DNase I to obtain an affinity chromatography eluent;
s5: performing composite chromatography on the affinity chromatography eluent, further purifying the recombinant DNase I, and removing the RNase to obtain a composite chromatography eluent;
s6: and (3) carrying out post-treatment on the composite chromatography eluent to obtain the separated and purified recombinant DNase I.
Preferably, in the step S2, a low-salt buffer solution is adopted for multiple ultrafiltration and displacement, so that the ionic strength of the clarified solution of the crude enzyme solution of the recombinant DNase I is reduced, and the recombinant DNase I is favorably bound to an anion exchange medium; wherein, the low salt buffer solution: 0.02-0.05M Tris-HCl, 0-10% glycerol, 0-0.2% Tween 20, pH 6.8-8.5.
Preferably, the step S3 of anion exchange chromatography is specifically:
regeneration: washing the chromatographic column with NaOH of not less than 0.5M at flow rate of 80-300cm/h for at least 30min for sterilization and pyrogen removal;
balancing: adopting a low-salt buffer solution to balance the volume of the column bed by 10-20 times at a flow speed of 80-300 cm/h;
loading: taking the one-step sample loading liquid to carry out ion column sample loading at the flow speed of 80-300 cm/h;
rebalancing: continuously adopting low-salt buffer solution to balance and wash the loaded chromatographic column by 5-10 times of column volume at the flow speed of 80-300 cm/h;
impurity washing: washing the column bed with a buffer solution with a specific salt concentration at a flow speed of 80-300cm/h for 5-10 times of the column volume; wherein the buffer of specific salt concentration: 0.05-0.15M NaCl,0.02-0.05M Tris-HCl, 0-10% glycerol, 0-0.2% Tween 20, pH6.8-8.5;
and (3) elution: adopting eluent with specific salt concentration to flush the column bed at the flow speed of 80-300cm/h for 5-10 times of the column volume; wherein, the eluent with specific salt concentration: 0.2-0.4M NaCl,0.02-0.05M Tris-HCl, 0-10% glycerol, 0-0.2% Tween 20 and pH6.8-8.5;
CIP: CIP cleaning is carried out on the chromatographic column by adopting 2M NaCl and 0.5-1M NaOH.
Preferably, the Anion Exchange chromatography column comprises a strong Anion exchanger comprising a strong Anion exchanger type Q and a weak Anion exchanger type DEAE, said strong Anion exchanger comprising Q bestar XL, Q bestar FF, Q Sepharose HP, Q sephafe FF, uniGel-80Q and Nuvia Q Anion Exchange Media; the weak anion exchanger comprises DEAE Sepharose FF, DEAE Sepharose HP and DEAE Sephalife.
Preferably, the step S4 affinity chromatography is specifically:
regeneration: washing the chromatographic column with 0.1M EDTA to remove metal ions, washing the chromatographic column with 0.5M NaOH for at least 30min to sterilize and depyrogenation, washing the chromatographic column with 2M NaCl for 5 times of column volume, washing the chromatographic column with pure water to pH below 10.0, washing the chromatographic column with 0.5M metal ion soluble salt for 5 times of column volume to realize hanging metal ion ligands, and washing the chromatographic column with pure water for 10 times of column volume after finishing:
balancing: adopting buffer solution with high salt and low imidazole to balance 10-20 times of column volume of the column bed at the flow rate of 80-400 cm/h; wherein, the buffer solution with high salt and low imidazole: 0.5M NaCl,0.005-0.03M imidazole, 0.02-0.05M Tris-HCl, 0-10% glycerol, 0-0.2% Tween 20, pH7.0-8.0;
sampling: respectively adding 0.5M imidazole and 2M NaCl mother liquor into the anion chromatography eluent to ensure 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 buffer solution with high salt and low imidazole, and carrying out metal column affinity chromatography sample loading at the flow rate of 80-400cm/h after fully and uniformly mixing;
rebalancing: after the sample loading is finished, continuously washing the column bed by 5-6 times of the column volume by using a buffer solution with high salt and low imidazole at the flow rate of 80-400 cm/h;
impurity washing: washing the column bed by adopting high-salt impurity-washing buffer solution containing a certain amount of surfactant at the flow speed of 80-400cm/h for 5-10 times of the column volume; wherein, the high-salt impurity-washing buffer solution containing a certain amount of surfactant: 0.8 to 1.5M NaCl,2% Triton X100, 0.02 to 0.05M Tris-HCl, pH6.8 to 8.5;
rebalancing: after the impurity washing is finished, continuously washing the column bed by 5-6 times of the column volume by using a buffer solution with high salt and low imidazole at the flow rate of 80-400 cm/h;
and (3) elution: eluting the chromatographic column bed by using elution buffer solution with specific imidazole concentration at the flow rate of 80-400cm/h for 5-10 times of the column volume; wherein, the elution buffer solution with specific imidazole concentration is 0.05-0.2M imidazole, 0.2-0.5M NaCl,0.02-0.05M Tris-HCl, 0-10% glycerol, 0-0.2% Tween 20, pH6.8-8.5;
CIP: the column was washed with 0.5M imidazole.
Preferably, the affinity chromatography is metal ion chelating His chromatography, the metal ion being a transition metal ion capable of providing an electron donor coordination site, including Cu 2+ 、Co 2+ 、Ni 2+ 、Zn 2+ 、Fe 2+ And Ca 2+ 。
Preferably, the affinity chromatography is Ni affinity chromatography, using Ni NTA or Ni TED types, the Ni chromatography column types including Ni Sepharose excel, ni Sepharose FF, ni Bestarose FF, uni NTA-80Ni and Ni NTA Beads 6FF.
