CN114410613A - Purification method and application of recombinant proteinase K - Google Patents
Purification method and application of recombinant proteinase K Download PDFInfo
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- 108010067770 Endopeptidase K Proteins 0.000 title claims abstract description 48
- 238000000746 purification Methods 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 28
- 239000007853 buffer solution Substances 0.000 claims abstract description 101
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 47
- 239000003480 eluent Substances 0.000 claims abstract description 29
- 230000002572 peristaltic effect Effects 0.000 claims abstract description 27
- 239000006228 supernatant Substances 0.000 claims abstract description 27
- 238000002156 mixing Methods 0.000 claims abstract description 21
- 238000000855 fermentation Methods 0.000 claims abstract description 20
- 230000004151 fermentation Effects 0.000 claims abstract description 20
- 238000003756 stirring Methods 0.000 claims abstract description 20
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 14
- 238000001042 affinity chromatography Methods 0.000 claims abstract description 12
- 150000002500 ions Chemical class 0.000 claims abstract description 12
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 12
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 12
- 108091005804 Peptidases Proteins 0.000 claims abstract description 9
- 239000004365 Protease Substances 0.000 claims abstract description 8
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims abstract description 8
- 229910021645 metal ion Inorganic materials 0.000 claims abstract description 8
- 235000019419 proteases Nutrition 0.000 claims abstract description 8
- 238000005086 pumping Methods 0.000 claims abstract description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 64
- 239000011780 sodium chloride Substances 0.000 claims description 32
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 18
- 239000000872 buffer Substances 0.000 claims description 15
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 12
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 7
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims description 7
- 239000001110 calcium chloride Substances 0.000 claims description 7
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 7
- 235000011148 calcium chloride Nutrition 0.000 claims description 7
- 239000012528 membrane Substances 0.000 claims description 7
- 239000000919 ceramic Substances 0.000 claims description 6
- 239000012501 chromatography medium Substances 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 238000002955 isolation Methods 0.000 claims 1
- 238000005265 energy consumption Methods 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 238000000605 extraction Methods 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108010022999 Serine Proteases Proteins 0.000 description 2
- 102000012479 Serine Proteases Human genes 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- NRTLIYOWLVMQBO-UHFFFAOYSA-N 5-chloro-1,3-dimethyl-N-(1,1,3-trimethyl-1,3-dihydro-2-benzofuran-4-yl)pyrazole-4-carboxamide Chemical compound C=12C(C)OC(C)(C)C2=CC=CC=1NC(=O)C=1C(C)=NN(C)C=1Cl NRTLIYOWLVMQBO-UHFFFAOYSA-N 0.000 description 1
- 241000222122 Candida albicans Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102000011782 Keratins Human genes 0.000 description 1
- 108010076876 Keratins Proteins 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 241001523956 Parengyodontium album Species 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108090000787 Subtilisin Proteins 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- -1 aromatic amino acids Chemical class 0.000 description 1
- 229940095731 candida albicans Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000005341 cation exchange Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000003028 enzyme activity measurement method Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012510 hollow fiber Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000008235 industrial water Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21064—Peptidase K (3.4.21.64)
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Abstract
The invention provides a purification method of recombinant proteinase K, which 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 the ultrafiltrate obtained in the step S1 into an ion affinity chromatography gel balanced by a buffer solution A, placing the chromatography gel in a purification barrel, subsequently stirring to facilitate the combination of the chromatography gel and protease k, subsequently settling, removing the supernatant by a peristaltic pump, subsequently rebalancing by the buffer solution A, standing, removing the supernatant by the peristaltic pump, adding the buffer solution B, stirring and settling, removing the supernatant by the peristaltic pump, adding the buffer solution C, stirring and settling, removing the supernatant by the peristaltic pump, eluting by the buffer solution D, and collecting the eluent; adding buffer solution E into the eluent, mixing uniformly, and performing secondary ultrafiltration. The purification method of the recombinant proteinase K provided by the invention has rapid purification speed; and has low requirements on equipment and low energy consumption.
Description
Technical Field
The invention relates 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 is a serine proteinase related to subtilisin and is purified from Candida albicans (Tritirachium album Limber). It is a highly active broad-spectrum protease, can grow in an environment with keratin (Kerantin) as the only carbon and nitrogen source, and is named as proteinase K.
