CN107446905B - Method for purifying recombinant human lysozyme - Google Patents
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Abstract
The invention discloses a recombinant human lysozyme purification method, which comprises the steps of firstly carrying out coarse purification on a large batch of recombinant human lysozyme by using a hydrophobic chromatographic column, and then directly carrying out fine purification by using a weak cation exchange chromatographic column.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a purification method of recombinant human lysozyme.
Background
The first research on lysozyme was from the lytic seed of Bacillus subtilis published in Nicole 1907, and Fleming et al 1922 found that there is an enzyme in human saliva and tears that can dissolve the cell wall to kill bacteria, and thus was named lysozyme. In 1937 Abraham and Robinson isolated lysozyme crystals from egg proteins, revealing the historical chapter on the study of lysozyme. In 1965, the lysozyme was analyzed by X-ray diffraction, such as Phillips, UK, and the first completely clarified the three-dimensional structure of lysozyme.
The lysozyme has antibacterial, antiviral and anti-inflammatory effects, and can be used in combination with antibiotic to enhance the therapeutic effect of antibiotic. It is also a non-specific immune factor in human body, can improve immunity of organism, and has good synergistic effect with other natural defense factors of cation antibacterial peptides. In addition, the protein is a natural protein, can be digested and absorbed as a nutrient substance in the gastrointestinal tract, has no toxicity, does not remain in the body, and is a food preservative, a nutritional health-care product and a medicine with high safety. Lysozyme is commonly used in various processed foods and beverages and integrates three functions of pharmacology, health care and antisepsis.
The lysozyme is not fully applied in China, and mainly the lysozyme produced in China has small quantity and poor quality, and the lysozyme for medicines, foods and scientific researches still depends on import. At present, the lysozyme is mainly extracted by taking chicken protein as a raw material, but the cost is high, the investment is large, and the process is complex. With the rapid development of food industry, microbial industry and bioengineering technology, the demand of lysozyme is increasing with the popularization and application of lysozyme in medicine. In addition to eggs, the development of new lysozyme resources is a new topic to be researched urgently. Meanwhile, the quality requirement of lysozyme is higher and higher, so a new lysozyme separation and purification process must be found, the purity and the enzyme activity of the lysozyme product are improved, and the social requirement is met to the utmost extent.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for purifying the recombinant human lysozyme, which has simple process, less required solvent and high product purity and recovery rate and is suitable for mass production.
The technical scheme adopted for solving the technical problems comprises the following steps:
1. coarse and pure ultrafiltration
And (3) carrying out centrifugal separation on the recombinant human lysozyme fermentation liquor, carrying out ultrafiltration degerming on the obtained supernatant through a 0.2-micron hollow fiber membrane, and collecting filtrate.
2. Hydrophobic chromatography
And (3) adding NaCl into the filtrate obtained in the step (1) to ensure that the concentration of the NaCl in the filtrate is 1.0-1.5 mol/L, purifying by using a hydrophobic chromatography column, eluting by using a mobile phase A at first and then by using a mobile phase B at an elution flow rate of 180-240 mL/min, and collecting the eluent of the mobile phase B.
3. Ion exchange chromatography
Adjusting the pH value of the eluate collected in the step 2 to 7.0-8.0, purifying by using a weak cation exchange chromatography column, eluting by using a mobile phase C during purification, then eluting by using a mixed solution of the mobile phase C and the mobile phase D in a volume ratio of 84:16, finally eluting by using a mixed solution of the mobile phase C and the mobile phase D in a volume ratio of 80: 20-50: 50 at an elution flow rate of 30-40 mL/min, and collecting the eluate when the volume ratio of the mobile phase C and the mobile phase D is 80: 20-50: 50 to obtain the recombinant human lysozyme with the purity of more than 98%.
The mobile phase A is a PB buffer solution with the pH value of 7.0-8.5 and containing 1.0-1.5 mol/L NaCl; the mobile phase B is a PB buffer solution with the pH value of 7.0-8.5; the mobile phase C is a PB buffer solution with the pH value of 7.0-8.0; the mobile phase D is PB buffer solution containing 1mol/L NaCl and having a pH value of 7.0-8.0.
The filler of the hydrophobic chromatographic column is preferably phenyl high-flow-rate agarose filler with the particle size of 45-160 mu m, the adsorption capacity of the filler on protein is not less than 30mg/mL, and the linear flow rate is not higher than 8 mL/cm.