Preferably, the step S5 of composite chromatography specifically comprises:
regeneration: washing the chromatographic column with 0.5M NaOH for at least 30min for sterilization and depyrogenation;
balancing: adopting low-salt buffer solution to balance the volume of the chromatographic column bed by 10-20 times at the flow speed of 50-400 cm/h; wherein, the low salt buffer solution: 0.02-0.05M Tris-HCl, 0-10% glycerol, 0-0.2% Tween 20, pH6.8-8.5;
sampling: diluting the affinity chromatography eluent by adopting a low-salt buffer solution, wherein the end point of dilution is in the conductance range of 5-10 mS/cm, and carrying out composite chromatography sampling at the flow rate of 50-400 cm/h;
rebalancing: adopting low-salt buffer solution to balance the volume of the chromatographic column bed by 5-6 times at the flow speed of 50-400 cm/h;
impurity washing: washing the column bed by 5-10 times of the column volume by using buffer solution with specific salt concentration at the flow speed of 50-400cm/h, and collecting the washing impurity peak; wherein the buffer solution with specific salt concentration: 0 to 0.2M NaCl or KCl,0.02 to 0.05M Tris-HCl,0 to 10 percent of glycerin, 0 to 0.2 percent of Tween 20, and pH6.8 to 8.5;
and (3) elution: eluting the column bed by 5-10 times of column volume by using eluent with specific salt concentration at the flow speed of 50-400cm/h, and collecting an elution peak; wherein, the eluent with specific salt concentration: 0.4-0.5M NaCl or KCl,0.02-0.05M Tris-HCl, 0-10% glycerol, 0-0.2% Tween 20, pH6.8-8.5;
CIP: CIP cleaning is carried out on the chromatographic column by adopting 2M NaCl and 0.5-1M NaOH.
Preferably, the conditions of the complex chromatography are hydrophobic and ionic complex mode packing, said complex packing comprising MMC HF, MMC FF, MMC Bestarose 6FF, capto MMC, diamond MMC and NM90 Agarose.
Preferably, the post-processing of step S6 is specifically:
film-wrapping pyrogen-removing treatment: measuring an ultrafiltration membrane package with a proper area according to a sample amount, wherein the molecular weight cut-off of the ultrafiltration membrane package is not more than 10KD, washing the ultrafiltration membrane package with 0.5M NaOH for at least 30min for disinfection and pyrogen removal, and then washing the membrane package with ultrapure water until the pH value is neutral;
film moistening and washing: adopting a replacement buffer solution to rinse a membrane package as required until the pH value of the filtered solution is 7.6 +/-0.5, and preparing to start concentration; wherein, the buffer solution for replacement: 0.02M Tris-HCl,4mM CaCl 2 ,pH7.6±0.05;
Concentration: putting an inlet and an interception end of a membrane package into the composite chromatographic eluent for concentration, measuring the concentration of the recombinant DNase I by using nano at any time in the concentration process, wherein the concentration of the recombinant DNase I is required to be not more than 20mg/mL, and recording the volume of a sample as V when the concentration is finished, wherein the V comprises the volume of the membrane package;
and (3) replacement: adding 3-5 times of V displacement buffer solution for displacement, wherein the volume concentration is the first time when the volume is reduced to the original volume V, the displacement times are 3-5 times, the displacement end point is pH7.6 +/-0.5, and the deviation between the conductance and the displacement buffer solution is not more than 1mS/cm, thus obtaining an ultrafiltration displacement semi-finished product;
blending: accurately measuring a certain volume of the ultrafiltration displacement semi-finished product, adding glycerol according to the volume ratio of 1:1, fully and uniformly mixing, and filtering by adopting 0.22 mu m to obtain a separated and purified recombinant DNase I finished product.
The industrial separation and purification method of DNase I has the advantages of simple steps, high yield, low cost and easy industrial amplification production, the purity of the recombinant DNase I 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, no RNase pollution exists, the method can be well applied to DNA removal (such as gDNA removal before RNA extraction or reverse transcription, template DNA removal through in vitro transcription, rRNA removal: application to RNA library building sequencing), DNA labeling by a notch translation method, DNase I footprint analysis experiments and the like, and the method has wide application and good economic value and social significance.
Drawings
FIG. 1 is a schematic flow chart of the industrial separation and purification method of DNase I of the present invention;
FIG. 2 is a SDS-PAGE graph of the purification process of 500 mL-fraction fermentation supernatant DNase I in example 1 of the present invention;
FIG. 3 is a SDS-PAGE graph showing the purification process of 10L-stage fermentation supernatant DNase I in example 2 of the present invention;
FIG. 4 is a SDS-PAGE image of recombinant DNase I products obtained in examples 1 and 2 of the present invention; in the figure, 1 is a SDS-PAGE picture of the recombinant DNase I finished product of example 1, and 2 is a SDS-PAGE picture of the recombinant DNase I finished product of example 2;
FIG. 5 is a graph showing the results of RNase residue detection for the recombinant DNase I products obtained in examples 1 and 2 and DNase I products labeled RNase free from 3 different manufacturers on the market; in the figure, 1 and 2 represent the results of example 1, 3 and 4 represent the results of example 2, 5 and 6 represent market samples 1,7 and 8 represent market samples 2,9 and 10 represent market samples 3, and 11 represents a negative control.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, embodiments accompanying figures are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as broadly as the present invention is capable of modification in various respects, all without departing from the spirit and scope of the present invention.
As shown in FIG. 1, the invention provides an industrial separation and purification method of DNase I, which comprises the following steps:
s1: centrifuging cell fermentation liquor or cell lysate for expressing the recombinant DNase I gene to obtain supernatant, thereby obtaining crude DNase I enzyme supernatant;
s2: concentrating the crude DNase I enzyme clear solution, and performing ultrafiltration to obtain a one-step sample solution;
s3: enriching and recombining DNase I by the one-step sample loading solution by adopting anion exchange chromatography, improving specific activity, and reducing endotoxin content to obtain anion chromatography eluent;
s4: carrying out affinity chromatography on the anion chromatography eluent, and further purifying the recombinant DNase I to obtain an affinity chromatography eluent;
s5: carrying out composite chromatography on the affinity chromatography eluent, further purifying the recombinant DNase I, and removing the RNase to obtain a composite chromatography eluent;
s6: and (3) carrying out post-treatment on the composite chromatography eluent to obtain the separated and purified recombinant DNase I.
