Purification method of high-purity label-free recombinant bovine lactoferrin

Abstract

The invention relates to a purification method of high-purity label-free recombinant bovine lactoferrin, belongs to the technical field of biochemistry, and solves the technical problems of industrial and large-scale preparation of label-free bovine lactoferrin with the purity of more than 95%, high safety and good activity. The invention relates to a purification method of high-purity label-free recombinant bovine lactoferrin. The purification method comprises the following steps: S1, expressing recombinant bovine lactoferrin; step S2, performing centrifugal treatment; step S3, carrying out resuspension treatment; step S4, performing high-pressure homogenization; step S5, performing centrifugal treatment; step S6, filtering treatment; s7, balancing the nickel column; step S8, loading a nickel column sample; s9, washing the nickel column for the first time; step S10, carrying out enzyme digestion on the tag; step S11, washing the nickel column for the second time; step S12, carrying out column balance by using a cation column; step S13, loading a cation column sample; step S14, washing the cation column; and step S15, cation column elution. The method provided by the invention provides an example for industrial and large-scale production of lactoferrin.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemistry, and particularly relates to a method for purifying high-purity label-free recombinant bovine lactoferrin. Background Art

[0002] Lactoferrin is a glycoprotein present in the exocrine secretions of mammals, which has the ability to chelate iron. Its biological properties include regulating iron adsorption in the gastrointestinal tract, regulating polymorphonuclear formation, and antibacterial and antifungal activities. Bovine lactoferrin has similar activities to lactoferrin. Therefore, lactoferrin has broad application prospects in the fields of biomedicine, health foods, cosmetics, etc.

[0003] At present, the production of lactoferrin at home and abroad is mainly extracted from bovine milk, with limited quantity, great difficulty in separation and high cost. With the development of gene editing technology, lactoferrin can be expressed from a variety of genetically engineered bacteria, which makes it possible for the industrial and large-scale production of lactoferrin. There are many studies on the separation and purification methods of recombinant lactoferrin, mainly including salting-out method, ultrafiltration method, chromatography method, etc. The main advantages of salting-out method and ultrafiltration method are that a large number of dairy samples can be processed in a short time, but the purity of the obtained lactoferrin is low, which can only be applied to industries with low requirements for the purity of lactoferrin; 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 is difficult to obtain a protein with a purity of not less than 95% in one-step purification, and it is only suitable for lactoferrin production with a small number of tags, low tag molecular weight, and no need to remove the tag. However, for use as medicines, special foods and health products, bovine lactoferrin with no tags, high safety and good activity is required. Therefore, finding a high-purity and tag-free recombinant protein purification method has become a technical problem that needs to be solved. Summary of the invention

[0004] In view of the above technical problems, the present invention provides a method for purifying high-purity tag-free recombinant bovine lactoferrin, which is used to solve the technical problems faced in the process of industrial and large-scale preparation of bovine lactoferrin with a purity exceeding 95%, no label, high safety and good activity.

[0005] The purpose of the present invention is mainly achieved through the following technical solutions:

[0006] The present invention provides a method for purifying high-purity label-free recombinant bovine lactoferrin, comprising the following steps:

[0007] Step S1, expression of recombinant bovine lactoferrin: the bovine lactoferrin gene with N-terminal fusion histidine tag gene and PAOX1 promoter are connected to the vector pPIC9K and then transformed into GS115 type Pichia pastoris, and the Pichia pastoris fermentation broth is obtained by high-density cell fermentation technology;

[0008] Step S2, centrifuging the Pichia pastoris fermentation broth to collect the Pichia pastoris containing the recombinant bovine lactoferrin;

[0009] Step S3, mixing the Pichia pastoris with a resuspension buffer to form a resuspension treatment solution, adding ethylenediaminetetraacetic acid to the resuspension treatment solution, and stirring to dissolve evenly; wherein the ratio of the amount of the Pichia pastoris to the resuspension buffer is 1 g: (10-30) ml, and the ratio of the amount of the resuspension buffer to the ethylenediaminetetraacetic acid is 1 liter: (1-2) mmol;

[0010] Step S4, high-pressure homogenizing the treated resuspended solution to obtain a high-pressure homogenized solution;

[0011] Step S5, centrifuging the high-pressure homogenized liquid to collect the supernatant containing the recombinant bovine lactoferrin;

[0012] Step S6, filtering the collected supernatant using a liquid filter with a pore size of 0.22 to 0.45 microns to obtain a filtrate containing recombinant bovine lactoferrin;

[0013] Step S7, preparing an equilibrium buffer, and then using 3 to 5 column volumes of the equilibrium buffer to balance the nickel column chromatography column, and controlling the linear velocity of the equilibrium buffer in the column to be 60 to 200 cm / h during column balancing;

[0014] Step S8, loading the filtrate in step S6 onto a nickel column chromatography column, controlling the linear velocity of the filtrate in the column to be 60-200 cm / h, and cyclically loading until the mass percentage of recombinant bovine lactoferrin in the nickel column loading effluent is less than 5%, and the loading is completed;

[0015] Step S9, using 3 to 5 column volumes of the nickel column equilibration buffer in step S7 to perform a first flush on the nickel column chromatography column, and controlling the linear velocity of the nickel column flushing liquid in the column to be 60 to 200 cm / h;

[0016] Step S10, loading the enzyme cleavage buffer onto a nickel column chromatography column, performing enzyme cleavage on the recombinant bovine lactoferrin on the column, controlling the linear velocity of the enzyme cleavage buffer in the column to be 150-200 cm / h, detecting the enzyme cleavage effluent at the same time, observing the UV280 curve, and starting to collect the enzyme cleavage effluent after the main peak of lactoferrin appears, and stopping the collection until the UV280 curve descends steadily;

[0017] Step S11, using 3 to 5 times the column volume of the nickel column equilibration buffer in step S7 to rinse the nickel column chromatography column for the second time; controlling the linear velocity of the rinsing buffer in the column to be 60 to 200 cm / h, detecting the rinsing effluent at the same time, observing the UV280 curve, and starting to collect the rinsing effluent after the main peak of lactoferrin appears, and stopping collecting until the UV280 curve descends steadily;

[0018] Step S12, using 3 to 5 column volumes of the equilibration buffer in step S7 to equilibrate the cationic chromatography column, and controlling the linear velocity of the elution buffer in the column to be 60 to 200 cm / h;

[0019] Step S13, loading the enzyme cleavage effluent and the flushing effluent collected in step S10 and step S11 onto a cationic chromatography column, controlling the linear velocity to be 60-200 cm / h, and cyclically loading until the mass percentage of recombinant bovine lactoferrin in the effluent loaded onto the cationic chromatography column is less than 5%, and the loading is completed;

[0020] Step S14, flushing the cation chromatography column with 3 to 5 column volumes of the nickel column equilibration buffer in step S7, and controlling the linear velocity of the cation column flushing liquid in the column to be 60 to 200 cm / h;

[0021] Step S15, prepare an elution buffer, use the elution buffer to flush the cationic chromatography column, control the linear velocity of the elution buffer in the column to be 60-200 cm / h, detect the elution effluent at the same time, observe the UV280 curve, and start collecting the flow-through protein solution after the main peak of lactoferrin appears, and stop collecting until the UV280 curve descends steadily.

[0022] Furthermore, in step S1, the histidine tag gene includes, in order of initial sequence, 6 to 10 consecutive histidine codons, and 4 consecutive aspartate codons and 1 lysine codon following the histidine codons.

[0023] Furthermore, in step S2, the centrifugal treatment is performed using a centrifuge at a rotation speed of 8000 to 12000 rpm for 20 to 30 minutes.

[0024] Furthermore, in step S3, the resuspension buffer is PB buffer, and the pH of the resuspension buffer at room temperature is pH 6.5 to pH 7.0.

[0025] Furthermore, in step S4, the high-pressure homogenization control pressure is 1000-1500 bar, and the high-pressure homogenization is performed 2-3 times.

