JP2006213678A - Microcapsule composition and method for exposing enzyme or protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To develop a method for stabilizing an enzyme or a protein with a microcapsule and a polysaccharide. <P>SOLUTION: The microcapsule composition comprises embedding a polysaccharide such as gum arabic, carrageenan, gelatin, trehalose, chitosan, etc., in a microcapsule obtained by combining a virus capsid protein with an enzyme or a protein and contributes to stabilization of an enzyme or a protein by mixing them. An enzyme catalytic reaction is carried out by exposing the embedded enzyme and the composition is applied to a detergent, etc. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention uses a virus capsid or the like, produces a microcapsule in which an enzyme or protein is embedded, and prepares a composition mixed with a polysaccharide to stably hold the enzyme or protein inside. Provide technology.

Enzymes are widely used in various industrial fields such as detergents, refining of textile products, decomposition of fats and oils, food processing, pharmaceuticals, clinical tests, cosmetics, and conversion and production of useful substances. The problem in measuring such utilization is that the enzyme stability is generally low and the requirements are often unsatisfactory. Attempts have been made to chemically modify enzymes as a way to deal with these problems. For example, a method of improving the thermal stability by modifying a proteolytic enzyme with polyethylene glycol (Patent Document 1), or a method of encapsulating the enzyme in a polymer shell and measuring stabilization (Patent Document 2) has been proposed. ing.
JP 2000-017299 A Special table 2000-503051 gazette

  The present invention uses genetic recombination technology to synthesize viruses, especially human hepatitis B virus capsid protein or a fusion protein of a capsid protein and a target protein, and encapsulates the enzyme or protein in microcapsules obtained under appropriate conditions. An object of the present invention is to provide a technique that makes it possible to stably retain an internal enzyme or protein by embedding and further preparing a composition mixed with a polysaccharide. Among them, enzymes are generally low in stability and often cannot satisfy the requirements. That is, most enzymes are easily denatured and inactivated by long-term storage under the coexistence of a mixture of a surfactant, an organic solvent, or the like under extremely high pH conditions. In particular, when the enzyme is a protease, in the medium with a high water content or in a dosage form such as an aqueous solution, in addition to denaturation, autolytic digestion occurs, and it is quickly deactivated during storage at room temperature. There is a problem that is difficult. For application to the conversion and production of useful substances, it has been desired to develop an enzyme with higher activity and stability.

  In the present invention, a microcapsule formed by a capsid protein of a virus embeds an enzyme or protein therein, thereby stably retaining the activity of the internal protein. When the enzyme is a protease, the microcapsules prevent self-digestion degradation even in solution, and the enzyme activity is not inactivated even when stored at room temperature. Moreover, it can be stably maintained even in a solution state by allowing the polysaccharide to coexist.

  The present invention provides the following.

  1. A microcapsule composition comprising a protein having a particle-forming ability and a polysaccharide.

  2. 2. The microcapsule composition according to 1 above, wherein an enzyme or protein is embedded in the microcapsule.

  3. 3. The microcapsule composition according to 2 above, wherein a protein having particle forming ability and an enzyme or protein are fused.

  4). 4. The microcapsule composition according to any one of 1 to 3 above, wherein the polysaccharide is at least one selected from gum arabic, carrageenan, trehalose and chitosan.

    5. 5. The microcapsule composition according to any one of 1 to 4 above, wherein the protein having particle-forming ability is a viral capsid protein or a mutant protein thereof.

  6). 6. The microcapsule composition according to 5 above, wherein the capsid protein is a capsid protein of Hepadnaviridae virus.

  7). 7. The microcapsule composition according to 6 above, wherein the hepadnavirus is hepatitis B virus.

  8). An enzyme characterized by expressing an enzyme or protein embedded in a microcapsule by adding a surfactant or an alkaline solution to the microcapsule composition according to any one of 2 to 7 above Or protein expression method.

