Method for purifying bovine lactoferrin from fermentation liquor by using heparin affinity chromatography column
By using a heparin affinity chromatography column and specific pH values and eluent components, the problem of low purification efficiency of lactoferrin was solved, and efficient one-step purification was achieved, reaching 95% purity, simplifying the steps and improving efficiency.
Patent Information
- Application Number
- CN202411193198.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-28
- Publication Date
- 2025-04-25
AI Technical Summary
In the prior art, the purification efficiency of lactoferrin is low, and multiple steps are required to achieve a purity of more than 95%. The purification steps are many, the cycle is long, and the purification efficiency is low.
The purity and purification efficiency of lactoferrin was improved by using heparin affinity chromatography columns to improve the purity and purification efficiency of lactoferrin through steps such as bacterial centrifugation, bacteria resuspension, high-pressure homogeneity, centrifugal filtration, ultrafiltration concentration, column equilibrium, loading, column rinsing and elution.
The efficient purification of lactoferrin is achieved, and the purity of more than 95% can be achieved in one step, simplifying the purification steps, shortening the purification cycle, and improving the purification efficiency.
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Figure BDA0005015648800000161
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of protein purification, and in particular to a method for purifying bovine lactoferrin from fermentation broth by using a heparin affinity chromatography column. Background Art
[0002] Bovine lactoferrin has multiple biological functions such as antibacterial, antiviral and antioxidant, and is widely used in food science, medicine and biotechnology. Bovine lactoferrin is mainly extracted from cow's milk, but separation is difficult and costly.
[0003] With the development of gene editing technology, bovine lactoferrin can be expressed from a variety of genetically engineered bacteria, and the main separation methods include salting-out, ultrafiltration, chromatography, etc. The main advantage of salting-out and ultrafiltration methods is that they can process a large number of dairy samples in a short time, but the purity of the bovine lactoferrin obtained is low, and it is only suitable for industries such as health products and food additives that do not require high purity of lactoferrin. Purifying lactoferrin using the principle of heparin or nickel column affinity chromatography is currently considered to be a better method for purifying lactoferrin, but it still requires multiple steps of purification to obtain a protein with a purity of more than 95%. There are many purification steps, a long cycle, and low purification efficiency. Summary of the invention
[0004] In view of this, the object of the present invention is to provide a method for purifying bovine lactoferrin from a fermentation broth using a heparin affinity chromatography column, so as to overcome the technical problem of low bovine lactoferrin purification efficiency in the prior art.
[0005] In a first aspect, the present invention provides a method for purifying bovine lactoferrin from a fermentation broth using a heparin affinity chromatography column, comprising the following steps:
[0006] (1) Bacterial cell centrifugation: centrifuging the Pichia pastoris fermentation broth containing bovine lactoferrin to collect the bacterial cells;
[0007] (2) Resuspension of the cells: Resuspend the cells in a citric acid buffer with a pH of 6.5 to 7;
[0008] (3) High-pressure homogenization: The bacterial solution obtained after resuspension is subjected to high-pressure homogenization;
[0009] (4) Centrifugal filtration: centrifuge the bacterial solution after high-pressure homogenization, collect the supernatant, and filter the supernatant;
[0010] (5) Ultrafiltration and concentration: The filtered supernatant is ultrafiltered and concentrated to 1 / 4 to 1 / 3 of the initial volume, and then the pH value of the concentrated solution is adjusted to 6.5 to 7.0 using citrate;
[0011] (6) Column equilibration: Use 4 to 6 column volumes of equilibration buffer to equilibrate the heparin affinity chromatography column;
[0012] (7) Loading: The concentrated solution after pH adjustment in step (5) is loaded onto a heparin affinity chromatography column as a loading solution;
[0013] (8) Column flushing: flush the heparin affinity chromatography column with 4 to 6 column volumes of equilibration buffer;
[0014] (9) Elution: using an eluent composed of a citrate buffer, NaCl and arginine to elute the bovine lactoferrin in the heparin affinity chromatography column, controlling the linear velocity of the eluent to be 100 to 120 cm / h, observing the UV280 curve, and starting to collect the eluent after the main peak of bovine lactoferrin appears, and stopping the collection when the baseline drops steadily;
[0015] The concentration of the citric acid buffer in the eluent is 20-50 mmol / L, the concentration of NaCl is 1-2 mol / L, and the concentration of arginine is 0.2-0.3 mmol / L; and the pH value of the eluent is 5.5-6.
[0016] Compared with the prior art, the present invention uses citrate to adjust the pH value of the sample solution containing bovine lactoferrin to 6.5-7, so that the bovine lactoferrin in the sample solution can be firmly bound to the heparin affinity chromatography column. During elution, the pH value of the eluent is set to 5.5-6. On the one hand, this specific pH value will affect the charge state of heparin and bovine lactoferrin, greatly weaken the binding force between bovine lactoferrin and the heparin affinity chromatography column, and make bovine lactoferrin easier to be eluted; on the other hand, the eluent in the present invention contains arginine and NaCl. Within the specific pH value range of 5.5-6, arginine and NaCl further improve the elution efficiency of bovine lactoferrin, and the bovine lactoferrin bound to the heparin affinity chromatography column is efficiently eluted. In addition, at this pH value, other non-target proteins have a higher binding force with the heparin affinity chromatography column than bovine lactoferrin, and will not be eluted, thereby ensuring the purity of bovine lactoferrin after chromatography.