[0026] Furthermore, in step S5, the centrifugal treatment is carried out at a temperature of 2 to 8°C, a centrifugal speed of 8000 to 12000 rpm, and a centrifugal treatment time of 20 to 30 min.

[0027] Furthermore, in step S7, the main body of the balancing buffer is PB buffer, and NaCl is added to the PB buffer at a concentration of 0.16-0.24 mol / L to prepare the balancing buffer, and the pH value of the balancing buffer at room temperature is pH 6.5-pH 7.0.

[0028] Furthermore, in step S10, the enzyme used for enzymatic cleavage is a recombinant bovine enterokinase light chain expressed by genetically engineered Escherichia coli, and the amount of enzyme used per milligram of recombinant bovine lactoferrin is 40-60U, wherein 1U is defined as the amount of enzyme required to cleave 95% of the recombinant bovine lactoferrin tag of 0.5mg stored in an enzyme cleavage buffer at 25°C within 12h to 16h; the enzyme cleavage buffer is a Tris-HCl buffer with a concentration of 20-30mM and a pH of 6.5-7.0.

[0029] Furthermore, in step S15, the main body of the elution buffer is PB buffer, and then NaCl is added to the PB buffer at a concentration of 0.8 to 1.2 mol / L to prepare the elution buffer, and the pH of the elution buffer at room temperature is pH 6.5 to pH 7.0.

[0030] Furthermore, after step S15, there are steps S16 and S17:

[0031] Step S16, sampling and testing: taking part of the flow-through protein solution collected in step S15, and testing the purity of the recombinant bovine lactoferrin according to the requirements of the national standard GB1903.17-2016;

[0032] Step S17, concentration and drying: the flow-through protein solution collected in step S15 is replaced, concentrated and then dried to obtain a recombinant bovine lactoferrin solid.

[0033] Compared with the prior art, the present invention can achieve at least one of the following technical effects:

[0034] (1) The present invention provides a method for purifying recombinant bovine lactoferrin from Pichia pastoris fermentation broth, which provides a good example for the industrial and large-scale production of lactoferrin.

[0035] (2) The present invention expresses recombinant bovine lactoferrin with a histidine tag at the N-terminus by Pichia pastoris, collects the recombinant bovine lactoferrin from the fermentation broth by using a nickel column chromatography column according to the affinity adsorption principle between the tag and a nickel column chromatography column, and then selects a specific enterokinase to remove the His tag on the nickel column according to the special protein sequence carried by the histidine tag; finally, according to the difference in isoelectric points of different proteins, cationic chromatography is used to further separate the recombinant bovine lactoferrin from impurities to achieve a better protein purification effect, and finally obtains recombinant bovine lactoferrin with a purity of more than 95%, which meets the requirements of the national standard GB1903.17-2016.

[0036] (3) The present invention designs the composition, concentration, pH value, etc. of each buffer according to the characteristics of recombinant bovine lactoferrin, and obtains the optimal result through experimental exploration.

[0037] (4) The flow rate in the nickel column chromatography column and the cationic chromatography column of the present invention is based on the inherent characteristics of the chromatography column filler and the characteristics of recombinant bovine lactoferrin, and the optimal speed range is explored through a large number of experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The drawings are only for the purpose of illustrating particular embodiments and are not to be considered limiting of the present invention. Like reference numerals refer to like components throughout the drawings.

[0039] Figure 1 The figure is a schematic diagram of the process flow of the method for purifying high-purity label-free recombinant bovine lactoferrin of the present invention;

[0040] Figure 2 SEC-HPLC chart of purity detection of recombinant bovine lactoferrin in Example 1 of the present invention;

[0041] Figure 3 SEC-HPLC chart of purity detection of recombinant bovine lactoferrin in Example 2 of the present invention;

[0042] Figure 4 SEC-HPLC chart of purity detection of recombinant bovine lactoferrin in Example 3 of the present invention;

[0043] In the figure, 1-lactoferrin is the main peak. DETAILED DESCRIPTION

[0044] A method for purifying high-purity tag-free recombinant bovine lactoferrin is further described in detail below in conjunction with specific examples. These examples are only for comparison and explanation purposes, and the present invention is not limited to these examples.

[0045] In the prior art, Pichia pastoris is widely used in protein expression, mainly in the fields of medicine, industry, and biology. Through the Pichia pastoris expression system, efficient expression and large-scale production of various important proteins can be achieved. The recombinant bovine lactoferrin purified by the present invention is bovine lactoferrin expressed by Pichia pastoris eukaryotically. In the plasmid preparation stage of the Pichia pastoris of the recombinant bovine lactoferrin of the present invention, in order to improve product expression and promote protein stability, a gene fragment containing multiple functions is selected as a tag protein. At the same time, in order to ensure safety, according to the characteristics of the tag protein, in the subsequent purification process of the recombinant bovine lactoferrin, an enzyme with a specific effect is applied to remove the tag on the recombinant bovine lactoferrin.

[0046] The present invention provides a method for purifying high-purity tag-free recombinant bovine lactoferrin expressed by Pichia pastoris, comprising the following steps:

[0047] Step S1, expression of recombinant bovine lactoferrin: the bovine lactoferrin gene with N-terminal fusion histidine tag gene and PAOX1 promoter are connected to the vector pPIC9K and then transformed into GS115 type Pichia pastoris, and the Pichia pastoris fermentation broth is obtained by high-density cell fermentation technology;

[0048] Step S2, centrifuging the Pichia pastoris fermentation broth to separate the supernatant and the Pichia pastoris precipitate, thereby collecting the Pichia pastoris containing the recombinant bovine lactoferrin;

[0049] Step S3, adding Pichia pastoris to a resuspension buffer at a ratio of 1 gram of Pichia pastoris to 10 to 30 milliliters of resuspension buffer to form a resuspension treatment solution, and then adding ethylenediaminetetraacetic acid to the resuspension treatment solution at a ratio of 1 liter of resuspension buffer to 1 to 2 mmol of ethylenediaminetetraacetic acid, and stirring to dissolve evenly;

[0050] Step S4, high-pressure homogenizing the resuspended solution after the treatment in step S3 to obtain a high-pressure homogenized solution;

[0051] Step S5, centrifuging the high-pressure homogenized liquid after the treatment in step S4, and collecting the supernatant in which the recombinant bovine lactoferrin is dissolved;

[0052] Step S6, filtering the supernatant collected in step S5 using a liquid filter with a pore size of 0.22 to 0.45 microns to obtain a filtrate containing recombinant bovine lactoferrin;

[0053] Step S7, preparing an equilibrium buffer, and then using 3 to 5 column volumes of the equilibrium buffer to balance the nickel column chromatography column;

[0054] Step S8, loading the filtrate in step S6 onto a nickel column chromatography column, controlling the linear velocity of the filtrate in the column to be 60-200 cm / h, and cyclically loading until the mass percentage of recombinant bovine lactoferrin in the nickel column loading effluent is less than 5%, and the loading is completed;

[0055] Step S9, using 3 to 5 column volumes of the nickel column equilibration buffer in step S7 to perform a first flush on the nickel column chromatography column;

[0056] Step S10, loading the enzyme cleavage buffer onto a nickel column chromatography column, performing enzyme cleavage on the recombinant bovine lactoferrin on the column, controlling the linear velocity of the enzyme cleavage buffer in the column to be 150-200 cm / h, detecting the enzyme cleavage effluent at the same time, observing the UV280 curve, and starting to collect the enzyme cleavage effluent after the main peak of lactoferrin appears, and stopping the collection until the UV280 curve descends steadily;

[0057] Step S11, using 3 to 5 times the column volume of the nickel column equilibration buffer in step S7 to rinse the nickel column chromatography column for the second time; controlling the linear velocity of the rinsing buffer in the column to be 60 to 200 cm / h, detecting the rinsing effluent at the same time, observing the UV280 curve, and starting to collect the rinsing effluent after the main peak of lactoferrin appears, and stopping collecting until the UV280 curve descends steadily;