And (3) transforming the recombinant plasmid carrying the DNase I gene fragment into host cells for induced expression culture to obtain the cell strain of the recombinant DNase I, wherein the expression hosts comprise fungal cells and bacterial cells. The cell culture fluid is cell fermentation fluid of a target protein secretion type generated by the growth of fungus cells and bacterial cells in a culture medium. The cell lysate is cell thallus produced by fungal cells and bacterial cells in a culture medium and expressed intracellularly by target protein, and cell contents released after cell disruption by methods such as high-pressure homogenization or ultrasonic disruption and the like are disrupted after the cell thallus is resuspended in a lysis buffer. Wherein the buffer solution comprises a lysis buffer solution, and the buffer solution comprises pH6.8-8.5 buffer systems, such as disodium hydrogen phosphate-citric acid buffer system, tris-HCl buffer system, barbital buffer system and phosphate buffer system, preferably Tris-HCl (pH7.5) buffer system.
Because the ionic strength of the cell fermentation liquid or cell lysate is high and the components are complex, the low-salt buffer solution is adopted to carry out ultrafiltration displacement on the crude DNase I enzyme clear liquid for multiple times in the step S2, so that the ionic strength of the crude DNase I enzyme clear liquid is reduced, and the combination of the recombinant DNase I on an anion exchange medium is facilitated. Wherein, the low salt buffer solution: 0.02-0.05M Tris-HCl (pH 6.8-8.5), 0-10% glycerol, 0-0.2% Tween 20; preferably 0.02M Tris-HCl (pH6.8-8.5), 0-10% glycerol, 0-0.2% Tween 20.
In some embodiments, the step S3 anion exchange chromatography is specifically:
regeneration: washing the chromatographic column with NaOH of not less than 0.5M at flow rate of 80-300cm/h for at least 30min for sterilization and removing pyrogen;
balancing: adopting a low-salt buffer solution to balance the volume of the column bed by 10-20 times at a flow speed of 80-300 cm/h; wherein, the low salt buffer solution: 0.02-0.05M Tris-HCl (pH6.8-8.5), 0% -10% glycerol, 0-0.2% Tween 20, and the optimal balance flow rate is 140cm/h;
loading: taking the one-step sample loading liquid to perform ion column sample loading at the flow rate of 80-300cm/h, and preferably, the sample loading flow rate is 100cm/h;
rebalancing: continuously adopting low-salt buffer solution to balance and wash the loaded chromatographic column by 5-10 times of column volume at the flow rate of 80-300cm/h, and preferably, the rebalance flow rate is 100cm/h;
impurity washing: washing impurities on the column bed by 5-10 times of the column volume at the flow speed of 80-300cm/h by adopting buffer solution with specific salt concentration; wherein the buffer solution with specific salt concentration: 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 impurity washing flow rate is 140cm/h;
and (3) elution: adopting eluent with specific salt concentration to flush the column bed at the flow speed of 80-300cm/h for 5-10 times of the column volume; wherein the eluent with specific salt concentration: 0.2-0.4M NaCl,0.02-0.05M Tris-HCl (pH6.8-8.5), 0-10% glycerin, 0-0.2% Tween 20; preferably, the elution flow rate is 140cm/h;
CIP: CIP cleaning is carried out on the chromatographic column by adopting 2M NaCl and 0.5-1M NaOH.
Wherein the Anion Exchange chromatography column comprises Q strong Anion exchanger and DEAE weak Anion exchanger with different resolution, such as Q Bestarose XL, Q Bestarose FF, Q Sepharose FF, Q Sepharose HP, Q Sephalife FF, DEAE Sepharose HP, DEAE Sepharose, unigel-80Q, nuvia Q Anion Exchange Media, etc., preferably Q Sepharose FF.
In some embodiments, the step S4 affinity chromatography is specifically:
regeneration: washing the column with 0.1M EDTA to remove metal ions, washing the column with 0.5M NaOH for at least 30min for sterilization and depyrogenation, washing the column with 2M NaCl for 5 times the column volume, washing the column with pure water to pH below 10.0, and washing the column with 0.5M metal ion soluble salt (such as NiSO) 4 ) Washing the chromatographic column by 5 times of the column volume to realize hanging of the metal ion ligand, and washing the chromatographic column by 10 times of the column volume by pure water after the completion;
balancing: adopting buffer solution with high salt and low imidazole to balance 10-20 times of column volume of the column bed at the flow rate of 80-400 cm/h; wherein the buffer solution of high salt and low imidazole: 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 180cm/h;
loading: respectively adding 0.5M imidazole and 2M NaCl mother liquor into the anion chromatography eluent to ensure 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 buffer solution with high salt and low imidazole, fully and uniformly mixing, and then carrying out metal column affinity chromatography sampling at the flow rate of 80-400cm/h, preferably, the sampling flow rate is 150cm/h;
rebalancing: after the sample loading is finished, continuously washing the column bed by 5-6 times of the column volume by using a buffer solution with high salt and low imidazole at the flow rate of 80-400 cm/h;
impurity washing: washing the column bed by a high-salt impurity washing buffer solution containing a certain amount of surfactant at a flow rate of 80-400cm/h for 5-10 times of the column volume; wherein, the high-salt impurity-washing buffer solution containing a certain amount of surfactant: 0.8 to 1.5M NaCl,2% Triton X100, 0.02 to 0.05M Tris-HCl (pH 6.8 to 8.5); preferably, the impurity washing flow rate is 180cm/h;
rebalancing: after impurity washing is finished, continuously washing the column bed by using a buffer solution with high salt and low imidazole at the flow rate of 80-400cm/h for 5-6 times of the column volume;
and (3) elution: eluting the chromatographic column bed by using elution buffer solution with specific imidazole concentration at the flow rate of 80-400cm/h for 5-10 times of the column volume; wherein, the elution buffer solution with specific imidazole concentration is 0.05-0.2M imidazole, 0.2-0.5M NaCl,0.02-0.05M Tris-HCl (pH6.8-8.5), 0-10% glycerol, 0-0.2% Tween 20; preferably the elution flow rate is 180cm/h;
CIP: the column was washed with 0.5M imidazole.