Proteinase K has a wide range of enzymatic activities and substrate specificities, and is a serine Proteinase with a wide range of cleavage activities. The relative molecular weight of the protease K is about 29.3kDa, can decompose peptide bonds and ester bonds adjacent to the c-terminal of hydrophobic amino acids, sulfur-containing amino acids and aromatic amino acids, and can be applied to biological technologies such as degradation of proteins.
The ProteinaseK has wide application, and can be used for experiments such as nucleic acid separation and purification (extraction of DNA and RNA), western blotting, nuclease removal in preparation of DNA and RNA and the like; it can be used for food processing (meat tenderization application, etc.), and can also be used for the inactivation of industrial water. In summary, proteinase K has excellent properties, so that it plays a role in various aspects such as biomedicine, food processing, environmental protection, etc., and thus has very important research value.
But the production and purification process of the proteinase K is rarely reported, and the purification method in the early natural extraction process has the disadvantages of complex process, long purification period and low recovery rate, and is not beneficial to mass production; in view of the above situation, the present application provides a method for purifying recombinant proteinase K and applications thereof.
Disclosure of Invention
The invention aims to provide a purification method of recombinant proteinase K and application thereof, and the purification method has the advantages of high purification speed and good purification effect.
The invention adopts the following technical scheme to solve the technical problems:
a method for purifying recombinant proteinase K comprising the steps of:
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 ion affinity chromatography gel balanced by a buffer solution A, placing the chromatography gel in a purification barrel, subsequently stirring to facilitate the combination of the chromatography gel and protease k, then settling for 1-3 hours, removing supernatant by a peristaltic pump, then re-balancing by the buffer solution A, standing, removing supernatant by the peristaltic pump, adding the buffer solution B, stirring the solution for 10-30min, settling for 10-30min, removing supernatant by the peristaltic pump, adding the buffer solution C, stirring the solution for 10-30min, settling for 10-30min, removing supernatant by the peristaltic pump, eluting by the buffer solution D, and collecting eluent;
s3, adding a buffer solution E 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 6.5-7.5;
and (3) buffer solution B: 35-60mM MES, 1mM NaCl, 10mM DNAse, pH 6.5-7.5;
and (3) buffer C: 35-60mM MES, 1M NaCl, pH 6.5-7.5;
and (3) buffer solution D: 15-25mM Tris-HCl, 20-40mM NaCl, 100mM imidazole, pH 6.5-7.5;
and (3) buffer solution E: 15-25mM Tris-HCl, 20-40mM NaCl, 1.5-5mM CaCl2, pH 6.5-7.5.
Further, the concentration volume of the ultrafiltration after the buffer solution A is added in the step S1 is 1/10-1/20 of the initial liquid volume; preferably 1/20.
Further, the adding volume of the buffer solution A is 0.8-2 times of that of the recombinant proteinase K fermentation liquor; preferably 1 to 1.5 times.
Further, in the step S1, before adding the buffer solution a, mixing uniformly and performing primary ultrafiltration, the recombinant proteinase K fermentation broth is filtered by a ceramic membrane.
Further, in the step S3, the volume of the buffer E added is 3-6 times of the volume of the eluent; preferably 4 to 4.5 times.
Further, in the step S3, the concentration multiple of the secondary ultrafiltration after adding the buffer C is 2 to 6 times; preferably 3 to 4 times.
Further, the ion affinity chromatography medium is SP Bestarose FF, UNOspHere Rapid S and POROSTMXS.
The purification method of the recombinant proteinase K is applied to the separation and purification of the recombinant proteinase K. The invention has the advantages that:
the purification method of the recombinant proteinase K has rapid purification speed; the purified proteinase k contains low dna content, which is beneficial to the subsequent nucleic acid extraction; low requirement on equipment and low energy consumption.
Detailed Description
The invention is further illustrated by the following examples, which are intended to illustrate, but not to limit the invention further.
