Method for separating and purifying bovine lactoferrin by using ligand affinity chromatography technology

Abstract

The invention discloses a method for separating and purifying bovine lactoferrin by using a ligand affinity chromatography technology, and belongs to the technical field of protein purification. The method comprises the following steps: introducing a bovine lactoferrin gene into pichia pastoris, fermenting to obtain a pichia pastoris fermentation solution, centrifuging, resuspending, homogenizing at high pressure, centrifuging and filtering the pichia pastoris fermentation solution to obtain a filtrate, separating and purifying bovine lactoferrin in the filtrate by using an affinity chromatography technology, immobilizing a triazine dye ligand in a filler of a chromatographic column, and purifying the triazine dye ligand to obtain the bovine lactoferrin. An eluent is prepared, the concentration of NaSCN in the eluent is 0.2-0.5 mol / L, the volume concentration of ethylene glycol in the eluent is 15%-20%, the concentration of NaCl in the eluent is 1-2 mol / L, and the bovine lactoferrin in the chromatographic column is eluted. According to the method, the composition of the eluent is optimized, the three components in the eluent are matched with one another and have a synergistic effect, the stability of the bovine lactoferrin is protected, the purity of the bovine lactoferrin is guaranteed, meanwhile, the elution efficiency of the bovine lactoferrin is improved, and the dosage of the eluent is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of protein purification, and in particular to a method for separating and purifying bovine lactoferrin by using ligand affinity chromatography technology. Background Art

[0002] Lactoferrin is a natural antibacterial glycoprotein that has antibacterial properties against both Gram-positive and Gram-negative bacteria. At the same time, lactoferrin also has biological activities such as immunomodulation and anti-oxidation, and has important application value in infant nutrition, immunity enhancement, and medical and health product development.

[0003] Lactoferrin produced at home and abroad is mainly extracted from cow's milk, which is difficult to separate and has high cost. With the development of gene editing technology, lactoferrin can now be expressed from a variety of genetically engineered bacteria. The main separation methods include salting out, ultrafiltration, chromatography, etc.

[0004] The main advantage of the salting-out method and ultrafiltration method is that a large amount of dairy samples can be processed in a short time, but the purity of the obtained lactoferrin is low. The purification of 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 lactoferrin with a purity of more than 95%, the purification efficiency is low, and the affinity chromatography filler is expensive, and the production cost is high. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a method for separating and purifying bovine lactoferrin using ligand affinity chromatography technology, so as to overcome the problems of low purification efficiency and high purification cost of bovine lactoferrin in the prior art.

[0006] In a first aspect, the present invention provides a method for separating and purifying bovine lactoferrin using ligand affinity chromatography, comprising the following steps:

[0007] (1) introducing a bovine lactoferrin gene into Pichia pastoris, and fermenting the Pichia pastoris to obtain a Pichia pastoris fermentation broth;

[0008] (2) centrifuging the Pichia pastoris fermentation broth, removing the supernatant, and collecting the Pichia pastoris cells;

[0009] (3) resuspending the centrifuged Pichia pastoris cells using PB buffer with a pH value of 6 to 7;

[0010] (4) high-pressure homogenization of the resuspended bacterial solution;

[0011] (5) centrifuging the bacterial solution after high-pressure homogenization, collecting the supernatant, filtering the supernatant, and adjusting the pH value of the filtered solution to 6 to 7;

[0012] (6) Column equilibration: using 2 to 5 column volumes of an equilibration buffer to equilibrate the chromatography column, wherein the filler of the chromatography column has a triazine dye fixed therein;

[0013] (7) Loading: Load the filtrate after adjusting the pH in step (5) onto the chromatography column, and control the linear velocity of the filtrate to be 50 to 100 cm / h; observe the UV280 curve, and collect the effluent from the chromatography column after an absorption peak appears. After loading, repeat step (7) with the collected effluent and load it again;

[0014] (8) Column flushing: flush the chromatography column with 2 to 5 column volumes of flushing solution;

[0015] (9) Elution: preparing an eluent, wherein the concentration of NaSCN in the eluent is 0.2-0.5 mol / L, the volume concentration of ethylene glycol is 15%-20%, and the concentration of NaCl is 1-2 mol / L, and the pH value of the eluent is 6-7. The bovine lactoferrin in the chromatography column is eluted with the eluent, and the linear velocity of the eluent is controlled 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.

[0016] Compared with the prior art, the filler of the affinity chromatography column of the present invention is fixed with triazine dye as an affinity ligand, and a specific eluent is used, in which the concentration of NaSCN is 0.2-0.5 mol / L, the volume concentration of ethylene glycol is 15%-20%, and the concentration of NaCl is 1-2 mol / L. First, NaSCN dissociates in the solution, and the generated SCN- ions can reduce the binding strength between bovine lactoferrin and triazine dye ligands through the triple effects of electrostatic shielding, coordination competition and hydrogen bond interference during the elution process, thereby promoting the release of bovine lactoferrin. Furthermore, the hydroxyl groups in ethylene glycol can also interact with the amino acid residues on the surface of bovine lactoferrin, affecting the electrostatic interaction and hydrogen bond network between bovine lactoferrin and triazine dye ligands, thereby affecting their binding. In addition, ethylene glycol also helps to stabilize the protein structure, affecting the binding of bovine lactoferrin with triazine dye ligands, so that SCN -Ions are more likely to gain an advantage in coordination competition, further promoting the dissociation of bovine lactoferrin from triazine dye ligands. NaCl is used as an ion stabilizer in the eluent to enhance the solubility of bovine lactoferrin and maintain the stability of the protein. At the same time, the co-existence of NaSCN and NaCl salts will further increase the ionic strength of the solution, thereby reducing the electrostatic interaction between bovine lactoferrin and triazine dye ligands, which is conducive to the elution of the protein. The present invention reduces the concentration of sodium chloride, which reduces the pressure for the desalination and concentration of the subsequent protein solution. The three components in the eluent cooperate with each other and work synergistically, while protecting the stability of bovine lactoferrin and ensuring the purity of bovine lactoferrin, the elution efficiency of bovine lactoferrin is improved. Compared with heparin or nickel column affinity chromatography in the prior art, the filler and ligand used in this application greatly reduce the cost, and the elution rate is increased by 8-12%, and the amount of eluent is reduced by 20-30%.

