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CN113980093B - Method for promoting crystallization of protein medicine by polymer and application - Google Patents

Method for promoting crystallization of protein medicine by polymer and application Download PDF

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Publication number
CN113980093B
CN113980093B CN202111251647.8A CN202111251647A CN113980093B CN 113980093 B CN113980093 B CN 113980093B CN 202111251647 A CN202111251647 A CN 202111251647A CN 113980093 B CN113980093 B CN 113980093B
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protein
solution
polymer
crystallization
lysozyme
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CN113980093A (en
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苏敏
吕凤
张清洋
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Hebei University of Technology
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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
    • 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

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Endocrinology (AREA)
  • Diabetes (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The invention relates to a method for promoting crystallization of protein drugs by using a polymer. The method comprises the following steps: adding the polymer into the protein solution, regulating the supersaturation degree of the protein solution to be 10-15, and stirring for 0.5-6 hours to obtain protein crystals; the obtained protein crystal is used for protein structure analysis or biological pharmacy as the composition of a targeted pharmaceutical preparation. The method has the advantages of simple process flow and short operation time, can improve the crystallization efficiency of protein medicines, can shorten the process of protein separation and purification, and has good application prospect.

Description

Method for promoting crystallization of protein medicine by polymer and application
Technical Field
The invention relates to the technical field of biological medicines, in particular to a method for crystallizing macromolecular protein medicines and application thereof.
Background
The protein medicine crystal has wide application prospect in the fields of protein medicine production, purification and the like, but the crystallization time of a plurality of proteins is long and the efficiency is low due to the large molecular weight of the proteins, which limits the application of the protein medicine crystal in actual production.
In the biotechnology field of researching protein crystallization, CN107814831 adopts a hanging drop method, and oxygen-removing ribonucleic acid is used for promoting protein crystallization, so that the success rate of protein crystallization in hanging drops can be improved; CN105566443 uses the method of cross-linking protein crystal to promote protein crystallization, and improves the success rate of protein crystallization condition screening; in CN106188220, the use of cyclodextrin and its derivatives as seed crystals improves the success rate of protein crystallization. However, due to the types of additives and crystallization modes, the above methods have the defects of strict limit on crystallization temperature, longer crystallization time and the like, for example, in order to ensure the activity of DNA, the experiment needs to be performed in a constant temperature box at 4 ℃; the crystallization time of CN105566443 is 3-7 days.
Disclosure of Invention
The invention provides a method for promoting crystallization of protein drugs by using polymers in order to overcome the defects of the prior art. The method uses the interaction between the protein and the polymer to accelerate the nucleation and crystallization of the protein by adding the high molecular polymer into the protein solution as the additive. The method has the advantages of simple process flow and short operation time, can improve the crystallization efficiency of protein medicines, overcomes the defects of long protein crystallization time and low efficiency in the traditional technology, can shorten the process of protein separation and purification, and has good application prospect.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for promoting crystallization of protein drugs by using polymers, comprising the following steps:
Adding the polymer into the protein solution, then adjusting the supersaturation degree of the protein solution to be 10-15, obtaining supersaturated solution, and stirring for 0.5-6 hours; obtaining protein crystals;
Wherein the mass ratio of the protein to the polymer m Proteins /m Polymer =5-15; the concentration of protein in the supersaturated solution is 1-20 mg/mL;
the method for regulating the supersaturation degree of the protein solution comprises the steps of adding a precipitator, cooling, freeze drying or decompressing and evaporating;
the method for adding the precipitant for adjusting the supersaturation degree of the protein solution is preferably adding a salting-out precipitant;
the precipitant is one or more of NaCl solution, znSO 4 solution, znCl 2 solution, znAc solution, znBr 2 solution, acetone, phenol, m-cresol, o-cresol and p-cresol;
The polymer is a polymer with carboxyl, amino or hydroxyl;
the polymer is an alcohol, phenol, amine, or organic acid.
The polymer is preferably polyaspartic acid, polyethylenimine, polylactic acid or polyethylene glycol.
The protein is oligopeptide, polypeptide, enzyme, hormone, nucleic acid or monoclonal antibody.
The solvent of the protein solution is a buffer solution, preferably a sodium acetate buffer solution, a sodium citrate buffer solution or hydrochloric acid.
The protein medicine prepared by the method is applied to protein structure analysis or composition of a target medicine preparation in biological pharmacy.
The invention has the beneficial effects that:
According to the invention, the polymer is mixed with the solution containing the protein, so that protein crystallization is promoted, the protein crystallization time is effectively shortened, and the protein crystallization efficiency is improved. By the invention, the improvement of the protein crystallization induction rate can be more than 2 times of that of a control group without adding polymer in the prior art at least. The invention has wide application prospect in the fields of protein medicine production, purification, crystallization and the like.
Drawings
FIG. 1 is a crystal diagram of the crystals of the lysozyme promoted by polyaspartic acid obtained in example 1.
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 invention provides a method for promoting crystallization of protein drugs by using a polymer, which comprises the following steps:
mixing the polymer with the protein solution, and regulating the supersaturation degree of the protein solution to obtain protein crystals.
In the present invention, protein solution refers to a protein-containing solution system in which solutes include, but are not limited to, proteins.
According to the invention, the polymer is introduced into the protein solution to promote the crystallization of the protein, so that the crystallization time of the protein is effectively shortened, the crystallization efficiency of the protein is improved, and the technology has a wide application prospect in the field of protein medicine production.
