CN112552372B - Preparation method of magnetic protein crystal, magnetic protein crystal and application - Google Patents

Preparation method of magnetic protein crystal, magnetic protein crystal and application Download PDF

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CN112552372B
CN112552372B CN202011377415.2A CN202011377415A CN112552372B CN 112552372 B CN112552372 B CN 112552372B CN 202011377415 A CN202011377415 A CN 202011377415A CN 112552372 B CN112552372 B CN 112552372B
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magnetic
protein
crystal
solution
nucleating agent
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CN112552372A (en
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苏敏
张清洋
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Hebei University of Technology
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C07K1/306—Extraction; Separation; Purification by precipitation by crystallization
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
    • A61K9/0009—Galenical forms characterised by the drug release technique; Application systems commanded by energy involving or responsive to electricity, magnetism or acoustic waves; Galenical aspects of sonophoresis, iontophoresis, electroporation or electroosmosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2462—Lysozyme (3.2.1.17)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01017—Lysozyme (3.2.1.17)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes

Abstract

The invention relates to the technical field of biological medicines, in particular to a preparation method of a magnetic protein crystal, the magnetic protein crystal and application. The preparation method of the magnetic protein crystal comprises the following steps: mixing the magnetic nucleating agent with a solution system containing protein, and adjusting the supersaturation degree of the protein solution to enable the protein to be crystallized and separated out on the magnetic nucleating agent, so as to obtain magnetic protein crystals. The magnetic protein crystal is prepared by introducing the magnetic nucleating agent into a solution system containing protein, the crystallization time of the protein is effectively shortened, the separation process of the protein crystal is simplified, the crystallization efficiency of the protein crystal and the separation efficiency of the crystal are improved, the prepared magnetic protein crystal can be used as a drug intermediate product, in particular, the magnetic protein crystal can be used for producing targeted drugs, and the technology has wide application prospect in the field of production of protein drugs.

