CN112831487B - Purification method and application of recombinant proteinase K - Google Patents

Purification method and application of recombinant proteinase K Download PDF

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CN112831487B
CN112831487B CN202110112885.4A CN202110112885A CN112831487B CN 112831487 B CN112831487 B CN 112831487B CN 202110112885 A CN202110112885 A CN 202110112885A CN 112831487 B CN112831487 B CN 112831487B
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buffer solution
ultrafiltration
proteinase
recombinant proteinase
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CN112831487A (en
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赵骞
陈亮
李晋
王�锋
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Wuhan Abclonal Inc
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    • C12N9/14—Hydrolases (3)
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Abstract

The invention provides a purification method of recombinant proteinase K and application thereof, wherein the purification method adopts an ultrafiltration-ion exchange-ultrafiltration process, and specifically comprises the following steps: adding a buffer solution A into the recombinant protease K fermentation liquor, uniformly mixing, and performing primary ultrafiltration to obtain an ultrafiltrate from which metal ions and hybrid protein with the molecular weight of less than 20KDa are removed; pumping into a cation exchange column balanced by buffer solution A, then re-balancing with buffer solution A, eluting with buffer solution B, and collecting the eluent; adding buffer solution C into the eluent, mixing uniformly, and performing secondary ultrafiltration. The purification method provided by the invention can greatly improve the production scale by adopting a simple ultrafiltration-ion exchange-ultrafiltration purification process, the daily yield can reach about 500g, the SDS-PAGE purity, the enzyme activity and the specific activity of the product are respectively higher than 95%, 800U/ml and 40U/mg, the cost of the used raw materials is low, and the protein recovery rate is high.

Description

Purification method and application of recombinant proteinase K
Technical Field
The invention belongs to the technical field of separation and purification of enzymes, and particularly relates to a purification method of recombinant proteinase K and application thereof.
Background
Proteinase K was first discovered in the 1974 extracts of Candida albicans (tritirachium album) and was so named because it digests disulfide-rich native Keratin (Keratin) and grows with Keratin as the sole carbon and nitrogen source.
Proteinase K is a serine protease with proteolytic enzyme activity that has a broad spectrum of cleavage capabilities for natural proteins, preferentially cleaving the carboxy-terminal peptide bonds of aliphatic and aromatic amino acids, especially alanine.
Proteinase K is active in a wide range of environments, is also active in high-salt buffer solution at the pH of 4-12.5 and 70 ℃, is compatible with denaturants and detergents (such as urea, SDS, guanidine hydrochloride, guanidine isothiocyanate, Triton and Tween) and the like, and can improve the activity of proteinase K in a certain concentration. The proteinase K has applications in medical treatment (virus and microbe killing), food (meat tenderization), leather (hair softening), wine brewing (wine clarification), amino acid preparation (feather degradation), nucleic acid extraction, in-situ hybridization and other aspects.
With the increasing demand of proteinase K, the large-scale production thereof is important. Specifically, the large-scale production of the proteinase K comprises the processes of fermentation, bacteria liquid separation, filtration, purification, freeze-drying, subpackaging and the like.
In the early stage, because the expression level of the natural proteinase K is low and the natural proteinase K has certain cytotoxicity, the acquisition of enough crude proteinase K is the rate-limiting step; along with the screening of mutant strains, the screening of multi-copy number high-expression proteinase K recombinant yeast strains and the further optimization of fermentation processes, the problem of capacity of crude proteinase K fermentation liquid is gradually solved, so that the purification of the crude proteinase K fermentation liquid is the key for restricting the large-scale production of proteinase K.
The production and purification processes of proteinase K are reported a lot, and the purification method in the early natural extraction process is complex in process, long in purification period and low in recovery rate, so that the mass production is not facilitated; after the development of recombinant expression proteinase K, a 6xhis label is added at the C terminal of the proteinase K, so that the purification purpose is achieved by affinity purification, the purification medium cost of the purification method is high, the risk of Ni shedding exists, and an ideal purification effect can be obtained by three to four steps in actual industrial production.