[0017] Furthermore, the preparation method of the filler in the heparin affinity chromatography column is as follows:
[0018] (1) After washing the agarose with ultrapure water, the agarose is placed in a PBS buffer system containing glutaraldehyde, wherein the pH value of the PBS buffer system is 6 to 8, the volume fraction of glutaraldehyde is 0.5% to 1%, and the agarose is activated at a speed of 50 to 100 rpm for 2 to 5 hours; 50 to 100 g of agarose is added per liter of the buffer system;
[0019] (2) adding heparin to a buffer solution containing N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide for activation, wherein the pH value of the buffer solution is 7 to 8, reacting at 30 to 38° C. for 3 to 6 hours to obtain activated heparin; adding 20 to 50 g of heparin per liter of buffer solution;
[0020] The concentration of N-hydroxysuccinimide in the buffer is 0.5-0.8 mol / L, and the concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 0.3-0.5 mol / L;
[0021] (3) After rinsing the activated heparin and activated agarose with 20 mmol / L PBS buffer respectively, the activated heparin and activated agarose are added to 20 mmol / L PBS buffer, the pH value of the PBS buffer is 6.5-7.5, and the reaction is carried out at 15-25°C for 10-12 hours to covalently bind the activated heparin and activated agarose to obtain a heparin affinity chromatography column filler; 50-100 g agarose and 20-50 g heparin are added to each liter of PBS buffer.
[0022] The above technical scheme further defines a method for preparing a filler in a heparin affinity chromatography column, wherein agarose is activated with glutaraldehyde, heparin is activated with N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and then the activated heparin is covalently bound to the activated agarose to prepare a heparin affinity chromatography column filler, which has a strong binding force with bovine lactoferrin at a pH value of 6.5 to 7.0, and a weakened binding force with bovine lactoferrin at a pH value of 5.5 to 6. Therefore, by adjusting the pH values of the loading solution and the eluent and using a specific eluent, the bovine lactoferrin can be efficiently eluted and separated from foreign proteins to obtain high-purity bovine lactoferrin.
[0023] Furthermore, in step (1), the centrifugal speed is 5000-10000 rpm, and the centrifugal time is 20-25 min.
[0024] Furthermore, in step (2), the concentration of the citric acid buffer is 20 to 50 mmol / L; 5 to 20 ml of citric acid buffer is added for every gram of bacterial cells.
[0025] Furthermore, in step (3), the pressure of high-pressure homogenization is 1000-1500 bar, and the homogenization is performed 3-5 times.
[0026] Furthermore, in step (4), the centrifugal speed is 5000-10000 rpm, the centrifugal time is 20-30 min, and the pore size of the filter used for filtration is 0.22-0.65 um.
[0027] Furthermore, during the ultrafiltration concentration in step (5), the pressure is ≤0.15 MPa, the temperature is 2-8° C., and the pore size of the ultrafiltration membrane is 10-30 kDa.
[0028] The above technical solution defines the conditions for ultrafiltration and concentration, and ultrafiltration and concentration can further remove some small molecular impurities such as small molecular proteins and pigments in the sample solution, which is conducive to obtaining high-purity bovine lactoferrin after purification.
[0029] Furthermore, the equilibration buffer in steps (6) and (8) is composed of 20 to 50 mmol / L of citric acid buffer and 20 to 50 mmol / L of NaCl, and the pH value of the equilibration buffer is 6.5 to 7.0.
[0030] The equilibration buffer in the above technical solution can wash away impurities contained in the heparin affinity chromatography column filler during column equilibration in step (6), and adjust the ionic strength and pH value of the chromatography column filler to ensure the adsorption capacity of the chromatography column filler for the target protein bovine lactoferrin.
[0031] The above-mentioned equilibration buffer can wash away the unbound or weakly bound foreign proteins or other impurities in the heparin affinity chromatography column during column washing in step (8), thereby improving the purity of the target protein bovine lactoferrin.
[0032] Furthermore, in step (7), the linear velocity of the loading liquid is controlled to be 60-120 cm / h, and the temperature of the loading liquid is controlled to be 2-20°C.
[0033] Furthermore, in step (8), the linear velocity of the equilibration buffer is 60 to 120 cm / h. DETAILED DESCRIPTION
[0034] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
[0035] In a first aspect, an embodiment of the present invention provides a method for purifying bovine lactoferrin from a fermentation broth using a heparin affinity chromatography column, comprising the following steps:
[0036] S1. Bacterial cell centrifugation: centrifuge the Pichia pastoris fermentation broth containing bovine lactoferrin to collect the bacterial cells.
[0037] For example, the preparation method of the Pichia yeast fermentation broth containing bovine lactoferrin in the above steps can be referred to as follows:
[0038] The bovine lactoferrin gene is connected to the vector pPIC9K to prepare a recombinant expression vector, the recombinant expression vector is transferred into GS115 type Pichia pastoris to prepare a recombinant Pichia pastoris, and the recombinant Pichia pastoris is fermented to obtain a Pichia pastoris fermentation liquid containing bovine lactoferrin.
[0039] Furthermore, in the above steps, a centrifuge can be used to centrifuge the Pichia yeast fermentation broth containing bovine lactoferrin, and the centrifugal speed is 5000-10000rpm, and the centrifugal time is 20-25min. For example, the centrifugal speed can be 5000rpm, 6000rpm, 7000rpm, 8000rpm, 9000rpm or 10000rpm, and the centrifugal time can be 20min, 21min, 22min, 23min, 24min or 25min. After centrifugation, the supernatant is poured out and the Pichia yeast cells are collected.
[0040] S2. Resuspension of bacteria: Resuspend the bacteria in a citric acid buffer with a pH value of 6.5 to 7.
[0041] The citric acid buffer in the above step can be selected from citric acid-sodium citrate buffer. The concentration of the citric acid buffer is 20-50 mmol / L, for example, 20 mmol / L, 30 mmol / L, 40 mmol / L or 50 mmol / L, so as to maintain the pH value of the buffer at 6.5-7.