Description

Preparation method of magnetic protein crystal, magnetic protein crystal and application
Technical Field
The invention relates to the technical field of biological medicines, in particular to a preparation method of a magnetic protein crystal, the magnetic protein crystal and application.
Background
The development of biotechnology has prompted the preparation of macromolecular compounds, and therefore polypeptides, proteins, DNA analogs and the like have been developed as drugs (hormones, monoclonal antibodies, vaccines) for therapeutic use, especially protein drugs. However, protein drugs have high sensitivity to enzymes and short shelf life, resulting in low bioavailability and, in addition, have immunogenicity problems. These render the effect after administration poor. The protein in the crystal form can alleviate the problems and promote the development of protein medicines. Most of the current methods for purifying protein drugs in industry are chromatographic techniques, which are expensive in raw materials and use a large amount of buffer solution. These problems can be avoided by crystallization techniques.
In the biotechnology field of protein drug discovery, in order to obtain optimized protein crystallization conditions, CN107814831, CN105566443 and CN106188220 respectively take deoxyribonucleic acid, cross-linked protein crystals and cyclodextrin as additives to promote protein crystallization, so that the success rate of protein crystallization is improved.
In recent years, the field of pharmaceutical engineering has been focused on purifying proteins by crystallization of the proteins to replace costly chromatography or to improve the efficiency and product quality of conventional crystallization methods. CN104892749, CN106117345 and CN105753966 respectively improve the crystallization processes of the insulin, the insulin glargine and the recombinant human insulin by regulating the pH value of a system or adding a precipitant, thereby improving the process efficiency and the quality of the product.
In the method, a magnetic nucleating agent is prepared by adopting magnetic particles subjected to chemical modification to guide the self-assembly of protein molecules, so that the efficiency of protein crystallization can be improved, and a magnetic protein crystal is obtained as a product. The method can simplify the purification and separation process of the protein, reduce the separation cost, and the obtained protein crystal can be used as a pharmaceutical intermediate product or a final product, and particularly can be used as an important component of a targeted pharmaceutical preparation.
Disclosure of Invention
The invention provides a preparation method of a magnetic protein crystal. The method has the advantages of simple process flow, short operation time, convenient crystal separation, and good application prospect, and the crystal product can be used for targeted treatment means.
In order to achieve one of the above objects, the present invention adopts the following technical scheme: a method for preparing a magnetic protein crystal, comprising the steps of:
and mixing the magnetic nucleating agent with the protein solution, and adjusting the supersaturation degree of the protein solution to enable the protein to be crystallized and separated out on the magnetic nucleating agent, so as to obtain the magnetic protein crystal.
Further, the preparation method of the magnetic nucleating agent comprises the following steps: mixing magnetic particles with a polymer solution to coat the polymer on the outer surfaces of the magnetic particles, thereby obtaining a magnetic nucleating agent;
or alternatively, the first and second heat exchangers may be,
mixing magnetic particles with monomers, and forming high molecules on the magnetic particles through polymerization reaction by the monomers, so that the high molecules are coated on the outer surfaces of the magnetic particles, and the magnetic nucleating agent is obtained.
Further, the magnetic particles include, but are not limited to, mnFe 2 O 4 、Fe 3 O 4 、ZnFe 2 O 4 Or CoFe 2 O 4 Etc.
Further, the magnetic particles are nanoparticles with a particle size of 10-800 nm, preferably 50-400 nm.
Further, the high molecular compound is a polymer with a carboxyl, amino or hydroxyl structure;
preferably, the polymer compound includes, but is not limited to, polysaccharides, alcohols, phenols, amines, organic acids, and the like;
further, the manner of mixing the magnetic nanoparticles with the polymer solution includes, but is not limited to, shaking, stirring, or ultrasound;
preferably, the magnetic nanoparticles are mixed with the polymer solution for 2-12 hours.
Further, the magnetic protein crystal preparation method, the protein comprises, but is not limited to, polypeptide, or enzyme, or hormone, or monoclonal antibody, etc.;
further, the preparation method of the magnetic protein crystal can adjust the supersaturation degree of the protein solution by adopting modes including but not limited to adding a precipitating agent, cooling, freeze drying or reduced pressure evaporation.
Preferably, the precipitating agent includes, but is not limited to, naCl, znSO 4 、ZnCl 2 、ZnAc、ZnBr 2 Acetone, phenol, m-cresol, o-cresol, p-cresol, and the like.
The second purpose of the invention is to provide a magnetic protein crystal which is prepared by the preparation method provided by one of the purposes of the invention;
preferably, the magnetic protein crystals have paramagnetic properties;
preferably, the saturated magnetization of the magnetic protein crystal is 1-50 emu.g -1 。
The third object of the present invention is to provide the use of magnetic protein crystals in protein purification, or targeted drugs, or gene vectors.
The invention has the beneficial effects that:
the magnetic protein crystal is prepared by introducing the magnetic nucleating agent into the protein solution, so that the protein crystallization time is effectively shortened, the separation process of the protein crystal is simplified, the crystallization efficiency of the protein crystal and the separation efficiency of the crystal are improved, the prepared magnetic protein crystal can be used as a drug intermediate product, and particularly can be used for producing targeted drugs, and the technology has wide application prospect in the field of protein drug production.
Detailed Description
The following description of the present invention will be made clearly and fully, and it is apparent that the embodiments described are some, but not all, of the embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The preparation method of the magnetic protein crystal provided by the invention comprises the following steps:
and mixing the magnetic nucleating agent with the protein solution, and adjusting the supersaturation degree of the protein solution to enable the protein to be crystallized and separated out on the magnetic nucleating agent, so as to obtain the magnetic protein crystal.
In the present invention, protein solution refers to a protein-containing solution system in which solutes include, but are not limited to, proteins.
The magnetic protein crystal is prepared by introducing the magnetic nucleating agent into the protein solution, so that the protein crystallization time is effectively shortened, the separation process of the protein crystal is simplified, the crystallization efficiency of the protein crystal and the separation efficiency of the crystal are improved, the prepared magnetic protein crystal can be used as a drug intermediate product, and particularly can be used for producing targeted drugs, and the technology has wide application prospect in the field of production of protein crystallization drugs.
[ preparation of magnetic protein Crystal nucleating agent ]
In a preferred embodiment of the present invention, the preparation of the magnetic protein crystal nucleating agent comprises the steps of:
and fully mixing the magnetic particles with a high molecular solution to enable the high molecules to be coated on the outer surfaces of the magnetic nano particles, thereby obtaining the magnetic protein crystal nucleating agent.
Or, the preparation of the magnetic protein crystal nucleating agent comprises the following steps: the monomer forms a macromolecule on the magnetic particles through polymerization reaction, so that the macromolecule is coated on the outer surfaces of the magnetic particles, and the magnetic nucleating agent is obtained.
In one embodiment of the invention, the magnetic particles are paramagnetic particles, including but not limited to MnFe 2 O 4 Or Fe 3 O 4 Or ZnFe 2 O 4 Or CoFe 2 O 4 Etc.
In one embodiment of the present invention, the magnetic particles are nanoparticles, and the effect of promoting the formation of magnetic protein crystals is better when the particle diameter is 10 to 800 nm, preferably 50 to 400 nm.
In one embodiment of the present invention, the polymer compound is a polymer having a carboxyl group, an amino group, or a hydroxyl group structure. For example: polysaccharides, alcohols, phenols, amines, organic acids, and the like.
In one scheme of the invention, the magnetic protein crystal nucleating agent of the polymer coated magnetic nano particles is obtained by fully mixing the polymer and the magnetic nano particles by stirring, ultrasonic, vibration or the like, so that the polymer is modified on the outer surfaces of the magnetic nano particles.
[ preparation of precipitant solution ]
In a preferred mode of the present invention, the method for preparing the precipitant solution includes the steps of:
the precipitant and optional auxiliary agent are mixed with buffer solution to prepare precipitant solution.
In one embodiment of the invention, the precipitating agent is a solvent including, but not limited to, naCl, or ZnSO 4 Or ZnCl 2 Or ZnAc, or ZnBr 2 Etc.
In one embodiment of the invention, the auxiliary agent is an aqueous solution comprising, but not limited to, acetone, or phenol, or m-cresol, or o-cresol, or p-cresol, etc.
[ preparation of magnetic protein Crystal ]
In one embodiment of the invention, the protein-containing solution system is mixed with the magnetic nucleating agent and subsequently with the precipitant solution, the protein crystallizing out on the magnetic nucleating agent under the action of the precipitant. Separating the crystals by using a magnet, washing and drying to obtain the magnetic protein crystals.
In one embodiment of the invention, the protein is a polypeptide, or an enzyme, or a hormone, or a monoclonal antibody, or the like, including but not limited to.
The preparation method of the magnetic protein crystal provided by the invention is simple to operate, can effectively avoid the introduction of impurities, shortens the crystallization time of protein, can be used as a pharmaceutical intermediate product, and particularly can be used for producing targeted drugs, and has wide application prospects in the field of protein drug production. The technical solution provided by the present invention is further described below in connection with the embodiments for the convenience of understanding by those skilled in the art.
Example 1
The embodiment provides a magnetic lysozyme crystal, which is prepared by the following steps:
(1) Fe with particle size of 150-200 nm 3 O 4 The particles and polyaspartic acid were mixed in distilled water at a mass ratio of 8:1, and then the pH was precisely adjusted to 6.
(2) Nano Fe is added by ultrasonic wave and stirring 3 O 4 The system of particles and polyaspartic acid and deionized water was thoroughly mixed for 6 hours.
(3) Separating the black product obtained in the step (2) by using a magnet, and washing with distilled water for 4 times to remove excessive polyaspartic acid, thereby obtaining polyaspartic acid modified Fe 3 O 4 Microspheres for standby.
(4) Dissolving NaCl and lysozyme in 0.1mol/L sodium acetate buffer solution with pH=4.6 to reach NaCl concentration of 58.44mg/mL and lysozyme concentration of 18.75 mg/mL, filtering the lysozyme solution through a filter into a crystallizer, and adding polyaspartic acid modified Fe 3 O 4 Microspheres were thoroughly mixed with lysozyme and then added to the NaCl solution. The lysozyme-containing solution was stirred at a temperature of 20℃for 24 hours at a stirrer peripheral line speed of 0.52 m/s.
(5) Separating the magnetic lysozyme crystal prepared in the step (4) by using a magnet, and washing the magnetic lysozyme crystal with distilled water for 4 times to remove impurities attached to the lysozyme crystal, thereby obtaining the magnetic lysozyme crystal.
Example 2
The embodiment provides a magnetic lysozyme crystal, which is prepared by the following steps:
(1) ZnFe with particle size ranging from 250 to 300 nanometers 2 O 4 Mixing particles and polyethyleneimine in distilled water according to a mass ratio of 8:1, and then accurately adjusting the pH to 6;
(2) Ultrasound to make nano ZnFe 2 O 4 The system of particles and polyethylenimine and deionized water was thoroughly mixed for 12 hours.
(3) Separating the black product obtained in the step (2) by using a magnet, and washing with distilled water for 4 times to remove excessive polyethyleneimine, thereby obtaining polyethyleneimine-modified Fe 3 O 4 Microspheres for standby.