[0017] Furthermore, the preparation method of the chromatography column filler fixed with triazine dye in step (6) is as follows:

[0018] (1) Activating the agarose filler with 0.5-1 mol / L CNBr, adding 0.5-1 liter of 0.5-1 mol / L CNBr per liter of agarose filler, and the activation time is 2-3 hours; after the activation is completed, removing the upper solution to obtain an activated agarose filler;

[0019] (2) dissolving the triazine dye ligand in a buffer solution with a pH value of 7 to 8, adding the activated agarose filler to the buffer solution at a ratio of 1 g of the activated agarose filler to 1.5 to 2 g of the triazine dye ligand, and simultaneously adding N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to a final concentration of 0.1 mol / L of the N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the buffer solution;

[0020] (3) Stirring, setting the rotation speed to 20-50 rpm, controlling the temperature to 2-20° C., and reacting for 10-16 hours to couple the triazine dye ligand with the activated agarose filler.

[0021] The above technical scheme further defines a method for preparing a chromatography column filler fixed with a triazine dye ligand. After the agarose filler is activated with CNBr, the activated agarose filler is reacted with a triazine dye ligand, N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) in a buffer for 10 to 16 hours. The activated agarose filler interacts with the triazine dye ligand, NHS and EDC, which not only allows the triazine dye ligand to be fully coupled with the agarose filler to obtain a chromatography column filler fixed with a triazine dye ligand, but also allows the chromatography column filler to bind more firmly to the target protein bovine lactoferrin in the loading step, thereby improving the binding efficiency of bovine lactoferrin with the filler. Further, in combination with the specific eluent of the present invention, the bovine lactoferrin firmly adsorbed in the chromatography column filler can also be eluted efficiently, further ensuring the purity and elution efficiency of bovine lactoferrin.

[0022] Furthermore, in step (2), the centrifugation speed is 5000-10000 rpm and the time is 20-30 min.

[0023] Furthermore, in step (3), the concentration of the PB buffer is 10 to 20 mmol / L; 5 to 20 ml of PB buffer is added for every gram of bacterial cells.

[0024] Furthermore, the pressure of the high-pressure homogenization in step (4) is 1000-1500 bar, and the homogenization is performed 2-5 times.

[0025] Furthermore, in step (5), the rotation speed during centrifugation is 5000-10000 rpm, and the time is 20-30 min; the pore size of the filter for filtering the supernatant is 0.22-0.65 um, and the pH value of the filtered solution is adjusted to 6-7 using dihydrogen phosphate.

[0026] Furthermore, the equilibrium buffer in step (6) is a PB buffer having a pH value of 6 to 7 and a concentration of 10 to 20 mmol / L.

[0027] Furthermore, during the loading process of step (7), the temperature of the loading liquid and the effluent liquid is controlled to be 4 to 20°C.

[0028] Furthermore, in step (8), the concentration of NaSCN in the flushing liquid is 0.1-0.15 mol / L, the volume concentration of ethylene glycol is 4%-6%, and the concentration of NaCl is 0.04-0.06 mol / L; the linear velocity of the flushing liquid is 50-100 cm / h.

[0029] The above technical solution defines the specific composition and linear velocity of the flushing liquid. The flushing liquid can be used to remove non-specifically adsorbed impurities in the chromatography column filler, thereby ensuring the elution efficiency of the subsequent elution process and the quality of the target protein bovine lactoferrin.

[0030] Furthermore, the buffer for dissolving the triazine dye ligand is any one or two of 0.1-0.15 mol / L NaHCO3 and 0.1-0.15 mol / L Na2CO3; the triazine dye is any one or two of red HE-3B triazine dye and yellow HE-4R triazine dye.

[0031] The above technical solution defines the composition of the buffer solution for dissolving the triazine dye ligand, which can provide a suitable pH environment for the coupling of the triazine dye ligand and the activated agarose filler, ensuring that the two can be fully coupled. DETAILED DESCRIPTION

[0032] 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.

[0033] In a first aspect, an embodiment of the present invention provides a method for separating and purifying bovine lactoferrin using ligand affinity chromatography technology, comprising the following steps:

[0034] S1. Introducing bovine lactoferrin gene into Pichia pastoris, and fermenting to obtain Pichia pastoris fermentation broth.

