CN114181925A - Industrial purification and freeze-drying method for recombinant proteinase K - Google Patents
Industrial purification and freeze-drying method for recombinant proteinase K Download PDFInfo
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
-
- 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)
Abstract
The invention discloses an industrial purification and freeze-drying method for recombinant proteinase K. Clarifying and filtering recombinant proteinase K fermentation liquor expressed by pichia pastoris, adding diluent for dilution, concentrating and dialyzing through an ultrafiltration step to obtain proteinase K dialysate containing trace metal ions and pigments, and separating and purifying the dialysate through cation exchange chromatography to obtain an eluent containing the recombinant proteinase K; crystallizing and precipitating the eluent containing the recombinant proteinase K at room temperature for 4-6h, uniformly stirring and pouring the mixture into a positive pressure filter device, and performing filter pressing to obtain a proteinase K filter cake; and after redissolving the filter cake, adding a freeze-drying excipient, carrying out freeze-drying according to an optimized freeze-drying process, and packaging to obtain a finished product of the proteinase K. The method has the advantages of simple process steps, high yield, low cost and easy industrial amplification production. The obtained proteinase K target has high yield and good activity.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for industrial purification and freeze-drying of recombinant proteinase K.
Background
Proteinase K is a serine protease, which was first purified in 1974 from an extract of Candida albicans (Tritirachium album) strain. The main cleavage site is peptide bond at carboxyl terminal of hydrophobic amino acid such as aliphatic or aromatic. Researches show that the enzyme has activity under the conditions of pH 4-12 and 20-60 ℃, and also has the capability of cleaving protein in SDS, urea, chelating agents (such as EDTA) and sulfhydryl reagents (such as trypsin inhibitor or chymotrypsin inhibitor). Thus, it has wide applications including preparation of chromosomal DNA by pulsed electrophoresis, Western blotting, and removal of nucleases in DNA and RNA preparations.
The application of the proteinase K is not only limited to the fields of medical diagnosis and scientific research, but also can replace the traditional chemical method for treating raw materials with possible pollution in the fields of industrial tanning, paper making, feed and the like. Therefore, the proteinase K with high activity and low cost obtained by utilizing the genetic engineering technology and the protein engineering technology has higher social value and economic value.
At present, the gene recombinant proteinase K produced industrially takes prokaryotic cells (such as Escherichia coli) and eukaryotic cells (yeast) as hosts. The use of Escherichia coli as a host has the advantages of simplicity and economy. However, the proteinase K activity expressed by the system is not high, the yield is too low, and the market demand is difficult to meet. The hexa-polyhistidine tag added to the C end of the recombinant proteinase K by using yeast as a host is adopted, and fermentation liquor is subjected to centrifugation, metal chelating column chromatography and weak cation exchange column chromatography separation, macroporous decolorizing gel decolorization and other steps, so that the process steps are complex. In view of the application value of proteinase K, the development of a rapid, economical and separation and purification technology suitable for industrial scale and continuous production is needed.
Disclosure of Invention
The invention aims to provide an industrial purification and freeze-drying method for recombinant proteinase K.
In order to achieve the purpose, the invention provides the following technical scheme:
a chromatography method for separating and purifying recombinant proteinase K from pichia pastoris sequentially comprises the following steps:
1) clarifying the supernatant of fermentation liquor of recombinant proteinase K expressed by pichia pastoris, adding diluent with the same volume, diluting and uniformly mixing. And concentrating the diluted fermentation supernatant by using an ultrafiltration membrane package, adding an equal volume of dialysate after the concentration is finished, and dialyzing for multiple times to obtain the proteinase K dialysate containing trace metal ions and pigments. Wherein the ultrafiltration membrane package can be selected from Millipore 5kD and 10kD or Sartorius 5kD and 10 kD. Preferred membrane traps have a pore size of 5 kD.
2) Regulating pH and conductance of the dialysate containing a small amount of metal ions and pigment and performing cation exchange chromatography to obtain a proteinase K eluent with high concentration. Wherein the filler for cation exchange chromatography is selected from NanoGel 30/50SP, UniGel 30/80SP, SP Bestarose FF, SP Bestarose HP, Bestarose Diomon MMC, Uniphere S, MacroPrep S, POROS XS, SP-6FF, SP-6HP, SP SepharoseTMFast Flow. In one embodiment, the packing material for cation exchange chromatography is preferably SP Bestarose FF. The cation exchange chromatography can be performed by gradient elution with pH or salinity, preferably gradient elution with sodium chloride.
3) And crystallizing and precipitating the obtained proteinase K eluent at room temperature, and preferably adjusting the protein concentration of the proteinase K eluent to be within the range of 40-50 mg/ml before crystallizing and precipitating. And after the precipitation is finished, obtaining a proteinase K precipitation filter cake by adopting a positive pressure filtering device. Wherein the filtering paper board is Shenyang great wall filtering paper board, and the aperture of the filtering plate is 0.2 to 5 μm, preferably 2.5 to 5 μm.
4) Adding the obtained proteinase K precipitation filter cake into a re-dissolving buffer solution for re-dissolving, wherein in one embodiment, the re-dissolving buffer solution is 10-50 mM Tris and 0-5 mM CaCl2pH 7.5-9.0 or 10-50 mM NaAc, 0-5 mM CaCl2And the pH value is 4.5-5.5. And (3) uniformly mixing the components by adopting an optimal freeze-drying formula, and freeze-drying according to an optimal freeze-drying process. And packaging the obtained freeze-dried powder to finally obtain a finished product of the protease K freeze-dried powder.