Example 1
This example provides a method for purifying recombinant proteinase K, comprising 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 ion affinity chromatography gel balanced by a buffer solution A, placing the chromatography gel in a purification barrel, subsequently stirring to facilitate the combination of the chromatography gel and protease k, then settling for 2 hours, removing supernatant by a peristaltic pump, then re-balancing by the buffer solution A, standing, removing supernatant by the peristaltic pump, adding the buffer solution B, stirring the solution for 20min, settling for 20min, removing supernatant by the peristaltic pump, adding the buffer solution C, stirring the solution for 20min, settling for 20min, removing supernatant by the peristaltic pump, eluting by the buffer solution D, and collecting eluent;
s3, adding a buffer solution E into the eluent, mixing uniformly, and performing secondary ultrafiltration to obtain the eluent;
wherein:
and (3) buffer solution A: 45mM MES, 45mM NaCl, 2mM EDTA-2Na, pH 7;
and (3) buffer solution B: 45mM MES, 1mM NaCl, 10mM DNAse, pH 7;
and (3) buffer C: 45mM MES, 1M NaCl, pH 7;
and (3) buffer solution D: 20mM Tris-HCl, 30mM NaCl, 100mM imidazole, pH 7;
and (3) buffer solution E: 20mM Tris-HCl, 30mM NaCl, 3mM CaCl2, pH 7.
The concentration volume of the ultrafiltration after the buffer solution A is added in the step S1 is 1/20 of the initial liquid volume; the adding volume of the buffer solution A is 1.2 times of that of the recombinant proteinase K fermentation liquor; in the step S1, buffer solution A is added and mixed evenly and onceBefore ultrafiltration, filtering the recombinant proteinase K fermentation liquor by using a ceramic membrane; in the step S3, the volume of the buffer E added is 4.2 times of the volume of the eluent; in the step S3, the concentration multiple of the secondary ultrafiltration is 2-6 times after the buffer solution C is added; preferably 3 to 4 times; the ion affinity chromatography medium is POROSTMOne of XS; the primary ultrafiltration and the secondary ultrafiltration are membrane-packed ultrafiltration.
Example 2
This example provides a method for purifying recombinant proteinase K, comprising 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 ion affinity chromatography gel balanced by a buffer solution A, placing the chromatography gel in a purification barrel, subsequently stirring to facilitate the combination of the chromatography gel and protease k, then settling for 1 hour, removing supernatant by a peristaltic pump, then re-balancing by the buffer solution A, standing, removing supernatant by the peristaltic pump, adding the buffer solution B, stirring the solution for 10min, settling for 10min, removing supernatant by the peristaltic pump, adding the buffer solution C, stirring the solution for 10min, settling for 10min, removing supernatant by the peristaltic pump, eluting by the buffer solution D, and collecting eluent;
s3, adding a buffer solution E into the eluent, mixing uniformly, and performing secondary ultrafiltration to obtain the eluent;
wherein:
and (3) buffer solution A: 35mM MES, 30mM NaCl, 1.5mM EDTA-2Na, pH 6.5;
and (3) buffer solution B: 35mM MES, 1mM NaCl, 10mM DNAse, pH 6.5;
and (3) buffer C: 35mM MES, 1M NaCl, pH 6.5;
and (3) buffer solution D: 15mM Tris-HCl, 20-40mM NaCl, 100mM imidazole, pH 6.5;
and (3) buffer solution E: 15mM Tris-HCl, 20mM NaCl, 1.5M CaCl2, pH 6.5.
The concentration volume of the ultrafiltration after the buffer solution A is added in the step S1 is 1/10 of the initial liquid volume; the adding volume of the buffer solution A is 1 time of that of the recombinant protease K fermentation liquor; in the step S1, before adding the buffer solution A for uniform mixing and primary ultrafiltration, filtering the recombinant proteinase K fermentation liquor by using a ceramic membrane; in the step S3, the volume of the buffer solution E added is 4.5 times of that of the eluent; in the step S3, the concentration multiple of the secondary ultrafiltration is 3 times after the buffer solution C is added; the ion affinity chromatography medium is UNOspHere Rapid S; the primary ultrafiltration and/or the secondary ultrafiltration is hollow fiber ultrafiltration membrane ultrafiltration.