  In the present invention, a microcapsule formed by a capsid protein of a virus can be stably maintained by using a composition in which a polysaccharide is mixed with a polysaccharide. In particular, when an enzyme or protein is embedded in a microcapsule, the activity of the enzyme or protein can be stably maintained. Furthermore, the enzyme or protein embedded in the microcapsule can be expressed by adding a surfactant or an alkaline solution to the composition containing the microcapsule. As an application of this technology, when the enzyme is a protease, the microcapsule prevents autolysis and digestion even in a dosage form such as an aqueous solution, and supplies a stable product that does not deactivate the enzyme activity even when stored at room temperature. It becomes possible.

(1. Particle-forming protein)
As a protein having particle forming ability, a protein called polyhedrin encoded by an insect virus and further subviral particles obtained from various viruses can be applied. For example, a wide range of viruses such as bacteriophages that infect microorganisms, animal viruses, plant viruses, invertebrate viruses, and algal viruses can be used. More preferably, it is a capsid derived from a spherical virus, such as hepatitis B virus core protein, adenovirus, animal virus such as poliovirus, phage such as φX174, MS2, cauliflower mosaic virus, tomato bushy stunt virus, rice dwarf virus, etc. Capsid proteins of algal viruses such as plant viruses, invertebrate viruses such as goatboyidescent virus and black beetle virus, and Paramecium symbiotic algal virus can be used.

(2. About microcapsules)
A microcapsule is a very small container of a sphere having a sphere diameter of approximately several microns (1 micron = 1/1600 millimeter) to 1 millimeter, and is manufactured by various techniques. The microcapsule makes it possible to handle the substance embedded in the inside as follows.
・ Apparently it can be handled as a solid. (Modification of form).
-Reaction and mixing with other substances can be avoided (sequestration effect).
-The storage period can be extended (stabilization effect).
・ Unstable elements can be stabilized (protective effect).
・ Release can be controlled (release control).

  That is, by encapsulating the contents, the substance embedded inside is protected, and it is isolated so that it does not react with other substances or mix until necessary, or released to the outside. It becomes possible to control the speed.

(3. Selection of microcapsules)
The microcapsule used in the present invention is preferably composed of a capsid protein, and its origin may be derived from any virus. “Capsid” refers to a shell structure that encloses the nucleic acid of a virus and forms its external form. In addition, when a nucleic acid of a virus or a variant thereof is necessary for the formation of a shell structure, the nucleic acid is also referred to as “capsid”. When multiple shell structures are covered, such as hepatitis B virus, all shell structures are capsids. “Capsid protein” refers to a protein that is a constituent of capsid. However, microcapsules are not limited to capsid proteins. It may be a cylindrical aggregate such as phycocyanin, or may be a microcapsule having a space in which a target molecule can be introduced inside an artificially designed interior.

  Capsids and phycocyanins are known to have a stable and strong structure even in a state where nothing is contained therein, which is advantageous for producing the microcapsules of the present invention.

  Many capsid proteins are advantageous for the present invention because their amino acids and the nucleic acid sequences that encode them are known and can be prepared by genetic engineering techniques, and can contain molecules inside. In particular, the capsid protein of the hepadnaviridae virus to which hepatitis B virus belongs can be easily prepared and purified by genetic engineering techniques using Escherichia coli and the like, and is stable and strong even in a state where nothing is contained inside. Because of its structure, it is convenient for making the microcapsules of the present invention.

An appropriate virus capsid can be selected according to the size and shape of the enzyme or protein to be embedded. The size of the microcapsule can be measured by the atomic coordinates of the X-ray crystal structure analysis result of the microcapsule crystal. The size and shape of the entire encapsulated enzyme / protein molecule can be measured using an electron microscope, an atomic force microscope, X-ray small angle scattering, or the like. For example, it is known that the inner diameter of the core protein (HBcAg) of the hepatitis B virus, which is a microcapsule, that is, the inner radius is about 11.2 nm and the inner surface area is about 1576 nm ² as a result of X-ray crystal structure analysis. (Samantha A. Wynne et al. :) Molecular Cell, The Crystal Structure of the Human Hepatitetes B. Viros Capsid human hepatitis B virus capsid.), USA, 1999, Vol. 3, p. 771-780, Fig. 1). Since this microcapsule is formed of 240 capsid proteins, the surface area inside the microcapsule per molecule is about 6.6 nm ² . Therefore, it can be determined that molecules having a cross-sectional area smaller than 6.6 nm ² can be embedded by binding to the viral capsid protein. Further, the length of the molecule needs to be 11.2 nm or less of the inner diameter of the microcapsule. That is, it is preferable to bind molecules having a cross-sectional area of 6.6 nm ² or less and a length of 11.2 nm or less to the microcapsules. Whether or not molecules can be encapsulated inside the microcapsule can be determined by observing the atomic coordinates of the microcapsule using an appropriate computer program. When encapsulating a larger protein or the like, a microcapsule having a large surface area such as bacteriophage φX174 or rhinovirus can be used. When analyzing very small proteins, cylindrical microcapsules such as phycocyanin can also be used.