In conclusion, it is very important to research and develop a purification method of recombinant proteinase K with simple operation, low cost and high recovery rate, and apply the purification method to the industrial separation and purification of recombinant proteinase K, which is a key technical problem that those skilled in the art need to solve urgently.
Disclosure of Invention
The invention aims to provide a method for purifying recombinant proteinase K aiming at the technical problems in the prior art, and discusses the application of the method in the industrial separation and purification of the recombinant proteinase K on the basis of the technical problems.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method of purifying recombinant proteinase K comprising:
s1, adding a buffer solution A into the recombinant proteinase K fermentation liquor, uniformly mixing, and performing primary ultrafiltration to obtain an ultrafiltrate with metal ions removed and hybrid protein with the molecular weight less than 20 KDa;
s2, pumping the ultrafiltrate obtained in the step S1 into a cation exchange column balanced by a buffer solution A, controlling the flow rate to be 0.35-0.6L/min, then carrying out rebalancing by the buffer solution A, eluting by a buffer solution B, and collecting eluent;
and S3, adding a buffer solution C into the eluent, uniformly mixing, and performing secondary ultrafiltration.
Wherein, the buffer solution A, the buffer solution B and the buffer solution C are respectively as follows:
and (3) buffer solution A: 35-60mM MES, 30-55mM NaCl, 1.5-3mM EDTA-2Na, pH 5.5-6.5;
and (3) buffer solution B: 35-60mM MES, 135-170mM NaCl, pH 5.5-6.5;
and (3) buffer C: 15-25mM Tris-HCl, 20-40mM NaCl, 1.5-5mM CaCl2，pH6.0-6.8。
In detail, in the primary ultrafiltration of step S1, since the ionic strength in the fermentation broth is high, the ionic concentration of the protein before loading is reduced by exchanging the buffer a; proteinase K has an isoelectric point of about pH8.9, and at pH5.6-6.5, the protein is positively charged, so that it binds to cation exchange media in low salt ion buffers. In step S2, the protein is eluted with buffer B having a high ionic strength. Further, in order to meet the industrial requirement, the second ultrafiltration of step S3 is performed, and replaced with conventional buffer C, which is advantageous for the subsequent application of proteinase K.
Specifically, EDTA is added to the buffer solution A, so that metal ions which influence the quality of nucleic acid during nucleic acid extraction can be removed; if the pH value of the buffer solution C is adjusted to be about 7.4, precipitation is easy to occur in the step S3 in the solution changing process, so that the solution changing failure is caused; the reason why the buffer C is stable when changing the pH of the buffer C at pH6.0 to 6.8 is that the pH of the buffer C is far from the isoelectric point of proteinase K.
In the above technical solution, in step S1, the number of times of one ultrafiltration after adding buffer a is 2-6, preferably 4.
Further, in the above technical solution, in step S1, the volume of the buffer a added is 0.8-2 times, preferably 1-1.2 times of the recombinant proteinase K fermentation broth.
Specifically, in the single ultrafiltration in step S1, the total volume of buffer a changed from the original volume should be 16 times or more. If the total volume of the exchange solution is not enough, the ionic strength is not reduced (to Cond6.0 +/-0.5 mS/cm), which can affect the combination efficiency of the proteinase K and the ion exchange medium; if the total volume of the liquid change is 32 times or more of the original volume, the operation time of the step is increased, the product quality is not further improved, the cost is not cost-effective in terms of time, and as the liquid change times are increased, proteinase K is lost and the recovery rate is reduced.
In a preferred embodiment of the present invention, step S1 further includes filtering the recombinant proteinase K fermentation broth with a hydrophilic polyethersulfone bag filter before adding buffer a and mixing and performing primary ultrafiltration.