(4) Dissolving NaCl and lysozyme in 0.1mol/L sodium acetate buffer solution with pH=4.6 to reach NaCl concentration of 58.44mg/mL and lysozyme concentration of 25 mg/mL, filtering the lysozyme solution through a filter into a crystallizer, and adding polyethyleneimine modified ZnFe 2 O 4 Microspheres were thoroughly mixed with lysozyme and then added to the NaCl solution. The lysozyme-containing solution was stirred at a temperature of 20℃for 24 hours at a stirrer peripheral line speed of 0.52 m/s.
(5) Separating the magnetic lysozyme crystal prepared in the step (4) by using a magnet, and washing the magnetic lysozyme crystal with distilled water for 4 times to remove impurities attached to the lysozyme crystal, thereby obtaining the magnetic lysozyme crystal.
Example 3
The embodiment provides a magnetic lysozyme crystal, which is prepared by the following steps:
(1) CoFe with particle size of 200-250 nm 2 O 4 Mixing the particles and sodium chondroitin sulfate in distilled water according to a mass ratio of 8:3, and then accurately regulating the pH valueTo 10.
(2) Nano CoFe by stirring 2 O 4 The system of particles and sodium chondroitin sulfate and deionized water was thoroughly mixed for 8 hours.
(3) Separating the black product obtained in the step (2) by using a magnet, and washing the black product with distilled water for 4 times to remove excessive chondroitin sulfate sodium, thereby obtaining the chondroitin sulfate sodium modified CoFe 2 O 4 Microspheres for standby.
(4) Dissolving lysozyme in sodium acetate buffer solution with the concentration of 0.1mol/L and the pH value of 4.6, wherein the concentration of the lysozyme is 21.87mg/mL, filtering the lysozyme solution through a filter, and then adding sodium chondroitin sulfate modified CoFe 2 O 4 Microspheres, which are thoroughly mixed with lysozyme. Freezing the system at-20deg.C, and vacuum sublimating and drying to remove crystal water to obtain crystal and amorphous mixture of magnetic lysozyme.
Example 4
The embodiment provides a magnetic lysozyme crystal, which is prepared by the following steps:
(1) MnFe with particle size ranging from 350 to 400 nanometers 2 O 4 Mixing particles and polylactic acid in distilled water according to a mass ratio of 8:3, and then accurately regulating the pH value to 6;
(2) Nano MnFe is obtained by oscillation 2 O 4 The system of particles and polylactic acid and deionized water was thoroughly mixed for 7 hours.
(3) Separating the black product obtained in the step (2) by using a magnet, and washing the black product with distilled water for 4 times to remove excessive polylactic acid, thereby obtaining polylactic acid modified MnFe 2 O 4 Microspheres for standby.
(4) Dissolving lysozyme in sodium acetate buffer solution with the concentration of 0.1mol/L and the pH value of 4.6 to reach the concentration of 25 mg/mL of lysozyme, filtering the lysozyme solution into a crystallizer through a filter, and then adding polylactic acid modified MnFe 2 O 4 The microspheres were thoroughly mixed with lysozyme and then evaporated under reduced pressure at a pressure of 2.5kPa, and stirred for 8 hours at a peripheral linear velocity of 0.52 m/s.
(5) Separating the magnetic lysozyme crystal prepared in the step (4) by using a magnet, and washing the magnetic lysozyme crystal with distilled water for 4 times to remove impurities attached to the lysozyme crystal, thereby obtaining the magnetic lysozyme crystal.
Example 5
The embodiment provides a magnetic bovine insulin crystal, which is prepared by the following steps:
(1) Fe with particle size of 100-150 nm 3 O 4 The particles and polyaspartic acid were mixed in distilled water at a mass ratio of 8:3, and then the pH was precisely adjusted to 6.
(2) Nano Fe by ultrasonic wave and oscillation 3 O 4 The system of particles and polyaspartic acid and deionized water was thoroughly mixed for 12 hours.
(3) Separating the black product obtained in the step (2) by using a magnet, and washing with distilled water for 4 times to remove excessive polyaspartic acid, thereby obtaining polyaspartic acid modified Fe 3 O 4 Microspheres for standby.
(4) Adding bovine insulin into HCl solution with pH=2 to dissolve, adding ZnCl 2 And acetone are added into sodium citrate buffer solution with the concentration of 0.48mol/L and the pH value of 6.58, so that ZnCl is added into the sodium citrate buffer solution 2 The concentration of (C) is 0.005 mol.L -1 Mixing bovine insulin solution and precipitant solution, and finally adding polyaspartic acid modified Fe obtained in step (3) to obtain the final product 3 O 4 And (3) microspheres. The system was left to crystallize at room temperature for 8 hours.
(5) Separating the magnetic bovine insulin crystals prepared in the step (4) by using a magnet, and washing the magnetic bovine insulin crystals with a sodium citrate buffer solution for 4 times to remove impurities attached to protein crystals, thereby obtaining the magnetic bovine insulin crystals.
Example 6
The embodiment provides a magnetic bovine insulin crystal, which is prepared by the following steps:
(1) ZnFe with particle size ranging from 100 to 150 nanometers 2 O 4 Mixing the particles and the sodium chondroitin sulfate in distilled water according to a mass ratio of 8:3, and thenThe pH was then adjusted to exactly 10.
(2) Oscillating and stirring the nano ZnFe 2 O 4 The system of particles and sodium chondroitin sulfate and deionized water was thoroughly mixed for 12 hours.
(3) Separating the black product obtained in the step (2) by using a magnet, and washing the black product with distilled water for 4 times to remove excessive chondroitin sulfate sodium, thereby obtaining the ZnFe modified by the chondroitin sulfate sodium 2 O 4 Microspheres for standby.
(4) Adding bovine insulin into HCl solution with pH=2 to dissolve, and adding ZnSO 4 And phenol are added into sodium citrate buffer solution with the concentration of 0.48mol/L and the pH value of 6.58, so that ZnCl is added into the sodium citrate buffer solution 2 The concentration of (C) is 0.005 mol.L -1 Mixing bovine insulin solution and precipitant solution, adding ZnFe modified by chondroitin sulfate sodium obtained in step (3) at the concentration of phenol of 15% (V/V) 2 O 4 And (3) microspheres. The system was left to crystallize at room temperature for 8 hours.
(5) Separating the magnetic bovine insulin crystals prepared in the step (4) by using a magnet, and washing the magnetic bovine insulin crystals with a sodium citrate buffer solution for 4 times to remove impurities attached to the bovine insulin crystals, thereby obtaining the magnetic bovine insulin crystals.