[0035] For example, the bovine lactoferrin gene can be connected to the vector pPIC9K containing the PAOX1 promoter to prepare a recombinant expression vector, and the obtained recombinant expression vector is transformed into GS115 type Pichia pastoris to prepare a recombinant Pichia pastoris, and the recombinant Pichia pastoris is subjected to high-density cell fermentation to obtain a Pichia pastoris fermentation liquid. It should be understood that the fermentation method can refer to the high-density cell fermentation method commonly used in the prior art. For example, reference can be made to "High Cell Density Fermentation Technology" published by Chemical Industry Press in 2006. The GenbanK ID of the bovine lactoferrin gene is JX294418.1.

[0036] S2. Centrifuging the Pichia yeast fermentation broth, removing the supernatant, and collecting the Pichia yeast cells.

[0037] The speed during centrifugation in the above steps is 5000-10000rpm, and the time is 20-30min. For example, the speed during centrifugation can be 5000rpm, 6000rpm, 7000rpm, 8000rpm, 9000rpm or 10000rpm, preferably 6000-8000rpm. The centrifugation time can be 20min, 22min, 24min, 26min, 28min or 30min, preferably 22-28min. It should be understood that different centrifugation times can be selected according to the centrifugation speed, so as to collect the Pichia yeast cells.

[0038] S3. Resuspend the centrifuged Pichia pastoris cells using PB buffer with a pH of 6 to 7.

[0039] The concentration of the PB buffer in the above steps is 10-20 mmol / L; for example, it can be selected to be 10 mmol / L, 12 mmol / L, 14 mmol / L, 16 mmol / L, 18 mmol / L or 20 mmol / L, so as to maintain the pH value in the range of 6-7.

[0040] When resuspending the bacteria in the above step, 5 to 20 ml of PB buffer is added per gram of bacteria. For example, 5 ml, 10 ml, 15 ml or 20 ml of PB buffer can be added per gram of bacteria, preferably 10 to 15 ml, to ensure that the bacteria are evenly distributed in the buffer. When the cells are subsequently broken by high-pressure homogenization, the bacterial cells can be more evenly subjected to external force, thereby improving the breaking efficiency and uniformity.

[0041] S4. Perform high pressure homogenization on the resuspended bacterial solution.

[0042] In the above steps, a high-pressure homogenizer can be used to homogenize the resuspended bacterial solution under high pressure, with the pressure controlled at 1000-1500 bar and homogenized 2-5 times. Under the above homogenization pressure and times, the bacterial cells can be fully broken and the proteins in the bacterial cells can be released.

[0043] S5. Centrifuge the bacterial solution after high-pressure homogenization, collect the supernatant, filter the supernatant, and adjust the pH value of the filtered solution to 6-7.

[0044] In the above steps, the centrifugal speed is 5000-10000 rpm, and the time is 20-30 min. After centrifugation, bovine lactoferrin is dissolved in the supernatant, and cell debris and some other macromolecular substances form precipitation.

[0045] Next, the supernatant containing bovine lactoferrin is filtered using a depth filter, a glass fiber filter, a hollow fiber filter or an ultrafiltration membrane with a pore size of 0.22 to 0.65 μm to remove large particle impurities in the supernatant.

[0046] Then, the pH value of the filtered solution is adjusted to 6-7 using dihydrogen phosphate, wherein the dihydrogen phosphate can be disodium hydrogen phosphate or dipotassium hydrogen phosphate.

[0047] S6, column equilibration: Use 2 to 5 column volumes of equilibration buffer to equilibrate the chromatography column, wherein the filler of the chromatography column is fixed with triazine dye. The equilibration buffer is a PB buffer with a pH value of 6 to 7 and a concentration of 10 to 20 mmol / L, the purpose of which is to establish a stable buffer environment, reduce the impact of the sample on the column, and prevent abnormal adsorption or elution behavior of the target molecule due to sudden changes in the environment.

[0048] The preparation method of the chromatography column filler fixed with triazine dye in the above steps is as follows:

[0049] S61. Use 0.5-1 mol / L CNBr to activate the agarose filler. Add 0.5-1 liter of 0.5-1 mol / L CNBr per liter of agarose filler. The activation time is 2-3 hours. After the activation is completed, remove the upper solution to obtain an activated agarose filler.

[0050] S62. Dissolve the triazine dye ligand in a buffer solution with a pH value of 7 to 8, add the activated agarose filler to the buffer solution at a ratio of 1 g of activated agarose filler to 1.5 to 2 g of triazine dye ligand, and simultaneously add N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, until the final concentrations of the N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the buffer solution are both 0.1 mol / L.

[0051] Furthermore, in the above step, the buffer for dissolving the triazine dye ligand is any one or two of 0.1-0.15 mol / L NaHCO3 and 0.1-0.15 mol / L Na2CO3.

[0052] Furthermore, the triazine dye in the above step is any one or both of red HE-3B triazine dye and yellow HE-4R triazine dye.

[0053] S63, stirring, setting the rotation speed to 20-50 rpm, controlling the temperature to 2-20° C., reacting for 10-16 hours to couple the triazine dye ligand with the activated agarose filler.

[0054] In the above steps, the inventors found that after the CNBr-activated agarose filler is fully reacted with the triazine dye ligand, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in a buffer, the triazine dye ligand can be fully coupled with the agarose filler, and the chromatography column filler with the triazine dye fixed thereon obtained after such treatment is more firmly bound to bovine lactoferrin, and more bovine lactoferrin is bound during the loading process, thereby improving the binding efficiency of bovine lactoferrin with the filler.