Wherein the activity of the proteinase K of the recombinant proteinase K fermentation broth supernatant is 30-40U/mgP.
The invention further discloses a specific implementation mode, and the implementation mode is enlarged by the process, so that the protein purification yield of the obtained proteinase K freeze-dried powder is not less than 70%, and the activity is 30-40U/mgP. The specific implementation steps are as follows:
1) clarifying the fermentation broth supernatant of recombinant proteinase K expressed by pichia pastoris, measuring the pH and the conductance of the clarified supernatant, adding diluent with the same volume to dilute, and uniformly mixing; the diluent is as follows: 10 to 50mM Tris, 0 to 5mM CaCl2，pH 7.5～9.0；
Installing an ultrafiltration membrane package on an ultrafiltration system, controlling inlet pressure and reflux pressure, adjusting transmembrane pressure and measuring water flux, and performing ultrafiltration concentration after the membrane package is installed; the interception aperture of the ultrafiltration membrane is 5-10 kD, and the transmembrane pressure is 1-1.25 bar or 14.5-18 psi or 0.1-0.125 MPa.
Adding the concentrated solution concentrated to the end point into dialysis solution with the same volume for dialysis for multiple times until the end point of dialysis; the dialysate is: 10 to 50mM Tris, 0 to 5mM CaCl2pH of 7.5 to 9.0; and the color of the dialysate at the dialysis endpoint is colorless, the conductivity is 0.5-2 mS/cm, and the pH is 7.5-9.0.
2) Adopting a cation exchange chromatography medium as a Bogelong SP Bestarose FF cation exchange filler, wherein the chromatography steps are as follows:
the components with 5-15 times of column volume are 10-50 mM Tris, 0-5 mM CaCl2Balancing the chromatographic column with a pH 7.5-9.0 balance buffer solution at a flow rate of 50-200 cm/h;
taking the proteinase K dialysate obtained in the step 1) as a sample loading sample, and loading the sample at a flow speed of 50-200 cm/h; wherein the conductivity of a sample is 0.5-2 mS/cm, the pH is 7.5-9.0, and the loading capacity is 56-90 mg of protein/ml filler;
10～50mM Tris、0～5mM CaCl2rebalancing the chromatographic column with an equilibrium buffer solution with pH of 7.5-9.0 at a flow rate of 50-200 cm/h;
the adopted components are 10-50 mM Tris, 0-5 mM CaCl2Eluting the chromatographic column with 0-100 mM NaCl and an elution buffer solution with the pH value of 7.5-9.0 at the flow rate of 50-200 cm/h to finally obtain a proteinase K eluent;
and the protein recovery of the cation chromatography step-by-step amplification chromatography is more than 70 percent.
3) Cutting a filter paper board with proper size, putting the filter paper board into a positive pressure filter, and adopting 10-50 mM Tris and 0-5 mM CaCl as the components2Rinsing the positive pressure filter device and the filter paper plate with 0-100 mM NaCl and an elution buffer solution with pH of 7.5-9.0;
adjusting the pH of the proteinase K eluent obtained in the step 2) to 7.5-9.0, Cond to 0-10 mS/cm, and protein concentration to 40-50 mg/ml, pouring the eluent into a stainless steel barrel, controlling the environmental temperature to be 20-25 ℃, and standing for more than 4 hours until the proteinase K is completely crystallized and precipitated;
pouring the completely precipitated proteinase K into a positive pressure filter tank in batches, introducing compressed air into a filter-pressing tank, controlling a pressure gauge of the filter-pressing tank to be 0-0.15 MPa, and starting filter pressing to finally obtain a proteinase K filter cake;
the protein recovery in the proteinase K filter cake is greater than 90%.
4) Adding 10-50 mM Tris and 0-5 mM CaCl into the filter cake containing the recombinant protease K obtained in the step 3)2pH 7.5-9.0 or 10-50 mM NaAc, 0-5 mM CaCl2Re-dissolving at a pH of 4.5-5.5, adding mannitol according to a W/V ratio of 1:20, and uniformly mixing to obtain a mixed solution;
pouring the mixed solution into a freeze-drying tray, setting the pre-freezing temperature to be-40 to-45 ℃, and cooling for 30min for 3 to 6 hours;
setting the first-stage temperature of primary drying to be-35 to-40 ℃, the temperature rise time to be 30min, maintaining for 1 to 2h, and setting the vacuum degree to be 10 to 20 Pa; the temperature of the second stage is 0-10 ℃, the temperature rise time is 4-6h, and the temperature rise time is maintained for 40-45 h.
The invention adopts industrial process steps of ultrafiltration, cation exchange, filter pressing and the like, shortens the batch production time to 1-2 days, and ensures that the total yield of the proteinase K in the freeze-dried preparation prepared after purification is up to more than 70 percent, thereby realizing large-scale and continuous production.
Drawings
FIG. 1: the color of the solution changes before and after the protease K fermentation liquor is ultrafiltered.
FIG. 2: GE DEAE anion exchange chromatography.
FIG. 3: SP Bestarose FF cation exchange chromatography 1ml column Linear elution chromatogram.
FIG. 4: SP Bestarose FF cation exchange chromatography 84ml column gradient elution chromatogram.
FIG. 5: proteinase K crystallization precipitation was compared to delamination.
FIG. 6: form change after proteinase K freeze-drying.
FIG. 7: solubilization of proteinase K in sodium acetate-acetic acid buffers of different pH.