Preferably, in the technical scheme, the pore diameter of the hydrophilic polyethersulfone bladder-type filter element is 0.22 μm.
In the above technical solution, in step S3, the volume of the buffer C added is 2.5-4 times, preferably 3-3.5 times of the volume of the eluent.
Further, in the above technical solution, in step S3, the number of times of the second ultrafiltration after adding the buffer C is 2-4 times, preferably 3 times.
Still further, in the above technical solution, in step S2, the cation exchange medium of the cation exchange column is SP Bestarose FF, UNOsphere Rapid S and POROSTMXS.
Still further, in the above technical solution, the first ultrafiltration and/or the second ultrafiltration is one of membrane filtration ultrafiltration and hollow fiber ultrafiltration membrane ultrafiltration.
In one embodiment of the present invention, the method for purifying recombinant proteinase K specifically comprises the following steps:
(1) filtering the recombinant proteinase K fermentation liquor by using a plate frame, collecting the recombinant proteinase K fermentation liquor, placing the recombinant proteinase K fermentation liquor in a refrigerating way at 2-8 ℃, filtering the recombinant proteinase K fermentation liquor by using a hydrophilic polyether sulfone bag-type filter element with the diameter of 0.22um to further remove cell fragments and other impurities before purification, and mixing the filtrate with a buffer solution A according to the volume ratio of 1: 1 mixingAfter homogenization, the mixture was retained with a retention of 8kDa, a surface area of 2.5m2The ultrafiltration membrane is used for exchanging the solution and concentrating, when the volume is concentrated to half of the initial volume, the buffer solution A with the same volume is added again and mixed evenly, and then 8KDa is intercepted, the surface area is 2.5m2The ultrafiltration membrane is used for exchanging liquid and concentrating, and the steps are continuously repeated for 4 times to remove metal ions and heteroproteins with the molecular weight less than 20 KDa;
(2) cation exchange purification (Shanghai Bogelong SP Bestarose FF purification medium) is carried out by a sharp APPS pilot full-automatic protein purifier, specifically, after a system is cleaned by ultrapure water, a cation exchange column is balanced by a buffer solution A, after the balance is finished, sample loading is carried out at the flow rate of 0.5L/min, after the sample loading is finished, the buffer solution A is used for re-balance, finally, a buffer solution B is used for elution, and eluent is collected;
(3) diluting the eluate obtained in step (2) with 3 volumes of buffer C, and retaining 8kDa with a surface area of 2.5m2The ultrafiltration membrane is used for exchanging the liquid and concentrating, when the volume is concentrated to one fourth of the initial volume, the same volume of buffer solution C is added again and mixed evenly, and then 8KDa is retained, the surface area is 2.5m2And (3) continuously repeating the ultrafiltration membrane exchange solution and concentration for 3 times to obtain the purified recombinant proteinase K.
Wherein, the buffer solution A, the buffer solution B and the buffer solution C are respectively as follows:
and (3) buffer solution A: 50mM MES, 40mM NaCl, 2mM EDTA-2Na, pH 5.9-6.1;
and (3) buffer solution B: 50mM MES, 150mM NaCl, pH 5.9-6.1;
and (3) buffer C: 20mM Tris-HCl, 30mM NaCl, 2mM CaCl2，pH 6.2-6.4。
The invention also provides the application of the recombinant proteinase K purification method in the separation and purification of the recombinant proteinase K.
Compared with the prior art, the invention has the beneficial effects that:
(1) the purification method of the recombinant proteinase K provided by the invention meets the product requirements only by adopting a simple ultrafiltration-ion exchange-ultrafiltration purification process, is very suitable for industrial production, and can reach a production scale of about 500g per day when a Suzhou Li ear APPSpilot full-automatic protein purification instrument is adopted;
(2) the reagent used in the purification method of the recombinant proteinase K provided by the invention has low cost of raw materials and high protein recovery rate, and the recovery rate reaches about 69%;
(3) the SDS-PAGE purity of the proteinase K prepared by the purification method of the recombinant proteinase K provided by the invention is more than 95%, the enzyme activity is more than 800U/ml, and the specific activity is more than 40U/mg.