[0055] S7, loading: Load the filtrate after adjusting the pH in step S5 onto the chromatography column, control the linear velocity of the filtrate to 50-100 cm / h; observe the UV280 curve, collect the effluent from the chromatography column after an absorption peak appears, repeat step S7 with the collected effluent and load it again after loading. During the loading process, control the temperature of the loading liquid and the effluent to 4-20°C.

[0056] For example, the linear velocity of the filtrate can be 50 cm / h, 60 cm / h, 70 cm / h, 80 cm / h, 90 cm / h or 100 cm / h, preferably 60 to 80 cm / h.

[0057] In the above steps, 1 mL of supernatant containing bovine lactoferrin is loaded for every 10 to 100 mg of filler, and the content of bovine lactoferrin in the supernatant is 0.1 to 10 mg / mL.

[0058] The sample was loaded twice in the above steps in order to fully combine the bovine lactoferrin in the filtrate with the triazine dye ligand.

[0059] S8, column flushing: flush the chromatography column with 2 to 5 column volumes of flushing solution, the linear velocity of the flushing solution is 50 to 100 cm / h. For example, the linear velocity can be 50 cm / h, 60 cm / h, 70 cm / h, 80 cm / h, 90 cm / h or 100 cm / h, preferably 60 to 80 cm / h.

[0060] Wherein, the concentration of NaSCN in the flushing liquid is 0.1-0.15 mol / L, the volume concentration of ethylene glycol is 4%-6%, the concentration of NaCl is 0.04-0.06 mol / L, and the pH value of the flushing liquid is 6-7. For example, the concentration of NaSCN can be 0.1 mol / L, 0.11 mol / L, 0.12 mol / L, 0.13 mol / L, 0.14 mol / L or 0.15 mol / L, preferably 0.1-0.13 mol / L. The volume concentration of ethylene glycol can be 4%, 5% or 6%. The concentration of NaCl can be 0.04 mol / L, 0.05 mol / L or 0.06 mol / L.

[0061] The flushing solution in the above steps is composed of NaSCN, ethylene glycol and NaCl in a specific concentration range. In this concentration range, the flushing solution will not affect the bovine lactoferrin bound to the chromatography column, but can remove non-specifically bound proteins and other impurities in the chromatography column to prevent them from interfering with the subsequent elution step.

[0062] S9, elution: prepare an eluent, wherein the concentration of NaSCN in the eluent is 0.2-0.5 mol / L, the volume concentration of ethylene glycol is 15%-20%, the concentration of NaCl is 1-2 mol / L, the pH value of the eluent is 6-7, and the bovine lactoferrin in the chromatography column is eluted with the eluent, and the linear velocity of the eluent is controlled to be 100-120 cm / h; observe the UV280 curve, and start collecting the eluent after the main peak of bovine lactoferrin appears; stop collecting when the baseline drops steadily.

[0063] Illustratively, the linear velocity of the eluent may be 100 cm / h, 110 cm / h or 120 cm / h.

[0064] For example, the concentration of NaSCN in the eluent can be 0.2 mol / L, 0.3 mol / L, 0.4 mol / L or 0.5 mol / L, preferably 0.3-0.5 mol / L. The volume concentration of ethylene glycol can be 15%, 16%, 17%, 18%, 19% or 20%, preferably 17%-20%. The concentration of NaCl can be 1 mol / L, 1.5 mol / L or 2 mol / L.

[0065] In the above steps, 0.2-0.5 mol / L NaSCN will dissociate Na + and SCN - ions, first, SCN - Ions can bind to the positively charged areas on the surface of bovine lactoferrin, thereby changing the charge distribution on the protein, thereby shielding or reducing the charge interaction between bovine lactoferrin and the triazine dye ligand, and reducing the binding strength between the two. - Ions can compete with triazine dye ligands for binding sites on bovine lactoferrin, and form more stable complexes with iron ions in bovine lactoferrin, thereby replacing triazine dye ligands and destroying the original binding between triazine dye ligands and bovine lactoferrin. - Ions can also form hydrogen bonds with bovine lactoferrin or triazine dye ligands, thereby interfering with the hydrogen bond network between bovine lactoferrin and triazine dye ligands, further reducing the binding strength between the two.

[0066] The addition of ethylene glycol will change the polarity and dielectric constant of the solution. The hydroxyl groups in the ethylene glycol molecules can form hydrogen bonds with the amino acid residues on the surface of bovine lactoferrin, interfering with the hydrogen bonding between bovine lactoferrin and the triazine dye ligand. In addition, ethylene glycol can stabilize the three-dimensional structure of bovine lactoferrin and prevent SCN. - The effect of ions on protein conformation weakens or shields the binding site between bovine ferritin and triazine dye ligand, making SCN - Ions can bind to bovine lactoferrin more easily, further promoting the dissociation of bovine lactoferrin from the ligand.

[0067] 1-2 mol / L NaCl as an ion stabilizer can enhance the solubility of bovine lactoferrin, maintain the stability of bovine lactoferrin, and prevent it from unfolding, aggregation, degradation and inactivation during the purification process. In addition, the co-existence of NaSCN and NaCl will further increase the ionic strength of the solution, thereby reducing the electrostatic interaction between bovine lactoferrin and triazine dye ligands, thereby improving the elution efficiency and purity of the protein.