[0042] When resuspending the cells in the above step, 5 to 20 ml of citric acid buffer is added per gram of cells. For example, 5 ml, 10 ml, 15 ml or 20 ml of citric acid buffer can be added per gram of cells for resuspending, preferably 10 to 15 ml.
[0043] S3. High-pressure homogenization: The bacterial solution obtained after resuspension is subjected to high-pressure homogenization.
[0044] In the above steps, the pressure during high-pressure homogenization is 1000-1500 bar, and the number of homogenizations is 3-5 times. For example, the pressure can be selected as 1000 bar, 1100 bar, 1200 bar, 1300 bar, 1400 bar, or 1500 bar; the number of homogenizations can be selected as 3 times, 4 times, or 5 times, so that the Pichia in the bacterial solution can be fully broken, so that bovine lactoferrin can be released from the Pichia cells into the bacterial solution.
[0045] S4. Centrifugal filtration: centrifuge the bacterial solution after high-pressure homogenization, collect the supernatant, and filter the supernatant.
[0046] The above steps can be centrifuged using a centrifuge, with a centrifugal speed of 5000-10000rpm and a centrifugal time of 20-30min. For example, the centrifugal speed can be 5000rpm, 6000rpm, 7000rpm, 8000rpm, 9000rpm or 10000rpm, and the centrifugal time can be 20min, 22min, 24min, 26min, 28min or 30min. After centrifugation, bovine lactoferrin is dissolved in the supernatant, and cell fragments of Pichia pastoris and other macromolecular impurities form a precipitate, and the supernatant containing bovine lactoferrin is collected and the precipitate is discarded.
[0047] In the above steps, a filter with a pore size of 0.22 to 0.65 um can be used for filtration. For example, the pore size can be selected to be 0.22 um, 0.35 um, 0.45 um, 0.55 um or 0.65 um. Insoluble large particle impurities in the supernatant are removed by filtration.
[0048] S5. Ultrafiltration and concentration: The filtered supernatant is ultrafiltered and concentrated to 1 / 4 to 1 / 3 of the initial volume, and then the pH value of the concentrated solution is adjusted to 6.5 to 7.0 using citrate.
[0049] In the above steps, an ultrafiltration membrane with a pore size of 10 to 30 kDa can be used for ultrafiltration concentration, and the pressure during ultrafiltration concentration is ≤ 0.15 MPa and the temperature is 2 to 8° C. The citrate can be sodium citrate or potassium citrate. Impurities such as small molecular weight miscellaneous proteins and small molecular pigments in the supernatant are further removed by ultrafiltration concentration.
[0050] In the above step, citrate is used to adjust the pH value of the concentrated solution to 6.5-7.0. Within this pH range, bovine lactoferrin can be better adsorbed by the heparin affinity chromatography column filler when loading the sample.
[0051] S6. Column equilibration: Use 4 to 6 column volumes of equilibration buffer to equilibrate the heparin affinity chromatography column.
[0052] The balancing buffer in the above step is composed of 20-50 mmol / L citric acid buffer and 20-50 mmol / L NaCl, and the pH value of the balancing buffer is 6.5-7.0. Specifically, the citric acid buffer can be a citric acid-sodium citrate buffer.
[0053] Furthermore, the preparation method of the filler in the heparin affinity chromatography column in the above step is as follows:
[0054] S61, after washing the agarose with ultrapure water for 2-3 times, put it into a PBS buffer system containing glutaraldehyde, wherein the pH value of the PBS buffer system is 6-8, the volume fraction of glutaraldehyde is 0.5%-1%, and stir it at a speed of 50-100 rpm for 2-5 hours to obtain activated agarose. 50-100 g of agarose is added to each liter of the buffer system.
[0055] S62. Add heparin to a buffer solution containing N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide for activation, wherein the pH value of the buffer solution is 7 to 8, and react at 30 to 38° C. for 3 to 6 hours to obtain activated heparin. Add 20 to 50 g of heparin per liter of buffer solution.
[0056] The concentration of N-hydroxysuccinimide in the buffer solution is 0.5 to 0.8 mol / L, and the concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 0.3 to 0.5 mol / L.
[0057] The purpose of using a buffer in the above steps is to provide a suitable pH value to ensure the effective activation reaction of heparin. Therefore, there is no restriction on the type of buffer, as long as the pH value during the activation reaction can be maintained in the range of 7 to 8. For example, phosphate buffer or Tris-HCl buffer can be selected.
[0058] S63. After washing the activated heparin and activated agarose with 20mmol / L PBS buffer for 2-3 times, add them to 20mmol / L PBS buffer, the pH value of which is 6.5-7.5, and react at 15-25°C for 10-12h to covalently bind the activated heparin and activated agarose to obtain a heparin affinity chromatography column filler. Add 50-100g of activated agarose and 20-50g of activated heparin to each liter of PBS buffer.
[0059] S64. Washing: After the above reaction is completed, use PBS buffer to wash the filler to remove heparin and reaction byproducts that are not bound to agarose.
[0060] S65, end-capping: Add 0.2-0.5 mol / L ethanolamine reagent to the filler to block unreacted active sites and reduce nonspecific adsorption. Add 1-2 L ethanolamine per liter of filler.
[0061] S66. Storage: The prepared heparin affinity chromatography column filler is stored in a 20% by volume ethanol solution at a storage temperature of 2 to 30°C.
[0062] The heparin affinity chromatography column filler prepared by the above technical scheme can efficiently bind to bovine lactoferrin in the sample solution when the pH value is 6.5-7.0. When the pH value changes to 5.5-6, the binding force between the filler and the bovine lactoferrin becomes lower. The bovine lactoferrin bound in the chromatography column can be efficiently eluted with an eluent of a specific pH value, and the bovine lactoferrin with a purity greater than 95% can be obtained by one-step purification.