Example 3
This example provides a method for purifying recombinant proteinase K, comprising 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 ion affinity chromatography gel balanced by a buffer solution A, placing the chromatography gel in a purification barrel, subsequently stirring to facilitate the combination of the chromatography gel and protease k, then settling for 3 hours, removing supernatant by a peristaltic pump, then re-balancing by the buffer solution A, standing, removing supernatant by the peristaltic pump, adding the buffer solution B, stirring the solution for 30min, settling for 30min, removing supernatant by the peristaltic pump, adding the buffer solution C, stirring the solution for 30min, settling for 30min, removing supernatant by the peristaltic pump, eluting by the buffer solution D, and collecting eluent;
s3, adding a buffer solution E into the eluent, mixing uniformly, and performing secondary ultrafiltration to obtain the eluent;
wherein:
and (3) buffer solution A: 60mM MES, 55mM NaCl, 3mM EDTA-2Na, pH 7.5;
and (3) buffer solution B: 60mM MES, 1mM NaCl, 10mM DNAse, pH 7.5;
and (3) buffer C: 60mM MES, 1M NaCl, pH 7.5;
and (3) buffer solution D: 25mM Tris-HCl, 40mM NaCl, 100mM imidazole, pH 7.5;
and (3) buffer solution E: 25mM Tris-HCl, 240mM NaCl, 5mM CaCl2, pH 7.5.
The concentration volume of the ultrafiltration after the buffer solution A is added in the step S1 is 1/20 of the initial liquid volume; the adding volume of the buffer solution A is 1.5 times of that of the recombinant proteinase K fermentation liquor; in the step S1, before adding the buffer solution A for uniform mixing and primary ultrafiltration, filtering the recombinant proteinase K fermentation liquor by using a ceramic membrane; in the step S3, the volume of the buffer solution E added is 4.5 times of that of the eluent; in the step S3, the concentration multiple of the secondary ultrafiltration is 4 times after the buffer solution C is added; the ion affinity chromatography medium is SP Bestarose FF; the primary ultrafiltration and the secondary ultrafiltration are membrane-packed ultrafiltration.
Comparative example 1
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 0.5L/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: 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 above comparative example 1 is a patent of a purification method of recombinant proteinase K and application thereof (CN202110112885.4) example 1, and a purification method in the prior art is taken as reference.
Comparative example 2
The conditions in inventive example 1 were adjusted, i.e. without using buffer C as second comparative ratio:
this example provides a method for purifying recombinant proteinase K, comprising 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 ion affinity chromatography gel balanced by a buffer solution A, placing the chromatography gel in a purification barrel, subsequently stirring to facilitate the combination of the chromatography gel and protease k, then settling for 2 hours, removing supernatant by a peristaltic pump, then re-balancing by the buffer solution A, standing, removing supernatant by the peristaltic pump, adding the buffer solution B, stirring for 20min, settling for 20min, removing supernatant by the peristaltic pump, eluting by a buffer solution D, and collecting eluent;
s3, adding a buffer solution E into the eluent, mixing uniformly, and performing secondary ultrafiltration to obtain the eluent;
wherein:
and (3) buffer solution A: 45mM MES, 45mM NaCl, 2mM EDTA-2Na, pH 7;
and (3) buffer solution B: 45mM MES, 1mM NaCl, 10mM DNAse, pH 7;
and (3) buffer solution D: 20mM Tris-HCl, 30mM NaCl, 100mM imidazole, pH 7;
and (3) buffer solution E: 20mM Tris-HCl, 30mM NaCl, 3mM CaCl2, pH 7.
The concentration volume of the ultrafiltration after the buffer solution A is added in the step S1 is 1/20 of the initial liquid volume; the adding volume of the buffer solution A is 1.2 times of that of the recombinant proteinase K fermentation liquor; in the step S1, before adding the buffer solution A for uniform mixing and primary ultrafiltration, filtering the recombinant proteinase K fermentation liquor by using a ceramic membrane; in the step S3, the volume of the buffer E added is 4.2 times of the volume of the eluent; in the step S3, the concentration multiple of the secondary ultrafiltration is 2-6 times after the buffer solution C is added; preferably 3 to 4 times; the ion affinity chromatography medium is POROSTMOne of XS; the primary ultrafiltration and the secondary ultrafiltration are membrane-packed ultrafiltration.