FIG. 8: standard curve for L-tyrosine.
Detailed Description
The technical solutions of the present invention will be described in detail below by way of examples and figures to better illustrate the features and advantages of the present invention. The examples provided should be construed as illustrative of the method of the invention and not limiting the technical solutions disclosed in the invention in any way.
The reagents and instruments used in the following examples are all generally commercially available unless otherwise specified.
Example 1: optimization of protease K purification and freeze-drying process
1.1 Experimental reagents and instruments
Proteinase K fermentation broth, purchased from Jinan Baismig bioengineering, Inc.;
an ultrafiltration system, available from merck millipore, model number CUF 100;
ultrafiltration membrane package, purchased from sartorius;
chromatographic packing, SP Bestarose FF, purchased from shanghai bocgon; DEAE anion exchange packing, available from general electric company;
other chemical agents are all commercially available products.
1. Tangential flow ultrafiltration
1.1 Ultrafiltration Process exploration
The yeast fermentation liquor is dark reddish brown and contains a large amount of pigments, according to the molecular weight of the proteinase K, the proper ultrafiltration membrane is adopted to carry out pigment removal treatment on the yeast fermentation liquor, and the color of the solution after ultrafiltration is lighter relative to the fermentation liquor and before ultrafiltration, which shows that the pigments are removed in a large amount. The color change of the fermentation broth before and after ultrafiltration is shown in figure 1.
According to the invention, the ultrafiltration membrane packs with different interception aperture and membrane area are selected for comparison experiment, the results are shown in table 1, the protein recovery of the ultrafiltration membrane packs with different interception aperture and membrane area is more than 90%, and both the two types of membrane packs can meet the requirement of amplification production. In addition, after the area of the comparative experiment membrane is amplified to 33 times, the protein recovery after ultrafiltration is relatively stable, which shows that the process can be stably amplified.
TABLE 1 Ultrafiltration results of ultrafiltration membranes of different cut-off pore sizes and membrane areas
1.2 Ultrafiltration Process optimization
In order to adapt to larger-scale fermentation liquor treatment capacity, ultrafiltration process parameters need to be optimized. 500ml of fermentation liquor is taken, and is added with ultrapure water with the same volume for dilution for later use while stirring.
Adopts Sartorius 0.1m2The 5kD ultrafiltration membrane is used for ultrafiltration concentration, and the normal water flux of the membrane product is 42L/h/m2Pin (max) 4 bar. After optimization, finally, Pin (inlet pressure) is controlled to be 2bar, Pret (reflux pressure) is controlled to be 0.5bar, and Pout (permeate pressure) is controlled to be 0 bar. TMP (transmembrane pressure) of the membrane was calculated as (Pin + Pret)/2-Pout 1.25bar 18 psi.
And optimizing the technological parameters of the ultrafiltration concentration and the dialysis steps according to the transmembrane pressure. The results are shown in tables 2 and 3. And calculating the batch throughput and the actual membrane package using area according to the average water flux of the optimized concentration and ultrafiltration steps. The final result is: 250L/batch, 8h ultrafiltration required membrane area { (250L/17LMH) + (250L 7/28LMH }/8h 9.65m2The required number of film packages is 9.65m2/0.66m2And ≈ 15 blocks.
TABLE 2 control table of process parameters of concentration step
The average flux in the concentration step was 0.5L/(18/60) h/0.1m2≈17LMH
TABLE 3 dialysis procedure parameter control Table
Mean flux of dialysis step＝3.5L/(74/60)h/0.1m2≈28LMH
2. Ion exchange chromatography
According to the isoelectric point of the proteinase K, the separation and purification of the proteinase K can be realized by adopting two purification modes, namely an anion penetration mode and a cation combination mode.
2.1 anion exchange chromatography
And (3) adopting a GE DEAE chromatographic column to carry out anion exchange chromatography condition groping, taking the protease K fermentation liquor subjected to ultrafiltration treatment, adjusting the pH to 7.6, and loading the sample after the electric conductivity is 0.9 mS/cm. With 10mM Tris-HCl, 1mM CaCl2The pH value is 7.5, namely balance liquid; 10mM Tris-HCl, 1M NaCl, 1mM CaCl2And the pH7.5 is the eluent for chromatography. The results show that proteinase K only penetrates 69.3%, thus discarding the anion penetration mode for proteinase K purification. The chromatogram is shown in figure 2.
2.2 cation exchange chromatography
2.2.1 cationic chromatography Condition exploration
And (3) adopting a Bogelong SP Bestarose FF chromatographic column to perform cation exchange chromatography condition groping, taking the protease K fermentation liquor subjected to ultrafiltration treatment, adjusting the pH to 7.5, and loading the sample after the conductance is 0.8 mS/cm. With 10mM Tris-HCl, 1mM CaCl2The pH value is 7.5, namely balance liquid; 10mM Tris-HCl, 0-1M NaCl, 1mM CaCl2And the pH7.5 is the eluent for chromatography. The result shows that the proteinase K is mainly eluted under the gradient of 0-100 mM NaCl, the protein recovery is 92.7%, and the activity is more than 30U/mg. The chromatogram is shown in figure 3.