Claims (4)

1. A preparation method of magnetic protein crystals is characterized in that a magnetic nucleating agent is mixed with a protein solution, the supersaturation degree of the protein solution is regulated, so that protein is crystallized and separated on the magnetic nucleating agent, and the crystals are separated by a magnet, washed and dried to obtain the magnetic protein crystals; regulating supersaturation degree of protein solution by adding at least one of precipitant, cooling, freeze drying or reduced pressure evaporation; the precipitant is selected from NaCl, znSO 4 、ZnCl 2 、ZnAc、 ZnBr 2 At least one of acetone, phenol, m-cresol, o-cresol or p-cresol;
the protein is selected from at least one of enzyme, hormone or monoclonal antibody;
the preparation method of the magnetic nucleating agent comprises the following steps:
mixing magnetic particles with a polymer solution to coat the polymer on the outer surfaces of the magnetic particles, thereby obtaining a magnetic nucleating agent; or mixing the magnetic particles with a monomer, and forming a polymer on the magnetic particles through polymerization reaction to coat the polymer on the outer surfaces of the magnetic particles to obtain the magnetic nucleating agent;
the magnetic particles are nano particles with the particle size of 10-800 nanometers;
the magnetic particles are selected from MnFe 2 O 4 、Fe 3 O 4 、ZnFe 2 O 4 Or CoFe 2 O 4 One of the following;
the high molecular compound is selected from one of polyaspartic acid, polyethyleneimine, sodium chondroitin sulfate or polylactic acid.
2. The method for producing a magnetic protein crystal according to claim 1, wherein the particle size of the magnetic particles is 50 to 400 nm.
3. The method for preparing a magnetic protein crystal according to claim 1, wherein the mixing of the magnetic nanoparticles with the polymer solution comprises at least one of shaking, stirring and ultrasound.
4. A method for producing a magnetic protein crystal according to claim 3, wherein,
the mixing time of the magnetic nano particles and the macromolecule solution is 2-12 hours.
CN202011377415.2A 2020-12-01 2020-12-01 Preparation method of magnetic protein crystal, magnetic protein crystal and application Active CN112552372B (en)

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CN113980093A (en) * 2021-10-25 2022-01-28 河北工业大学 Method for promoting protein medicine crystallization by polymer and application
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US10705079B2 (en) * 2014-05-29 2020-07-07 Shenzhen New Industries Biomedical Engineering Co., Ltd. Method for preparing magnetic microsphere for separation of biological protein and use thereof
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