The filler of the weak cation exchange chromatographic column is preferably high-flow-rate agarose weak cation exchange filler with the particle size of 40-120 mu m, the total ion exchange capacity of the filler is not lower than 0.08mmol/mL, and the linear flow rate is not higher than 12 mL/cm.
In the step 1, it is further preferable that before the obtained supernatant is subjected to ultrafiltration sterilization by a 0.2 μm hollow fiber membrane, physiological saline is added to ensure the recovery rate of protein in the ultrafiltration process.
In the step 2, preferably, the elution is finally carried out by using a mixed solution of the mobile phase C and the mobile phase D in a volume ratio of 75: 25-70: 30.
In the step 3, preferably, the eluate collected in the step 2 is diluted with deionized water until the conductivity is 1.8-2.5 mS/cm, then the pH is adjusted to 7.0-8.0, and the eluate is purified by a weak cation exchange chromatography column, wherein the pH value is adjusted by NaOH or HCl.
The invention has the advantages that:
1. the invention adopts hollow fiber membrane ultrafiltration to remove insoluble impurities contained in the supernatant and a small amount of thalli and bacteria in the fermentation liquor.
2. The method has the advantages that the first step is hydrophobic chromatography purification, all target proteins can be captured at one time, the yield is high, the further desalting, concentrating and freeze-drying processes of supernatant obtained by the centrifugal separation of fermentation liquor are avoided, the time is saved, and the purification efficiency is improved. Then combining with ion exchange chromatography, the purity of the recombinant human lysozyme finally obtained is more than 98%, and the yield is more than 90%.
Detailed Description
The present invention will be described in further detail with reference to examples, but the scope of the present invention is not limited to these examples.
Example 1
1. Coarse and pure ultrafiltration
34L of recombinant human lysozyme fermentation liquor (pichia pastoris fermentation liquor containing recombinant human lysozyme) is put into a sedimentation centrifuge, centrifuged at 2000r/min for 60min, and 21L of supernatant is collected through a liquid skimming tube. And (3) passing 21L of the supernatant through a 0.2-micron hollow fiber membrane for ultrafiltration and sterilization, supplementing 5L of normal saline before the ultrafiltration is finished, ensuring the protein recovery rate in the ultrafiltration process, and collecting 24L of filtrate.
2. Hydrophobic chromatography
Adding NaCl into the 24L filtrate obtained in the step 1 to enable the concentration of the NaCl in the filtrate to be 1.2mol/L, then purifying the filtrate through a hydrophobic chromatography column by high performance liquid chromatography, wherein the filler is phenyl high-flow-rate agarose filler with protein adsorption capacity not less than 30mg/mL, linear flow rate not higher than 8mL/cm and particle size of 45-160 mu m, unbound protein and impurities are eluted by PB buffer solution with pH 8.0 containing 1.2mol/L NaCl during purification, target protein is eluted by PB buffer solution with pH 8.0, the elution flow rate is 200mL/min, and eluent eluted by PB buffer solution with pH 8.0 is collected.
3. Ion exchange chromatography
Diluting the eluent collected in the step 2 with deionized water to the conductivity of 1.94mS/cm, adjusting the pH value of the eluent to 8.0 with NaOH, passing through a weak cation exchange chromatographic column by high performance liquid chromatography, wherein the filler is a high flow rate agarose weak cation exchange filler with the total ion exchange amount of not less than 0.08mmol/mL, the linear flow rate of not more than 12mL/cm and the particle size of 40-120 mu m, eluting unbound impurities and hetero-proteins with a PB buffer solution with the pH of 8.0, eluting the hetero-proteins bound to the chromatographic column with a mixed solution of the PB buffer solution with the pH of 8.0 and the PB buffer solution with the pH of 8.0 at a volume ratio of 84:16 containing 1mol/L NaCl, finally eluting the target protein with a mixed solution of the PB buffer solution with the pH of 8.0 and the PB buffer solution with the pH of 8.0 at a volume ratio of 75:25 containing 1mol/L NaCl at a volume ratio of 35 mL/25, and collecting the eluted protein with the mixed solution of the PB buffer solution with the pH of 8.0 and the mixed solution with the NaCl containing 1mol/L at a volume ratio of 75:25 And eluting the eluent to obtain the recombinant human lysozyme with the purity of 99.0 percent, wherein the final yield of the recombinant human lysozyme is 90 percent.