[0063] S7, loading: The concentrated solution after adjusting the pH in step S5 is loaded onto the heparin affinity chromatography column as the loading solution.
[0064] In the above steps, the linear velocity of the loading liquid is controlled to be 60-120 cm / h, and the temperature of the loading liquid is controlled to be 2-20° C. For example, the linear velocity of the loading liquid can be 60 cm / h, 70 cm / h, 80 cm / h, 90 cm / h, 100 cm / h, 110 cm / h or 120 cm / h; the temperature can be 2° C., 8° C., 12° C., 16° C. or 20° C. Controlling the linear velocity and temperature of the loading liquid during loading helps to evenly distribute the loading liquid on the heparin affinity chromatography column, so that the bovine lactoferrin can fully combine with the filler in the chromatography column.
[0065] S8. Column flushing: flush the heparin affinity chromatography column with 4 to 6 column volumes of equilibration buffer.
[0066] The balancing buffer in the above step is composed of 20-50 mmol / L citric acid buffer and 20-50 mmol / L NaCl, with a pH value of 6.5-7.0. Specifically, the citric acid buffer may be a citric acid-sodium citrate buffer.
[0067] Furthermore, the linear velocity of the equilibration buffer during column washing is 60 to 120 cm / h. For example, the linear velocity of the equilibration buffer may be 60 cm / h, 70 cm / h, 80 cm / h, 90 cm / h, 100 cm / h, 110 cm / h or 120 cm / h. Using the above equilibration buffer for column washing can wash away the impurities with weak binding force to the filler without affecting the binding of bovine lactoferrin to the chromatography column, so as to separate bovine lactoferrin from some impurities.
[0068] S9, elution: using an eluent composed of a citrate buffer, NaCl and arginine to elute the bovine lactoferrin in the heparin affinity chromatography column, controlling the linear velocity of the eluent to be 100-120 cm / h, observing the UV280 curve, and starting to collect the eluent after the main peak of bovine lactoferrin appears, and stopping the collection when the baseline drops steadily. For example, the linear velocity of the eluent can be 100 cm / h, 110 cm / h or 120 cm / h.
[0069] Furthermore, the concentration of the citric acid buffer in the eluent is 20-50 mmol / L, the concentration of NaCl is 1-2 mol / L, and the concentration of arginine is 0.2-0.3 mmol / L; and the pH value of the eluent is 5.5-6.
[0070] For example, the concentration of the citric acid buffer may be 20 mmol / L, 30 mmol / L, 40 mmol / L or 50 mmol / L. The citric acid buffer may be a citric acid-sodium citrate buffer.
[0071] For example, the concentration of NaCl may be 1 mol / L, 1.5 mol / L or 2 mol / L.
[0072] For example, the concentration of arginine may be 0.2 mol / L, 0.25 mol / L or 0.3 mol / L.
[0073] The inventors found that when the pH value of the above-mentioned specific eluent is adjusted to 5.5-6, the elution efficiency of bovine lactoferrin on the heparin affinity chromatography column is the highest, the separation effect of bovine lactoferrin and other impurities is the best, and high-purity bovine lactoferrin can be obtained.
[0074] In order to better illustrate the technical solution of the present invention, the present invention also provides the following specific examples. It should be understood that the raw materials used in the following examples are all commercially available raw materials unless otherwise specified.
[0075] Example 1
[0076] This embodiment provides a method for purifying bovine lactoferrin from a fermentation broth using a heparin affinity chromatography column, comprising the following steps:
[0077] (1) The bovine lactoferrin gene is connected to the vector pPIC9K to prepare a recombinant expression vector, the recombinant expression vector is transferred into GS115 type Pichia pastoris to prepare a recombinant Pichia pastoris, and the recombinant Pichia pastoris is fermented to obtain a Pichia pastoris fermentation broth containing bovine lactoferrin.
[0078] The fermentation process of the recombinant Pichia pastoris is as follows: the recombinant Pichia pastoris is cultured using a basal inorganic salt medium (BSM) for Pichia pastoris. During the bacterial growth stage, the temperature is controlled at 30°C, the pH value is 6-7, and the DO is controlled at 20-60%. After culturing for about 3-5 hours, the DO rebounds. When it rebounds to above 100%, 50% glycerol is used for feeding at a flow rate of 30 g / L / h. After continuing to culture for 10 hours, the feeding is stopped, the bacteria are starved for 30 minutes, the temperature is controlled at 25°C, and 100% methanol is used for feeding at a flow rate of 1.5 mL / L / h. The Pichia pastoris fermentation broth is obtained by induction culture for 96 hours.
[0079] Centrifugation of cells: The Pichia yeast fermentation broth containing bovine lactoferrin was centrifuged at 7000 rpm for 22 min using a centrifuge. After the centrifugation was completed, the supernatant was discarded and the Pichia yeast cells were collected.
[0080] (2) Resuspension of the cells: Take 200 g of the Pichia cells prepared in the above step and resuspend the Pichia cells in 1000 ml of citric acid-sodium citrate buffer with a pH value of 6.7 and a concentration of 20 mmol / L.
[0081] (3) High-pressure homogenization: The resuspended bacterial solution was homogenized using a high-pressure homogenizer with the pressure controlled at 1200 bar for 4 times.
[0082] (4) Centrifugal filtration: The high-pressure homogenized bacterial solution was centrifuged at 7000 rpm for 25 min, the supernatant was collected, and the supernatant was filtered using a filter with a pore size of 0.45 μm.
[0083] (5) Ultrafiltration concentration: The filtered supernatant was ultrafiltered and concentrated using an ultrafiltration membrane with a pore size of 20 kDa. The pressure during ultrafiltration concentration was 0.1 MPa and the temperature was 5°C. After the volume was concentrated to 1 / 4 of the initial volume, the pH value of the concentrated solution was adjusted to 6.7 using sodium citrate.
