A purification method for recombinant proteinase K and its application

Abstract

The present invention provides a method for purifying recombinant proteinase K, comprising the following steps: S1, adding buffer A to the recombinant proteinase K fermentation broth to be purified, and performing ultrafiltration once after mixing to remove metal ions and miscellaneous proteins with a molecular weight less than 10kDa; S2, pumping the obtained ultrafiltrate into a cation exchange column balanced with buffer A, then rebalancing with buffer A, eluting with buffer B, and collecting the eluate; S3, adding buffer A to the obtained eluate, performing ultrafiltration and concentration, and obtaining a highly purified recombinant proteinase K; wherein, buffer A: 9-11mM HAc-NaAc, pH=5.0-5.5; buffer B: 9-11mM HAc-NaAc, 1.0-2.0M NaCl, pH=5.0-5.5. The method described in the present invention has simple process and high product yield and purity, and is suitable for industrial-grade production of products.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme separation and purification, and specifically relates to a purification method of recombinant proteinase K and application thereof. Background Art

[0002] Proteinase K is a serine protease with proteolytic activity. It was first discovered in the extract of Tritirachium album limber in 1974. Since the enzyme can digest natural keratin rich in disulfide bonds and can grow with keratin as the only carbon and nitrogen source, it has a broad-spectrum cleavage ability for natural proteins and prefers to cleave aliphatic amino acids and aromatic amino acids, especially the carboxyl-terminal peptide bond of alanine.

[0003] Proteinase K can maintain its activity in a wide range of environments. It is also active in pH 4-12.5, high salt buffer and 70°C. It is compatible with denaturants and detergents (such as urea, SDS, guanidine hydrochloride, guanidine isothiocyanate, Triton and Tween). Within a certain concentration, these denaturants and detergents can enhance the activity of proteinase K. Proteinase K is used in medicine, food, leather, winemaking, amino acid preparation, nucleic acid extraction, in situ hybridization and other fields. With the increasing demand for proteinase K, its large-scale production is particularly important. Specifically, the large-scale production of proteinase K includes fermentation, bacterial liquid separation, filtration, purification, freeze-drying and packaging processes.

[0004] With the development of biotechnology, due to the low expression level of natural proteinase K and certain cytotoxicity, the key to restricting the large-scale production of proteinase K has gradually shifted from the capacity problem of crude enzyme solution of proteinase K fermentation in the early stage to how to efficiently purify proteinase K. The purification method in the early natural extraction process was complex, the purification cycle was long and the recovery rate was low, which was not conducive to mass production; after the development of recombinant expression of proteinase K, the his tag was added to the C-terminus of proteinase K to facilitate affinity purification to achieve the purpose of purification. The purification medium cost of this purification method is relatively high, and there is a risk of Ni shedding, and three to four steps are required in actual industrial production to achieve the ideal purification effect.

[0005] The buffer used in the known proteinase K purification method is MES solution, which has a relatively expensive raw material price and a high cost of use.

[0006] In summary, it is particularly important to research and develop a purification method for recombinant proteinase K that is simple to operate, low in cost and has a high recovery rate, and to apply it to the industrial separation and purification of recombinant proteinase K. This is a key technical problem that technical personnel in this field urgently need to solve. Summary of the invention

[0007] In view of the problems existing in the above-mentioned prior art, the present invention optimizes the composition of the buffer solution to develop a purification method for recombinant carboxypeptidase B with simple operation and high purity, thereby avoiding product loss and cost loss caused by repeated purification.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] A method for purifying recombinant proteinase K comprises the following steps:

[0010] S1. Add buffer A to the fermentation broth of the recombinant proteinase K to be purified, mix well, and then perform ultrafiltration to remove metal ions and foreign proteins with a molecular weight less than 10 kDa;

[0011] S2, pumping the ultrafiltrate obtained in step S1 into a cation exchange column equilibrated with buffer A, then re-equilibrated with buffer A, and then eluted with buffer B, and collecting the eluate;

[0012] S3, adding buffer A to the eluate obtained in step S2, and performing ultrafiltration and concentration to obtain a high-purity recombinant proteinase K; wherein,

[0013] Buffer A: 9-11 mM HAc-NaAc, pH = 5.0-5.5;

[0014] Buffer B: 9-11 mM HAc-NaAc, 1.0-2.0 M NaCl, pH = 5.0-5.5.