[0058] Step S12, using 3 to 5 column volumes of the equilibration buffer in step S7 to equilibrate the cationic chromatography column;

[0059] Step S13, loading the enzyme cleavage effluent and the flushing effluent collected in step S10 and step S11 onto a cationic chromatography column, controlling the linear velocity to be 60-200 cm / h, and cyclically loading until the mass percentage of recombinant bovine lactoferrin in the effluent loaded onto the cationic chromatography column is less than 5%, and the loading is completed;

[0060] Step S14, flushing the cation chromatography column with 3 to 5 column volumes of the nickel column equilibration buffer in step S7;

[0061] Step S15, preparing an elution buffer, using the elution buffer to rinse the cationic chromatography column, detecting the elution effluent, observing the UV280 curve, and starting to collect the protein solution after the main peak of lactoferrin appears, and stopping the collection until the UV280 curve descends steadily;

[0062] Step S16, sampling and testing: taking part of the flow-through protein solution collected in step S15, and testing the purity of the recombinant bovine lactoferrin according to the requirements of the national standard GB1903.17-2016;

[0063] Step S17, concentration and drying: the flow-through protein solution collected in step S15 is replaced, concentrated and then dried to obtain a recombinant bovine lactoferrin solid.

[0064] Specifically, in step S1, a histidine tag, i.e., a His tag, is set at the N-terminus of the recombinant bovine lactoferrin. One advantage of setting the His tag at the N-terminus of the recombinant bovine lactoferrin is that the structure can utilize an effective translation start site on the tag, and another advantage is that the His tag can be removed more cleanly. The histidine tag gene includes 6 to 10 consecutive histidine codons in the order of starting, and 4 consecutive aspartic acid codons and 1 lysine codon located after the histidine codon, and the bovine lactoferrin gene is after the histidine tag gene. The bovine lactoferrin gene fused with the histidine tag gene, the PAOX1 promoter and the vector pPIC9K are connected and transformed into the GS115 type Pichia pastoris, and high-density cell fermentation technology is used to achieve industrial mass production, and a large amount of Pichia pastoris fermentation broth containing recombinant bovine lactoferrin with the same activity as human lactoferrin is obtained.

[0065] The gene sequence of the recombinant bovine lactoferrin with a histidine tag at the N-terminus is shown in SEQ ID NO.1, and the amino acid sequence of the recombinant bovine lactoferrin with a histidine tag at the N-terminus is shown in SEQ ID NO.2.

[0066] It should be noted that in step S1, the Pichia yeast fermentation broth contains, in addition to recombinant bovine lactoferrin, other substances such as pigments, miscellaneous proteins, nucleic acids, etc.

[0067] Specifically, in step S2, the Pichia yeast fermentation broth is centrifuged at a speed of 8000 to 12000 rpm for 20 to 30 minutes using a centrifuge to separate the clear liquid from the Pichia yeast precipitate. The clear liquid contains Pichia yeast metabolism, waste dissolved in the clear liquid and residual culture medium, and finally the Pichia yeast containing recombinant bovine lactoferrin is collected; the centrifuge can be a sedimentation type or a filtration type centrifuge.

[0068] Specifically, in step S3, the resuspension buffer is a PB buffer, i.e., a phosphate buffer, and a 20 mM NaH2PO4 solution and a 20 mM Na2HPO4 solution are mixed, or a 20 mM KH2PO4 solution and a 20 mM K2HPO4 solution are mixed to prepare a resuspension buffer, and the pH of the resuspension buffer at room temperature is pH 6.5 to pH 7.0. The purpose of adding ethylenediaminetetraacetic acid to the resuspension treatment solution is to prevent the decomposition and inactivation of recombinant bovine lactoferrin.

[0069] Specifically, in step S4, the high-pressure homogenization control pressure is 1000-1500 bar, and the high-pressure homogenization is performed 2-3 times. The high-pressure homogenization causes the Pichia pastoris containing recombinant bovine lactoferrin in a resuspended state to be broken under the action of ultra-high pressure, thereby finally achieving a homogenization effect.

[0070] Specifically, in step S5, when the high-pressure homogenized liquid containing the recombinant bovine lactoferrin precipitate is centrifuged, the temperature is controlled at 2-8°C, the centrifugal speed is 8000-12000rpm, the centrifugation is performed for 20-30min, and the centrifuge can be a sedimentation type or a filtration type centrifuge. The centrifugation separates the insoluble cell wall precipitate in the high-pressure homogenized liquid, thereby obtaining a supernatant in which the recombinant bovine lactoferrin is dissolved.

[0071] Specifically, in step S6, the filtration treatment removes the solid matter with larger particle size that is insoluble in the supernatant, so as to prepare the material for the next step of nickel column chromatography.

[0072] Specifically, in step S7, the nickel column chromatography column filler is Ni-NTA agarose purification resin, the filler particle size is 40 to 200 microns, and the column volume is the filler volume; the main body of the equilibration buffer is PB buffer, that is, phosphate buffer, and a 20mM NaH2PO4 solution and a 20mM Na2HPO4 solution are mixed, or a 20mM KH2PO4 solution and a 20mM K2HPO4 solution are mixed to prepare a PB buffer; then NaCl is added to the PB buffer at a concentration of 0.16 to 0.24 mol / L to prepare an equilibration buffer, and the pH of the equilibration buffer at room temperature is pH6.5 to pH7.0. When the column is equilibrated, the linear velocity of the equilibration buffer in the column is controlled to be 60 to 200 cm / h.

[0073] It should be noted that in step S8, the histidine residue carries an imidazole group, which can react with Ni 2+ ions form coordination bonds and selectively bind to metal ions. 2+ Ions can be fixed on the chromatography medium agarose purification resin using the chelating ligand NTA, so recombinant bovine lactoferrin with his tag can be assembled with Ni 2+ Ions can be selectively bound to the agarose purification resin, while other impurity proteins cannot bind or can only weakly bind. In addition, according to the inventor's experimental exploration and summary, it is found that 1L of Ni-NTA agarose purification resin filler can purify 20-40g of recombinant bovine lactoferrin. Therefore, through step S8, recombinant bovine lactoferrin can be accurately and quickly initially extracted onto a nickel column chromatography column, thereby achieving preliminary purification of recombinant bovine lactoferrin.

[0074] In addition, the mass percentage of recombinant bovine lactoferrin in the nickel column effluent is detected by liquid chromatography, such as size exclusion chromatography (SEC-HPLC), reverse phase high performance liquid chromatography (RP-HPLC), and ion exchange chromatography (IEC).

[0075] Specifically, in step S9, during flushing, the linear velocity of the nickel column flushing liquid in the column is controlled to be 60-200 cm / h, and the column volume is the filler volume of the nickel column chromatography column. Through this step, impurity proteins that are not bound to the nickel column chromatography column are flushed out of the nickel column chromatography column.

[0076] Specifically, in step S10, the enzyme is a recombinant bovine enterokinase light chain expressed by Escherichia coli cultured by genetic engineering; the enzyme cleavage buffer is a Tris-HCl buffer with a concentration of 20 to 30 mM and a pH of 6.5 to 7.0; the enzyme activity unit 1U in the present invention is defined as the amount of enzyme required to cut 95% of the recombinant bovine lactoferrin tag of 0.5 mg stored in the above enzyme cleavage buffer within 12h to 16h at 25°C, and the amount of enzyme used per milligram of recombinant bovine lactoferrin in the present invention is 40 to 60U.