[0084] (6) Column equilibration: The heparin affinity chromatography column was equilibrated using 5 column volumes of equilibration buffer, wherein the equilibration buffer consisted of 20 mmol / L citric acid-sodium citrate buffer and 35 mmol / L NaCl. The pH value of the equilibration buffer was 6.7. The linear velocity of the equilibration buffer was controlled at 60 cm / h. The volume of the heparin affinity chromatography column was 100 mL.
[0085] (7) Loading: The concentrated solution after adjusting the pH in step (5) is loaded onto the heparin affinity chromatography column as the loading solution. The linear velocity of the loading solution is controlled to be 90 cm / h and the temperature of the loading solution is controlled to be 10°C.
[0086] (8) Column flushing: The heparin affinity chromatography column was flushed with 5 column volumes of equilibration buffer at a linear velocity of 90 cm / h. The equilibration buffer was the same as that used in step (6) for column equilibration.
[0087] (9) Elution: The bovine lactoferrin in the heparin affinity chromatography column was eluted with an eluent consisting of 20 mmol / L citric acid-sodium citrate buffer, 1.5 mol / L NaCl and 0.25 mmol / L arginine at a pH of 5.7. The linear velocity of the eluent was controlled at 110 cm / h. The UV280 curve was observed. The eluent was collected after the main peak of bovine lactoferrin appeared and the collection was stopped when the baseline dropped steadily.
[0088] The preparation method of the filler in the heparin affinity chromatography column in this embodiment is as follows:
[0089] (1) After washing the agarose with ultrapure water for 3 times, the agarose was placed in a PBS buffer system containing glutaraldehyde, wherein the pH value of the PBS buffer system was 7 and the volume fraction of glutaraldehyde was 0.7%, and the agarose was obtained by stirring at a speed of 70 rpm for 3.5 hours. 75 g of agarose was added per liter of the buffer system.
[0090] (2) Heparin is activated by adding it to a PBS buffer solution containing N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, the pH value of the PBS buffer solution being 7.5, and reacting it at 34°C for 4.5 hours to obtain activated heparin. 35 g of heparin is added to each liter of PBS buffer solution.
[0091] The concentration of N-hydroxysuccinimide in the buffer solution was 0.65 mol / L, and the concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was 0.4 mol / L.
[0092] (3) After the activated heparin and activated agarose were washed three times with 20 mmol / L PBS buffer respectively, they were added to 20 mmol / L PBS buffer, the pH value of which was 7, and reacted at 20°C for 11 h to covalently bind the activated heparin and activated agarose to obtain a heparin affinity chromatography column filler. 75 g of activated agarose and 35 g of activated heparin were added to each liter of PBS buffer.
[0093] (4) Washing: After the above reaction is completed, the filler is washed with PBS buffer to remove heparin that is not bound to agarose and reaction byproducts.
[0094] (5) Capping: Add 0.35 mol / L ethanolamine reagent to the filler to block the unreacted active sites. Add 1.5 L ethanolamine per liter of filler.
[0095] (6) Storage: The heparin affinity chromatography column filler prepared above was stored in a 20% by volume ethanol solution at a storage temperature of 20°C.
[0096] Example 2
[0097] This embodiment provides a method for purifying bovine lactoferrin from a fermentation broth using a heparin affinity chromatography column, comprising the following steps:
[0098] (1) The bovine lactoferrin gene is connected to the vector pPIC9K to prepare a recombinant expression vector, the recombinant expression vector is transferred into GS115 type Pichia pastoris to prepare a recombinant Pichia pastoris, and the recombinant Pichia pastoris is fermented to obtain a Pichia pastoris fermentation broth containing bovine lactoferrin.
[0099] The fermentation process of the recombinant Pichia pastoris is as follows: the recombinant Pichia pastoris is cultured using a basal inorganic salt medium (BSM) for Pichia pastoris. During the bacterial growth stage, the temperature is controlled at 30°C, the pH value is 6-7, and the DO is controlled at 20-60%. After culturing for about 3-5 hours, the DO rebounds. When it rebounds to above 100%, 50% glycerol is used for feeding at a flow rate of 30 g / L / h. After continuing to culture for 10 hours, the feeding is stopped, the bacteria are starved for 30 minutes, the temperature is controlled at 25°C, and 100% methanol is used for feeding at a flow rate of 1.5 mL / L / h. The Pichia pastoris fermentation broth is obtained by induction culture for 96 hours.
[0100] Centrifugation of cells: The Pichia yeast fermentation broth containing bovine lactoferrin was centrifuged at 5000 rpm for 25 min using a centrifuge. After the centrifugation was completed, the supernatant was discarded and the Pichia yeast cells were collected.
[0101] (2) Resuspension of the cells: Take 150 g of the Pichia cells prepared in the above step and resuspend the Pichia cells in 1500 ml of citric acid-sodium citrate buffer with a pH value of 6.5 and a concentration of 30 mmol / L.
[0102] (3) High-pressure homogenization: The resuspended bacterial solution was homogenized using a high-pressure homogenizer with the pressure controlled at 1000 bar for 5 times.
[0103] (4) Centrifugal filtration: The high-pressure homogenized bacterial solution was centrifuged at 5000 rpm for 30 min, the supernatant was collected, and the supernatant was filtered using a filter with a pore size of 0.22 μm.
[0104] (5) Ultrafiltration concentration: The filtered supernatant was ultrafiltered and concentrated using an ultrafiltration membrane with a pore size of 10 kDa. The pressure during ultrafiltration concentration was 0.05 MPa and the temperature was 2°C. After the volume was concentrated to 1 / 3 of the initial volume, the pH value of the concentrated solution was adjusted to 6.5 using sodium citrate.