Drawings
FIG. 1 is a diagram showing the detection result of metal ion residues according to an embodiment of the present invention;
FIG. 2 is an electrophoretogram showing the result of DNase residual detection of proteinase K in the example of the present invention;
FIG. 3 is an electrophoretogram showing the result of RNase residue detection of proteinase K in the examples of the present invention.
Detailed Description
The present invention is further described in detail below with reference to specific examples so that those skilled in the art can more clearly understand the present invention.
The following examples are given for the purpose of illustration only and are not intended to limit the scope of the invention.
All other embodiments obtained by a person skilled in the art based on the specific embodiments of the present invention without any inventive step are within the scope of the present invention.
In the examples of the present invention, all the raw material components are commercially available products well known to those skilled in the art unless otherwise specified.
In the embodiments of the present invention, unless otherwise specified, all technical means are conventional and well-known to those skilled in the art.
In the examples of the present invention, the raw materials used and the relevant equipment information are shown in the following table.
Figure BDA0002919635380000061
Figure BDA0002919635380000071
Example 1
The embodiment of the invention provides a purification method of recombinant proteinase K, which specifically comprises the following steps:
(1) filtering the recombinant proteinase K fermentation liquor by using a plate frame, collecting the recombinant proteinase K fermentation liquor, placing the recombinant proteinase K fermentation liquor in a refrigerating way at 2-8 ℃, filtering the recombinant proteinase K fermentation liquor by using a hydrophilic polyether sulfone bag-type filter element with the diameter of 0.22um to further remove cell fragments and other impurities before purification, and mixing the filtrate with a buffer solution A according to the volume ratio of 1: 1 after mixing, the mixture is retained by 8KDa and the surface area is 2.5m2The ultrafiltration membrane is used for exchanging and concentrating, when the volume is concentrated to half of the initial volume, the same volume of buffer solution A is added again for mixing, and then 8KDa is retained, the surface area is 2.5m2The ultrafiltration membrane is used for exchanging liquid and concentrating, and the steps are continuously repeated for 4 times to remove metal ions and heteroproteins with the molecular weight less than 20 KDa;
(2) cation exchange purification (Shanghai Bogelong SP Bestarose FF purification medium) is carried out by a sharp APPS pilot full-automatic protein purifier, specifically, after a system is cleaned by ultrapure water, a cation exchange column is balanced by a buffer solution A, after the balance is finished, sample loading is carried out at the flow rate of 0.5L/min, after the sample loading is finished, the buffer solution A is used for re-balance, finally, a buffer solution B is used for elution, and eluent is collected;
(3) diluting the eluate obtained in step (2) with 3 volumes of buffer C, and retaining 8kDa with a surface area of 2.5m2The ultrafiltration membrane is used for exchanging the liquid and concentrating, when the volume is concentrated to one fourth of the initial volume, the same volume of buffer solution C is added again and mixed evenly, and then 8KDa is retained, the surface area is 2.5m2And (3) continuously repeating the ultrafiltration membrane exchange solution and concentration for 3 times to obtain the purified recombinant proteinase K.