2.2.2 progressive amplification by cation chromatography
On the basis of the exploration of cation chromatography conditions, various chromatography parameters of SP Bestarose FF are optimized and confirmed. Wherein the sample loading conductance is 0.5-2 mS/cm, the sample loading capacity is 56-90 mg protein/ml filler, and the elution conditions are 10mM Tris-HCl, 0-100 mM NaCl and 1-5 mM CaCl2And the pH value is 7.5-9.0. To verify the stability of the chromatographic process, the volume of the filler is sequentially enlarged from 1ml in the groping stage to 8.6ml, 28ml and 84ml under optimized chromatographic parameters. The results show that the protein recovery of the step-by-step amplification chromatography is more than 70 percent. The results of the stepwise amplification chromatography are shown in Table 4. An SP Bestarose FF chromatogram enlarged to 84ml is shown in the attached figureFig. 4.
TABLE 4 SP Bestarose FF progressive amplification chromatography results
3. Solid-liquid separation of proteinase K
Proteinase K is easy to crystallize and separate out under the conditions of low salt environment and high protein concentration. The method utilizes the characteristic of proteinase K to adjust the pH value of SP Bestarose FF chromatographic eluent to 7.5-9.0, Cond to 0-10 mS/cm and protein concentration to 40-50 mg/ml, and stands for 4-6h at 20-25 ℃ after uniformly stirring and mixing.
Under the above conditions, proteinase K crystallizes out and separates. The crystallization precipitation and delamination results are shown in FIG. 5.
The precipitated precipitate is collected by utilizing a solid-liquid separation technology platform and selecting a filtering paper board and a positive pressure filtering device with proper sizes. Wherein the filtering paper board is Shenyang great wall filtering paper board, the aperture of the filtering plate is 0.2-5 μm, the filtering paper board is adopted for filtering, protease K precipitate is trapped on the filtering paper board to form a filter cake, and the filtering liquid is clear and transparent. 30ml of the suspension after precipitation was subjected to pressure filtration for three experiments. And taking the pressure filtrate and the filter cake complex solution for protein content determination. The results show that the protein recovery after crystallization and precipitation is more than 90%, and the precipitation is collected by adopting the method, so that the purposes of removing impurities and concentrating can be achieved. The results are shown in Table 5.
TABLE 5 recovery results of protease K crystallized precipitated protein
4. Proteinase K freeze-drying process
Adding 10-50 mM Tris and 0-5 mM CaCl into the obtained filter cake containing the recombinant protease K2pH 7.5-9.0 or 10-50 mM NaAc, 0-5 mM CaCl2And re-dissolving at pH 4.5-5.5. Adding mannitol according to the proportion of W/V (1:20) and mixing uniformly. Pouring the mixed solution into a freeze-drying tray, and setting the pre-freezing temperatureThe temperature is between 40 ℃ below zero and 45 ℃ below zero, the cooling time is 30min, and the temperature is maintained for 3 to 6 hours. Setting the first-stage temperature of primary drying to be-35 to-40 ℃, the temperature rise time to be 30min, maintaining for 1 to 2h, and setting the vacuum degree to be 10 to 20 Pa. The temperature of the second stage is 0-10 ℃, the temperature rise time is 4-6h, and the temperature rise time is maintained for 40-45 h.
The preferable freeze-drying process has the following freeze-drying parameters: cooling at-45 deg.C for 30min for 6 h; setting the first stage temperature of primary drying at-40 ℃, the temperature rise time at 30min, maintaining for 1h, and setting the vacuum degree at 20 Pa; the temperature of the second stage is 0 ℃, the temperature rise time is 6 hours, and the temperature is maintained for 45 hours. The lyophilized proteinase K is shown in figure 6.
The freeze-dried powder obtained by the freeze-drying process is taken for activity determination (casein is taken as a substrate), and the results show that the activity determination values of the freeze-dried powder at different sampling points are all more than 30U/mg and are equivalent to the activity of Merck proteinase K. The results of the activity measurements are shown in Table 6.
TABLE 6 measurement results of proteinase K lyophilized powder activity at different sampling points
5. Proteinase K solubility study
5.1 proteinase K Freeze-dried powder dissolving buffer solution
In order to explore the optimal dissolving conditions of the proteinase K freeze-dried powder, 20mM Tris-HCl with the pH value of 9.68-7.15 is respectively adopted in the invention; 20mM PB, pH 8.02-5.60; dissolving the proteinase K freeze-dried powder by 20mM NaAc at the pH value of 7.60-5.20, and observing the dissolved state. The results show that proteinase K dissolves well in 20mM NaAc at pH 5.20-5.60.
To verify the result, accurately weighing 7 parts of proteinase K freeze-dried powder, putting 100mg of each part into a 10ml EP tube, respectively preparing 100ml of 20mM NaAc and 100ml of buffers with pH 5.58, 5.48, 5.40, 5.28 and 5.21, respectively adding 5ml of the buffers with different pH into 5 tubes, shaking and uniformly mixing, and observing the dissolution state. Two tubes were separately filled with 5ml of 50% glycerol and 5ml of ultrapure water as controls. The results of the activity measurement are shown in Table 7, and the results of the dissolution are shown in FIG. 7.