[0077] It should be noted that enterokinase is a specific protease, and the recognition site of enterokinase is also a cleavage site, and its recognition sequence is aspartic acid-aspartic acid-aspartic acid-aspartic acid-lysine, and the cleavage site is located after lysine, but the site after the recognition sequence is not cut, which is proline. The present invention sets a histidine tag at the N-terminus of recombinant bovine lactoferrin, and after protein expression, the end of the histidine tag protein is aspartic acid (D)-aspartic acid (D)-aspartic acid (D)-aspartic acid (D)-lysine (K), that is, DDDDK, and the recombinant bovine lactoferrin is after the histidine tag, so that the site recognized and cleaved by enterokinase is located just between the His tag and the recombinant bovine lactoferrin protein, and from the sequence of the recombinant bovine lactoferrin, it can be seen that there is no DDDDK enzyme cleavage site inside the recombinant bovine lactoferrin, so that the His tag at the N-terminus of the recombinant bovine lactoferrin is cut off by enterokinase.

[0078] Specifically, in step S11, during flushing, the linear velocity of the nickel column flushing liquid in the column is controlled to be 60-200 cm / h, and the column volume is the filler volume of the nickel column chromatography column. This step is used to flush the recombinant bovine lactoferrin remaining on the nickel column chromatography column.

[0079] It should be noted that the nickel column chromatography column is connected to the protein chromatography system, and the protein chromatography system can be used to control the nickel column equilibration, nickel column loading, nickel column flushing and enzyme label cleavage in steps S7 to S11, such as controlling the column midline velocity and detecting the effluent.

[0080] Specifically, in step S12, the filler of the cationic chromatography column is SP agarose gel FF, which is a strong cation exchange medium formed by bonding sulfopropyl groups to high-flow agarose gel, with a particle size of 70 to 120 microns, and the column volume is the filler volume. When the column is balanced, the linear velocity of the elution buffer in the column is controlled to be 60 to 200 cm / h.

[0081] It should be noted that in step S13, the isoelectric point of lactoferrin is 8.7, and the pH values ​​of step S10 enzyme digestion buffer and step S11 flushing solution are 6.5-7.0, so recombinant bovine lactoferrin is positively charged in solution, and the ion exchange resin of the cationic chromatography column is negatively charged, so when the collected enzyme digestion effluent and flushing effluent pass through the cationic chromatography exchange column, they will be attracted by the negatively charged resin and adsorbed onto the column, while other uncharged or negatively charged impurity proteins etc. flow through the cationic column. In addition, according to the groping summary of the inventor's experiment, it is found that 1L of SP agarose gel FF filler can purify 20-40g of recombinant bovine lactoferrin. The purity of recombinant bovine lactoferrin is further improved by this step to make it meet the requirements of national standard GB1903.17-2016.

[0082] Specifically, in step S14, during flushing, the linear velocity of the cationic column flushing liquid in the column is controlled to be 60-200 cm / h, and the column volume is the filler volume of the cationic chromatography column. Through this step, impurity proteins that are not bound to the cationic chromatography column and are retained are flushed out of the cationic chromatography column.

[0083] Specifically, in step S15, the main body of the elution buffer is PB buffer, i.e., phosphate buffer, and a 20mM NaH2PO4 solution and a 20mM Na2HPO4 solution are mixed, or a 20mM KH2PO4 solution and a 20mM K2HPO4 solution are mixed to prepare a PB buffer; then, NaCl is added to the PB buffer at a concentration of 0.8 to 1.2 mol / L to prepare an elution buffer, and the pH of the elution buffer at room temperature is pH 6.5 to pH 7.0. During elution, the linear velocity of the elution buffer in the column is controlled to be 60 to 200 cm / h.

[0084] It should be noted that the cationic chromatography column is connected to the protein chromatography system, and the protein chromatography system can be used to control the cation column equilibration, cation column loading, cation column flushing and cation column elution in steps S12 to S15, such as controlling the column midline velocity, detecting the effluent, etc.

[0085] Specifically, in step S16, the national standard GB1903.17-2016 requires that the food nutrient enhancer lactoferrin accounts for a percentage of total protein mass (w / %) ≥ 95%, and the purity detection method is high performance liquid chromatography. The purity detection also reflects the effect of label removal.

[0086] Specifically, in step S17, the flow-through protein solution of step S15 is first ultrafiltered and concentrated to 1 / 5 of the initial total volume, and then the replacement fluid is added in batches to be ultrafiltered and concentrated to 1 / 5 of the initial total volume to obtain a high-concentration desalted recombinant bovine lactoferrin solution, and the total volume of the replacement fluid added in batches is equal to the initial total volume of the flow-through protein solution.

[0087] The main body of the replacement fluid is PB buffer, i.e., phosphate buffer. A 20mM NaH2PO4 solution and a 20mM Na2HPO4 solution are mixed, or a 20mM KH2PO4 solution and a 20mM K2HPO4 solution are mixed to prepare a PB buffer; then ethylenediaminetetraacetic acid and TWEEN80 are added to the PB buffer at a concentration of 0.08 to 0.12mM, respectively, to prepare the replacement fluid. The pH value of the replacement fluid at room temperature is pH 6.5 to pH 7.0. TWEEN80 is polyoxyethylene ether of sorbitan monooleate, referred to as emulsifier T-80.

[0088] Subsequently, the high-concentration desalted recombinant bovine lactoferrin solution is spray-dried or freeze-dried to obtain protein solids.

[0089] Figure 1 The figure is a schematic diagram of the process flow of the method for purifying high-purity label-free recombinant bovine lactoferrin of the present invention.

[0090] The present invention expresses recombinant bovine lactoferrin with a histidine tag at the N-terminus by Pichia yeast, collects recombinant bovine lactoferrin from fermentation broth by using a nickel column chromatography column according to the affinity adsorption principle of the His tag and a nickel column chromatography column, and then selects a specific enterokinase to remove the His tag on the nickel column according to the special protein sequence carried by the histidine tag; finally, according to the difference in isoelectric points of different proteins, a cation chromatography is used to further separate the recombinant bovine lactoferrin from impurities to achieve a better protein purification effect. In addition, according to the characteristics of the recombinant bovine lactoferrin, the present invention designs the component composition, concentration, pH value, etc. of each buffer, and obtains the optimal result through experimental exploration; wherein, the flow rate in the column of the nickel column chromatography column and the cation chromatography column is based on the inherent characteristics of the chromatography column filler, combined with the characteristics of the recombinant bovine lactoferrin, and the optimal speed range is explored through a large number of experiments, and finally the recombinant bovine lactoferrin with a purity of more than 95% is obtained.

[0091] Example 1

[0092] A method for purifying high-purity label-free recombinant bovine lactoferrin comprises the following steps:

[0093] Step S1, expression of recombinant bovine lactoferrin: the bovine lactoferrin gene with N-terminal fusion histidine tag gene and PAOX1 promoter are connected to the vector pPIC9K and then transformed into GS115 type Pichia pastoris, and the Pichia pastoris fermentation broth is obtained by high-density cell fermentation technology;

[0094] The histidine tag gene includes, in order of initial sequence, 6 consecutive histidine codons, and 4 consecutive aspartic acid codons and 1 lysine codon after the histidine codons. The bovine lactoferrin gene is located after the histidine tag gene.

[0095] Step S2, centrifuging the Pichia pastoris fermentation broth to separate the clear liquid and the Pichia pastoris precipitate, thereby collecting 2 g of Pichia pastoris containing recombinant bovine lactoferrin;

[0096] The Pichia yeast fermentation liquid is centrifuged for 20 minutes at a rotation speed of 8000 rpm, and the centrifuge is a sedimentation centrifuge.

[0097] Step S3, 2 grams of Pichia pastoris are prepared with 20 ml of resuspension buffer, the Pichia pastoris is added to the resuspension buffer to form a resuspension treatment solution, and then ethylenediaminetetraacetic acid is added to a final concentration of 1 mmol / L, and stirred to dissolve evenly;

[0098] The resuspension buffer is PB buffer, and a 20 mM NaH2PO4 solution and a 20 mM Na2HPO4 solution are mixed to prepare the resuspension buffer, wherein the volume of the NaH2PO4 solution accounts for 68.5%, the volume of the Na2HPO4 solution accounts for 31.5%, and the pH value of the resuspension buffer at room temperature is pH 6.5.