Example 2
1. Coarse and pure ultrafiltration
30L of recombinant human lysozyme fermentation liquor (pichia pastoris fermentation liquor containing recombinant human lysozyme) is put into a sedimentation centrifuge, centrifuged at 2000r/min for 60min, and 18L of supernatant is collected through a liquid skimming pipe. And (3) passing 18L of the supernatant through a 0.2-micron hollow fiber membrane for ultrafiltration and sterilization, supplementing 5L of normal saline before the ultrafiltration is finished, ensuring the protein recovery rate in the ultrafiltration process, and collecting 21L of filtrate.
2. Hydrophobic chromatography
Adding NaCl into the 21L filtrate obtained in the step 1 to enable the concentration of the NaCl in the filtrate to be 1.0mol/L, then purifying the filtrate through a hydrophobic chromatography column by high performance liquid chromatography, wherein the filler is phenyl high-flow-rate agarose filler with protein adsorption capacity not less than 30mg/mL, linear flow rate not higher than 8mL/cm and particle size of 45-160 mu m, firstly eluting unbound protein and impurities by using PB buffer solution with pH value of 7.0 and containing 1.5mol/L NaCl, then eluting target protein by using PB buffer solution with pH value of 7.0, and collecting the eluent, and the elution flow rate is 180 mL/min.
3. Ion exchange chromatography
Diluting the eluent collected in the step 2 with deionized water to the conductivity of 2.45ms/cm, adjusting the pH value of the eluent to 7.5 with NaOH, passing through a high performance liquid chromatography weak cation exchange chromatographic column, wherein the filler is a high flow rate agarose weak cation exchange filler with the total ion exchange amount of not less than 0.08mmol/mL, the linear flow rate of not more than 12mL/cm and the particle size of 40-120 mu m, eluting unbound impurities and hetero-proteins with a PB buffer solution with the pH of 7.5, eluting the hetero-proteins bound to the chromatographic column with a mixed solution of the PB buffer solution with the pH of 7.5 and the PB buffer solution with the pH of 7.5 at a volume ratio of 84:16, finally eluting with a mixed solution of the PB buffer solution with the pH of 7.5 and the PB buffer solution with the pH of 1mol/L NaCl at a volume ratio of 50:50, eluting with the flow rate of 40mL/min, and collecting the mixed solution of the PB buffer solution with the pH of 7.5 and the PB buffer solution with the pH of 1mol/L of NaCl at a volume ratio of 50:50 Eluting to obtain the recombinant human lysozyme with the purity of 98.3 percent, wherein the final yield of the recombinant human lysozyme is 95 percent.
Example 3
1. Coarse and pure ultrafiltration
Putting 36L of recombinant human lysozyme fermentation liquor (pichia pastoris fermentation liquor containing recombinant human lysozyme) into a sedimentation centrifuge, centrifuging at 2000r/min for 60min, and collecting 20L of supernatant through a liquid skimming pipe. And (3) passing 20L of the supernatant through a 0.2-micron hollow fiber membrane for ultrafiltration and sterilization, supplementing 5L of normal saline before the ultrafiltration is finished, ensuring the protein recovery rate in the ultrafiltration process, and collecting 23L of filtrate.
2. Hydrophobic chromatography
Adding NaCl into 23L of filtrate obtained in the step 1 to enable the concentration of the NaCl in the filtrate to be 1.5mol/L, then purifying the filtrate through a hydrophobic chromatography column by high performance liquid chromatography, wherein the filler is phenyl high-flow-rate agarose filler with the protein adsorption capacity of not less than 30mg/mL, the linear flow rate of not more than 8mL/cm and the particle size of 45-160 mu m, firstly eluting unbound protein and impurities by using a PB buffer solution with the pH value of 8.5 and containing 1.0mol/L NaCl, then eluting the target protein by using a PB buffer solution with the pH value of 8.5, and collecting the eluent, wherein the elution flow rate is 240 mL/min.