Wherein, the buffer solution A, the buffer solution B and the buffer solution C are respectively as follows:
and (3) buffer solution A: 50mM MES, 40mM NaCl, 2mM EDTA-2Na, pH 5.9-6.1;
and (3) buffer solution B: 50mM MES, 150mM NaCl, pH 5.9-6.1;
and (3) buffer C: 20mM Tris-HCl, 30mM NaCl, 2mM CaCl2，pH 6.2-6.4。
Comparative example 1
Compared with the embodiment 1, the purification method of the recombinant proteinase K is only different in that the buffer solution A1 does not contain EDTA-2Na, and specifically comprises the following steps:
(1) filtering the recombinant proteinase K fermentation liquor by using a plate frame, collecting the recombinant proteinase K fermentation liquor, placing the recombinant proteinase K fermentation liquor in a refrigerating way at 2-8 ℃, filtering the recombinant proteinase K fermentation liquor by using a hydrophilic polyether sulfone bag-type filter element with the diameter of 0.22um to further remove cell fragments and other impurities before purification, and mixing the filtrate with a buffer solution A1 according to the volume ratio of 1: 1 after mixing, the mixture is retained by 8KDa and the surface area is 2.5m2The ultrafiltration membrane is used for exchanging the liquid and concentrating, when the volume is concentrated to half of the initial volume, the same volume of buffer solution A1 is added again for mixing, and then 8KDa is retained, the surface area is 2.5m2The ultrafiltration membrane is used for exchanging liquid and concentrating, and the steps are continuously repeated for 4 times to remove metal ions and heteroproteins with the molecular weight less than 20 KDa;
(2) cation exchange purification (Shanghai Bogelong SP Bestarose FF purification medium) is carried out by a sharp APPS pilot full-automatic protein purifier, specifically, after a system is cleaned by ultrapure water, a cation exchange column is balanced by a buffer solution A1, sample loading is carried out at the flow rate of 0.5L/min after the balance is finished, the sample loading is carried out again by a buffer solution A1 after the sample loading is finished, finally, elution is carried out by a buffer solution B, and eluent is collected;
(3) diluting the eluate obtained in step (2) with 3 volumes of buffer C, and retaining 8kDa with a surface area of 2.5m2The ultrafiltration membrane is used for exchanging the liquid and concentrating, when the volume is concentrated to one fourth of the initial volume, the same volume of buffer solution C is added again and mixed evenly, and then 8KDa is retained, the surface area is 2.5m2And (3) continuously repeating the ultrafiltration membrane exchange solution and concentration for 3 times to obtain the purified recombinant proteinase K.
Wherein the buffer solution A1, the buffer solution B and the buffer solution C are respectively as follows:
buffer a 1: 50mM MES, 40mM NaCl, pH 5.9-6.1;
and (3) buffer solution B: 50mM MES, 150mM NaCl, pH 5.9-6.1;
and (3) buffer C: 20mM Tris-HCl, 30mM NaCl, 2mM CaCl2，pH 6.2-6.4。
Comparative example 2
The comparative example of the invention provides a purification method of recombinant proteinase K, compared with example 1, the only difference is that the pH of the buffer C1 is adjusted to 7.3-7.5, and the purification method specifically comprises the following steps:
(1) filtering the recombinant proteinase K fermentation liquor by using a plate frame, collecting the recombinant proteinase K fermentation liquor, placing the recombinant proteinase K fermentation liquor in a refrigerating way at 2-8 ℃, filtering the recombinant proteinase K fermentation liquor by using a hydrophilic polyether sulfone bag-type filter element with the diameter of 0.22um to further remove cell fragments and other impurities before purification, and mixing the filtrate with a buffer solution A according to the volume ratio of 1: 1 after mixing, the mixture is retained by 8KDa and the surface area is 2.5m2The ultrafiltration membrane is used for exchanging and concentrating, when the volume is concentrated to half of the initial volume, the same volume of buffer solution A is added again for mixing, and then 8KDa is retained, the surface area is 2.5m2The ultrafiltration membrane is used for exchanging liquid and concentrating, and the steps are continuously repeated for 4 times to remove metal ions and heteroproteins with the molecular weight less than 20 KDa;
(2) performing cation exchange purification (Shanghai Bogelong SP Bestarose FF purification medium) by using a sharp APPS pilot full-automatic protein purification instrument, specifically, cleaning the system by using ultrapure water, balancing a cation exchange column by using a buffer solution A, loading at a flow rate of 0.5L/min after balancing, re-balancing by using the buffer solution A after loading, finally eluting by using a buffer solution B, and collecting eluent;
(3) diluting the eluate obtained in step (2) with 3 volumes of buffer C1, and retaining 8kDa with a surface area of 2.5m2The ultrafiltration membrane of (2) is exchanged and concentrated, during which a precipitate generally does not reach half the initial volume and the exchange step is terminated with a failure.