[0068] The eluent of the invention comprises NaSCN, ethylene glycol and NaCl. By further limiting the concentration of NaSCN in the eluent to 0.2-0.5 mol / L, the volume concentration of ethylene glycol to 15%-20%, and the concentration of NaCl to 1-2 mol / L, the three components act synergistically to elute bovine lactoferrin firmly adsorbed in the chromatography column filler, thereby greatly improving the elution efficiency and reducing the amount of the eluent while protecting the stability of the bovine lactoferrin and ensuring the purity of the bovine protein.

[0069] 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.

[0070] Example 1

[0071] This embodiment provides a method for separating and purifying bovine lactoferrin using ligand affinity chromatography technology, comprising the following steps:

[0072] (1) The bovine lactoferrin gene was connected to the vector pPIC9K containing the PAOX1 promoter to prepare a recombinant expression vector, the obtained recombinant expression vector was transformed into GS115 type Pichia pastoris to prepare a recombinant Pichia pastoris, and the recombinant Pichia pastoris was subjected to high-density cell fermentation to obtain a Pichia pastoris fermentation broth.

[0073] (2) The Pichia yeast fermentation broth was centrifuged at 7000 rpm for 25 min, the supernatant was removed, and 20 g of Pichia yeast cells were collected.

[0074] (3) Resuspend the centrifuged Pichia pastoris cells in 150 ml of PB buffer with a pH of 6.5 and a concentration of 15 mmol / L.

[0075] (4) Use a high-pressure homogenizer to homogenize the resuspended bacterial solution, control the pressure at 1300 bar, and homogenize three times.

[0076] (5) 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 depth filter with a pore size of 0.35 μm. The pH value of the filtered solution was adjusted to 6.5 using disodium hydrogen phosphate.

[0077] (6) Column equilibration: Prepare PB buffer with a pH value of 6.5 and a concentration of 15 mmol / L as the equilibration buffer, and use 4 times the column volume of the equilibration buffer to equilibrate the chromatography column. The linear velocity of the equilibration buffer is controlled to be 50 cm / h. The filler of the chromatography column is agarose filler fixed with yellow HE-4R triazine dye, and the volume of the filler is 4.8 ml.

[0078] (7) Loading: Load the filtrate after adjusting the pH in step (5) onto the chromatography column, and control the linear velocity of the filtrate to 80 cm / h; observe the UV280 curve, collect the effluent from the chromatography column after an absorption peak appears, and repeat step (7) with the collected effluent for re-loading after loading. During the loading process, control the temperature of the loading liquid and the effluent to be 10° C. After loading, detect the bovine lactoferrin content in the flow-through liquid.

[0079] (8) Column flushing: The column was flushed with a flushing solution of 3 column volumes, and the linear velocity of the flushing solution was controlled to be 80 cm / h. The concentration of NaSCN in the flushing solution was 0.1 mol / L, the volume concentration of ethylene glycol was 5%, the concentration of NaCl was 0.05 mol / L, and the pH value of the flushing solution was 6-7.

[0080] (9) Elution: preparing an eluent, wherein the concentration of NaSCN in the eluent is 0.5 mol / L, the volume concentration of ethylene glycol is 20%, and the concentration of NaCl is 2 mol / L, and the pH value of the eluent is 6-7. The bovine lactoferrin in the chromatography column is eluted with the eluent, and the linear velocity of the eluent is controlled to be 100 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.

[0081] The preparation method of the agarose filler fixed with yellow HE-4R triazine dye in this embodiment is as follows:

[0082] (1) Use 0.5 mol / L CNBr to activate the agarose filler. Add 0.5 liters of 0.5 mol / L CNBr per liter of agarose filler. The activation time is 2.5 hours. After the activation is completed, remove the upper solution to obtain the activated agarose filler.

[0083] (2) The yellow HE-4R triazine dye ligand is dissolved in a buffer solution with a pH value of 7 to 8 and a concentration of 0.1 mol / L NaHCO3. The activated agarose filler is added to the buffer solution at a ratio of 1 g of the activated agarose filler to 2 g of the yellow HE-4R triazine dye ligand. N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide are also added simultaneously, until the final concentrations of the N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the buffer solution are both 0.1 mol / L.

[0084] (3) Stirring, setting the speed to 20 rpm, controlling the temperature to 10° C., and reacting for 13 h to couple the yellow HE-4R triazine dye ligand with the activated agarose filler.

[0085] Example 2

[0086] This embodiment provides a method for separating and purifying bovine lactoferrin using ligand affinity chromatography technology, comprising the following steps:

[0087] (1) The bovine lactoferrin gene was connected to the vector pPIC9K containing the PAOX1 promoter to prepare a recombinant expression vector, the obtained recombinant expression vector was transformed into GS115 type Pichia pastoris to prepare a recombinant Pichia pastoris, and the recombinant Pichia pastoris was subjected to high-density cell fermentation to obtain a Pichia pastoris fermentation broth.

[0088] (2) The Pichia yeast fermentation broth was centrifuged at 5000 rpm for 30 min using a centrifuge, the supernatant was removed, and 20 g of Pichia yeast cells were collected.

[0089] (3) Resuspend the centrifuged Pichia pastoris cells in 200 ml of PB buffer with a pH of 6 and a concentration of 10 mmol / L.

[0090] (4) Use a high-pressure homogenizer to homogenize the resuspended bacterial solution, control the pressure at 1000 bar, and homogenize 5 times.