[0015] In the present invention, it is further preferred that the buffer B is: 9-11 mM HAc-NaAc, 1.0-1.5 M NaCl, pH=5.0-5.5.

[0016] The present invention is further preferred that the recombinant proteinase K fermentation broth is obtained by introducing proteinase K into Pichia pastoris and fermenting it using BMMY medium, and the isoelectric point of the recombinant proteinase K is detected to be most stable at a pH value of about 8.9, and its isoelectric point is 4.5. At pH = 5.0, the protein is negatively charged, so it can be bound to an anion exchange medium in a low-salt ion buffer. In step S2, the protein is eluted using a buffer B with a high ionic strength.

[0017] Furthermore, in the ultrafiltration concentration of step S3, the eluate is replaced and concentrated with buffer A, which is beneficial to maintaining the stability of proteinase K. In step S3, the sample obtained by purification and elution is concentrated with an ultrafiltration concentration tube, and buffer A is added during the concentration to achieve the purpose of replacing the eluate.

[0018] It is further preferred that the fermentation broth of the recombinant proteinase K to be purified has been centrifuged and filtered to remove cell debris and other impurities before purification, and has been refrigerated and stored at low temperature.

[0019] In the present invention, it is further preferred that the storage temperature is 2 to 10°C, preferably 4±0.5°C.

[0020] The present invention is further preferably configured such that in step S1, the total volume of the buffer A added is 0.8 to 2 times the volume of the recombinant proteinase K fermentation broth.

[0021] The present invention is further preferably that before step S2, CaCl is added to the ultrafiltrate obtained in step S1 2 , placed at low temperature overnight for self-activation treatment, the CaCl 2 The concentration is 2±0.5mM.

[0022] Furthermore, after replacing the buffer A in step S1 to reduce the ion concentration of the protein before loading, 2 mM CaCl was added. 2 , and placed at 4°C overnight for self-activation.

[0023] It should be noted that the overnight placement generally refers to a placement time of ≥12 hours.

[0024] The present invention is further preferably that in step S2, the flow rate of the buffer solution B is controlled to be 10-20 mL / min.

[0025] The present invention is further preferably configured such that in step S3, the total volume of the buffer A added is 2 to 4 times that of the eluent.

[0026] The present invention is further preferably that in step S3, the number of secondary ultrafiltration after adding buffer A is 2 to 4 times.

[0027] It is further preferred that the anion exchange medium of the cation exchange column is selected from QFF anion column filler.

[0028] The present invention also provides an application of the purification method in the separation and purification of recombinant proteinase K.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The purification method of recombinant proteinase K provided by the present invention only uses a simple ultrafiltration-ion exchange-ultrafiltration concentration purification process to meet product requirements, and is very suitable for industrial-grade production.

[0031] (2) The present invention found that when the pH is 5.0, the purity and yield of the sample are the highest.

[0032] (3) The raw material cost of the reagents used in the purification method of the recombinant proteinase K provided by the present invention is low and the SDS-PAGE purity of the obtained protein is as high as over 95%. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is the chromatogram of Example 1 of the present invention.

[0034] Figure 2 It is the chromatogram of Comparative Example 1 of the present invention.

[0035] Figure 3 Shown is the electrophoresis analysis diagram of the crude enzyme and the elution peak of Example 1 of the present invention. DETAILED DESCRIPTION

[0036] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with the embodiments, but the present invention is not limited to the following embodiments.

[0037] In the following examples, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in the field or the product instructions are used; if the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased.

[0038] In the following examples, unless otherwise specified, the reagents and materials involved are all commercially available products well known to those skilled in the art. In the following examples, unless otherwise specified, the technical means used are all technical means well known to those skilled in the art.

