CN101352673A - An affinity chromatography medium for separating lysozyme and its preparation method - Google Patents

Abstract

The invention belongs to the field of protein purification reagent, and particularly relates to an affinity chromatography medium taking chitin as raw material and a preparation method thereof. The method adopts the chitin as the raw material to prepare the affinity chromatography medium for separation and purification of lysozyme. The preparation method adopted by the invention is convenient and easy for operation with low cost; the absorbability of the chitin affinity medium is of specificity, and velocity of flow is quick; purification of human lysozyme by the affinity chromatography medium has the advantages of simple steps and high efficiency, and the lysozyme with the purity near 100 % can be separated from zymotic fluid just by one time of purification, thus the purification efficiency is obviously higher than the purification efficiency of ion exchange chromatography. Therefore, the preparation technology of the chitin affinity medium is applicable to large-scaled industrialized production.

Description

An affinity chromatography medium for separating lysozyme and its preparation method

technical field

The invention belongs to the field of protein purification reagents. Specifically, the invention relates to an affinity chromatography medium using chitin as a raw material and a preparation method thereof.

Background technique

Affinity chromatography is a chromatographic system that applies the principle of reversible binding between biopolymers and ligands, and binds the ligands firmly to the carrier through covalent bonds. The role of this reversible combination is mainly based on the recognition of the spatial structure of its ligand by the biopolymer. Commonly used biological affinity relationships are enzyme-substrate, substrate analog, inhibitor, activator, cofactor, antibody-antigen, hormone-receptor protein, carrier protein, lectin-polysaccharide, glycoprotein, cell Surface receptors, nucleic acid-complementary nucleotide sequences, histones, nucleic acid binding proteins, etc.

In affinity chromatography, the magnitude of the adsorption between the ligand and the substance to be separated is mainly related to the space structure of the ligand, so the equilibrium constant KD of the intermolecular interaction can be used to measure the strength of the affinity. However, their binding force is nothing more than the hydrogen bond, electrostatic interaction, hydrophobic interaction, etc. between the corresponding functional groups. Therefore, in addition to changing the ligand to change the strength of the affinity chromatography, the affinity between the ligand and the biomacromolecule can also be changed by changing the pH and ionic strength, so as to achieve the purpose of elution. However, due to the diversity of affinity chromatography, the elution conditions often need to be obtained through experiments. In addition, other substances such as ligands and inhibitors can also be used to compete with ligands or biomacromolecules on the chromatographic agent to achieve the purpose of elution. This method is called specific elution . Specific elution can obtain high resolving power, but the price of eluent is higher, so it is often used in conjunction with ordinary elution. It is worth noting that during elution, a small amount of ligands will be eluted together with the protein, so a gel chromatography is often added afterwards to remove small molecular ligands.

The uses of lysozyme are quite extensive. In foreign countries, lysozyme is widely used in food sterilization, fresh-keeping, antisepsis, microbial fermentation and extraction of bacterial contents such as nucleic acid, protein, antibiotics, enzymes, etc. in bioengineering technology. In medicine and clinical practice, various drugs made from lysozyme have the functions of antibacterial and antiviral, anti-inflammation, detumescence, and enhancing the curative effect of antibiotics. In China, lysozyme has not been fully used, mainly due to the low yield and poor quality of domestically produced lysozyme, and the lysozyme used in medicine, food and scientific research still relies on imports. With the rapid development of food industry, microbial industry and bioengineering technology, and the popularization and application of lysozyme in the field of medicine, people's demand for lysozyme is increasing day by day, and at the same time, the requirements for the quality of lysozyme are also getting higher and higher, so Many researchers are looking for new lysozyme separation and purification technology to improve the purity and activity of lysozyme products.

So far, there has been no report on the use of chitin as an affinity chromatography medium, that is, as a ligand, for the separation of lysozyme.

Contents of the invention

The purpose of the present invention is to provide an affinity chromatography medium for lysozyme separation with simple operation, low cost and high efficiency.

Another object of the present invention is to provide a preparation method of the above medium.

The invention provides an affinity chromatography medium, which is alkalized chitin.