  The outer shell of the Hepadnaviridae virus is composed of a double capsid with an outer envelope containing a lipid membrane and an inner core. In particular, the inner core capsid is known to form stable microcapsules and is therefore advantageous as a microcapsule for use in the present invention. Among them, the core protein of hepatitis B virus (HBcAg) and its mutant protein have known conditions for forcibly forming microcapsules, and their atomic coordinates are registered and published in PDB. It is particularly convenient to use as a microcapsule to be used. For example, those encoded by the DNA described in SEQ ID NO: 1 are preferred. A mutant protein is a protein in which part of the amino acid constituting the protein is substituted with another amino acid, or part of the sequence is inserted or deleted, Represents a protein that retains its function as a core protein. Similarly, a nucleic acid in which a partial sequence of nucleobases constituting a nucleic acid is replaced with another nucleobase, or a partial sequence is inserted or deleted is referred to as a “mutant (nucleic acid)”.

(4. Embedding enzymes or proteins in microcapsules)
The “inside of the microcapsule” used in the present invention means a space that does not affect the interaction between proteins constituting the microcapsule in the microcapsule. In addition, the state in which the protein is arranged inside the microcapsule is said to be “embedded in the microcapsule”. For example, when a cylindrical microcapsule such as phycocyanin is used as the aggregate, the molecule to be bound does not need to be completely covered with the microcapsule, and the interaction between the viral capsid proteins is affected in the microcapsule. If it exists inside the cylinder that is not given, the bound molecule is “inside the microcapsule” and is “embedded in the microcapsule”. In addition, the “enzyme or protein” used in the present invention represents one embedded in the viral capsid protein. As a method of embedding an enzyme or protein inside the microcapsule, the microcapsule formed only from the capsid protein is decomposed under a low salt or alkaline condition, placed in a solution of a high concentration enzyme or protein, and then again in a high salt condition. Then, by reconstituting the capsid protein under low acid conditions, a microcapsule in which an enzyme or protein is embedded can be obtained. The morphogenic linker peptide of HBV capsid protein forms a mobile from the EMBO Journal vol21 No5 pp876-884 2002 In the array on the interior surface, the capsid protein expressed in E. coli is purified under alkaline conditions, and reconstituted into microcapsules by treatment in acidic conditions (pH 5) at 21 ° C. for 1 hour in the presence of 60 mM MgCl 2. However, in this report, no enzyme or protein is actually embedded in the microcapsule.

(5. Embedding enzymes and proteins in microcapsules using fusion proteins)
The binding between the capsid protein of the microcapsule and the target protein can be achieved by preparing a fusion protein obtained by fusing the capsid protein, the target protein, and the protein constituting each of the capsid protein by conventional genetic manipulation. The term “fusion” used in the present invention means that both proteins are bound by a peptide bond. Usually, DNA that encodes a protein or peptide is inserted before or after or in the middle of DNA encoding the protein, and the fusion protein is prepared using the DNA. However, the fusion protein can also be obtained by other methods, for example, chemical synthesis. . The site where the target protein is fused to the capsid protein is preferably immediately before or after the amino acid exposed inside the microcapsule.

  In fusion binding, as long as there is a gene, the target molecule for analysis can be easily linked by conventional genetic manipulation, and the expression vector incorporating the gene can be easily amplified. Create fused proteins. Therefore, there is no need to control the binding site, and the fusion bond creates microcapsules having the same molecular species, the same number of molecules, and the same three-dimensional structure as compared with the bond that forms the covalent bond described later. It is one of the effective means for this.