TABLE 7 determination of the solubility Activity of proteinase K in sodium acetate-acetic acid buffers at different pH values
Example 2: proteinase K Process amplification
2.1 production of raw materials and instrumentation
Proteinase K fermentation broth, purchased from Jinan Baismig bioengineering, Inc.;
tris (hydroxymethyl) aminomethane, purchased from Nanjing chemical reagents, Inc., and analytically pure;
anhydrous calcium chloride, available from north Hebei Huachen pharmaceutical Co., Ltd, pharmaceutical grade;
sodium chloride, available from Hebei North China pharmaceutical Co., Ltd, pharmaceutical grade;
sodium hydroxide, available from Chengdu Mali hupi Reynaudi pharmaceutical adjuvant manufacturing, LLC, pharmaceutical grade;
ethanol, available from Nanjing chemical reagents, Inc., pharmaceutical grade;
mannitol, available from Shijiazhuang Huaxu pharmaceutical industry, pharmaceutical grade;
the thimerosal sodium is purchased from chemical reagents of national medicine group, and is analytically pure;
an ultrafiltration system, available from merck millipore, model number CUF 100;
an ultrafiltration membrane package purchased from sartorius and having a model of HYDRO, 5 KD;
industrial grade chromatography column, available from general electric company, model number chromaflow 800;
chromatographic packing purchased from Shanghai Bogelong, model SP Bestarose FF;
a freeze dryer, available from Beijing Suzhongtian scientific and technology Co., Ltd, model number: YLDZ-0.5
2.2 stock production
Step (1): and (4) pretreating raw materials.
Sterilizing and filtering 10 barrels of 25L/barrel of fermentation liquor by using a 10-inch sterilizing filter with the diameter of 0.22 mu m, and subpackaging the fermentation liquor into two barrels, wherein each barrel is about 125L;
step (2): and (5) primarily concentrating.
Using a 5KD film to cover each barrel of fermentation liquor obtained in the step (1) and using a dialysate 1 (the formula is 0.111g/L CaCl)2Aqueous solution) is dialyzed for 1-2 times in equal volume, then each barrel of fermentation liquor is concentrated to 75L, then two barrels are combined into one barrel of 150L, dialysis for 3-4 times in equal volume is continued by dialysis 1, and then dialysate 2 (formula: 1.21g/L Tris, 0.111g/L CaCl2pH7.5 +/-0.1, Cond 0.8-1mS/cm), dialyzing for 3-5 times in equal volume;
and (3): and (5) blending the sample liquid.
Adjusting the pH of the concentrated feed solution obtained in step (2) to 7.5 + -0.1 with Tris, adjusting the conductance to 0.8-1.2mS/cm, and adding dialysate 2 (formula: 1.21g/L Tris, 0.111g/L CaCl)2pH7.5 +/-0.1, Cond 0.8-1mS/cm) to dilute the protein content of the feed liquid to 10-15mg/ml, thus forming a chromatography sample loading liquid;
and (4) carrying out chromatographic purification.
Performing chromatographic purification according to the following steps 1) to 7), and collecting chromatographic eluent as a target component.
1) Column assembling: the method adopts a GE Chromflow 800 chromatographic column and SP Bestarose FF packing, the height of the packed column is 16-18cm, the volume of the packed chromatographic packing is 80-86L, and the column efficiency is detected to be more than 3000.
2) Washing the filler with water: the chromatography column is washed with purified water at the flow rate of about 145cm/h for more than 30min (350-420L).
3) Balancing: equilibration buffer (1.21g/L Tris, 0.111g/L CaCl)2pH7.5 +/-0.1, Cond 0.8-1mS/cm) of the chromatographic column 600-700L, the flow rate is about 145cm/h, and the balance is carried out until the pH of the permeate at the outlet of the chromatographic column and the baseline of the conductivity are stable.
4) Loading: and (4) loading with the chromatographic loading solution in the step (3), wherein the loading capacity is less than 67.5mg of total protein/ml of filler. The sample flow rate is about 145cm/h, and the inlet pressure of the sample chromatographic column is less than 2.0 bar.
5) Rebalancing: the column was equilibrated again for 550-650L with the above equilibration buffer at a flow rate of about 145 cm/h. And then balancing until the ultraviolet absorption value of the permeation liquid at the chromatography outlet is less than 20 mv.
6) Elution and collection of target components: eluting with elution buffer (1.21g/L Tris, 0.555g/L CaCl)25.844g/L NaCl, pH7.5 +/-0.1 and Cond 12 +/-1 mS/cm), eluting the chromatographic column, wherein the elution flow rate is about 145cm/h, collecting when the peak is chromatographed and the ultraviolet absorption value is more than 30mv, stirring uniformly while collecting, and stopping collecting when the ultraviolet absorption value is less than 30mv, thus obtaining the target component.
7) Regeneration and preservation: after the elution, the chromatography column was regenerated with 2M NaCl and 0.5M NaOH, respectively, at a regeneration flow rate of 145cm/h and a regeneration volume of 300-420L. After regeneration, the column was stored with 20% ethanol.
And (5): and (4) separating precipitates from filter pressing.
And (3) standing and precipitating the target component which is purified and collected in the step (4) for more than 5 hours, starting to perform positive pressure filtration and separation by using a 75-micron filter paper board, collecting precipitate (filter cake), and collecting clear supernatant. The inlet pressure during the filter pressing does not exceed 4 bar.
And (6): and (4) preparing stock solution.
Adding 20L of the clarified supernatant obtained in the step (5) into the filter cake for dissolving, adding 10L of stock solution to prepare mother solution (2.5kg of mannitol and 5g of thimerosal for dissolving by the clarified supernatant) into the filter cake, and finally adding the clarified supernatant to a constant volume of 50L to obtain the proteinase k stock solution.