Chitin is 2-N-acetylamino-2-deoxy-β-1,4-glucose, which is a straight-chain nitrogen-containing compound polymerized by 2-N-acetylamino-D-glucose through β-1,4 glycosidic bonds. Polysaccharides are natural polymers that are very similar in structure to cellulose. The chitin used in the present invention can be raw material with various commercially available products at present, for example, (C7170) of sigma company, and Jinan Haidebei Marine Bioengineering Co., Ltd., Qingdao Haipu Biotechnology Co., Ltd., Weifang Kehai chitin Co., Ltd. etc. products.

In the present invention, the chitin is basified at 10-30° C. for 6-36 hours, then left to stand and washed until neutral, so as to prepare the chromatographic medium. The alkali used is one or more of sodium hydroxide and potassium hydroxide, and the mass molar ratio of chitin to ^OH- in the alkali is 25-200:1, and the unit is gram/mole.

Preferably, after the chitin is alkalized and left to stand, it is filtered and washed until it becomes neutral for later use. The method of filtration can be degreasing gauze filtration, etc. Here, washing to neutrality refers to washing away the alkali adsorbed on the surface of chitin, so that it is neutral in water, that is, the pH is about 7.

Even better, after the chitin is alkalized, it can be treated with an acid and left to wash until it becomes neutral for later use. The acid used may be weak acids such as acetic acid, citric acid, and acetic acid. The concentration is 1-5M.

After treatment, the medium particles can precipitate rapidly without flocculent floating matter. When the empty column is eluted with low concentration of acetic acid, the absorbance value of the eluent at 280nm should be small.

In the present invention, the calculation method of medium swelling (i.e. medium yield) is: a certain mass of medium is alkalized, filtered, washed or after alkalized, filtered, washed, frozen, immersed in distilled water, and placed in a graduated cylinder Settle naturally for 12 hours, read the wet volume V of the medium, and the swelling ratio of the medium = V/mass of the medium.

In the present invention, the method for calculating the shrinkage ratio of the medium is as follows: a certain volume of fully swollen medium is packed into a column, and after equilibrating the column bed with 0.02MPBS of pH 6.40, the column volume V _a is calculated, and the column bed is balanced with 0.1M acetic acid solution, To calculate column volume _Vb , media shrinkage ratio = 1- _Vb / _Va .

In the present invention, the method for measuring the relative flow rate ratio of the medium is: under the pressure of 0.05 MPa, measure the flow rate v _α of the 0.1M acetic acid solution in the empty bed per unit time. Pack the media of each group with the same volume into the column, use 0.1M acetic acid solution to balance the column bed, and measure the flow rate v _β of each media per unit time under the pressure of 0.05MPa. Medium relative flow rate ratio = v _β /v _α .

Among the present invention, the medium adsorption capacity comparison method is: adopt static adsorption method: measure 1ml to fully swell each group of medium, add the chicken lysozyme (c-LYZ) that the concentration of 3ml distilled water and 1ml is 10mg/ml, mix, wait Slightly settle, take 0.5ml of supernatant, centrifuge at 5000rpm for 10min, take 0.4ml of supernatant, dilute to 2ml with distilled water, measure the absorbance at 280nm, record it as _A1 ; Calculate the supernatant volume V, take 0.5ml of supernatant, centrifuge at 5000rpm for 10min, take 0.4ml of supernatant, dilute to 2ml with distilled water, let it stand for 6h, measure the absorbance at 280nm, record it as A ₂ , A _Δ = A ₁ - A ₂ , the adsorption capacity of the medium Q _r-hlyz = A _Δ × V × medium swelling ratio.

In the present invention, the calculation method for the water absorption of the chitin affinity medium is: take 0.05 g of the chitin affinity medium freeze-dried in vacuum, add 0.5 ml of double distilled water, and after swelling at room temperature for 6 hours, centrifuge at 3000 rpm for 30 minutes, and calculate the volume of the supernatant , and calculate the water absorption per unit mass of chitin affinity medium.