[ 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 aspect of the invention, the precipitant 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.
[ Polymer promotes crystallization of protein ]
In one embodiment of the present invention, the polymer is mixed with a protein solution to adjust the supersaturation degree of the protein solution, thereby obtaining protein crystals. Filtering, washing and drying the crystals to obtain protein crystals.
In one embodiment of the invention, the polymer is a polymer including, but not limited to, alcohols, or phenols, or amines, or organic acids, and the like.
In one embodiment of the invention, the protein is a polypeptide, or an enzyme, or a hormone, or a nucleic acid, or a monoclonal antibody, or the like, including but not limited to.
In one embodiment of the invention, the final concentration of the protein solution includes, but is not limited to, 1mg/mL, or 5mg/mL, or 10mg/mL, or 15mg/mL, or 20mg/mL, etc.
The method for promoting protein crystallization by using the polymer provided by the invention is simple to operate, shortens the crystallization time of the protein, improves the crystallization efficiency of the protein, and has wide application prospect in the field of protein medicine 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 method for promoting lysozyme crystallization by polyaspartic acid, which comprises the following steps:
(1) NaCl and lysozyme (300 mg) were dissolved in a sodium acetate buffer solution (8 mL, pH value was adjusted with acetic acid) having a concentration of 0.1mol/L and pH=4.6, the dissolved lysozyme solution was filtered through a filter having a pore size of 0.2 μm into a crystallizer, then 30mg of polyaspartic acid was filtered into the crystallizer so as to be sufficiently mixed with lysozyme (i.e., the mass ratio of protein to polymer was m Proteins /m Polymer =10), and finally the dissolved NaCl solution was filtered into the crystallizer (i.e., the volume ratio was 1:1, at this time, the supersaturation degree value was 13.02), to obtain a protein solution having a final concentration of lysozyme of 18.75 mg/mL. The lysozyme-containing solution was stirred at 20℃and a peripheral linear velocity of 0.52m/s at a stirring paddle, and the solution began to be cloudy for about 2 hours, and small crystals were precipitated, and a large amount of crystals were obtained for 6 hours.
(2) Filtering the lysozyme crystal prepared in the step (1), and washing with distilled water for 4 times to remove impurities attached to the lysozyme crystal, thereby obtaining the lysozyme crystal.
As can be seen from FIG. 1, the lysozyme obtained by the invention has consistent crystal morphology and uniform granularity (the particle size is 10-20 μm), which shows that the addition of the polymer can effectively promote the crystallization of protein and improve the crystallization efficiency of protein. The protein crystal with the quality completely meets the requirements of stable crystal property, uniform granularity and easy storage, and can be applied to protein structure analysis or bio-pharmaceuticals as a targeted pharmaceutical preparation.
Example 2
The embodiment provides a method for promoting lysozyme crystallization by polyethylene glycol, which comprises the following steps:
(1) NaCl and lysozyme (300 mg) were dissolved in a sodium acetate buffer solution (8 mL, pH value was adjusted with acetic acid) having a concentration of 0.1mol/L and pH=4.6, the dissolved lysozyme solution was filtered through a filter having a pore size of 0.2 μm into a crystallizer, then 30mg of polyethylene glycol was filtered into the crystallizer so as to be thoroughly mixed with lysozyme (i.e., the mass ratio of protein to polymer was m Proteins /m Polymer =10), and finally the dissolved NaCl solution was filtered into the crystallizer (i.e., the volume ratio was 1:1, at this time, the supersaturation degree value was 13.02), to obtain a protein solution having a final concentration of lysozyme of 18.75 mg/mL. The lysozyme-containing solution was cloudy at a peripheral line speed of 0.52m/s at 20℃for about 2 hours, and small crystals were precipitated and a large amount of crystals were obtained for 6 hours.
(2) Filtering the lysozyme crystal prepared in the step (1), and washing with distilled water for 4 times to remove impurities attached to the lysozyme crystal, thereby obtaining the lysozyme crystal.
Example 3
The embodiment provides a method for promoting insulin crystallization by polyaspartic acid, which comprises the following steps:
(1) Bovine insulin (40 mg) was dissolved in HCl solution (8 mL) at ph=2 to obtain bovine insulin solution having a concentration of 5.00 mg/mL. Then acetone and zinc sulfate were added to a sodium citrate buffer solution (8 mL, pH was adjusted with NaOH) at a concentration of 0.48mol/L, pH =6.58 to obtain a precipitant solution at an acetone concentration of 10.0% (v/v) and a zinc sulfate concentration of 0.1% (w/w), bovine insulin solution and precipitant solution were mixed (i.e., volume ratio of 1:1), then 4mg polyaspartic acid was added to the mixed solution (i.e., mass ratio of protein to polymer m Proteins /m Polymer =10), and the system was left to crystallize at room temperature for 3.5 hours to obtain a large amount of crystals.
(2) Filtering the insulin crystal prepared in the step (1), and washing with a sodium citrate buffer solution for 4 times to remove impurities attached to the insulin crystal, thereby obtaining the insulin crystal.
Comparative example 1
NaCl and lysozyme (300 mg) were dissolved in a sodium acetate buffer (8 mL, pH adjusted with acetic acid) at a concentration of 0.1mol/L and pH=4.6, respectively, and the dissolved lysozyme solution and NaCl solution were filtered through a filter having a pore size of 0.2 μm into a crystallizer to obtain a protein solution having a final concentration of lysozyme of 18.75 mg/mL. The lysozyme-containing solution was stirred at a temperature of 20℃for 15 hours at a stirrer peripheral line speed of 0.52m/s to obtain lysozyme crystals.
Comparative example 2
Bovine insulin (40 mg) was first dissolved in HCl solution (8 mL) at ph=2 to obtain bovine insulin solution having a concentration of 5.00 mg/mL. Then, acetone and zinc sulfate were added to a sodium citrate buffer solution (8 mL, pH was adjusted with NaOH) having a concentration of 0.48mol/L, pH =6.58 to obtain a precipitant solution having an acetone concentration of 10.0% (v/v) and a zinc sulfate concentration of 0.1% (w/w), the bovine insulin solution and the precipitant solution were mixed, and the system was left to crystallize at room temperature for 8 hours to obtain insulin crystals.
As can be seen from the above examples and comparative examples, the crystallization of proteins can be promoted by mixing the polymer with a solution containing proteins, thereby effectively shortening the crystallization time of proteins and improving the crystallization efficiency of proteins. The protein crystallization induction rate is more than 2 times of that of a control group without the added polymer.
The invention is not a matter of the known technology.