Wherein the affinity chromatography is metal ion chelating His chromatography, the purification tag of the fusion protein is His, and the metal ion is transition metal ion capable of providing electron donor coordination sites, including Cu 2+ 、Co 2+ 、Ni 2+ 、 Zn 2+ 、Fe 2+ 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, preferably Ni Sepharose FF.
In some embodiments, the step S5 of complex chromatography is specifically:
regeneration: washing the chromatographic column with 0.5M NaOH for at least 30min for sterilization and depyrogenation;
balancing: adopting low-salt buffer solution to balance the volume of the chromatographic column bed by 10-20 times at the flow speed of 50-400 cm/h; wherein, the low salt buffer solution: 0.02-0.05M Tris-HCl (pH6.8-8.5), 0-10% glycerol, 0-0.2% Tween 20; preferably the equilibrium flow rate is 130cm/h;
loading: diluting the affinity chromatography eluent by using a low-salt buffer solution, wherein the end point of the dilution is in the conductance range of 5-10 mS/cm, and performing composite chromatography sample loading at the flow rate of 50-400cm/h, preferably at the sample loading flow rate of 120cm/h;
rebalancing: adopting low-salt buffer solution to balance the volume of the chromatographic column bed by 5-6 times at the flow speed of 50-400 cm/h;
impurity washing: washing the column bed by 5-10 times of column volume by using buffer solution with specific salt concentration at the flow speed of 50-400cm/h, and collecting a washing impurity peak; wherein the buffer of specific salt concentration: 0 to 0.2M NaCl or KCl,0.02 to 0.05M Tris-HCl (pH6.8 to 8.5), 0 to 10 percent glycerol, 0 to 0.2 percent Tween 20; preferably, the impurity washing flow rate is 130cm/h;
and (3) elution: eluting the column bed by 5-10 times of column volume by using eluent with specific salt concentration at the flow speed of 50-400cm/h, and collecting an elution peak; wherein, the eluent with specific salt concentration: 0.4-0.5M NaCl or KCl,0.02-0.05M Tris-HCl (pH6.8-8.5), 0-10% glycerin, 0-0.2% Tween 20; preferably the elution flow rate is 130cm/h;
CIP: CIP cleaning is carried out on the chromatographic column by adopting 2M NaCl and 0.5-1M NaOH.
Wherein the conditions of the complex chromatography comprise complex fillers with different resolutions, such as MMC HF, MMC FF, MMC Bestarose 6FF, capto MMC, diamond MMC and NM90 Agarose, etc., preferably MMC Bestarose 6FF.
In some embodiments, the post-processing of step S6 specifically includes:
film packaging and pyrogen removing treatment: measuring an ultrafiltration membrane package with a proper area according to a sample amount, wherein the molecular weight cut-off of the ultrafiltration membrane package is not more than 10KD, washing the ultrafiltration membrane package with 0.5M NaOH for at least 30min for disinfection and pyrogen removal, and then washing the membrane package with ultrapure water until the pH value is neutral;
film moistening and washing: adopting a replacement buffer solution to rinse the membrane package as required until the pH value of the filtered solution is 7.6 +/-0.5, and preparing to start concentration; among them, preferred is a replacement buffer: 0.02M Tris-HCl (pH7.6. + -. 0.05), 4mM CaCl 2 ;
And (3) concentrating: putting an inlet and an interception end of a membrane package into the composite chromatographic eluent for concentration, measuring the concentration of the recombinant DNase I by using nano at any time in the concentration process, wherein the concentration of the recombinant DNase I is required to be not more than 20mg/mL, and recording the volume of a sample as V when the concentration is finished, wherein the V comprises the volume of the membrane package;
and (3) replacement: adding 3-5 times of V displacement buffer solution for displacement, wherein the volume concentration is the first time when the volume is reduced to the original volume V, the displacement times are 3-5 times, the displacement end point is pH7.6 +/-0.5, and the deviation between the conductance and the displacement buffer solution is not more than 1mS/cm, thus obtaining an ultrafiltration displacement semi-finished product;
blending: accurately measuring a certain volume of the ultrafiltration displacement semi-finished product, adding glycerol according to the volume ratio of 1:1, fully and uniformly mixing, and filtering by adopting 0.22 mu m to obtain a separated and purified recombinant DNase I finished product.
The industrial separation and purification method of DNase I has the advantages of simple steps, high yield, low cost and easy industrial amplification production, the purity of the recombinant DNase I 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, no RNase pollution exists, the method can be well applied to DNA removal (such as gDNA removal before RNA extraction or reverse transcription, template DNA removal through in vitro transcription, rRNA removal: application to RNA library building sequencing), DNA labeling by a notch translation method, DNase I footprint analysis experiments and the like, and the method has wide application and good economic value and social significance.
Example 1
An industrial separation and purification method of DNase I comprises the following steps:
1. obtaining recombinant DNase I yeast fermentation supernatant
With reference to the method described in patent document CNl06520724B, wild-type gene is used to construct plasmid, and the plasmid is induced and expressed in Pichia pastoris host to obtain recombinant DNase I yeast fermentation broth.
Taking 500mL yeast fermentation liquor for expressing the recombinant DNase I, and centrifuging at 7000rpm for 20min to collect fermentation liquor supernatant.