2.3 Freeze drying
Pre-freezing: shaking the feed liquid uniformly, pouring the solution into a stainless steel freeze-drying tray, and pre-freezing for 4 hours at-45 ℃;
cooling the rear box in the step (2): after pre-freezing, the rear box starts to cool, the temperature of the product is kept at minus 45 ℃, and the rear box is cooled for 30 min;
step (3), vacuumizing: after the temperature of the rear box is reduced, vacuumizing the box body, wherein the vacuum is controlled to be below 25 pa;
step (4), heating: heating the plate layer to 0 ℃ within 6 hours, and controlling the vacuum degree to be 20-30 pa;
step (5), sublimation for one time: maintaining the temperature of the plate layer at 0 ℃, controlling the vacuum degree at 15-30pa, and entering the next section after the temperature of the product reaches more than-1.5 ℃;
step (6) heating: heating the plate layer to 10 ℃ within 30min, and controlling the vacuum degree to be 10-15 pa;
step (7) secondary sublimation: maintaining the temperature of the plate layer at 10 ℃ and controlling the vacuum degree at 5-10 pa;
and (8) judging an end point: and after the vacuum degree is less than 5pa, closing the valves of the front box and the rear box, wherein the vacuum degree of the front box is less than 5pa for 3min, and finishing freeze-drying.
2.4 packaging finished products:
step (1), crushing the freeze-dried powder: after the freeze-drying is finished, crushing the proteinase K and collecting the crushed proteinase K into a double-layer sterile bag to obtain freeze-dried fine powder.
Step (2) sub-packaging: weighing and packaging the protease K freeze-dried fine powder in a clean room isolator to obtain a recombinant protease K finished product.
The process is scaled up according to the above examples, the process is stable and reliable, and the results of the multiple batches of the produced products are shown in table 8:
TABLE 8 production and purification yield of proteinase K from different preparation batches
| Batches of | Yield (%) |
| Preparation of batch 1 | 70.5％ |
| Preparation of |
73.3％ |
| Preparation of batch 3 | 70.5％ |
| Preparation of batch 4 | 70.0％ |
| Preparation of |
70.5％ |
| Preparation of batch 6 | 70.5％ |
Example 3: proteinase K Activity assay
3.1 experimental principle: the method takes bovine hemoglobin as a substrate for detecting the activity of proteinase K. The bovine hemoglobin solution is hydrolyzed into peptides and amino acids which are soluble in trichloroacetic acid under the action of proteinase K, the peptides and the amino acids reduce a phenol reagent under the alkaline condition to generate blue compounds, and the color depth of the compounds at the wavelength of 750nm is in direct proportion to the concentration of enzyme hydrolysate.
Definition of activity units: at 37 ℃ pH7.5, 1 unit proteinase K per minute hydrolyzed the color produced by the action of the urea-denatured bovine hemoglobin product with the Folin phenol reagent, which was equivalent to the color produced by the action of 1.0. mu. mol tyrosine with the Folin phenol reagent.
3.2 Main instrumentation
PH meter (Sartorius PB-10)
Enzyme mark instrument (Molecular Device Versamax)
3.3 preparation of reagents and solutions
Potassium dihydrogen phosphate, bovine hemoglobin, sodium hydroxide, urea, calcium chloride, trichloroacetic acid, phenol reagent (Folin phenol), L-tyrosine, and hydrochloric acid
3.3.1 preparation of reagents
The reagent is prepared by using high-purity water (not less than 18M omega xcm resistance at 25 ℃)
(1)1M potassium dihydrogen phosphate solution: 13.61g of potassium dihydrogen phosphate were weighed out and dissolved in 50ml of water, and the pH was adjusted to 7.5 with 1M KOH at 37 ℃ to 100 ml.
(2) Substrate (100mM potassium dihydrogen phosphate-2.0% (W/V) bovine hemoglobin-6M Urea): weighing 2.0g bovine hemoglobin, dissolving in 40ml water at 37 deg.C, stirring for 30min, adding 8.0ml 1M NaOH, stirring for 20min at 37 deg.C, adding 36.0g urea, stirring for 60min at 37 deg.C, adding 10ml 1M potassium dihydrogen phosphate, adjusting pH to 7.5 with 5M HCl at 37 deg.C, and diluting to 100 ml.
(3)20mM CaCl2Solution: 0.295g of calcium chloride is weighed out and dissolved in water and the volume is made to 100 ml.
(4)305mM TCA solution (trichloroacetic acid solution): 24.9g of trichloroacetic acid are weighed out and dissolved in water and the volume is made up to 500 ml.
(5) NaOH solution:
1M NaOH solution: 20g of sodium hydroxide are weighed, dissolved in water and made up to 500 ml.
b.0.5M NaOH solution: diluting 1M NaOH solution with water in the same volume.
(6) Hydrochloric acid solution:
1M hydrochloric acid: 8.33ml of hydrochloric acid was diluted with water and made up to 100 ml.
b.0.5M hydrochloric acid: 1M hydrochloric acid was diluted with an equal volume of water.
3.3.2 Standard Curve preparation
Weighing 20mg of L-tyrosine, heating to 70-80 ℃ with 80ml of water to completely dissolve the L-tyrosine, cooling to room temperature, and diluting to 100ml with water. Can be stored in a refrigerator at 2-8 deg.C for 6 months.
The preparation of the L-tyrosine standard curve was carried out according to the preparation procedure in Table 9, and the contents of the respective standards in the final reaction system were 0.0088, 0.0264, 0.044, 0.0616, 0.0792, 0.0968, 0.1144, and 0. mu. mol.