[0099] Step S4, high-pressure homogenizing the resuspended solution after the treatment in step S3 to obtain a high-pressure homogenized solution;

[0100] The high-pressure homogenization control pressure is 1000 bar, and the high-pressure homogenization is performed twice.

[0101] Step S5, centrifuging the high-pressure homogenized liquid after the treatment in step S4, and collecting the supernatant in which the recombinant bovine lactoferrin is dissolved;

[0102] During the centrifugal treatment, the temperature was controlled at 2°C, the centrifugal speed was 8000 rpm, the centrifugal treatment was performed for 20 minutes, and a decanter centrifuge was used.

[0103] Step S6, filtering the supernatant collected in step S5 using a liquid filter with a pore size of 0.22 to 0.45 microns to obtain a filtrate containing recombinant bovine lactoferrin;

[0104] Step S7, preparing an equilibration buffer, and then using 3 column volumes, i.e. 15 ml of the equilibration buffer, to equilibrate the nickel column chromatography column;

[0105] The nickel column chromatography column filler is Ni-NTA agarose purification resin, the filler particle size is 40 to 200 microns, and the filler volume is 5 ml; the main body of the equilibration buffer is PB buffer, and a 20mM NaH2PO4 solution and a 20mM Na2HPO4 solution are mixed to prepare the PB buffer, wherein the volume of the NaH2PO4 solution accounts for 68.5%, and the volume of the Na2HPO4 solution accounts for 31.5%, and then NaCl is added to the PB buffer at a concentration of 0.16 mol / L to prepare the equilibration buffer, and the pH value of the equilibration buffer at room temperature is pH 6.5. When the column is equilibrated, the linear velocity of the equilibration buffer in the column is controlled to be 60 cm / h.

[0106] Step S8, loading the filtrate in step S6 onto a nickel column chromatography column, controlling the linear velocity of the filtrate in the column to be 60 cm / h, and cyclically loading the sample twice until the mass percentage of recombinant bovine lactoferrin in the nickel column loading effluent is 4.2%, and the loading is completed;

[0107] Step S9, prepare a nickel column flushing buffer, and use 3 times the column volume, i.e. 15 ml of the nickel column equilibration buffer in step S7 to flush the nickel column chromatography column for the first time; during flushing, control the linear velocity of the nickel column flushing buffer in the column to be 60 cm / h.

[0108] Step S10, loading the enzyme cleavage buffer onto a nickel column chromatography column, performing enzyme cleavage on the recombinant bovine lactoferrin on the column, controlling the linear velocity of the enzyme cleavage buffer in the column to be 150 cm / h, detecting the enzyme cleavage effluent at the same time, observing the UV280 curve, and starting to collect the enzyme cleavage effluent after the main peak of lactoferrin appears, and stopping the collection until the UV280 curve descends steadily, and a total of 9.6 ml is collected;

[0109] The enzyme is a recombinant bovine enterokinase light chain expressed by genetically engineered Escherichia coli; the enzyme cleavage buffer is a 20 mM Tris-HCl buffer with a pH of 6.5, and the amount of enzyme used per milligram of recombinant bovine lactoferrin is 40 U.

[0110] Step S11, using 3 times column volume, i.e. 15 ml of the nickel column equilibration buffer in step S7, to rinse the nickel column chromatography column for the second time; controlling the linear velocity of the rinsing buffer in the column to be 60 cm / h, detecting the rinsing effluent at the same time, observing the UV280 curve, and starting to collect the rinsing effluent after the main peak of lactoferrin appears, and stopping collecting until the UV280 curve descends steadily, and a total of 10.2 ml is collected;

[0111] Step S12, using 3 column volumes, i.e. 15 ml, of the equilibration buffer in step S7 to equilibrate the cationic chromatography column;

[0112] The filler of the cationic chromatography column is SP agarose gel FF, with a particle size of 70 to 120 microns and a filler volume of 5 ml.

[0113] Step S13, loading the enzyme digestion effluent and the flushing effluent collected in step S10 and step S11 onto a cationic chromatography column, controlling the linear velocity to be 60 cm / h, and cyclically loading until the mass percentage of recombinant bovine lactoferrin in the effluent loaded onto the cationic chromatography column is 4.2%, and the loading is completed;

[0114] Step S14, washing the cation chromatography column with 3 column volumes, i.e., 15 ml of the nickel column equilibration buffer in step S7;

[0115] Step S15, prepare an elution buffer, use the elution buffer to rinse the cationic chromatography column, detect the elution effluent, observe the UV280 curve, and start collecting the flow-through protein solution after the main peak of lactoferrin appears, and stop collecting until the UV280 curve descends steadily, and a total of 28.6 ml is collected;

[0116] The main body of the elution buffer is PB buffer, and a 20mM NaH2PO4 solution and a 20mM Na2HPO4 solution are mixed to prepare the PB buffer, wherein the volume of the NaH2PO4 solution accounts for 68.5%, and the volume of the Na2HPO4 solution accounts for 31.5%, and then NaCl is added to the PB buffer at a concentration of 0.8mol / L to prepare the elution buffer, and the pH value of the elution buffer at room temperature is pH 6.5. During elution, the linear velocity of the elution buffer in the column is controlled to be 60cm / h.

[0117] Step S16, sampling and testing: taking part of the flow-through protein solution, and using size exclusion chromatography (SEC-HPLC) to test the purity of recombinant bovine lactoferrin according to the requirements of national standard GB1903.17-2016, the purity is 97.3%; Figure 2 This is a SEC-HPLC chart of the purity detection of recombinant bovine lactoferrin in Example 1, wherein reference numeral 1 is the main peak of lactoferrin.

[0118] Step S17, concentration and drying: the flow-through protein solution collected in step S15 is replaced, concentrated and then dried to obtain a recombinant bovine lactoferrin solid.

[0119] First, the protein solution was ultrafiltered and concentrated to 1 / 5 of the initial total volume, i.e., 5.72 ml. Then, the replacement fluid was added five times and ultrafiltered and concentrated to 1 / 5 of the initial total volume, i.e., 5.72 ml, to obtain a high-concentration desalted recombinant bovine lactoferrin solution. The volume of the replacement fluid added each time was 5.72 ml.

[0120] The main body of the replacement fluid is PB buffer. PB buffer is prepared by mixing 20mM NaH2PO4 solution and 20mM Na2HPO4 solution, in which the volume of NaH2PO4 solution accounts for 68.5% and the volume of Na2HPO4 solution accounts for 31.5%. Then, ethylenediaminetetraacetic acid and TWEEN80 are added to the PB buffer at a concentration of 0.08mM respectively to prepare the replacement fluid. The pH value of the replacement fluid is pH 6.5 at room temperature.

[0121] Subsequently, the high-concentration desalted recombinant bovine lactoferrin solution was spray-dried to obtain 0.18 g of protein solid.

[0122] Example 2

[0123] A method for purifying high-purity label-free recombinant bovine lactoferrin comprises the following steps:

[0124] Step S1, expression of recombinant bovine lactoferrin: the bovine lactoferrin gene with N-terminal fusion histidine tag gene and PAOX1 promoter are connected to the vector pPIC9K and then transformed into GS115 type Pichia pastoris, and the Pichia pastoris fermentation broth is obtained by high-density cell fermentation technology;

[0125] The histidine tag gene includes, in order of initial sequence, 6 consecutive histidine codons, and 4 consecutive aspartic acid codons and 1 lysine codon after the histidine codons. The bovine lactoferrin gene is located after the histidine tag gene.