2. Ultrafiltration liquid exchange
Ultrafiltering and concentrating the fermentation supernatant with 10kDa ultrafiltration membrane, and adding 3 times of displacement Buffer A when concentrating to 1/3 of the original volume 0 Continuously carrying out ultrafiltration until the volume is 1/3 of the original volume; repeating the steps for 3 times until the conductivity of the concentrated solution is 1.1mS/cm, and performing top membrane by using a displacement Buffer A0 to obtain a one-step sample solution. Buffer A 0 :0.05M Tris-HCl (pH 7.50), 5% glycerol, 0.1% Tween 20.
As shown in Table 1, the treatment capacity of the volume of the fermentation supernatant is amplified by 20 times, and the recovery rates of the protein and the enzyme activity after ultrafiltration are stable after the area of the ultrafiltration membrane is linearly amplified, which indicates that the process can be stably amplified.
TABLE 1 summary analysis results of ultrafiltration section parameters during process scale-up
3. Anion exchange chromatography (one step chromatography)
Regeneration: the column was washed with 0.5M NaOH at a flow rate of 140cm/h for 30min for sterilization and depyrogenation.
Balancing: at a flow rate of 140cm/h using Buffer A 1 Equilibrating Q Sepharose FF column (column height 10cm, column volume 50 mL) until effluent pH is 7.50, preparing for loading when conductance is within 1mS/cm of equilibration buffer conductance, bufferA 1 :0.02M Tris-HCl (pH7.50), 5% glycerol, 0.1% Tween 20.
Loading: and loading the one-step loading solution obtained by ultrafiltration to an ion chromatography column at the flow rate of 100 cm/h. The loading capacity of one-step loading is controlled to be not more than 30mL of loading solution per mL of filler.
Rebalancing: continuing with Buffer A at a flow rate of 140cm/h 1 The buffer equilibrated the bed for 5 column volumes.
Impurity washing: at a flow rate of 140cm/h with Buffer B 1 The buffer washes out 5 column volumes. Buffer B 1 : 0.15M NaCl,0.02M Tris-HCl (pH 7.5), 5% glycerol, 0.1% Tween 20.
And (3) elution: at a flow rate of 140cm/h using Buffer B 2 Eluting with buffer solution by column chromatography. The eluted peak was collected using a pyrogen-free vessel. Buffer B 2 :0.4M NaCl,0.02M Tris-HCl (pH 7.5), 5% glycerol, 0.1% Tween 20.
CIP: the column was CIP washed 5 times the column volume with 2M NaCl and 0.5M NaOH.
As shown in Table 2, after the anion exchange chromatography is linearly amplified by 20 times, the recovery rates of the protein and the enzyme activity of the eluent are stable, which shows that the process amplification is stable and feasible, wherein the enzyme activity is influenced by the salt concentration, so that when a sample with salt is tested, sampling and desalting are required to detect the activity.
TABLE 2Q FF chromatographic purification Table
4. Affinity chromatography (two-step chromatography)
Regenerating a nickel column: washing Ni Sepharose FF (NTA type) column chromatography with 0.1M EDTA at pump speed of 180cm/h for removing nickel, washing the column chromatography with 0.5M NaOH, sterilizing for at least 30min, removing pyrogen, washing the column chromatography with 2M NaCl for 5 times of column volume, washing the column chromatography with pure water to pH below 0.0, and washing with 0.5M NiSO 4 And washing the chromatographic column at the pump speed of 80cm/h for 5 times of the column volume to hang nickel, and washing the chromatographic column with pure water for 10 times of the column volume after finishing hanging nickel.
Balancing: using Buffera 2 The Ni FF column (column height 12cm, column volume 25 mL) was equilibrated at a pump speed of 180cm/h for 20 column volumes, and the effluent was measured to pH7.51 and prepared for loading. BufferA 2 :0.5M NaCl, 0.03M imidazole, 0.02M Tris-HCl (pH 7.50) 5% glycerol, 0.1% Tween 20.
Sampling: slowly adding 2M NaCl solution into the Q FF eluent to enable the final concentration of NaCl to be 0.5M, adding 2M imidazole solution to enable the final concentration of imidazole to be 30mM, fully and uniformly mixing, loading at the flow rate of 150cm/h, and controlling the loading amount of the second step to be not more than 2mL of loading liquid per mL of filler.
Rebalancing: continuing to use Buffer A at a flow rate of 180cm/h 2 Equilibrating the chromatography column for 5 column volumes.
Impurity washing: using Buffer B 3 Washing the column bed 10 times the column volume at a flow rate of 180cm/h, buffer B 3 : 0.8~1.5M NaCl,2%Triton X 100,0.02M Tris-HCl(pH7.50)。
Rebalancing: after the impurities are washed, buffer A is continuously used 2 The bed was equilibrated at a flow rate of 180cm/h for 5 column volumes.
And (3) elution: using Buffer B 4 Eluting at a flow rate of 180cm/h for chromatography, buffer B 4 :0.2M imidazole, 0.2M NaCl,0.02M Tris-HCl (pH 7.50), 5% glycerol, 0.1% Tween 20, and the eluted peaks were collected in a pyrogen-free vessel.
CIP: the column was washed with 0.5M imidazole.
As shown in Table 3, the recovery rates of protein and enzyme activity of the eluate after linear amplification of affinity chromatography were stable, indicating that the process amplification was stable and feasible. The salt concentration influences the determination of enzyme activity, so when a salt-containing sample is tested, the activity can be detected after the sample is subjected to desalting treatment.
TABLE 3 chromatographic purification of Ni FF
5. Compound chromatography (three-step chromatography)
Regeneration: the column was washed with 0.5M NaOH at a linear flow rate of 130cm/h for 30min for sterilization and depyrogenation.
Balancing: using Buffer A 3 Balancing MMC Bestarose 6FF chromatographic column (height of column 10cm, volume of column 5 mL) at flow rate of 130cm/h for 20 times of column volume, determining effluent pH to 7.54, preparing sample loading, and buffering A 3 :0.02M Tris-HCl (pH7.50), 5% glycerol, 0.1% Tween 20.