Wherein the buffer solution A, the buffer solution B and the buffer solution C1 are respectively as follows:
and (3) buffer solution A: 50mM MES, 40mM NaCl, 2mM EDTA-2Na, pH 5.9-6.1;
and (3) buffer solution B: 50mM MES, 150mM NaCl, pH 5.9-6.1;
and (3) buffer C: 20mM Tris-HCl, 30mM NaCl, 2mM CaCl2，pH 7.3-7.5。
Comparative example 3
The comparative example of the present invention provides a method for purifying recombinant proteinase K, which is different from example 1 only in that the number of times of one ultrafiltration in step S1 is 7, and specifically comprises the following steps:
(1) filtering the recombinant proteinase K fermentation liquor by using a plate frame, collecting the recombinant proteinase K fermentation liquor, refrigerating the recombinant proteinase K fermentation liquor at the temperature of-4 ℃, filtering the recombinant proteinase K fermentation liquor by using a hydrophilic polyether sulfone bag-type filter element of 0.22um to further remove cell fragments and other impurities before purification, and mixing the filtrate with a buffer solution A1 according to the volume ratio of 1: 1 after mixing, the mixture is retained by 8KDa and the surface area is 2.5m2The ultrafiltration membrane is used for exchanging the liquid and concentrating, when the volume is concentrated to half of the initial volume, the same volume of buffer solution A1 is added again for mixing, and then 8KDa is retained, the surface area is 2.5m2The ultrafiltration membrane of (2) is exchanged and concentrated, repeated 7 times continuously, and metal ions and heteroproteins with molecular weight lower than about 20KD are removed;
(2) cation exchange purification (Shanghai Bogelong SP Bestarose FF purification medium) is carried out by a sharp APPS pilot full-automatic protein purifier, specifically, after a system is cleaned by ultrapure water, a cation exchange column is balanced by a buffer solution A1, sample loading is carried out at the flow rate of 0.5L/min after the balance is finished, the sample loading is carried out again by a buffer solution A1 after the sample loading is finished, finally, elution is carried out by a buffer solution B, and eluent is collected;
(3) diluting the eluate obtained in step (2) with 3 volumes of buffer C, and retaining 8kDa with a surface area of 2.5m2The ultrafiltration membrane is used for exchanging the liquid and concentrating, when the volume is concentrated to one fourth of the initial volume, the same volume of buffer solution C is added again and mixed evenly, and then 8KDa is retained, the surface area is 2.5m2And (3) continuously repeating the ultrafiltration membrane exchange solution and concentration for 3 times to obtain the purified recombinant proteinase K.
Wherein the buffer solution A1, the buffer solution B and the buffer solution C are respectively as follows:
buffer A: 50mM MES, 40mM NaCl, 2mM EDTA-2Na, pH 5.9-6.1;
and (3) buffer solution B: 50mM MES, 150mM NaCl, pH 5.9-6.1;
and (3) buffer C: 20mM Tris-HCl, 30mM NaCl, 2mM CaCl2，pH 6.2-6.4。
Test results
(1) Calculation of recovery
The amount of total protein was calculated from the volume and the concentration of total protein, and then the recovery after each step was calculated by comparison with the original initial enzyme solution (recovery was calculated as 100%).
The specific results are shown in the following table:
Figure BDA0002919635380000111
analysis of the table shows that the increase of the times of ultrafiltration liquid exchange in the comparative example 3 affects the recovery rate of the final product and also increases the time of the step; meanwhile, the recovery rate of the proteinase K in the embodiment of the invention is about 69%.