In the present invention, the calculation method of the medium yield: After a certain mass of medium is alkalized, filtered and washed, it is immersed in distilled water, placed in a measuring cylinder for natural settlement for 12 hours, and the wet volume V ₁ of the medium is read, and the swelling ratio of the medium = V ₁ /medium quality: After the above medium was treated with 0.1M acetic acid, filtered, washed, immersed in distilled water, placed in a graduated cylinder for natural settlement for 12 hours, read the medium wet volume V ₂ , medium yield = V ₂ /medium quality.

In the present invention, the loss ratio of the medium=1-medium yield/medium swelling ratio.

In the present invention, the adsorption capacity of the medium Q _r-hlyz = A _Δ × V × medium swelling ratio.

In another aspect, the present invention provides a method for preparing the above-mentioned medium, that is, after the chitin is alkalized at 10-30° C. for 3 minutes to 24 hours, it is left to stand and washed until neutral.

In the preparation method of the present invention, the alkali used is one or more of sodium hydroxide and potassium hydroxide. The concentration of the alkali used can be 1-10M, and the dosage can be a liquor ratio of 1:5-20.

In the preparation method of the present invention, the alkalization time may be 6-18 hours. For example, 6 hours, 10 hours, 12 hours, 15 hours, 18 hours, etc.

In the preparation method of the present invention, the mass molar ratio of chitin to ^OH- in the alkali can be 25-200:1, and the unit is g/mol.

In the preparation method of the present invention, after the chitin is alkalized and left to stand, it is filtered and washed until it is neutral for later use.

In the preparation method of the present invention, the reaction temperature is 15-25°C. For example, 15°C, 18°C, 20°C, 25°C and so on. It can also be 10°C, 13°C, 28°C, 30°C, etc.

In the preparation method of the present invention, after the chitin is alkalized, it is treated with a weak acid and then left to wash. The acid used can be weak acids such as acetic acid, citric acid, and acetic acid.

In the preparation method of the present invention, during the acid treatment, the molar ratio of chitin to H ⁺ in the acid is 25-200:1, and the unit is g/mol.

In the present invention, the unmentioned reaction conditions can be carried out using conventional conditions according to specific experimental conditions.

The invention adopts chitin as a raw material to prepare an affinity chromatographic medium for separating and purifying lysozyme. The preparation method adopted in the present invention is simple and easy, and the cost is low; the chitin affinity medium has specific adsorption and the flow rate is fast; the chitin affinity medium is used to purify human lysozyme, the steps are simple and efficient, and after one purification, it can be obtained from fermentation Nearly 100% pure lysozyme was isolated from the liquid, and the purification efficiency was significantly higher than that of ion exchange chromatography. Therefore, the preparation process of chitin affinity media in this study can be applied to large-scale industrial production.

Detailed ways

Below in conjunction with embodiment the present invention is further described.

The main reagent involved in the embodiment has:

30% acrylamide gel storage solution: Take 29g of acrylamide and 1g of methylenebisacrylamide, dissolve in 60ml of double-distilled water, dilute to 100ml, pH is less than 7.0, and store in a brown bottle at 4°C.

10% ammonium persulfate: Dissolve 1 g of ammonium persulfate in 10 ml of double distilled water and store at 4°C.

1.5M Tris-HCl buffer solution (pH8.8): Take 18.16g of Tris base, fully dissolve in 50ml of double distilled water, adjust the pH to 8.0 with concentrated hydrochloric acid, and dilute to 100ml.

1M Tris-HCl buffer (pH6.8): Take 12.11g of Tris base, fully dissolve in 80ml of double distilled water, adjust the pH to 6.8 with concentrated hydrochloric acid, and dilute to 100ml.

Loading buffer for protein electrophoresis: take 5ml of 0.5mol/L Tris Cl (pH6.8), 1g of SDS, 0.6ml of β-mercaptoethanol, 8ml of 50% glycerol, 1ml of 0.05% bromophenol blue, and 4.4ml of double distilled water .

Electrophoresis buffer: take Tris 3.03g, glycine 14.41g, distilled water to 1ml, add SDS 1g, stir to dissolve.