3. Ion exchange chromatography
Diluting the eluent collected in the step 2 with deionized water to the conductivity of 1.85ms/cm, adjusting the pH value of the eluent to 7.0 with HCl, passing through a weak cation exchange chromatographic column of high performance liquid chromatography, wherein the filler is a high flow rate agarose weak cation exchange filler with the total ion exchange amount of not less than 0.08mmol/mL, the linear flow rate of not more than 12mL/cm and the particle size of 40-120 mu m, eluting unbound impurities and hetero-proteins with a PB buffer solution with the pH of 7.0, eluting the hetero-proteins bound to the chromatographic column with a mixed solution of the PB buffer solution with the pH of 7.0 and the PB buffer solution with the pH of 7.0 in a volume ratio of 84:16 containing 1mmol/L NaCl, eluting with a mixed solution of the PB buffer solution with the pH of 7.0 and the PB buffer solution with the pH of 1mol/L NaCl in a volume ratio of 80:20, eluting with the flow rate of 30mL/min, and collecting the mixed solution of the PB buffer solution with the pH of 7.0 and the PB buffer solution with the pH of 1mol/L of 7.0 and the PB buffer solution with the pH of 7.0 in a volume ratio of 20 of Eluting to obtain the recombinant human lysozyme with the purity of 98.5 percent, wherein the final yield of the recombinant human lysozyme is 92 percent.
Claims (5)
1. A method for purifying recombinant human lysozyme is characterized in that:
(1) coarse and pure ultrafiltration
Carrying out centrifugal separation on the recombinant human lysozyme fermentation liquor, carrying out ultrafiltration degerming on the obtained supernatant through a hollow fiber membrane of 0.2 mu m, and collecting filtrate;
(2) hydrophobic chromatography
Adding NaCl into the filtrate obtained in the step (1) to enable the concentration of the NaCl in the filtrate to be 1.0-1.5 mol/L, then purifying through a hydrophobic chromatography column, eluting with a mobile phase A at first and then with a mobile phase B at an elution flow rate of 180-240 mL/min, and collecting the eluent of the mobile phase B;
the filler of the hydrophobic chromatographic column is phenyl high-flow-rate agarose filler with the particle size of 45-160 mu m, the adsorption capacity of the filler on protein is not less than 30mg/mL, and the linear flow rate is not higher than 8 mL/cm; the mobile phase A is a PB buffer solution with the pH = 7.0-8.5 and containing 1.0-1.5 mol/L NaCl; the mobile phase B is a PB buffer solution with the pH = 7.0-8.5;
(3) ion exchange chromatography
Adjusting the pH value of the eluate collected in the step (2) to 7.0-8.0, purifying by using a weak cation exchange chromatography column, eluting by using a mobile phase C during purification, then eluting by using a mixed solution of the mobile phase C and the mobile phase D in a volume ratio of 84:16, finally eluting by using a mixed solution of the mobile phase C and the mobile phase D in a volume ratio of 80: 20-50: 50, wherein the elution flow rate is 30-40 mL/min, and collecting the eluate when the volume ratio of the mobile phase C and the mobile phase D is 80: 20-50: 50 to obtain recombinant human lysozyme with the purity of more than 98%;
the filler of the weak cation exchange chromatographic column is a high-flow-rate agarose weak cation exchange filler with the particle size of 40-120 mu m, the total ion exchange capacity of the filler is not lower than 0.08mmol/mL, and the linear flow rate is not higher than 12 mL/cm; the mobile phase C is a PB buffer solution with the pH = 7.0-8.0; the mobile phase D is PB buffer solution with the pH = 7.0-8.0 and containing 1mol/L NaCl.
2. The method for purifying recombinant human lysozyme according to claim 1, wherein: and (3) in the step (1), before the obtained supernatant passes through a hollow fiber membrane of 0.2 mu m for ultrafiltration and sterilization, adding physiological saline to ensure the protein recovery rate in the ultrafiltration process.
3. The method for purifying recombinant human lysozyme according to claim 1, wherein: and (3) finally eluting with a mixed solution of the mobile phase C and the mobile phase D in a volume ratio of 75: 25-70: 30 in the step (2).
4. The method for purifying recombinant human lysozyme according to claim 1, wherein: in the step (3), the eluent collected in the step (2) is diluted by deionized water until the conductivity is 1.8-2.5 mS/cm, then the pH is adjusted to 7.0-8.0, and the eluent is purified by a weak cation exchange chromatography column.
5. The method for purifying recombinant human lysozyme according to claim 4, wherein: in the step (3), the pH of the eluent B obtained in the step (2) is adjusted to 7.0-8.0 by NaOH or HCl.
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| CN109799335B (en) * | 2019-01-31 | 2022-04-01 | 陕西慧康生物科技有限责任公司 | Method for detecting pichia host protein residue in recombinant human lysozyme |
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