[0091] (5) 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 depth filter with a pore size of 0.22 μm. The pH value of the filtered solution was adjusted to 6 using disodium hydrogen phosphate.

[0092] (6) Column equilibration: Prepare PB buffer with a pH value of 6 and a concentration of 10 mmol / L as the equilibration buffer, and use 2 times the column volume of the equilibration buffer to equilibrate the chromatography column. The linear velocity of the equilibration buffer is controlled at 100 cm / h. The filler of the chromatography column is agarose filler fixed with red HE-3B triazine dye, and the volume of the filler is 4.8 ml.

[0093] (7) Loading: Load the filtrate after adjusting the pH in step (5) onto the chromatography column, and control the linear velocity of the filtrate to 50 cm / h; observe the UV280 curve, and collect the effluent from the chromatography column after an absorption peak appears. After loading, repeat step (7) with the collected effluent and load it again. During the loading process, control the temperature of the loading liquid and the effluent to 4° C. After loading, detect the bovine lactoferrin content in the flow-through liquid.

[0094] (8) Column flushing: The column was flushed with a flushing solution of 3 column volumes, and the linear velocity of the flushing solution was controlled to be 50 cm / h. The concentration of NaSCN in the flushing solution was 0.15 mol / L, the volume concentration of ethylene glycol was 4%, the concentration of NaCl was 0.04 mol / L, and the pH value of the flushing solution was 6-7.

[0095] (9) Elution: preparing an eluent, wherein the concentration of NaSCN in the eluent is 0.2 mol / L, the volume concentration of ethylene glycol is 15%, and the concentration of NaCl is 1 mol / L, and the pH value of the eluent is 6-7. The bovine lactoferrin in the chromatography column is eluted using the eluent, and the linear velocity of the eluent is controlled to be 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.

[0096] The preparation method of the agarose filler fixed with red HE-3B triazine dye in this embodiment is as follows:

[0097] (1) Activate the agarose filler with 1 mol / L CNBr. Add 1 liter of 1 mol / L CNBr to each liter of agarose filler. The activation time is 2 h. After activation, remove the upper solution to obtain an activated agarose filler.

[0098] (2) The red HE-3B triazine dye ligand is dissolved in a buffer solution with a pH value of 7 to 8 and a concentration of 0.1 mol / L Na2CO3. The activated agarose filler is added to the buffer solution at a ratio of 1 g of activated agarose filler to 1.5 g of red HE-3B triazine dye ligand. N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide are also added to the buffer solution until the final concentrations of the N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in the buffer solution are both 0.1 mol / L.

[0099] (3) Stirring, setting the speed to 50 rpm, controlling the temperature to 2° C., and reacting for 16 h to couple the red HE-3B triazine dye ligand with the activated agarose filler.

[0100] Example 3

[0101] This embodiment provides a method for separating and purifying bovine lactoferrin using ligand affinity chromatography technology, comprising the following steps:

[0102] (1) The bovine lactoferrin gene was connected to the vector pPIC9K containing the PAOX1 promoter to prepare a recombinant expression vector, the obtained recombinant expression vector was transformed into GS115 type Pichia pastoris to prepare a recombinant Pichia pastoris, and the recombinant Pichia pastoris was subjected to high-density cell fermentation to obtain a Pichia pastoris fermentation broth.

[0103] (2) The Pichia yeast fermentation broth was centrifuged at 10,000 rpm for 20 min, the supernatant was removed, and 20 g of Pichia yeast cells were collected.

[0104] (3) Resuspend the centrifuged Pichia pastoris cells in 300 ml of PB buffer with a pH of 7 and a concentration of 20 mmol / L.

[0105] (4) Use a high-pressure homogenizer to homogenize the resuspended bacterial solution, control the pressure at 1500 bar, and homogenize twice.

[0106] (5) 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 depth filter with a pore size of 0.65 μm. The pH value of the filtered solution was adjusted to 7 using disodium hydrogen phosphate.

[0107] (6) Column equilibration: PB buffer with a pH value of 7 and a concentration of 20 mmol / L was prepared as an equilibration buffer. The chromatographic column was equilibrated with 5 column volumes of the equilibration buffer. The linear velocity of the equilibration buffer was controlled to be 100 cm / h. The filler of the chromatographic column was an agarose filler fixed with a triazine dye. The volume of the filler was 4.8 ml. The triazine dye was a mixed triazine dye containing 70 wt% yellow HE-4R and 30 wt% red HE-3B.

[0108] (7) Loading: Load the filtrate after pH adjustment in step (5) onto the chromatography column, and control the linear velocity of the filtrate to be 100 cm / h; observe the UV280 curve, and collect the effluent from the chromatography column after an absorption peak appears. After loading, repeat step (7) with the collected effluent and load it again. During the loading process, control the temperature of the loading liquid and the effluent to be 20° C. After loading, detect the bovine lactoferrin content in the flow-through liquid.

[0109] (8) Column flushing: The column was flushed with a flushing solution of 3 times the column volume, and the linear velocity of the flushing solution was controlled to be 100 cm / h. The concentration of NaSCN in the flushing solution was 0.15 mol / L, the volume concentration of ethylene glycol was 6%, the concentration of NaCl was 0.06 mol / L, and the pH value of the flushing solution was 6-7.