The main instrument involved in the embodiment has:

Protein Gel Electrophoresis System US Bio-Rad Company

Gel Imaging Analysis System US UVP Company

752 UV-Vis Spectrophotometer Shanghai Precision Scientific Instrument Co., Ltd.

Agilent series 1100 HPLC analyzer United States Hewlett-Packard Company

PHSJ-4A pH Meter Shanghai Precision Scientific Instrument Co., Ltd.

Constant temperature shaker Shanghai Institute of Centrifugal Machinery

Magnetic Stirrer Shanghai Precision Scientific Instrument Co., Ltd.

Clean bench Suzhou Antai Air Technology Co., Ltd.

Dialysis bag Huamei Bioengineering Company

The influence of embodiment 1 alkalization on medium performance

A1, take 5g of chitin, dissolve it in 100ml of distilled water, stir magnetically at room temperature for 6 hours, let it stand for 6 hours, and set aside; A2, take 5g of chitin, dissolve it in 100ml of distilled water, stir it magnetically for 6 hours at room temperature, and filter it with two layers of degreasing gauze 1. Wash, re-soak with 100ml distilled water, stand still, and set aside; A3, take 5g chitin, dissolve in 100ml 10M NaOH solution, stir magnetically at room temperature, alkalinize for 6 hours, filter, wash until neutral, and immerse in distilled water , stand still, spare. The results are shown in Table 1.

Table 1 The effect of alkalization on medium performance

The influence of embodiment 2 dilute acid treatment on medium performance

Take 4 parts of 5g chitin, dissolve in 100ml of 10M NaOH solution, stir magnetically at room temperature, after alkalization for 6h, filter and wash until neutral, C1, the treatment method is the same as step A3; C2, after step A3, add to the medium solution Add an equal amount of 0.04MPBS with a pH of 6.40, stir for 5 minutes, filter, wash until neutral, soak in distilled water, let stand, and set aside; C3, after step A3, add an equal amount of 0.2M acetic acid to the medium solution , after stirring for 5 minutes, filter and wash until neutral, soak in distilled water, let stand, and set aside. The results are shown in Table 2.

Table 2 Effect of acetic acid treatment on media performance

The influence of embodiment 3NaOH concentration on medium performance

Get 3 parts of 5g chitin, E1, the treatment method is the same as D12 (see Example 5); E2, adopt 5M NaOH solution to alkalize, other treatment methods are the same as D12; E3, adopt 2.5M NaOH solution to alkalize, other treatment methods are the same as D12. The detection indicators are the same as above. The results are shown in Table 3.

The influence of table 3 NaOH concentration on medium performance

The influence of embodiment 4 bath ratio on medium performance

Take three parts of 5g chitin, F1, dissolve in 50ml of 2.5M NaOH solution, other treatment methods are the same as E3; F2, dissolve in 100ml of 2.5M NaOH solution, other treatment methods are the same as E3; F3, dissolve in 200ml of 2.5M NaOH solution Among them, other processing methods are the same as E3. The detection indicators are the same as above. The results are shown in Table 4.

The influence of table 4 bath ratio on medium performance

The influence of embodiment 5 alkalization time on medium performance

Take 4 parts of 5g chitin, dissolve in 100ml of 10M NaOH solution, D11, the treatment method is the same as step C2, D12, magnetic stirring at room temperature, alkalization for 12h, other treatment methods are the same as C2; D13, room temperature magnetic stirring, alkalization for 18h, others The treatment method is the same as C2; D14, magnetic stirring at room temperature, alkalization for 24 hours, other treatment methods are the same as C2. The detection indicators are the same as above. The specific results are shown in Table 5.