[0105] (6) Column equilibration: The heparin affinity chromatography column was equilibrated using 4 column volumes of equilibration buffer, the equilibration buffer consisting of 30 mmol / L citric acid-sodium citrate buffer and 20 mmol / L NaCl. The pH value of the equilibration buffer was 6.5. The linear velocity of the equilibration buffer was controlled at 100 cm / h. The volume of the heparin affinity chromatography column was 60 mL.
[0106] (7) Loading: The concentrated solution after adjusting the pH in step (5) is loaded onto the heparin affinity chromatography column as the loading solution. The linear velocity of the loading solution is controlled to be 60 cm / h and the temperature of the loading solution is controlled to be 2°C.
[0107] (8) Column flushing: The heparin affinity chromatography column was flushed with 4 column volumes of equilibration buffer at a linear velocity of 60 cm / h. The equilibration buffer was the same as that used for column equilibration in step (6).
[0108] (9) Elution: The bovine lactoferrin in the heparin affinity chromatography column was eluted with an eluent consisting of 30 mmol / L citric acid-sodium citrate buffer, 1 mol / L NaCl and 0.2 mmol / L arginine at a pH of 5.5. The linear velocity of the eluent was controlled at 100 cm / h. The UV280 curve was observed. The eluent was collected after the main peak of bovine lactoferrin appeared and the collection was stopped when the baseline dropped steadily.
[0109] The preparation method of the filler in the heparin affinity chromatography column in this embodiment is as follows:
[0110] (1) After washing the agarose with ultrapure water for 3 times, the agarose was placed in a PBS buffer system containing glutaraldehyde, wherein the pH value of the PBS buffer system is 7 and the volume fraction of glutaraldehyde is 0.5%, and stirred at a speed of 50 rpm for 2 hours to obtain activated agarose. 50 g of agarose was added per liter of the buffer system.
[0111] (2) Heparin is activated by adding it to a PBS buffer solution containing N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, the pH value of the PBS buffer solution being 7, and reacting at 30° C. for 3 h to obtain activated heparin. 20 g of heparin is added to each liter of PBS buffer solution.
[0112] The concentration of N-hydroxysuccinimide in the buffer solution was 0.5 mol / L, and the concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was 0.3 mol / L.
[0113] (3) After the activated heparin and activated agarose were washed three times with 20 mmol / L PBS buffer respectively, they were added to 20 mmol / L PBS buffer, the pH value of which was 7, and reacted at 15°C for 10 h to covalently bind the activated heparin and activated agarose to obtain a heparin affinity chromatography column filler. 50 g of activated agarose and 20 g of activated heparin were added to each liter of PBS buffer.
[0114] (4) Washing: After the above reaction is completed, the filler is washed with PBS buffer to remove heparin that is not bound to agarose and reaction byproducts.
[0115] (5) Capping: Add 0.35 mol / L ethanolamine reagent to the filler to block the unreacted active sites. Add 1.5 L ethanolamine per liter of filler.
[0116] (6) Storage: The heparin affinity chromatography column filler prepared above was stored in a 20% by volume ethanol solution at a storage temperature of 20°C.
[0117] Example 3
[0118] This embodiment provides a method for purifying bovine lactoferrin from a fermentation broth using a heparin affinity chromatography column, comprising the following steps:
[0119] (1) The bovine lactoferrin gene is connected to the vector pPIC9K to prepare a recombinant expression vector, the recombinant expression vector is transferred into GS115 type Pichia pastoris to prepare a recombinant Pichia pastoris, and the recombinant Pichia pastoris is fermented to obtain a Pichia pastoris fermentation broth containing bovine lactoferrin.
[0120] The fermentation process of the recombinant Pichia pastoris is as follows: the recombinant Pichia pastoris is cultured using a basal inorganic salt medium (BSM) for Pichia pastoris. During the bacterial growth stage, the temperature is controlled at 30°C, the pH value is 6-7, and the DO is controlled at 20-60%. After culturing for about 3-5 hours, the DO rebounds. When it rebounds to above 100%, 50% glycerol is used for feeding at a flow rate of 30 g / L / h. After continuing to culture for 10 hours, the feeding is stopped, the bacteria are starved for 30 minutes, the temperature is controlled at 25°C, and 100% methanol is used for feeding at a flow rate of 1.5 mL / L / h. The Pichia pastoris fermentation broth is obtained by induction culture for 96 hours.
[0121] Centrifugation of cells: The Pichia yeast fermentation broth containing bovine lactoferrin was centrifuged at 10,000 rpm for 20 min using a centrifuge. After the centrifugation was completed, the supernatant was discarded and the Pichia yeast cells were collected.
[0122] (2) Resuspension of the cells: Take 300 g of the Pichia cells prepared in the above step and resuspend the Pichia cells in 4500 ml of citric acid-sodium citrate buffer with a pH value of 7 and a concentration of 50 mmol / L.
[0123] (3) High-pressure homogenization: Use a high-pressure homogenizer to homogenize the bacterial solution obtained after resuspension, control the pressure at 1500 bar, and homogenize 3 times.
[0124] (4) Centrifugal filtration: The high-pressure homogenized bacterial solution was centrifuged at 10,000 rpm for 20 min, the supernatant was collected, and the supernatant was filtered using a filter with a pore size of 0.65 μm.
[0125] (5) Ultrafiltration concentration: The filtered supernatant was ultrafiltered and concentrated using an ultrafiltration membrane with a pore size of 30 kDa. The pressure during ultrafiltration concentration was 0.15 MPa and the temperature was 8°C. After the volume was concentrated to 1 / 4 of the initial volume, the pH value of the concentrated solution was adjusted to 7 using sodium citrate.