[0110] (9) Elution: preparing an eluent, wherein the concentration of NaSCN in the eluent is 0.4 mol / L, the volume concentration of ethylene glycol is 17%, and the concentration of NaCl is 1.5 mol / L, and the pH value of the eluent is 6-7. The bovine lactoferrin in the chromatography column is eluted using the eluent, and the linear velocity of the eluent is controlled to be 110 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.

[0111] The preparation method of the agarose filler fixed with a mixed triazine dye containing 70 wt% yellow HE-4R and 30 wt% red HE-3B in this embodiment is as follows:

[0112] (1) Activate the agarose filler with 0.5 mol / L CNBr, add 1 liter of 0.5 mol / L CNBr per liter of agarose filler, and the activation time is 3 hours; after the activation is completed, remove the upper solution to obtain the activated agarose filler.

[0113] (2) A mixed triazine dye ligand containing 70 wt% yellow HE-4R and 30 wt% red HE-3B was dissolved in a buffer solution with a pH value of 7-8 and a concentration of 0.15 mol / L Na2CO3. The activated agarose filler was added to the buffer solution at a ratio of 1 g of the activated agarose filler to 1.7 g of the mixed triazine dye ligand. N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide were also added to the buffer solution until the final concentrations of the N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the buffer solution were both 0.1 mol / L.

[0114] (3) Stirring, setting the speed to 35 rpm, controlling the temperature to 20° C., and reacting for 10 h to couple the mixed triazine dye ligand containing 70% yellow HE-4R and 30% red HE-3B with the activated agarose filler.

[0115] Comparative Example 1

[0116] The bovine lactoferrin in the Pichia yeast fermentation broth was separated and purified using a conventional nickel column chromatography column in the prior art. Compared with Example 1, the difference is that:

[0117] Step (6) column equilibration: prepare an equilibration buffer, wherein the concentration of Tris buffer in the equilibration buffer is 10 mmol / L, the concentration of NaCl is 100 mmol / L, and the pH value is 7-7.5. Use 5 times the column volume of the equilibration buffer to perform column equilibration on the nickel column chromatography column, control the linear velocity of the equilibration buffer to 100 cm / h, and the filler in the chromatography column is Ni-NTA agarose resin.

[0118] Step (8) column flushing: flush the chromatography column with 5 column volumes of flushing solution, and control the linear velocity of the flushing solution to be 100 cm / h. The concentration of Tris buffer in the flushing solution is 10 mmol / L, the concentration of NaCl is 100 mmol / L, and the pH value of the flushing solution is 7.5.

[0119] Step (9) elution: preparing an eluent, wherein the concentration of Tris buffer in the eluent is 20 mmol / L, the concentration of NaCl is 100 mmol / L, the concentration of imidazole is 350 mmol / L, the pH value of the eluent is 7.5, and the bovine lactoferrin in the chromatography column is eluted using the eluent, and the linear velocity of the eluent is controlled to be 100 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.

[0120] Comparative Example 2

[0121] Bovine lactoferrin was separated and purified according to the method of Example 1, except that the agarose filler immobilized with yellow HE-4R triazine dye in step (6) was replaced by an equal amount of Ni-NTA agarose resin.

[0122] Comparative Example 3

[0123] Bovine lactoferrin was isolated and purified according to the method of Example 1, except that an equal amount of NaSCN in the eluent of step (9) was replaced by NaCl.

[0124] Comparative Example 4

[0125] Bovine lactoferrin was separated and purified according to the method of Example 1, except that the ethylene glycol in the eluent of step (9) was replaced by ethanol in an equal amount.

[0126] Comparative Example 5

[0127] Bovine lactoferrin was isolated and purified according to the method of Example 1, except that when preparing the agarose filler immobilized with yellow HE-4R triazine dye, the N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in step (2) were replaced by 0.1 mol / L NaHCO3 buffer in equal amounts.

[0128] Comparative Example 6

[0129] Bovine lactoferrin was separated and purified according to the method of Example 1, except that when preparing the agarose filler fixed with yellow HE-4R triazine dye, the activation in step (1) was not performed, and the agarose activated by CNBr in step (2) was replaced by an equal amount of carboxylated agarose (provided by Shanghai Cent Biotechnology).

[0130] Test Case

[0131] The bovine lactoferrin content in the flow-through of Examples 1 to 3 and Comparative Examples 1 to 6, the bovine lactoferrin content and purity in the eluate collected after elution, the amount of eluate used and the bovine lactoferrin elution rate were detected. The results are shown in Table 1.

[0132] Table 1

[0133]

[0134] From the above test results, it can be seen that in Comparative Example 1, a conventional nickel column chromatography column in the prior art was used to separate and purify bovine lactoferrin in the Pichia yeast fermentation broth. In Comparative Example 2, after the agarose filler fixed with yellow HE-4R triazine dye in step (6) was replaced with an equal amount of Ni-NTA agarose resin, the content, purity and elution rate of bovine lactoferrin in the eluate decreased, indicating that the use of triazine dye as an affinity chromatography ligand in combination with a specific eluent in the present application can improve the content, purity and elution rate of bovine lactoferrin.

[0135] In Comparative Example 3, after an equal amount of NaSCN in the eluent was replaced with NaCl, the content, purity and elution rate of bovine lactoferrin in the eluent decreased, indicating that there is a synergistic effect between NaSCN and ethylene glycol in the eluent of the present application, which can further improve the content, purity and elution rate of bovine lactoferrin.