TABLE 9 formulation table of L-tyrosine standard curve
3.3.3 proteinase K sample preparation
a solid sample: weighing 0.5g of solid powder, dissolving with high-purity water, fixing the volume to 25ml, and adding CaCl2The solution is diluted to the activity range of 0.075-0.175U/mlAnd (4) measuring.
b liquid sample: diluting the protein concentration to 20mg/ml with high purity water, and adding CaCl2The solution was diluted to an activity range of 0.075-0.175U/ml for the assay.
3.4 detection step
3.4.1 protein concentration measurement of proteinase K and Merck proteinase K (BCA method)
200mg of proteinase K freeze-dried powder is accurately weighed, added into 10ml of high-purity water and shaken to be fully dissolved. Then diluted 40-fold with high purity water and Merck proteinase K to within the standard curve.
And adding 25 mul of diluted sample and BSA standard substance into an ELISA plate, continuously adding 200 mul of BCA working solution, incubating at 37 ℃ for 30min, and setting the wavelength of an ELISA reader to be 562nm for reading. Reference to "Reference BCA Plus" for the concentration determination procedureTMThe Assay was performed on protocol in a Protein Assay Reagent for measurement, protocol #1856210, Thermo "kit.
Merck proteinase K concentration was determined in the same manner.
3.4.2 determination of proteinase K Activity
3.4.2.1 according to the following table, 0.50ml of substrate was added to each tube.
3.4.2.237 deg.C for 10min, adding 0.10ml diluted enzyme solution, and adding 0.10ml 20mM CaCl in blank control tube2And (3) solution.
3.4.2.3 vortex and mix well, incubate 10min at 37 ℃ and add 1.00ml of 305mM TCA solution.
3.4.2.4 vortex and mix well, and let stand for 20min at room temperature.
3.4.2.513000 rpm for 1min, taking out all supernatant, and shaking and mixing uniformly.
3.4.2.6 centrifuging, adding 0.40ml 0.5M sodium hydroxide solution into 0.20ml supernatant, covering, and mixing by vortex.
3.4.2.7 vortex each concentration of L-tyrosine standard with a standard blank.
3.4.2.8 putting 0.6mL of L-tyrosine standard substance prepared by 3.3.2 into a 2mL EP tube, adding 0.12mL of forinophenol reagent into the tube, shaking and mixing uniformly, synchronously adding 0.12mL of forinophenol reagent into the 3.4.2.6 processed sample to be detected, shaking and mixing uniformly, and reacting at room temperature for 30 min.
3.4.2.9 and taking 300 mul of reacted standard substance and sample to be detected respectively, adding into each hole of the enzyme label plate, setting the wavelength of the enzyme label instrument to 750nm for reading.
Note that: if the solution is turbid, it is filtered through a 0.45 μm filter head and the reading is again taken.
3.4.3 data processing
3.4.3.1 standard curve
ΔA750A is the standard750Standard substance-A750Blank space
A linear fit was made with tyrosine content (. mu. mol) as the X-axis and the average OD as the Y-axis, and the formula is as follows: y ═ A + BX, R2Not less than 0.99, and the fitting standard curve is shown in figure 8.
3.4.4 analysis of results
3.4.4.1 the results of the multiple tests are shown in Table 10 below:
TABLE 10 control proteinase K Activity assays, self-made batches and commercially available
3.4.4.2 sample calculation formula:
sample Δ A750＝A750Sample A750Blank space
Tyrosine content (sample) ═ sample Δ a750-A)/B
Sample enzyme activity ═ tyrosine content (sample) × 1.6 × dilution factor/(0.1 × 10 × 0.2)
1.6 volume to complete stop reaction (ml)
0.2 volume (ml) to stop reaction with F & C reagent
df is dilution factor
0.10 Bettva add enzyme solution (mL)
Experiment incubation time (minutes)
Weight of enzyme to be tested (mg)
V ═ volume of dissolved enzyme
Total activity ═ sample enzyme activity/(m/V)
Protein content measured by sample enzyme activity/BCA method
Therefore, the specific activities of the two batches of home-made proteinase K detected by the method are respectively 40U/mgP and 43U/mgP, which are equivalent to the specific activity (41U/mgP) of a commercial Merck proteinase K product, and the detection method is reliable and stable.
Claims (10)
1. An industrial purification and freeze-drying method of recombinant proteinase K sequentially comprises the following steps:
1) clarifying supernatant of recombinant proteinase K fermentation liquor expressed by yeast, adding diluent with the same volume, diluting and uniformly mixing; concentrating the diluted fermentation supernatant by using an ultrafiltration membrane package with the cut-off pore size of 5-10 kD, and then adding an isovolumetric dialysate for dialysis for multiple times to obtain a proteinase K dialysate;
2) regulating the pH value and the electric conductivity of the proteinase K dialysate, then carrying out cation exchange chromatography, and carrying out gradient elution by adopting pH or salinity to obtain a high-concentration proteinase K eluent; wherein the filler for cation exchange chromatography is selected from NanoGel 30/50SP, UniGel 30/80SP, SP Bestarose FF, SP Bestarose HP, Bestarose modular MMC, Uniphere S, MacroPrep S, POROS XS, SP-6FF, SP-6HP, or SP SepharoseTM Fast Flow；
3) Crystallizing and precipitating the proteinase K eluent at room temperature, and obtaining a proteinase K precipitation filter cake by adopting a positive pressure filtering device after precipitation is finished;
4) adding the proteinase K precipitation filter cake into a redissolution buffer solution for redissolving, and freeze-drying to finally obtain a finished proteinase K freeze-dried powder product;
the method is amplified by a process, the protein purification yield of the obtained proteinase K freeze-dried powder is not less than 70%, and the proteinase K activity is 30-40U/mgP.