[0126] Step S2, centrifuging the Pichia pastoris fermentation broth to separate the clear liquid and the Pichia pastoris precipitate, thereby collecting Pichia pastoris containing recombinant bovine lactoferrin, taking 16 g;

[0127] The Pichia yeast fermentation broth is centrifuged at a speed of 10,000 rpm for 25 minutes to separate the clear liquid from the Pichia yeast precipitate, and finally the Pichia yeast containing recombinant bovine lactoferrin is collected; the centrifuge is a sedimentation type or a filtration type centrifuge.

[0128] Step S3, 16 grams of Pichia pastoris are prepared with 320 ml of resuspension buffer, the Pichia pastoris is added to the resuspension buffer to form a resuspension treatment solution, and then ethylenediaminetetraacetic acid is added to a final concentration of 1.5 mmol / L, and stirred to dissolve evenly;

[0129] The resuspension buffer is PB buffer, i.e., phosphate buffer, and a 20 mM NaH2PO4 solution and a 20 mM Na2HPO4 solution are mixed to prepare the resuspension buffer, wherein the volume of the NaH2PO4 solution accounts for 53.8%, the volume of the Na2HPO4 solution accounts for 46.2%, and the pH value of the resuspension buffer at room temperature is pH 6.75.

[0130] Step S4, high-pressure homogenizing the resuspended solution after the treatment in step S3 to obtain a high-pressure homogenized solution;

[0131] The high-pressure homogenization control pressure is 1250 bar, and the high-pressure homogenization is performed 3 times.

[0132] Step S5, centrifuging the high-pressure homogenized liquid after the treatment in step S4, and collecting the supernatant in which the recombinant bovine lactoferrin is dissolved;

[0133] During the centrifugal treatment, the temperature was controlled at 5°C, the centrifugal speed was 10000 rpm, the centrifugal treatment was 25 minutes, and the centrifuge used was a filter centrifuge.

[0134] Step S6, filtering the supernatant collected in step S5 using a liquid filter with a pore size of 0.22 to 0.45 microns to obtain a filtrate containing recombinant bovine lactoferrin;

[0135] Step S7, preparing an equilibration buffer, and then using 4 column volumes, i.e. 160 ml of the equilibration buffer, to equilibrate the nickel column chromatography column;

[0136] The nickel column chromatography column filler is Ni-NTA agarose purification resin, the filler particle size is 40 to 200 microns, and the filler volume is 40 ml; the main body of the equilibration buffer is PB buffer, and a 20 mM NaH2PO4 solution and a 20 mM Na2HPO4 solution are mixed to prepare the PB buffer, wherein the volume of the NaH2PO4 solution accounts for 53.8%, and the volume of the Na2HPO4 solution accounts for 46.2%, and then NaCl is added to the PB buffer at a concentration of 0.20 mol / L to prepare the equilibration buffer, and the pH value of the equilibration buffer at room temperature is pH 6.75. When the column is equilibrated, the outflow linear velocity of the equilibration buffer is controlled to be 130 cm / h.

[0137] Step S8, loading the filtrate in step S6 onto a nickel column chromatography column, controlling the linear velocity of the filtrate in the column to be 130 cm / h, and cyclically loading the sample twice until the mass percentage of recombinant bovine lactoferrin in the nickel column loading effluent is 4.7%, and the loading is completed;

[0138] Step S9, prepare a nickel column flushing buffer, and use 4 times the column volume, i.e. 160 ml of the nickel column equilibration buffer in step S7 to flush the nickel column chromatography column for the first time; during flushing, control the linear velocity of the nickel column flushing buffer in the column to be 130 cm / h.

[0139] Step S10, loading the enzyme cleavage buffer onto a nickel column chromatography column, performing enzyme cleavage on the recombinant bovine lactoferrin on the column, controlling the linear velocity of the enzyme cleavage buffer in the column to be 175 cm / h, detecting the enzyme cleavage effluent at the same time, observing the UV280 curve, and starting to collect the enzyme cleavage effluent after the main peak of lactoferrin appears, and stopping the collection until the UV280 curve descends steadily, and a total of 88.6 ml is collected;

[0140] The enzyme is a recombinant bovine enterokinase light chain expressed by genetically engineered Escherichia coli; the enzyme cleavage buffer is a 25 mM Tris-HCl buffer with a pH of 6.75, and the amount of enzyme used per milligram of recombinant bovine lactoferrin is 50 U.

[0141] Step S11, using 4 times column volume, i.e. 160 ml of the nickel column equilibration buffer in step S7, to rinse the nickel column chromatography column for the second time; controlling the linear velocity of the rinsing buffer in the column to be 130 cm / h, detecting the rinsing effluent at the same time, observing the UV280 curve, and starting to collect the rinsing effluent after the main peak of lactoferrin appears, and stopping collecting until the UV280 curve descends steadily, and a total of 113.6 ml is collected;

[0142] Step S12, using 4 column volumes, i.e. 160 ml, of the equilibration buffer in step S7 to equilibrate the cation chromatography column;

[0143] The filler of the cationic chromatography column is SP agarose gel FF, the particle size is 70 to 120 microns, and the filler volume is 40 ml.

[0144] Step S13, loading the digestion effluent and the flushing effluent collected in step S10 and step S11 onto a cationic chromatography column, controlling the linear velocity to be 130 cm / h, and cyclically loading until the mass percentage of recombinant bovine lactoferrin in the effluent loaded onto the cationic chromatography column is 4.7%, and the loading is completed;

[0145] Step S14, flushing the cation chromatography column with 4 column volumes, i.e., 160 ml of the nickel column equilibration buffer in step S7;

[0146] Step S15, prepare elution buffer, use elution buffer to rinse the cationic chromatography column, detect the elution effluent at the same time, observe the UV280 curve, and start collecting the flow-through protein solution after the main peak of lactoferrin appears, and stop collecting until the UV280 curve descends steadily, and a total of 222.5 ml is collected;

[0147] The main body of the elution buffer is PB buffer, and a 20mM NaH2PO4 solution and a 20mM Na2HPO4 solution are mixed to prepare the PB buffer, wherein the volume of the NaH2PO4 solution accounts for 53.8%, and the volume of the Na2HPO4 solution accounts for 46.2%. Then, NaCl is added to the PB buffer at a concentration of 1.0 mol / L to prepare the elution buffer, and the pH of the elution buffer at room temperature is pH 6.75. During elution, the linear velocity of the elution buffer in the column is controlled to be 130 cm / h.

[0148] Step S16, sampling and testing: taking part of the flow-through protein solution, and using size exclusion chromatography (SEC-HPLC) to test the purity of recombinant bovine lactoferrin to be 98.3% in accordance with the requirements of national standard GB1903.17-2016; Figure 3 This is a SEC-HPLC chart of the purity detection of recombinant bovine lactoferrin in Example 2, wherein reference numeral 1 in the figure indicates the main peak of lactoferrin.

[0149] Step S17, concentration and drying: the flow-through protein solution collected in step S15 is replaced, concentrated and then dried to obtain a recombinant bovine lactoferrin solid.

[0150] First, the protein solution was ultrafiltered and concentrated to 1 / 5 of the initial total volume, i.e., 44.5 ml. Then, the replacement fluid was added in five times and ultrafiltered and concentrated to 1 / 5 of the initial total volume, i.e., 44.5 ml, to obtain a high-concentration desalted recombinant bovine lactoferrin solution. The volume of the replacement fluid added each time was 44.5 ml.

[0151] The main body of the replacement fluid is PB buffer. PB buffer is prepared by mixing 20mM NaH2PO4 solution and 20mM Na2HPO4 solution, in which the volume of NaH2PO4 solution accounts for 53.8% and the volume of Na2HPO4 solution accounts for 46.2%. Then, ethylenediaminetetraacetic acid and TWEEN80 are added to the PB buffer at a concentration of 0.10mM respectively to prepare the replacement fluid. The pH value of the replacement fluid is pH6.75 at room temperature.

[0152] Subsequently, the high-concentration desalted recombinant bovine lactoferrin solution was freeze-dried to obtain 1.5 g of protein solid.