[0039] In the embodiment of the present invention, the raw materials and related equipment information used are shown in Table 1 below:

[0040] Table 1 Raw materials and equipment related information

[0041] raw material factory Part Number Tris(hydroxymethyl)aminomethane, Tris Shanghai Jizhi Biochemical Technology Co., Ltd. 77-86-1 Sodium chloride, NaCl Tianjin Komiou Chemical Reagent Co., Ltd. 7647-14-5 <![CDATA[Calcium chloride, CaCl 2 > Tianjin Komiou Chemical Reagent Co., Ltd. 10043-52-4 Concentrated hydrochloric acid, HCl Sinopharm Chemical Reagent Co., Ltd. 7647-01-0 EDTA-2Na Tianjin Komiou Chemical Reagent Co., Ltd. KMO0652 Protein Purification Instrument Inseth Unique AutoPure Protein Purification System QFF anion column packing Saifen Technology 250490990 Concentrator Centrifuge Tube Millipore UFC901008

[0042] The recombinant proteinase K fermentation broth used in the examples of the present invention is obtained by introducing proteinase K into Pichia pastoris and fermenting it using BMMY medium. The recombinant proteinase K fermentation broth was homogenized and centrifuged, and the supernatant was taken and refrigerated at 4° C. for standby use. Before purification, the low-temperature stored proteinase K fermentation broth was filtered using a 0.2 μm ultrafiltration tube to obtain a proteinase K fermentation broth from which cell debris and other impurities were further removed, which was used for purification in the following examples.

[0043] Example 1

[0044] This embodiment provides a method for purifying recombinant proteinase K, which specifically comprises the following steps:

[0045] (1) Liquid exchange and concentration: After the fermentation broth of recombinant proteinase K is mixed with buffer A at a volume ratio of 1:1, the broth is exchanged and concentrated using an ultrafiltration membrane with a cutoff of 10 kDa to remove metal ions and foreign proteins with a molecular weight of less than 10 kDa to obtain a concentrated solution;

[0046] (2) Self-activation: Add CaCl to the concentrated solution obtained in step (1) 2 , CaCl in concentrate 2 The concentration was 2 mM and the cells were placed at 4°C overnight for self-activation.

[0047] (3) Chromatographic purification: QFF anion chromatography filler was taken and loaded into a column. The QFF column was regenerated using 2 mol / NaCl. After repeated washing with purified water, buffer A was used to balance 2-3 column volumes. Buffer B was used for gradient elution. The flow rate of buffer B for elution was 15 ml / min. Buffer A: 10 mM HAc-NaAc, pH = 5.0; buffer B: 10 mM HAc-NaAc, 1 M NaCl, pH = 5.0. The eluates at different elution peaks were collected respectively. The chromatograms obtained were as follows: Figure 1 shown.

[0048] (4) Buffer A was added to the collected eluate, and ultrafiltration and concentration were performed three times to obtain high-purity recombinant proteinase K. The eluate after ultrafiltration and concentration was subjected to electrophoresis analysis. The analysis results were as follows: Figure 3 As shown in the figure, lane 1 is the supernatant of the crude proteinase K (i.e., the proteinase K fermentation broth to be purified), lane 2 is the proteinase K after the liquid is changed, lane 3 is the QFF flow-through of proteinase K, lane 4 is the QFF elution peak of proteinase K, and lane 5 is the marker. It can be seen from the figure that the purity of the proteinase K sample obtained after purifying proteinase K using the method of Example 1 can reach more than 95%.

[0049] It can be seen from the results that electrophoretically pure recombinant proteinase K can be obtained through the above purification process; the activity of the purified enzyme was measured, and the results are shown in Table 2. The yield of the recombinant proteinase K obtained in the above steps is calculated as shown in Table 2.

[0050] Example 2

[0051] Compared with Example 1, the difference is that in step (1), the fermentation broth of the recombinant proteinase K to be purified is mixed with the mixture in a volume ratio of 1:1.5 and then ultrafiltered and concentrated to replace the liquid; at the same time, buffer A: 10mM HAc-NaAc, pH = 4.0; buffer B: 10mM HAc-NaAc, 1M NaCl, pH = 4.0. The purity of the obtained recombinant proteinase K is 90%, and the activity of the purified enzyme is measured, and the results are shown in Table 2. The yield of the recombinant proteinase K obtained in the above steps is calculated as shown in Table 2.