[0126] (6) Column equilibration: The heparin affinity chromatography column was equilibrated using 6 column volumes of equilibration buffer, wherein the equilibration buffer consisted of 50 mmol / L citric acid-sodium citrate buffer and 50 mmol / L NaCl. The pH value of the equilibration buffer was 7. The linear velocity of the equilibration buffer was controlled at 120 cm / h. The volume of the heparin affinity chromatography column was 130 mL.
[0127] (7) Loading: The concentrated solution after adjusting the pH in step (5) is loaded onto the heparin affinity chromatography column as the loading solution. The linear velocity of the loading solution is controlled to be 120 cm / h and the temperature of the loading solution is controlled to be 20°C.
[0128] (8) Column flushing: The heparin affinity chromatography column was flushed with 6 column volumes of equilibration buffer at a linear velocity of 120 cm / h. The equilibration buffer was the same as that used for column equilibration in step (6).
[0129] (9) Elution: The bovine lactoferrin in the heparin affinity chromatography column was eluted with an eluent composed of 50 mmol / L citric acid-sodium citrate buffer, 2 mol / L NaCl and 0.3 mmol / L arginine at a pH of 6. The linear velocity of the eluent was controlled at 120 cm / h. The UV280 curve was observed. The eluent was collected after the main peak of bovine lactoferrin appeared and the collection was stopped when the baseline dropped steadily.
[0130] The preparation method of the filler in the heparin affinity chromatography column in this embodiment is as follows:
[0131] (1) After washing the agarose with ultrapure water for 3 times, the agarose was placed in a PBS buffer system containing glutaraldehyde, wherein the pH value of the PBS buffer system is 7 and the volume fraction of glutaraldehyde is 1%, and the agarose was activated by stirring at a speed of 100 rpm for 5 hours. 100 g of agarose was added to each liter of the buffer system.
[0132] (2) Heparin is activated by adding it to a PBS buffer solution containing N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, the pH value of the PBS buffer solution being 8, and reacting it at 38°C for 6 hours to obtain activated heparin. 50 g of heparin is added to each liter of PBS buffer solution.
[0133] The concentration of N-hydroxysuccinimide in the buffer solution was 0.8 mol / L, and the concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was 0.5 mol / L.
[0134] (3) After the activated heparin and activated agarose were washed three times with 20 mmol / L PBS buffer respectively, they were added to 20 mmol / L PBS buffer, the pH value of which was 7, and reacted at 25°C for 12 h to covalently bind the activated heparin and activated agarose to obtain a heparin affinity chromatography column filler. 100 g of activated agarose and 50 g of activated heparin were added to each liter of PBS buffer.
[0135] (4) Washing: After the above reaction is completed, the filler is washed with PBS buffer to remove heparin that is not bound to agarose and reaction byproducts.
[0136] (5) Capping: Add 0.35 mol / L ethanolamine reagent to the filler to block the unreacted active sites. Add 1.5 L ethanolamine per liter of filler.
[0137] (6) Storage: The heparin affinity chromatography column filler prepared above was stored in a 20% by volume ethanol solution at a storage temperature of 20°C.
[0138] Comparative Example 1
[0139] Bovine lactoferrin was purified from the fermentation broth using a heparin affinity chromatography column according to the method of Example 1, except that the pH value of the eluent was different, and the pH value of the eluent was set to 6.5.
[0140] Comparative Example 2
[0141] Bovine lactoferrin was purified from the fermentation broth using a heparin affinity chromatography column according to the method of Example 1, except that the pH value of the eluent was different, and the pH value of the eluent was set to 5.0.
[0142] Comparative Example 3
[0143] Bovine lactoferrin was purified from the fermentation broth using a heparin affinity chromatography column according to the method of Example 1, except that an equal amount of arginine in the eluate was replaced by lysine.
[0144] Comparative Example 4
[0145] Bovine lactoferrin was purified from the fermentation broth using a heparin affinity chromatography column according to the method of Example 1, except that the pH value of the loading solution was set to 6.0.
[0146] Comparative Example 5
[0147] Bovine lactoferrin was purified from the fermentation broth using a heparin affinity chromatography column according to the method of Example 1, except that the pH value of the loading solution was set to 7.5.
[0148] Comparative Example 6
[0149] Bovine lactoferrin was purified from the fermentation broth using a heparin affinity chromatography column according to the method of Example 1, except that the filler in the heparin affinity chromatography column was replaced with heparin-agarose gel prepared by another method.
[0150] The preparation method of heparin-agarose gel in this comparative example is as follows:
[0151] (1) Take 5 g of agarose gel from which water has been filtered out, add 7.5 mL of 0.8 mol / L NaOH, 10 mg of NaBH4, and 2 mL of epichlorohydrin, and react at 25°C for 8 h to obtain epichlorohydrin-activated agarose gel.
[0152] (2) Add 1 g of dried epichlorohydrin-activated agarose gel into a sealed reactor, then add 1 mL of concentrated ammonia water, and react at 40° C. for 4 h to obtain amino agarose gel.
[0153] (3) Take 1 g of the drained amino agarose gel and add it to the reactor, add 30 mg of heparin, 6 mg of sodium borohydride, and 1 mL of 0.2 mol / L phosphate buffer with a pH of 7, and react at 30°C for 48 h to obtain heparin-agarose gel.
[0154] Test Case
[0155] The content and purity of bovine lactoferrin in the eluates collected after purification in the above Examples 1 to 3 and Comparative Examples 1 to 5 were detected, and the results are shown in Table 1.