[0153] Example 3

[0154] A method for purifying high-purity label-free recombinant bovine lactoferrin comprises the following steps:

[0155] Step S1, expression of recombinant bovine lactoferrin: the bovine lactoferrin gene with N-terminal fusion histidine tag gene and PAOX1 promoter are connected to the vector pPIC9K and then transformed into GS115 type Pichia pastoris, and the Pichia pastoris fermentation broth is obtained by high-density cell fermentation technology;

[0156] The histidine tag gene includes, in order of initial sequence, 6 consecutive histidine codons, and 4 consecutive aspartic acid codons and 1 lysine codon after the histidine codons. The bovine lactoferrin gene is located after the histidine tag gene.

[0157] Step S2, centrifuging the Pichia pastoris fermentation broth to separate the supernatant and the Pichia pastoris precipitate, thereby collecting 400 g of the Pichia pastoris containing recombinant bovine lactoferrin;

[0158] The Pichia yeast fermentation broth was centrifuged at 12000 rpm for 30 minutes to separate the clear liquid from the Pichia yeast precipitate, and finally the Pichia yeast containing recombinant bovine lactoferrin was collected; the centrifuge used was a sedimentation centrifuge.

[0159] Step S3, adding Pichia to the resuspension buffer at a ratio of 400 g of Pichia to 12 L of resuspension buffer to form a resuspension treatment solution, then adding ethylenediaminetetraacetic acid to a final concentration of 2 mmol / L, and stirring to dissolve evenly;

[0160] The resuspension buffer is PB buffer, i.e., phosphate buffer, and a 20 mM KH2PO4 solution and a 20 mM K2HPO4 solution are mixed to prepare the resuspension buffer, wherein the volume of the KH2PO4 solution accounts for 39%, the volume of the K2HPO4 solution accounts for 61%, and the pH value of the resuspension buffer at room temperature is pH 7.0.

[0161] Step S4, high-pressure homogenizing the resuspended solution after the treatment in step S3 to obtain a high-pressure homogenized solution;

[0162] The high-pressure homogenization control pressure is 1500 bar, and the high-pressure homogenization is performed twice.

[0163] Step S5, centrifuging the high-pressure homogenized liquid after the treatment in step S4, and collecting the supernatant in which the recombinant bovine lactoferrin is dissolved;

[0164] During the centrifugal treatment, the temperature is controlled at 8°C, the centrifugal speed is 12000rpm, the centrifugal treatment is carried out for 30 minutes, and the centrifuge can be a filter centrifuge.

[0165] Step S6, filtering the supernatant collected in step S5 using a liquid filter with a pore size of 0.22 to 0.45 microns to obtain a filtrate containing recombinant bovine lactoferrin;

[0166] Step S7, preparing an equilibration buffer, and then using 5 column volumes, i.e. 5 L of the equilibration buffer, to equilibrate the nickel column chromatography column;

[0167] The nickel column chromatography column filler is Ni-NTA agarose purification resin, the filler particle size is 40 to 200 microns, and the filler volume is 1L; the main body of the equilibrium buffer is PB buffer, and a 20mM KH2PO4 solution and a 20mM K2HPO4 solution are mixed to prepare the PB buffer, wherein the volume of the KH2PO4 solution accounts for 39%, and the volume of the K2HPO4 solution accounts for 61%, and then NaCl is added to the PB buffer at a concentration of 0.24mol / L to prepare the equilibrium buffer, and the pH of the equilibrium buffer at room temperature is pH7.0. When the column is balanced, the linear velocity of the equilibrium buffer outflow is controlled to be 200cm / h.

[0168] Step S8, loading the filtrate in step S6 onto a nickel column chromatography column, controlling the linear velocity of the filtrate in the column to be 200 cm / h, and cyclically loading the sample twice until the mass percentage of recombinant bovine lactoferrin in the nickel column loading effluent is 3.5%, until the loading is completed;

[0169] Step S9, prepare a nickel column flushing buffer, and use 5 times the column volume, i.e. 5 L of the nickel column equilibration buffer in step S7, to flush the nickel column chromatography column for the first time; during flushing, control the linear velocity of the nickel column flushing buffer in the column to be 200 cm / h.

[0170] Step S10, loading the enzyme cleavage buffer onto a nickel column chromatography column, performing enzyme cleavage labeling on the recombinant bovine lactoferrin on the column, controlling the linear velocity of the enzyme cleavage buffer in the column to be 200 cm / h, detecting the enzyme cleavage effluent at the same time, observing the UV280 curve, and starting to collect the enzyme cleavage effluent after the main peak of lactoferrin appears, and stopping the collection until the UV280 curve descends steadily, and a total of 3.6 L is collected;

[0171] The enzyme is a recombinant bovine enterokinase light chain expressed by genetically engineered Escherichia coli; the enzyme cleavage buffer is a 30 mM Tris-HCl buffer with a pH of 7.0, and the amount of enzyme used per milligram of recombinant bovine lactoferrin is 60 U.

[0172] Step S11, using 5 times column volume, i.e. 5L of nickel column equilibration buffer in step S7 to rinse the nickel column chromatography column for the second time; controlling the linear velocity of the rinsing buffer in the column to 200cm / h, detecting the rinsing effluent at the same time, observing the UV280 curve, and starting to collect the rinsing effluent after the main peak of lactoferrin appears, and stopping collecting until the UV280 curve descends steadily, and a total of 4.5L is collected;

[0173] Step S12, using 5 column volumes, i.e. 5 L of the equilibration buffer in step S7, to equilibrate the cationic chromatography column;

[0174] The filler of the cationic chromatography column is SP agarose gel FF, the particle size is 70 to 120 microns, and the filler volume is 1L.

[0175] Step S13, loading the enzyme cleavage effluent and the flushing effluent collected in step S10 and step S11 onto a cationic chromatography column, controlling the linear velocity to be 200 cm / h, and cyclically loading until the mass percentage of recombinant bovine lactoferrin in the effluent loaded onto the cationic chromatography column is 3.5%, and the loading is completed;

[0176] Step S14, using 5 column volumes, i.e., 5 L of the nickel column equilibration buffer in step S7, to rinse the cation chromatography column;

[0177] Step S15, prepare an elution buffer, use the elution buffer to rinse the cationic chromatography column, detect the elution effluent at the same time, observe the UV280 curve, and start collecting the flow-through protein solution after the main peak of lactoferrin appears, and stop collecting until the UV280 curve descends steadily, and a total of 6L is collected;

[0178] The main body of the elution buffer is PB buffer, and a 20mM KH2PO4 solution and a 20mM K2HPO4 solution are mixed to prepare the PB buffer, wherein the volume of the KH2PO4 solution accounts for 39% and the volume of the K2HPO4 solution accounts for 61%, and then NaCl is added to the PB buffer at a concentration of 1.2mol / L to prepare the elution buffer, and the pH of the elution buffer at room temperature is pH 7.0. During elution, the linear velocity of the elution buffer in the column is controlled to be 200cm / h.

[0179] Step S16, sampling and testing: taking part of the flow-through protein solution, and using size exclusion chromatography (SEC-HPLC) to test the purity of recombinant bovine lactoferrin to be 96.6% in accordance with the requirements of national standard GB1903.17-2016; Figure 4 This is a SEC-HPLC chart of the purity detection of recombinant bovine lactoferrin in Example 3, wherein reference numeral 1 is the main peak of lactoferrin.

[0180] Step S17, concentration and drying: the flow-through protein solution collected in step S15 is replaced, concentrated and then dried to obtain a recombinant bovine lactoferrin solid.

[0181] First, the protein solution was ultrafiltered and concentrated to 1 / 5 of the initial total volume, i.e., 1.2 L. Then, the replacement fluid was added in five times and ultrafiltered and concentrated to 1 / 5 of the initial total volume, i.e., 1.2 L, to obtain a high-concentration desalted recombinant bovine lactoferrin solution. The volume of the replacement fluid added each time was 1.2 L ml.