Claims (1)

1. A method for promoting crystallization of protein medicines by using polymers, which is characterized by comprising the following steps:
adding a polymer into a protein solution, adjusting the supersaturation degree of the protein solution to be 10-15, obtaining a supersaturated solution, and stirring for 0.5-6 hours to obtain protein crystals;
wherein the mass ratio of the protein to the polymer is m Proteins /m Polymer = 5-15; the concentration of protein in the supersaturated solution is 1-20 mg/mL;
the polymer is polyaspartic acid;
the method for regulating the supersaturation degree of the protein solution is to add a precipitator;
the precipitant is one or more of NaCl solution, znSO 4 solution, znCl 2 solution, znAc solution, znBr 2 solution, acetone, phenol, m-cresol, o-cresol and p-cresol;
the solvent of the protein solution is buffer solution, which is sodium acetate buffer solution, sodium citrate buffer solution or hydrochloric acid;
the protein is an enzyme or hormone.
CN202111251647.8A 2021-10-25 2021-10-25 Method for promoting crystallization of protein medicine by polymer and application Active CN113980093B (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102060907A (en) * 2010-11-04 2011-05-18 西北工业大学 96 kit for screening protein crystallization
CN113015748A (en) * 2018-10-31 2021-06-22 默沙东公司 Anti-human PD-1 antibody crystals and methods of use thereof

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7229500B2 (en) * 2000-11-20 2007-06-12 Parallel Synthesis Technologies, Inc. Methods and devices for high throughput crystallization
WO2008102469A1 (en) * 2007-02-23 2008-08-28 Kwansei Gakuin Educational Foundation Protein crystallizing agent and method of crystallizing protein therewith
CN103333219A (en) * 2013-04-17 2013-10-02 西北工业大学 Method for selecting buffer solution for protein crystallization
CN105255847A (en) * 2015-11-17 2016-01-20 天津大学 Method for using ionic liquid for regulating and controlling solubility of lysozyme to prepare lysozyme crystals
US10655168B2 (en) * 2017-12-22 2020-05-19 Pacific Biosciences Of California, Inc. Modified biotin-binding proteins for immobilization
CN111450403A (en) * 2020-04-03 2020-07-28 南开大学 Microneedle array for rapid transdermal delivery of protein drugs and preparation method thereof
CN112552372B (en) * 2020-12-01 2023-05-02 河北工业大学 Preparation method of magnetic protein crystal, magnetic protein crystal and application

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102060907A (en) * 2010-11-04 2011-05-18 西北工业大学 96 kit for screening protein crystallization
CN113015748A (en) * 2018-10-31 2021-06-22 默沙东公司 Anti-human PD-1 antibody crystals and methods of use thereof

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