Loading: using Buffer A 3 The Ni FF eluted sample was diluted to an end point of 8mS/cm conductivity. After being fully mixed, the mixture is loaded on an MMC Bestarose 6FF chromatographic column at the flow rate of 120 cm/h. The two-step loading is controlled so that the loading amount of each mL of filler does not exceed 5mL of loading solution.
Rebalancing: continuing at a flow rate of 130cm/h with Buffer A 3 The bed was equilibrated by 5 column volumes.
Impurity washing: continuing the flow rate of 130cm/h with Buffer B 5 Washing the chromatographic column with 5 times of column volume, buffer B 5 :0.1M NaCl,0.02M Tris-HCl (pH 7.5), 5% glycerol, 0.1% Tween 20.
And (3) elution: using Buffer B 6 Eluting with 130cm/h flow rate, collecting eluate, buffer B 6 :0.4M NaCl,0.02M Tris-HCl (pH 7.5), 5% glycerol, 0.1% Tween 20.
CIP: CIP cleaning is carried out on the chromatographic column by adopting 2M NaCl and 0.5-1M NaOH.
As shown in Table 4, after the compound chromatography is linearly amplified by 20 times, the recovery rates of the protein and the enzyme activity of the eluent are stable, which indicates that the process amplification is stable and feasible. Wherein the salt concentration can influence the determination of enzyme activity, therefore, when a salt-containing sample is tested, the activity can be detected after the sample is desalted.
TABLE 4 MMC Bestarose 6FF chromatography purification Table
6. Post-treatment
Film-wrapping pyrogen-removing treatment: according to the sample amount, an ultrafiltration membrane package with a proper area is measured, the molecular weight cut-off of the ultrafiltration membrane package is 10KD, the ultrafiltration membrane package is flushed by 0.5M NaOH for at least 30min for sterilization and pyrogen removal, and then the membrane package is flushed by ultrapure water until the pH value is 7.0.
Film moistening and washing: the membrane was then rinsed with displacement Buffer C. Until the pH 7.6. + -. 0.5 is filtered off, concentration is started. Buffer C:0.02M Tris-HCl (pH 7.62), 4mM CaCl 2 。
Concentration: and (4) putting the inlet and the interception end of the membrane package into the eluted protein solution of the composite chromatography in the step (5) for concentration. Concentrating to 1/4 of the original volume, measuring protein concentration at any time by nano during concentration process, wherein the protein concentration is required to be not more than 20mg/mL, and recording the sample volume as V when concentration is completed 1 。
And (3) replacement: adding 3-5 times of V 1 Volume replacement Buffer C was replaced and the volume was concentrated to V 1 The time is the first pass, the number of substitutions is 4, and the end point of the substitutions is pH7.63.
Blending: accurately measuring a certain volume of the ultrafiltration displacement semi-finished product, adding glycerol according to the volume ratio of 1:1, fully and uniformly mixing, and filtering by adopting a 0.22 mu m filter to obtain the finished product.
The SDS-PAGE pattern of the purification process of this example is shown in FIG. 2.
Example 2
The difference from example 1 is that 10L of yeast fermentation broth expressing recombinant DNase I was taken, and the SDS-PAGE pattern of the purification process of 10L of yeast fermentation broth expressing recombinant DNase I is shown in FIG. 3.
Example 3
Purity tests were carried out on the products obtained in examples 1 and 2
The recombinant DNase I obtained by respectively separating and purifying 500mL and 10L of yeast fermentation liquor for expressing the recombinant DNase I is detected and analyzed by SDS-PAGE, and the result is shown in figure 4, the purity is over 99.0 percent by adopting a gel imager to scan gray and adopting a peak area normalization method.
Example 4
The products obtained in examples 1 and 2 were tested for endotoxin content
The determination results of endotoxin content of the recombinant DNase I products obtained in example 1 and example 2 before preparation were determined by using limulus tridentatus kit, and the determination results showed that the content of endotoxin in the recombinant DNase I product obtained in example 1 was 0.21-0.53EU/mg, and the content of endotoxin in the recombinant DNase I product obtained in example 2 was 0.30-0.80EU/mg, which were far below the limit of 10EU/mg of the mRNA vaccine enzyme raw material universal standard.
Example 5
RNase contamination assay was performed on the products obtained in examples 1 and 2
mu.L of DNase I sample and 1.6. Mu.g of MS2 RNA were incubated at 37 ℃ for 4h to detect contamination. As shown in FIG. 5, the recombinant DNase I products obtained in examples 1 and 2 were free of RNase contamination, and 3 DNase I products labeled with RNase free from different manufacturers were purchased from the market and subjected to RNase detection, wherein 1 of the DNase products was free of RNase, and the other two of the DNase products were contaminated to different degrees. Therefore, the DNase I product prepared by the separation and purification method has obvious advantages compared with the same type of samples in the market.
Example 6
Activity measurement was performed on the products obtained in examples 1 and 2
Definition of unit enzyme activity of DNase I: the amount of enzyme required to increase the absorbance at 260nm per mL of the reaction system by 0.01 at 37 ℃ and pH 5.0 in 1min using calf thymus DNA at a final concentration of 33.33. Mu.g/mL as a substrate was defined as 1 enzyme activity unit (U).
Specific enzyme activity: specific enzyme activity is defined as the unit of activity per mg of enzyme protein, expressed in U/mg.