(2) Activity assay
After purifying the product of the bioscience GmbH of Bautake, Wuhan, Egyoka, by the methods of example 1 and comparative example 1, respectively, a 20mg/ml solution was prepared with commercially available product 1 (Kingsu Kinuoan proteinase K product) and commercially available product 2(sigma proteinase K product), respectively, and then the enzyme activity and specific activity were measured.
The activity determination method refers to a spectrophotometric method of Folin & Ciocalteu's Phenol reagent with casein as a substrate (specifically referring to sigma official network).
The test results are shown in the following table:
Figure BDA0002919635380000112
from the activity measurement, it can be seen that the activity of proteinase K purified in the present invention is equivalent to that of product 1, and slightly higher than that of product 2.
(3) Metal ion residue detection
Using the proteinase K products obtained in example 1 and comparative example 1, respectively, commercially available product 1 (Jiangsu Kinuoan proteinase K product) was incubated with plasmid pUC19, and whether the DNA was stable or not was observed. Namely: the proteinase K solution and pUC19 plasmid without DNase pollution are incubated for 16 hours at 37 ℃, agarose gel electrophoresis is carried out to detect whether the plasmid is complete, and the 20ul reaction system is as follows: unit (ul)
Figure BDA0002919635380000121
The results of the detection are shown in FIG. 1. In the figure, lanes 1 and 2 correspond to the samples obtained by purifying the product of the bioscience, Wuhan-ai-Bordetec Biotech Co., Ltd. by the method of example 1, lanes 3 and 4 correspond to the samples obtained by purifying the product of the bioscience, Wuhan-ai-Bordetec Biotech Co., Ltd. by the method of comparative example 1, and lane A corresponds to the commercially available product 1 (Jiangsu Kinopanan proteinase K product); as can be seen from the figure, if EDTA-2Na is not added to buffer A1, the excess of metal ions may be caused, resulting in non-specific DNA fragmentation.
(4) DNase residue detection of proteinase K
Samples obtained by purifying products of the botaike biotechnology limited, wuhan ai by the method of example 1 were incubated with proteinase K solutions of commercially available products 1 (the product of keiskei kininorage proteinase K) and 2 (the product of sigma proteinase K) and pUC19 plasmid without dnase contamination at 37 ℃ for 16 hours, respectively, to determine whether there was dnase residue, and a blank control was set, and 20ul of the reaction system was as follows: unit (ul)
Figure BDA0002919635380000122
After the reaction was completed, 6 XDNA loading buffer was added to terminate the reaction, and the reaction was run on 0.7% agarose gel.
The electrophoretogram is shown in FIG. 2; in the figure, lanes 1 and 2 correspond to the commercial product 1 (Jiangsu Kinoprofen proteinase K product); 3. lane 4 corresponds to commercial product 2(sigma proteinase K product); 5. lane 6 corresponds to a sample purified from the product of boc-itake biotechnology limited, wuhan using the method of example 1; 7. lane 8 corresponds to a blank control group; m is a DNA Marker. As can be seen from the figure, the proteinase K samples obtained after purification of the product of the Biotech Co., Ltd, Bauitak, Egyptian, in Wuhan, by the method of example 1, were free from residual contamination of DNase.
(5) RNase residue detection of proteinase K
Samples obtained by purifying products of the bioscience ltd, bocek, e.g., wuhan, by the method of example 1, were incubated with proteinase K solutions of commercially available products 1 (kegsu kininoran proteinase K product) and 2(sigma proteinase K product) and newly extracted RNA without rnase contamination at 37 ℃ for 16 hours, respectively, to determine whether rnase residues were present, and a blank control was set, and 20ul of the reaction system was as follows: unit (ul)
Figure BDA0002919635380000131
After the reaction was completed, 6 XRNA loading buffer was added to terminate the reaction, and the reaction was run on 0.7% agarose gel.