The impact of table 5 alkalization time on medium performance

Embodiment 6 chitin affinity medium adsorption capacity

Take 16ml of chitin medium, add 250ml of fermentation broth supernatant, magnetically stir and absorb at room temperature for 0.5h, precipitate for 10min, pour off the supernatant, add 500ml of double distilled water, stir and wash, precipitate for 10min, repeat washing once, and pour off the supernatant . Put the chitin affinity medium into a 10mm×20cm chromatographic column, wash the column with 0.02MPBS of pH 6.24, and after the OD _280nm value of the effluent drops below 0.010, replace it with 0.05M acetic acid solution of pH 3.27 for dissociation, collect the washed Eluate, and stop collecting after the OD _280nm of the eluate drops below 0.010. Adjust the pH of the eluate to 8.0 with 1M carbonate buffer, perform DEAE-Sephadex A-25 anion exchange chromatography, collect the eluate, and adjust the pH of the eluate to 6.24 with 1M acetic acid. Put the enzyme solution into a dialysis zone with a cut-off range of 4000-6000 of an appropriate length, dialyze with 10 times the volume of double-distilled water at 4°C for 12 hours, and change the water 3-4 times in the middle. Divide the enzyme solution into subpackages, carry out vacuum freeze-drying, and calculate the product quality and the adsorption capacity of the chitin affinity medium. The results show that 6.5 mg of rh-LYZ can be purified from the supernatant of 250 ml of fermentation broth using 16 ml of chitin affinity medium. Therefore, under this adsorption condition, the adsorption capacity of each ml of chitin affinity medium is about 0.4 mg .

The preparation method of embodiment 7 chitin affinity chromatography medium

A certain mesh of chitin (usually 60 mesh) is basified with 2M NaOH solution at a bath ratio of 1:10 at room temperature for 12 hours under stirring → filtered with two layers of absorbent cotton gauze, washed with 50 times the volume of distilled water for 3 times, and reimmersed in a certain Volume distilled water → add an equal volume of 0.2M acetic acid solution, stir magnetically at room temperature for 5 minutes → filter with two layers of absorbent cotton gauze, wash with distilled water until neutral, re-immerse in a certain volume of distilled water, add absolute ethanol to 20% concentration, store at 4°C ,spare.

The purification of embodiment 8 chicken lysozyme (c-LYZ)

Take 15ml of chitin medium, add the supernatant of the fermentation broth, magnetically stir and adsorb at room temperature for 0.5h, after 10min of precipitation, pour off the supernatant, put the medium into a 1cm×20cm chromatographic column, wash the column bed with 0.02MPBS of pH 6.4, wash After the OD _280nm value of the eluate drops below 0.010, use a 0.05M acetic acid solution with pH 3.27 for dissociation, collect the eluate, and stop collecting after the OD _280nm of the eluate drops below 0.010. Use 0.2M sodium acetate buffer solution with pH 5.42 for dissociation, collect the eluate, and stop collecting after the OD _280nm of the eluate drops below 0.010; The eluate was collected as a unit, and the collection was stopped after the OD _280nm of the eluate dropped below 0.010.

The crude product of c-LYZ purified by one-step elution was analyzed by SDS-PAGE using BandScan V4.30 software. The protein contents (according to the molecular weight) in the crystals were 24.5%, 28.1%, and 47.4%, respectively, and the purity of c-LYZ in the supernatant was 100%.

Claims (10)

1. an affinity chromatography medium that is used to separate lysozyme is characterized in that, this chromatography media is the chitin after the alkalization.

2. the preparation method of medium according to claim 1 is characterized in that, chitin is left standstill and be washed till neutrality in alkalization under the 7-30 ℃ of condition after 6-36 hour, gets final product.

3. preparation method as claimed in claim 2 is characterized in that, used alkali is one or several in NaOH, the potassium hydroxide.

4. preparation method as claimed in claim 2 is characterized in that, alkalization time is 8-24 hour.

5. preparation method as claimed in claim 2 is characterized in that, OH in chitin and the alkali ^-The quality mol ratio be 8-200: 1, unit is a gram/mole.

6. preparation method as claimed in claim 2 is characterized in that, OH in chitin and the alkali ^-The quality mol ratio be 25-150: 1, unit is a gram/mole.

7. preparation method as claimed in claim 2 is characterized in that, after the chitin alkalization is left standstill, through filtering washing again to neutral standby.

8. preparation method as claimed in claim 2 is characterized in that, reaction temperature is 15-25 ℃.

9. preparation method as claimed in claim 2 is characterized in that, after the chitin alkalization, leaves standstill washing again with acetic acid or citric acid treatment to neutrality.

10. preparation method as claimed in claim 9 is characterized in that, during acid treatment, adds H in chitin and the acid ⁺The quality mol ratio be 25-200: 1, unit is a gram/mole.