The specific operation method for the activity determination is described in patent document CNl 06520724B. Through determination, the specific activity of the recombinant DNase I product obtained in the embodiment 1 is 13655U/mg, and the specific activity of the recombinant DNase I product obtained in the embodiment 1 is 13782U/mg, so that the market requirements can be met.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. An industrial separation and purification method of DNase I is characterized by comprising the following steps:
s1: centrifuging cell fermentation liquor or cell lysate expressing recombinant DNase I gene to obtain supernatant, and obtaining crude enzyme supernatant of the recombinant DNase I;
s2: concentrating the crude DNase I enzyme solution clarified liquid, and performing ultrafiltration liquid exchange to obtain a one-step sample solution;
s3: enriching and recombining DNase I by the one-step sample loading solution by adopting anion exchange chromatography, improving specific activity, and reducing endotoxin content to obtain anion chromatography eluent;
s4: carrying out affinity chromatography on the anion chromatography eluent, and further purifying the recombinant DNase I to obtain an affinity chromatography eluent;
s5: performing composite chromatography on the affinity chromatography eluent, further purifying the recombinant DNase I, and removing the RNase to obtain a composite chromatography eluent;
s6: and (3) carrying out post-treatment on the composite chromatographic eluent to obtain the separated and purified recombinant DNase I.
2. The method for industrial separation and purification of DNase I as claimed in claim 1, wherein said step S2 is performed by multiple ultrafiltration and displacement with low-salt buffer solution, so as to reduce the ionic strength of the clarified solution of crude DNase I enzyme solution, and facilitate the binding of recombinant DNase I to anion exchange medium; wherein, the low salt buffer solution: 0.02-0.05M Tris-HCl, 0-10% glycerol, 0-0.2% Tween 20, pH 6.8-8.5.
3. The industrial separation and purification method of DNase I according to claim 1, characterized in that said anion exchange chromatography of step S3 is specifically:
regeneration: washing the chromatographic column with NaOH of not less than 0.5M at flow rate of 80-300cm/h for at least 30min for sterilization and pyrogen removal;
balancing: adopting a low-salt buffer solution to balance the volume of the column bed by 10-20 times at a flow speed of 80-300 cm/h;
loading: taking the one-step sample loading liquid to carry out ion column sample loading at the flow speed of 80-300 cm/h;
rebalancing: continuously adopting low-salt buffer solution to balance and wash the loaded chromatographic column by 5-10 times of column volume at the flow speed of 80-300 cm/h;
impurity washing: washing impurities on the column bed by 5-10 times of the column volume at the flow speed of 80-300cm/h by adopting buffer solution with specific salt concentration; wherein the buffer solution with specific salt concentration: 0.05-0.15M NaCl,0.02-0.05M Tris-HCl, 0-10% glycerol, 0-0.2% Tween 20, pH6.8-8.5;
and (3) elution: adopting eluent with specific salt concentration to flush the column bed at the flow speed of 80-300cm/h for 5-10 times of the column volume; wherein, the eluent with specific salt concentration: 0.2-0.4M NaCl,0.02-0.05M Tris-HCl, 0-10% glycerol, 0-0.2% Tween 20, pH 6.8-8.5;
CIP: CIP cleaning is carried out on the chromatographic column by adopting 2M NaCl and 0.5-1M NaOH.
4. The method for industrial separation and purification of DNase I as claimed in claim 3, wherein the Anion Exchange chromatography column comprises strong Anion exchanger type Q and weak Anion exchanger type DEAE, and the strong Anion Exchange product comprises Q Bestarose XL, Q Bestarose FF, Q Sepharose FF, Q Sepharose HP, Q Sephalife FF, unigel-80Q and Nuvia Q Anion Exchange Media; the weak anion exchanger comprises DEAE Sepharose FF, DEAE Sepharose HP and DEAE Sephafe.
5. The method for industrial separation and purification of DNase I according to claim 1, wherein said step S4 of affinity chromatography is specifically:
regeneration: washing the chromatographic column with 0.1M EDTA to remove metal ions, washing the chromatographic column with 0.5M NaOH for at least 30min for sterilization and depyrogenation, washing the chromatographic column with 2M NaCl for 5 times the column volume, washing the chromatographic column with pure water to pH below 10.0, washing the chromatographic column with 0.5M metal ion soluble salt for 5 times the column volume to realize metal ion ligand hanging, and washing the chromatographic column with pure water for 10 times the column volume;
balancing: adopting buffer solution with high salt and low imidazole to balance 10-20 times of column volume of the column bed at the flow rate of 80-400 cm/h; wherein, the buffer solution with high salt and low imidazole: 0.5M NaCl,0.005-0.03M imidazole, 0.02-0.05M Tris-HCl,0% -10% glycerol, 0-0.2% Tween 20, pH7.0-8.0;
loading: respectively adding 0.5M imidazole and 2M NaCl mother liquor into the anion chromatography eluent to ensure 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 buffer solution with high salt and low imidazole, and carrying out metal column affinity chromatography sample loading at the flow rate of 80-400cm/h after fully and uniformly mixing;
rebalancing: after the sample loading is finished, continuously washing the column bed by 5-6 times of the column volume by using a buffer solution with high salt and low imidazole at the flow rate of 80-400 cm/h;
impurity washing: washing the column bed by a high-salt impurity washing buffer solution containing a certain amount of surfactant at a flow rate of 80-400cm/h for 5-10 times of the column volume; wherein, the high-salt impurity washing buffer solution containing a certain amount of surfactant: 0.8 to 1.5M NaCl,2% Triton X100, 0.02 to 0.05M Tris-HCl, pH6.8 to 8.5;
rebalancing: after the impurity washing is finished, continuously washing the column bed by 5-6 times of the column volume by using a buffer solution with high salt and low imidazole at the flow rate of 80-400 cm/h;
and (3) elution: eluting the chromatographic column bed by using elution buffer solution with specific imidazole concentration at the flow rate of 80-400cm/h for 5-10 times of the column volume; wherein, the elution buffer solution with specific imidazole concentration is 0.05-0.2M imidazole, 0.2-0.5M NaCl,0.02-0.05M Tris-HCl, 0-10% glycerol, 0-0.2% Tween 20, pH6.8-8.5;
CIP: the column was washed with 0.5M imidazole.