The electrophoretogram is shown in FIG. 3; in the figure, lanes 1 and 2 correspond to the commercial product 1 (Jiangsu Kinoprofen proteinase K product); 3. lane 4 corresponds to commercial product 2(sigma proteinase K product); 5. lane 6 corresponds to a sample purified from the product of boc-itake biotechnology limited, wuhan using the method of example 1; 7. lane 8 corresponds to a blank control group; and 9 is a DNA Marker. As can be seen from the figure, the proteinase K sample obtained after purifying the product of the Biotech company Limited, Bauitaike, Wuhan by the method of example 1 has no RNase residue contamination.
Finally, the method of the present invention is only a preferred embodiment and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (14)

1. A method for purifying recombinant proteinase K,
the method comprises the following steps:
s1, adding a buffer solution A into the recombinant proteinase K fermentation liquor, uniformly mixing, and performing primary ultrafiltration to obtain an ultrafiltrate with metal ions removed and hybrid protein with the molecular weight less than 20 KDa;
s2, pumping the ultrafiltrate obtained in the step S1 into a cation exchange column balanced by a buffer solution A, controlling the flow rate to be 0.35-0.6L/min, then carrying out rebalancing by the buffer solution A, eluting by a buffer solution B, and collecting eluent;
s3, adding a buffer solution C into the eluent, mixing uniformly, and performing secondary ultrafiltration to obtain the eluent;
wherein:
and (3) buffer solution A: 35-60mM MES, 30-55mM NaCl, 1.5-3mM EDTA-2Na, pH 5.5-6.5;
and (3) buffer solution B: 35-60mM MES, 135-;
and (3) buffer C: 15-25mM Tris-HCl, 20-40mM NaCl, 1.5-5mM CaCl2，pH 6.0-6.8。
2. The method for purifying recombinant proteinase K according to claim 1,
in step S1, the number of times of ultrafiltration after adding buffer A is 2-6.
3. The method for purifying recombinant proteinase K according to claim 2,
the number of times of ultrafiltration after addition of buffer A was 4.
4. The method of purifying recombinant proteinase K according to claim 2,
in step S1, the volume of the buffer solution A added is 0.8-2 times of the recombinant proteinase K fermentation liquid.
5. The method for purifying recombinant proteinase K according to claim 4,
the adding volume of the buffer solution A is 1-1.2 times of the recombinant proteinase K fermentation liquor.
6. The method for purifying recombinant proteinase K according to claim 1,
and step S1, filtering the recombinant proteinase K fermentation liquor by using a hydrophilic polyether sulfone bag type filter element before adding the buffer solution A, uniformly mixing and carrying out primary ultrafiltration.
7. The method for purifying recombinant proteinase K according to claim 6,
the aperture of the hydrophilic polyether sulfone bag type filter element is 0.22 mu m.
8. The method for purifying recombinant proteinase K according to claim 1,
in step S3, the volume of buffer C added is 2.5-4 times of the volume of the eluent.
9. The method for purifying recombinant proteinase K according to claim 8,
the volume of the buffer solution C added is 3-3.5 times of the eluent.
10. The method of purifying recombinant proteinase K according to claim 8,
in step S3, the number of times of the secondary ultrafiltration after adding the buffer C is 2-4.
11. The method for purifying recombinant proteinase K according to claim 10,
the number of times of the secondary ultrafiltration after the addition of buffer C was 3.
12. The method for purifying recombinant proteinase K according to any one of claims 1 to 11,
in step S2, the cation exchange medium of the cation exchange column is SP Bestarose FF, UNOsphere Rapid S and POROS™XS.
13. The method for purifying recombinant proteinase K according to any one of claims 1 to 11,
the primary ultrafiltration and/or the secondary ultrafiltration is one of membrane-packed ultrafiltration and hollow fiber ultrafiltration membrane ultrafiltration.
14. Use of the method of purifying recombinant proteinase K according to any one of claims 1 to 13 for the isolation and purification of recombinant proteinase K.
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