The total protein amount was calculated from the volume and total protein concentration and then compared to the original initial enzyme solution to calculate the recovery for each step, as shown in the following table:
the enzyme activity measurements of the purified products of example 1 and comparative example 1 showed that the enzyme activity of 981U/ml in example 1 was higher than that of 932981U/ml in comparative example 1, and was superior to that of the commercially available product of the prior art. Therefore, the purification method of the recombinant proteinase K provided by the invention has excellent enzyme activity on the basis of ensuring the recovery rate of total protein.
Finally, it should be noted that: the above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; it will be understood by those skilled in the art that the present invention may be modified and equivalents may be substituted; all such modifications and variations are intended to be included herein within the scope of this disclosure and the present invention and protected by the following claims.
Claims (8)
1. A method for purifying recombinant proteinase K, comprising the steps of:
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 ion affinity chromatography gel balanced by a buffer solution A, placing the chromatography gel in a purification barrel, subsequently stirring to facilitate the combination of the chromatography gel and protease k, then settling for 1-3 hours, removing supernatant by a peristaltic pump, then re-balancing by the buffer solution A, standing, removing supernatant by the peristaltic pump, adding the buffer solution B, stirring the solution for 10-30min, settling for 10-30min, removing supernatant by the peristaltic pump, adding the buffer solution C, stirring the solution for 10-30min, settling for 10-30min, removing supernatant by the peristaltic pump, eluting by the buffer solution D, and collecting eluent;
s3, adding a buffer solution E 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 6.5-7.5;
and (3) buffer solution B: 35-60mM MES, 1mM NaCl, 10mM DNAse, pH 6.5-7.5;
and (3) buffer C: 35-60mM MES, 1M NaCl, pH 6.5-7.5;
and (3) buffer solution D: 15-25mM Tris-HCl, 20-40mM NaCl, 100mM imidazole, pH 6.5-7.5;
and (3) buffer solution E: 15-25mM Tris-HCl, 20-40mM NaCl, 1.5-5mM CaCl2, pH 6.5-7.5.
2. The method of claim 1, wherein the concentrated volume of the ultrafiltration after the buffer A is added in step S1 is 1/10-1/20 of the initial liquid volume.
3. The method for purifying recombinant proteinase K according to claim 2, wherein the volume of the buffer solution A added is 0.8-2 times of the recombinant proteinase K fermentation broth.
4. The method for purifying recombinant proteinase K according to claim 1, wherein in step S1, the fermentation broth of recombinant proteinase K is filtered by ceramic membrane before adding buffer A for mixing and performing primary ultrafiltration.
5. The method of claim 1, wherein in step S3, the volume of buffer E added is 3-6 times of the volume of the eluent.
6. The method of claim 1, wherein the concentration of the second ultrafiltration in step S3 is 2-6 times after the buffer C is added.
7. The method of claim 1, wherein said ionic affinity is forThe chromatography medium is SP Bestarose FF, UNOspHere Rapid S and POROSTMXS.
8. Use of the method of purifying recombinant proteinase K according to any one of claims 1 to 7 for the isolation and purification of recombinant proteinase K.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210011678.4A CN114410613A (en) | 2022-01-06 | 2022-01-06 | Purification method and application of recombinant proteinase K |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2006327991A (en) * | 2005-05-26 | 2006-12-07 | Nippon Zenyaku Kogyo Kk | New method for purifying recombinant protein |
| CN112831487A (en) * | 2021-01-27 | 2021-05-25 | 武汉爱博泰克生物科技有限公司 | Purification method and application of recombinant proteinase K |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006327991A (en) * | 2005-05-26 | 2006-12-07 | Nippon Zenyaku Kogyo Kk | New method for purifying recombinant protein |
| CN112831487A (en) * | 2021-01-27 | 2021-05-25 | 武汉爱博泰克生物科技有限公司 | Purification method and application of recombinant proteinase K |
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| Title |
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| 刘瑞芳 等主编, 徐州：中国矿业大学出版社 * |
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