[0136] In Comparative Example 4, after an equal amount of ethylene glycol in the eluent was replaced with ethanol, the content, purity and elution rate of bovine lactoferrin in the eluent decreased, indicating that compared with ethanol, ethylene glycol can more accurately and effectively regulate the interaction between bovine lactoferrin and triazine dye ligands, thereby improving the recovery rate of bovine lactoferrin.

[0137] Comparative Example 5 does not contain N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and Comparative Example 6 does not perform CNBr activation. The bovine lactoferrin content in the flow-through of Comparative Examples 5 and 6 increases, indicating that the chromatography column filler prepared by compounding N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide with CNBr-activated agarose filler in the present application has a higher binding rate with bovine lactoferrin.

[0138] Moreover, the amount of eluent used in comparative examples 1 to 6 is significantly higher than that in embodiments 1 to 3 of the present application, indicating that the ratio of NaSCN, NaCl and ethylene glycol in the eluent of the present application is reasonable and synergistic, and has a better elution effect on the chromatography column with triazine dye as ligand obtained by the specific method of the present invention, thereby reducing the amount of eluent used.

[0139] 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 separating and purifying bovine lactoferrin using ligand affinity chromatography, characterized in that: The following steps are involved: (1) introducing a bovine lactoferrin gene into Pichia pastoris, and fermenting the Pichia pastoris to obtain a Pichia pastoris fermentation broth; (2) centrifuging the Pichia pastoris fermentation broth, removing the supernatant, and collecting the Pichia pastoris cells; (3) resuspending the centrifuged Pichia pastoris cells using PB buffer with a pH value of 6 to 7; (4) high-pressure homogenization of the resuspended bacterial solution; (5) centrifuging the bacterial solution after high-pressure homogenization, collecting the supernatant, filtering the supernatant, and adjusting the pH value of the filtered solution to 6 to 7; (6) Column equilibration: using 2 to 5 column volumes of an equilibration buffer to equilibrate the chromatography column, wherein the filler of the chromatography column has a triazine dye fixed therein; (7) Loading: Load the filtrate after adjusting the pH in step (5) onto the chromatography column, and control the linear velocity of the filtrate to be 50 to 100 cm / h; observe the UV280 curve, and collect the effluent from the chromatography column after an absorption peak appears. After loading, repeat step (7) with the collected effluent and load it again; (8) Column flushing: flush the chromatography column with 2 to 5 column volumes of flushing solution; (9) Elution: preparing an eluent, wherein the concentration of NaSCN in the eluent is 0.2-0.5 mol / L, the volume concentration of ethylene glycol is 15%-20%, and the concentration of NaCl is 1-2 mol / L, and the pH value of the eluent is 6-7. The bovine lactoferrin in the chromatography column is eluted with the eluent, and the linear velocity of the eluent is controlled 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.

2. The method according to claim 1, characterized in that The preparation method of the chromatography column filler fixed with triazine dye in step (6) is as follows: (1) Activating the agarose filler with 0.5-1 mol / L CNBr, adding 0.5-1 liter of 0.5-1 mol / L CNBr per liter of agarose filler, and the activation time is 2-3 hours; after the activation is completed, removing the upper solution to obtain an activated agarose filler; (2) dissolving the triazine dye ligand in a buffer solution with a pH value of 7 to 8, adding the activated agarose filler to the buffer solution at a ratio of 1 g of the activated agarose filler to 1.5 to 2 g of the triazine dye ligand, and simultaneously adding N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to a final concentration of 0.1 mol / L of the N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the buffer solution; (3) Stirring, setting the rotation speed to 20-50 rpm, controlling the temperature to 2-20° C., and reacting for 10-16 hours to couple the triazine dye ligand with the activated agarose filler.

3. The method according to claim 1, characterized in that In step (2), the centrifugal speed is 5000-10000 rpm and the time is 20-30 min.

4. The method according to claim 1, characterized in that: The concentration of the PB buffer in step (3) is 10-20 mmol / L; 5-20 ml of PB buffer is added for every gram of bacterial cells.

5. The method according to claim 1, characterized in that The pressure of the high-pressure homogenization in step (4) is 1000-1500 bar, and the homogenization is performed 2-5 times.

6. The method according to claim 1, characterized in that In step (5), the centrifugation speed is 5000-10000 rpm, and the time is 20-30 min; the pore size of the filter used to filter the supernatant is 0.22-0.65 um, and the pH value of the filtered solution is adjusted to 6-7 using dihydrogen phosphate.

7. The method according to claim 1, characterized in that The equilibrium buffer in step (6) is a PB buffer with a pH value of 6 to 7 and a concentration of 10 to 20 mmol / L.

8. The method according to claim 1, characterized in that During the loading process of step (7), the temperature of the loading liquid and the effluent liquid is controlled at 4 to 20°C.

9. The method according to claim 1, characterized in that: In step (8), the concentration of NaSCN in the flushing liquid is 0.1-0.15 mol / L, the volume concentration of ethylene glycol is 4%-6%, and the concentration of NaCl is 0.04-0.06 mol / L; the linear velocity of the flushing liquid is 50-100 cm / h.

10. The method according to claim 2, characterized in that The buffer solution for dissolving the triazine dye ligand is any one or two of 0.1-0.15 mol / L NaHCO3 and 0.1-0.15 mol / L Na2CO3; the triazine dye is any one or two of red HE-3B triazine dye and yellow HE-4R triazine dye.

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