2. The method according to claim 1, wherein the proteinase K activity of the supernatant of the recombinant proteinase K fermentation broth is 30-40U/mgP.
3. The method of claim 1, wherein the ultrafiltration membrane module is selected from the group consisting of Millipore 5kD, 10kD, Sartorius 5kD, 10 kD.
4. The process of claim 1, wherein the packing for cation exchange chromatography is SP Bestarose FF.
5. The method according to claim 1, wherein in the step 3), the proteinase K eluate is adjusted to have a protein concentration in the range of 40 to 50mg/ml before the crystallization precipitation, and the pore size of a filter paper plate used in a filter device is 2.5 to 5 μm.
6. The method according to claim 1, wherein the reconstitution buffer in step 4) is 10-50 mM Tris, 0-5 mM CaCl2pH 7.5-9.0 or 10-50 mM NaAc, 0-5 mM CaCl2，pH 4.5～5.5。
7. The method according to claim 1, wherein the step 1) is:
7a) pichia pastoris expressed recombinant proteinase KClarifying the supernatant of the fermentation liquor, measuring the pH and the conductance of the clarified supernatant, adding diluent with the same volume to dilute, and uniformly mixing; the diluent is as follows: 10 to 50mM Tris, 0 to 5mM CaCl2，pH 7.5～9.0；
7b) Installing an ultrafiltration membrane package on an ultrafiltration system, controlling inlet pressure and reflux pressure, adjusting transmembrane pressure and measuring water flux, and performing ultrafiltration concentration after the membrane package is installed; the interception aperture of the ultrafiltration membrane is 5-10 kD, and the transmembrane pressure is 1-1.25 bar or 14.5-18 psi or 0.1-0.125 MPa.
7c) Adding the concentrated solution concentrated to the end point into dialysis solution with the same volume for dialysis for multiple times until the end point of dialysis; the dialysate is: 10 to 50mM Tris, 0 to 5mM CaCl2pH of 7.5 to 9.0; and the color of the dialysate at the dialysis endpoint is colorless, the conductivity is 0.5-2 mS/cm, and the pH is 7.5-9.0.
8. The method as claimed in claim 1, wherein in the step 2), the cation exchange chromatography medium is a Bogelong SP Bestarose FF cation exchange filler, and the chromatography step is as follows:
8a) the components with 5-15 times of column volume are 10-50 mM Tris, 0-5 mM CaCl2Balancing the chromatographic column with a pH 7.5-9.0 balance buffer solution at a flow rate of 50-200 cm/h;
8b) taking the proteinase K dialysate obtained in the step 1) as a sample loading sample, and loading the sample at a flow speed of 50-200 cm/h; wherein the conductivity of a sample is 0.5-2 mS/cm, the pH is 7.5-9.0, and the loading capacity is 56-90 mg of protein/ml filler;
8c)10～50mM Tris、0～5mM CaCl2rebalancing the chromatographic column with an equilibrium buffer solution with pH of 7.5-9.0 at a flow rate of 50-200 cm/h;
8d) the adopted components are 10-50 mM Tris, 0-5 mM CaCl2Eluting the chromatographic column with 0-100 mM NaCl and an elution buffer solution with the pH value of 7.5-9.0 at the flow rate of 50-200 cm/h to finally obtain a proteinase K eluent;
and the protein recovery of the cation chromatography step-by-step amplification chromatography is more than 70 percent.
9. The method according to claim 1, wherein the step 3) is:
9a) cutting a filter paper board with proper size, putting the filter paper board into a positive pressure filter, and adopting 10-50 mM Tris and 0-5 mM CaCl as the components2Rinsing the positive pressure filter device and the filter paper plate with 0-100 mM NaCl and an elution buffer solution with pH of 7.5-9.0;
9b) adjusting the pH of the proteinase K eluent obtained in the step 2) to 7.5-9.0, Cond to 0-10 mS/cm, and protein concentration to 40-50 mg/ml, pouring the eluent into a stainless steel barrel, controlling the environmental temperature to be 20-25 ℃, and standing for more than 4 hours until the proteinase K is completely crystallized and precipitated;
9c) pouring the completely precipitated proteinase K into a positive pressure filter tank in batches, introducing compressed air into a filter-pressing tank, controlling a pressure gauge of the filter-pressing tank to be 0-0.15 MPa, and starting filter pressing to finally obtain a proteinase K filter cake;
the protein recovery in the proteinase K filter cake is greater than 90%.
10. The method according to claim 1, wherein the step 4) is:
10a) adding 10-50 mM Tris and 0-5 mM CaCl into the filter cake containing the recombinant protease K obtained in the step 3)2pH 7.5-9.0 or 10-50 mM NaAc, 0-5 mM CaCl2Re-dissolving at a pH of 4.5-5.5, adding mannitol according to a W/V ratio of 1:20, and uniformly mixing to obtain a mixed solution;
10b) pouring the mixed solution into a freeze-drying tray, setting the pre-freezing temperature to be-40 to-45 ℃, and cooling for 30min for 3 to 6 hours;
10c) setting the first-stage temperature of primary drying to be-35 to-40 ℃, the temperature rise time to be 30min, maintaining for 1 to 2h, and setting the vacuum degree to be 10 to 20 Pa; the temperature of the second stage is 0-10 ℃, the temperature rise time is 4-6h, and the temperature rise time is maintained for 40-45 h.
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