[0156] Table 1
[0157]
[0158]
[0159] It can be seen from the above test results that after the pH value of the eluent was changed in Comparative Examples 1 and 2, the content and purity of bovine lactoferrin in the eluent were significantly reduced, indicating that the pH value of 5.5 to 6 in the eluent of the present application is the most suitable pH value for the elution of bovine lactoferrin. In Comparative Example 3, arginine in the eluent was replaced with lysine in equal amounts, and the purity of bovine lactoferrin in the eluent decreased, indicating that at a pH value of 5.5 to 6, the use of an eluent containing arginine and NaCl helps to better separate bovine lactoferrin from other impurities and improve the purity of bovine lactoferrin. After the pH value of the loading solution was changed in Comparative Examples 4 and 5, the content and purity of bovine lactoferrin in the eluent were significantly reduced, indicating that when the pH value of the loading solution is controlled to 6.5 to 7 in the present application, bovine lactoferrin can be better adsorbed by the heparin affinity chromatography column filler prepared in the present invention when loading. In Comparative Example 6, after the filler in the heparin affinity chromatography column was replaced with heparin-agarose gel prepared by another method, the purity of bovine lactoferrin in the eluate decreased, indicating that the heparin affinity chromatography column filler prepared in the present invention combined with the eluate in a specific pH range can better separate bovine lactoferrin from other impurities, thereby improving the purity of bovine lactoferrin.
[0160] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A method for purifying bovine lactoferrin from a fermentation broth using a heparin affinity chromatography column, characterized in that: The following steps are involved: (1) Bacterial cell centrifugation: centrifuging the Pichia pastoris fermentation broth containing bovine lactoferrin to collect the bacterial cells; (2) Resuspension of the cells: Resuspend the cells in a citric acid buffer with a pH of 6.5 to 7; (3) High-pressure homogenization: The bacterial solution obtained after resuspension is subjected to high-pressure homogenization; (4) Centrifugal filtration: centrifuge the bacterial solution after high-pressure homogenization, collect the supernatant, and filter the supernatant; (5) Ultrafiltration and concentration: The filtered supernatant is ultrafiltered and concentrated to 1 / 4 to 1 / 3 of the initial volume, and then the pH value of the concentrated solution is adjusted to 6.5 to 7.0 using citrate; (6) Column equilibration: Use 4 to 6 column volumes of equilibration buffer to equilibrate the heparin affinity chromatography column; (7) Loading: The concentrated solution after pH adjustment in step (5) is loaded onto a heparin affinity chromatography column as a loading solution; (8) Column flushing: flush the heparin affinity chromatography column with 4 to 6 column volumes of equilibration buffer; (9) Elution: using an eluent composed of a citrate buffer, NaCl and arginine to elute the bovine lactoferrin in the heparin affinity chromatography column, controlling the linear velocity of the eluent to be 100 to 120 cm / h, observing the UV280 curve, and starting to collect the eluent after the main peak of bovine lactoferrin appears, and stopping the collection when the baseline drops steadily; The concentration of the citric acid buffer in the eluent is 20-50 mmol / L, the concentration of NaCl is 1-2 mol / L, and the concentration of arginine is 0.2-0.3 mmol / L; and the pH value of the eluent is 5.5-6.
2. The method according to claim 1, characterized in that The preparation method of the filler in the heparin affinity chromatography column is as follows: (1) After washing the agarose with ultrapure water, placing it in a PBS buffer system containing glutaraldehyde, wherein the pH value of the PBS buffer system is 6 to 8, the volume fraction of the glutaraldehyde is 0.5% to 1%, and stirring at a speed of 50 to 100 rpm for 2 to 5 hours to obtain activated agarose; 50 to 100 g of agarose is added per liter of the buffer system; (2) adding heparin to a buffer solution containing N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide for activation, wherein the pH value of the buffer solution is 7 to 8, reacting at 30 to 38° C. for 3 to 6 hours to obtain activated heparin; adding 20 to 50 g of heparin per liter of buffer solution; The concentration of N-hydroxysuccinimide in the buffer is 0.5-0.8 mol / L, and the concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 0.3-0.5 mol / L; (3) After the activated heparin and activated agarose are washed with 20 mmol / L PBS buffer respectively, they are added to 20 mmol / L PBS buffer, wherein the pH value of the PBS buffer is 6.5-7.5, and reacted at 15-25° C. for 10-12 hours to allow the activated heparin to be covalently bonded to the activated agarose to obtain a heparin affinity chromatography column filler; 50-100 g agarose and 20-50 g heparin are added to each liter of PBS buffer.
3. The method according to claim 1, characterized in that In step (1), the centrifugal speed is 5000-10000 rpm, and the centrifugal time is 20-25 min.
4. The method according to claim 1, characterized in that: The concentration of the citric acid buffer in step (2) is 20 to 50 mmol / L; 5 to 20 ml of citric acid buffer is added for every gram of bacterial cells.
5. The method according to claim 1, characterized in that: In step (3), the pressure of high pressure homogenization is 1000-1500 bar, and homogenization is performed 3-5 times.
6. The method according to claim 1, characterized in that In step (4), the centrifugal speed is 5000-10000 rpm, and the centrifugal time is 20-30 min; the pore size of the filter used during filtration is 0.22-0.65 um.
7. The method according to claim 1, characterized in that During the ultrafiltration concentration in step (5), the pressure is ≤0.15 MPa, the temperature is 2-8° C., and the pore size of the ultrafiltration membrane is 10-30 kDa.
8. The method according to claim 1, characterized in that: The equilibration buffer in steps (6) and (8) is composed of 20-50 mmol / L citric acid buffer and 20-50 mmol / L NaCl, and the pH value of the equilibration buffer is 6.5-7.
0.
9. The method according to claim 1, characterized in that: In step (7), the linear velocity of the loading liquid is controlled to be 60-120 cm / h, and the temperature of the loading liquid is controlled to be 2-20°C.
10. The method according to claim 1, characterized in that The linear velocity of the equilibration buffer in step (8) is 60 to 120 cm / h.