6. The method for industrial separation and purification of DNase I as claimed in claim 5, wherein the affinity chromatography is metal ion chelating His chromatography, the metal ion is a transition metal ion capable of providing electron donor coordination sites, including Cu 2+ 、Co 2 + 、Ni 2+ 、Zn 2+ 、Fe 2+ And Ca 2+ 。
7. The method for industrial separation and purification of DNase I according to claim 6, characterized in that the affinity chromatography is Ni affinity chromatography using Ni NTA or Ni TED types, and the Ni chromatography column types include Ni Sepharose excel, ni Sepharose FF, ni Bestarose FF, uniNTA-80Ni and Ni NTA Beads 6FF.
8. The method for industrial separation and purification of DNase I according to claim 1, wherein said step S5 of complex chromatography is specifically:
regeneration: washing the chromatographic column with 0.5M NaOH for at least 30min for sterilization and depyrogenation;
balancing: adopting low-salt buffer solution to balance the volume of the chromatographic column bed by 10-20 times at the flow speed of 50-400 cm/h; wherein, the low salt buffer solution: 0.02-0.05M Tris-HCl, 0-10% glycerol, 0-0.2% Tween 20, pH6.8-8.5;
sampling: diluting the affinity chromatography eluent by adopting a low-salt buffer solution, wherein the end point of dilution is in the conductance range of 5-10 mS/cm, and carrying out composite chromatography sampling at the flow speed of 50-400 cm/h;
rebalancing: adopting low-salt buffer solution to balance the volume of the chromatographic column bed by 5-6 times at the flow speed of 50-400 cm/h;
impurity washing: washing the column bed by 5-10 times of column volume by using buffer solution with specific salt concentration at the flow speed of 50-400cm/h, and collecting a washing impurity peak; wherein the buffer of specific salt concentration: 0 to 0.2M NaCl or KCl,0.02 to 0.05M Tris-HCl,0 to 10 percent of glycerin, 0 to 0.2 percent of Tween 20, and pH6.8 to 8.5;
and (3) elution: eluting the column bed by 5-10 times of column volume by using eluent with specific salt concentration at the flow speed of 50-400cm/h, and collecting an elution peak; wherein, the eluent with specific salt concentration: 0.4-0.5M NaCl or KCl,0.02-0.05M Tris-HCl, 0-10% glycerol, 0-0.2% Tween 20, pH6.8-8.5;
CIP: CIP cleaning is carried out on the chromatographic column by adopting 2M NaCl and 0.5-1M NaOH.
9. The method for industrial separation and purification of DNase I as claimed in claim 8, wherein the conditions of complex chromatography are ionic and hydrophobic complex mode packing, said complex packing comprising MMC HF, MMC FF, MMC Bestarose 6FF, capto MMC, diamond MMC and NM90 Agarose.
10. The method for industrial separation and purification of DNase I as claimed in claim 1, wherein the post-treatment of step S6 comprises:
film packaging and pyrogen removing treatment: weighing an ultrafiltration membrane package with an appropriate area according to a sample, wherein the molecular weight cut-off of the ultrafiltration membrane package is not more than 10KD, flushing the ultrafiltration membrane package with 0.5M NaOH for at least 30min for disinfection and pyrogen removal, and then flushing the membrane package with ultrapure water until the pH value is neutral;
film-coating and rinsing: adopting a replacement buffer solution to rinse a membrane package as required until the pH value of the filtered solution is 7.6 +/-0.5, and preparing to start concentration; wherein, the buffer solution for replacement: 0.02M Tris-HCl,4mM CaCl 2 ,pH7.6±0.05;
Concentration: putting an inlet and an interception end of a membrane package into the composite chromatographic eluent for concentration, measuring the concentration of the recombinant DNase I by using nano at any time in the concentration process, wherein the concentration of the recombinant DNase I is required to be not more than 20mg/mL, and recording the volume of a sample as V when the concentration is finished, wherein the V comprises the volume of the membrane package;
and (3) replacement: adding 3-5 times of V displacement buffer solution for displacement, wherein the volume concentration is the first time when the volume is reduced to the original volume V, the displacement times are 3-5 times, the displacement end point is pH7.6 +/-0.5, and the deviation between the conductance and the displacement buffer solution is not more than 1mS/cm, thus obtaining an ultrafiltration displacement semi-finished product;
blending: accurately measuring the ultrafiltration displacement semi-finished product with a certain volume, adding glycerol according to the volume ratio of 1.
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1366042A (en) * | 2001-01-19 | 2002-08-28 | 北京华大基因研究中心 | Process for preparing recombinant deoxyribonuclease I |
CN101294150A (en) * | 2007-04-28 | 2008-10-29 | 福建农林大学 | Ganoderma lucidum deoxyribonuclease and extraction purification process |
CN105001299A (en) * | 2015-07-14 | 2015-10-28 | 天津瑞普生物技术股份有限公司 | Method for removing endotoxin in protein |
US20150329618A1 (en) * | 2012-12-21 | 2015-11-19 | Wuhan Healthgen Biotechnology Corp | Chromatographic method for isolating and purifying high-purity recombined human serum albumin |
CN112266415A (en) * | 2020-10-30 | 2021-01-26 | 江苏艾迪药业股份有限公司 | Method for large-scale production of thrombin regulatory protein |
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Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1366042A (en) * | 2001-01-19 | 2002-08-28 | 北京华大基因研究中心 | Process for preparing recombinant deoxyribonuclease I |
CN101294150A (en) * | 2007-04-28 | 2008-10-29 | 福建农林大学 | Ganoderma lucidum deoxyribonuclease and extraction purification process |
US20150329618A1 (en) * | 2012-12-21 | 2015-11-19 | Wuhan Healthgen Biotechnology Corp | Chromatographic method for isolating and purifying high-purity recombined human serum albumin |
CN105001299A (en) * | 2015-07-14 | 2015-10-28 | 天津瑞普生物技术股份有限公司 | Method for removing endotoxin in protein |
CN112266415A (en) * | 2020-10-30 | 2021-01-26 | 江苏艾迪药业股份有限公司 | Method for large-scale production of thrombin regulatory protein |
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