[0052] Example 3

[0053] Compared with Example 1, the difference is that in step (1), the fermentation broth of the recombinant proteinase K to be purified is mixed with the mixture in a volume ratio of 1:2 and then ultrafiltered and concentrated to replace the liquid; at the same time, buffer A: 10mM HAc-NaAc, pH = 6.0; buffer B: 10mM HAc-NaAc, 1M NaCl, pH = 6.0. The purity of the obtained recombinant proteinase K is 85%, and the activity of the purified enzyme is measured, and the results are shown in Table 2. The yield of the recombinant proteinase K obtained in the above steps is calculated as shown in Table 2.

[0054] Example 4

[0055] Compared with Example 1, the difference is: buffer A: 10mM HAc-NaAc, pH = 4.0; buffer B: 10mM HAc-NaAc, 1M NaCl, pH = 4.0. The purity of the obtained recombinant proteinase K is 80%, and the activity of the purified enzyme is measured, and the results are shown in Table 2. The yield of the recombinant proteinase K obtained in the above steps is calculated as shown in Table 2.

[0056] Example 5

[0057] Compared with Example 1, the difference is: buffer A: 10mM HAc-NaAc, pH = 6.0; buffer B: 10mM HAc-NaAc, 1M NaCl, pH = 6.0. The purity of the obtained recombinant proteinase K is 85%, and the activity of the purified enzyme is measured, and the results are shown in Table 2. The yield of the recombinant proteinase K obtained in the above steps is calculated as shown in Table 2.

[0058] Example 6

[0059] Compared with Example 1, the difference is: buffer A: 10mM HAc-NaAc, pH = 5.0; buffer B: 10mM HAc-NaAc, 0.5M NaCl, pH = 5.0. The purity of the obtained recombinant proteinase K is 85%, and the activity of the purified enzyme is measured, and the results are shown in Table 2. The yield of the recombinant proteinase K obtained in the above steps is calculated as shown in Table 2.

[0060] Example 7

[0061] Compared with Example 1, the difference is: buffer A: 10mM HAc-NaAc, pH = 5.0; buffer B: 10mM HAc-NaAc, 2M NaCl, pH = 5.0. The purity of the obtained recombinant proteinase K is 90%, and the activity of the purified enzyme is measured, and the results are shown in Table 1. The yield of the recombinant proteinase K obtained in the above steps is calculated as shown in Table 2.

[0062] Example 8

[0063] Compared with Example 1, the difference is that in step (3), the number of ultrafiltrations after adding buffer A is 2. The purity of the obtained recombinant proteinase K is 90%, and the activity of the purified enzyme is measured, and the results are shown in Table 2. The yield of the recombinant proteinase K obtained in the above steps is calculated as shown in Table 2.

[0064] Example 9

[0065] Compared with Example 1, the difference is that in step (3), the number of ultrafiltrations after adding buffer A is 4. The purity of the obtained recombinant proteinase K is 95%, and the activity of the purified enzyme is measured, and the results are shown in Table 2. The yield of the recombinant proteinase K obtained in the above steps is calculated as shown in Table 2.

[0066] Example 10

[0067] Compared with Example 1, the difference is that in step (3), the number of ultrafiltrations after adding buffer A is 5. The purity of the obtained recombinant proteinase K is 95%, and the activity of the purified enzyme is measured, and the results are shown in Table 2. The yield of the recombinant proteinase K obtained in the above steps is calculated as shown in Table 2.

[0068] Embodiment 11

[0069] Compared with Example 1, the difference is: (2) Self-activation: Add CaCl to the concentrated solution obtained in step (1) 2 , CaCl in concentrate 2 The concentration of the purified proteinase K was 2.5 mM and the proteinase K was placed at 4°C overnight for self-activation. The purity of the obtained recombinant proteinase K was 85%. The activity of the purified enzyme was measured and the results are shown in Table 2. The yield of the recombinant proteinase K obtained in the above steps was calculated as shown in Table 2.