[0182] The main body of the replacement fluid is PB buffer. A 20mM KH2PO4 solution and a 20mM K2HPO4 solution are mixed to prepare the PB buffer, wherein the volume of the KH2PO4 solution accounts for 39% and the volume of the K2HPO4 solution accounts for 61%. Then, ethylenediaminetetraacetic acid and TWEEN80 are added to the PB buffer at a concentration of 0.12mM respectively to prepare the replacement fluid. The pH value of the replacement fluid is pH 7.0 at room temperature.

[0183] Subsequently, the high-concentration desalted recombinant bovine lactoferrin solution was freeze-dried to obtain 31 g of protein solid.

[0184] It can be seen from the gradually enlarged embodiments of the present invention that purification of unlabeled recombinant bovine lactoferrin from Pichia pastoris can achieve industrialized mass production; according to the requirements of national standard GB1903.17-2016, the purity of recombinant bovine lactoferrin detected by size exclusion chromatography (SEC-HPLC) is above 96%, which meets the requirements of national standard GB1903.17-2016 that the content of lactoferrin as a food nutrient enhancer in total protein mass percentage (w / %) is ≥95%.

[0185] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for purifying high-purity tag-free recombinant bovine lactoferrin, characterized in that: The method comprises the following steps: Step S1, expression of recombinant bovine lactoferrin: the bovine lactoferrin gene with N-terminal fusion histidine tag gene and PAOX1 promoter are connected to the vector pPIC9K and then transformed into GS115 type Pichia pastoris, and the Pichia pastoris fermentation broth is obtained by high-density cell fermentation technology; Step S2, centrifuging the Pichia pastoris fermentation broth to collect the Pichia pastoris containing recombinant bovine lactoferrin; Step S3, mixing the Pichia pastoris with a resuspension buffer to form a resuspension treatment solution, adding ethylenediaminetetraacetic acid to the resuspension treatment solution, and stirring to dissolve evenly; wherein the ratio of the amount of the Pichia pastoris to the resuspension buffer is 1 g: (10-30) ml, and the ratio of the amount of the resuspension buffer to the ethylenediaminetetraacetic acid is 1 liter: (1-2) mmol; Step S4, high-pressure homogenizing the treated resuspended solution to obtain a high-pressure homogenized solution; Step S5, centrifuging the high-pressure homogenized liquid to collect the supernatant containing the recombinant bovine lactoferrin; Step S6, filtering the collected supernatant using a liquid filter with a pore size of 0.22 to 0.45 microns to obtain a filtrate containing recombinant bovine lactoferrin; Step S7, preparing an equilibrium buffer, and then using 3 to 5 column volumes of the equilibrium buffer to balance the nickel column chromatography column, and controlling the linear velocity of the equilibrium buffer in the column to be 60 to 200 cm / h during column balancing; Step S8, loading the filtrate in step S6 onto a nickel column chromatography column, controlling the linear velocity of the filtrate in the column to be 60-200 cm / h, and cyclically loading until the mass percentage of recombinant bovine lactoferrin in the nickel column loading effluent is less than 5%, and the loading is completed; Step S9, using 3 to 5 column volumes of the nickel column equilibration buffer in step S7 to perform a first flush on the nickel column chromatography column, and controlling the linear velocity of the nickel column flushing liquid in the column to be 60 to 200 cm / h; Step S10, loading the enzyme cleavage buffer onto a nickel column chromatography column, performing enzyme cleavage on the recombinant bovine lactoferrin on the column, controlling the linear velocity of the enzyme cleavage buffer in the column to be 150-200 cm / h, detecting the enzyme cleavage effluent at the same time, observing the UV280 curve, and starting to collect the enzyme cleavage effluent after the main peak of lactoferrin appears, and stopping the collection until the UV280 curve descends steadily; Step S11, using 3 to 5 times the column volume of the nickel column equilibration buffer in step S7 to rinse the nickel column chromatography column for the second time; controlling the linear velocity of the rinsing buffer in the column to be 60 to 200 cm / h, detecting the rinsing effluent at the same time, observing the UV280 curve, and starting to collect the rinsing effluent after the main peak of lactoferrin appears, and stopping collecting until the UV280 curve descends steadily; Step S12, using 3 to 5 column volumes of the equilibration buffer in step S7 to equilibrate the cationic chromatography column, and controlling the linear velocity of the elution buffer in the column to be 60 to 200 cm / h; Step S13, loading the enzyme cleavage effluent and the flushing effluent collected in step S10 and step S11 onto a cationic chromatography column, controlling the linear velocity to be 60-200 cm / h, and cyclically loading until the mass percentage of recombinant bovine lactoferrin in the effluent loaded onto the cationic chromatography column is less than 5%, and the loading is completed; Step S14, flushing the cation chromatography column with 3 to 5 column volumes of the nickel column equilibration buffer in step S7, and controlling the linear velocity of the cation column flushing liquid in the column to be 60 to 200 cm / h; Step S15, prepare an elution buffer, use the elution buffer to flush the cationic chromatography column, control the linear velocity of the elution buffer in the column to be 60-200 cm / h, detect the elution effluent at the same time, observe the UV280 curve, and start collecting the flow-through protein solution after the main peak of lactoferrin appears, and stop collecting until the UV280 curve descends steadily.

2. The method according to claim 1, characterized in that In the step S1, the histidine tag gene includes, in order of initial sequence, 6 to 10 consecutive histidine codons, and 4 consecutive aspartic acid codons and 1 lysine codon located after the histidine codons.

3. The method according to claim 1, characterized in that In the step S2, the centrifugal treatment is performed using a centrifuge at a rotation speed of 8000 to 12000 rpm for 20 to 30 minutes.

4. The method according to claim 1, characterized in that: In step S3, the resuspension buffer is PB buffer, and the pH of the resuspension buffer is pH 6.5 to pH 7.0 at room temperature.

5. The method according to claim 1, characterized in that In step S4, the high-pressure homogenization control pressure is 1000-1500 bar, and the high-pressure homogenization is performed 2-3 times.

6. The method according to claim 1, characterized in that In step S5, the centrifugal treatment is performed at a temperature of 2 to 8°C, a centrifugal speed of 8000 to 12000 rpm, and a centrifugal treatment time of 20 to 30 minutes.

7. The method according to claim 1, characterized in that In step S7, the main body of the balancing buffer is PB buffer, and NaCl is added to the PB buffer at a concentration of 0.16-0.24 mol / L to prepare the balancing buffer. The pH value of the balancing buffer at room temperature is pH 6.5-pH 7.

0.

8. The method according to claim 1, characterized in that In the step S10, the enzyme used for enzymatic cleavage is a recombinant bovine enterokinase light chain expressed by Escherichia coli cultured by genetic engineering, and the amount of enzyme used per milligram of recombinant bovine lactoferrin is 40-60U, wherein 1U is defined as the amount of enzyme required to cleave 95% of the recombinant bovine lactoferrin tag of 0.5mg stored in the enzymatic cleavage buffer at 25°C within 12h to 16h; the enzymatic cleavage buffer is a Tris-HCl buffer with a concentration of 20-30mM and a pH of 6.5-7.

0.

9. The method according to claim 1, characterized in that: In step S15, the main body of the elution buffer is PB buffer, and then NaCl is added to the PB buffer at a concentration of 0.8-1.2 mol / L to prepare the elution buffer. The pH value of the elution buffer is pH 6.5-pH 7.0 at room temperature.

10. The method according to claim 1, characterized in that Step S15 is followed by steps S16 and S17: Step S16, sampling and testing: taking part of the flow-through protein solution collected in step S15, and testing the purity of the recombinant bovine lactoferrin according to the requirements of the national standard GB1903.17-2016; Step S17, concentration and drying: the flow-through protein solution collected in step S15 is replaced, concentrated and then dried to obtain a recombinant bovine lactoferrin solid.

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