[0070] Example 12

[0071] Compared with Example 1, the difference is: (2) Self-activation: Add CaCl to the concentrated solution obtained in step (1) 2 , CaCl in concentrate 2The concentration of the purified proteinase K was 1.5 mM and the proteinase K was placed at 4°C overnight for self-activation. The purity of the obtained recombinant proteinase K was 90%. The activity of the purified enzyme was measured and the results are shown in Table 2. The yield of the recombinant proteinase K obtained in the above steps was calculated as shown in Table 2.

[0072] Comparative Example 1

[0073] Compared with Example 1, the difference is that step (2) uses CaCl 2 The self-activation treatment step was changed to using 5 mM EDTA-2Na and leaving it at room temperature for 20 min to chelate excess metal ions. The purity of the obtained recombinant proteinase K was 60%. The activity of the purified enzyme was measured, and the results are shown in Table 2. The yield of the recombinant proteinase K obtained in the above steps was calculated as shown in Table 2.

[0074] Comparative Example 2

[0075] Compared with Example 1, the difference is that: self-activation is not performed, and the concentrated solution obtained in step (1) is directly purified by anion chromatography. The purity of the obtained recombinant proteinase K is 50%, and the activity of the purified enzyme is measured, and the results are shown in Table 2. The yield of the recombinant proteinase K obtained in the above steps is calculated as shown in Table 2.

[0076] Control group 1 proteinase K was a commercially available product purchased from Yisheng Biotechnology.

[0077] Control group 2 proteinase K was a commercial product purchased from Merck.

[0078] Table 2 Enzyme activity, purity and yield after purification in each example and comparative example

[0079]

[0080] The results show that the recombinant proteinase K obtained by the purification method of the present invention has excellent purity and total protein recovery rate; at the same time, it has higher enzyme activity than the comparative example and the commercially available products in the prior art; and the purification method of the present invention has a simple process and is therefore suitable for industrial production.

[0081] It should be noted that the above-described embodiments should be understood as illustrative rather than limiting the scope of protection of the present invention, and the scope of protection of the present invention shall be subject to the claims. For those skilled in the art, some non-essential improvements and adjustments made to the present invention still fall within the scope of protection of the present invention without departing from the essence and scope of the present invention.

Claims

1. A method for purifying recombinant proteinase K, characterized in that: The steps include: S1. Add buffer A to the fermentation broth of the recombinant proteinase K to be purified, mix well, and then perform ultrafiltration to remove metal ions and foreign proteins with a molecular weight less than 10 kDa; S2, pumping the ultrafiltrate obtained in step S1 into a cation exchange column equilibrated with buffer A, then re-equilibrated with buffer A, and then eluted with buffer B, and collecting the eluate; S3, adding buffer A to the eluate obtained in step S2, and performing ultrafiltration and concentration to obtain a high-purity recombinant proteinase K; wherein, Buffer A: 9-11 mM HAc-NaAc, pH = 5.0-5.5; Buffer B: 9-11 mM HAc-NaAc, 1.0-2.0 M NaCl, pH = 5.0-5.5; Before step S2, CaCl2 is added to the ultrafiltrate obtained in step S1 and placed at 4°C overnight for self-activation treatment, wherein the concentration of CaCl2 in the ultrafiltrate is 2±0.5m.

2. The method for purifying recombinant proteinase K according to claim 1, characterized in that: In step S1, the recombinant proteinase K fermentation broth to be purified has been centrifuged and filtered to remove cell debris and other impurities before purification, and has been refrigerated and stored at low temperature.

3. The method for purifying recombinant proteinase K according to claim 1, characterized in that: In step S1, the total volume of the buffer A added is 0.8 to 2 times the volume of the recombinant proteinase K fermentation broth.

4. The method for purifying recombinant proteinase K according to claim 1, characterized in that: In step S3, the total volume of the buffer A added is 2-4 times that of the eluent.

5. The method for purifying recombinant proteinase K according to claim 1, characterized in that: In step S3, the number of secondary ultrafiltration after adding buffer A is 2 to 4 times. 6 . Use of the method for purifying recombinant proteinase K according to any one of claims 1 to 5 in the separation and purification of recombinant proteinase K.

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