(6. Sequence design of fusion protein)
Design of DNA for producing a fusion protein in which a capsid protein is fused with an enzyme or a protein can be performed by conventional genetic manipulation as follows. For example, by synthesizing a DNA that is partially complementary to the HBcAg gene using a known genetic engineering technique, a portion (restriction enzyme site) that can be cleaved with a specific restriction enzyme at any part of the HBcAg gene The DNA encoding any protein can be introduced before or after the introduced restriction enzyme site or the introduced restriction enzyme site.

(7. Obtaining capsid protein sequence information and genes)
The capsid protein gene, that is, the DNA encoding the amino acid sequence, can be isolated from a virus-infected patient, animal, cell, or microorganism by PCR. For example, in the case of the hepatitis B virus core protein (HBcAg), from a cDNA library extracted from the serum of chronic active hepatitis B infected patients, for example, literature (Antoine Touze et al., Journal)・ Journal of Clinical Microbiology, Baculobilos Expression of Chimeric Hepatitetes B Biros Core Particles with Hepatitetes E Biroth Epitopes and There Youth・ Baculovirus Expression of Chimeric Hepatitis B Virus Core Particles with Hepatitis E Virus Epitopes and Their Use in a Hepatitis E Immunoassay, USA, 1999, Vol. 37, p.438- 441) can be used for isolation by PCR. Capsid protein genes of viruses other than hepatitis B virus can be isolated in the same manner. Primers necessary for isolation can be designed using the sequence information of each virus gene. Viral DNA and amino acid sequence information is registered in the NCBI genome database, for example, and published on the Internet. Genes other than the viral capsid can be obtained in the same manner. For example, the phycocyanin gene of Example X can be isolated by PCR using a cDNA library extracted from cyanobacteria.

  In addition, if it cannot be isolated by PCR, or if it is artificially designed, DNA that has been partially chemically synthesized according to the DNA or amino acid sequence information of the protein that forms the microcapsule can be joined using DNA polymerase, etc. The gene can be created. The bacteriophage gene can be obtained, for example, from the National Institute of Technology and Evaluation / National Biological Resource Center (NBRC) for a fee.

(8. Obtaining the target protein)
The target protein may be obtained by any means. It can usually be obtained by using any of the following methods. These include chemical synthesis, isolation / extraction from substances containing the target protein, such as animals, plants and microorganisms, and protein expression using a gene encoding the protein that is the target protein. The same applies when producing the target protein by fusing it with the capsid protein. The gene of the target protein can also be obtained in the same manner as the capsid gene.

(9. Preparation of expression vector)
A gene, that is, a PCR product or chemically synthesized DNA can be cut out and purified using an appropriate restriction enzyme and incorporated into an expression vector. In the case of PCR method, the expression vector can be created more easily by incorporating the sequence (restriction enzyme site) that can be cleaved with a specific restriction enzyme into the primer used in the case of chemical synthesis. become. As the expression vector, an expression vector suitable for the host is preferably used depending on the type of host into which the expression vector is to be incorporated.

(10. Preparation of microcapsules)
A microcapsule can be prepared using a gene recombinant for conventional protein production. For example, an expression vector into which a capsid protein gene has been incorporated is incorporated into a host such as a microorganism such as Escherichia coli, yeast, a plant body or plant cell, an animal cell or transgenic animal, an insect cell or an insect together with a liposome, etc. It is possible to convert and protein expression. Moreover, it can also create using a cell-free protein expression system, without using a host. A cell-free protein expression kit is commercially available from, for example, Roche Research, and is a useful means that can produce a protein simply and in a short time.

  When the microcapsule is a virus capsid, the microcapsule can be formed using specific conditions depending on the type of virus capsid. For example, microcapsules are formed by expressing a large amount of capsid protein (HBcAg) in Escherichia coli cells at a high concentration. In addition, when the microcapsule is a capsid of bacteriophage MS2, it can be forcibly formed by binding to RNA containing a specific sequence consisting of 19 bases and binding to that RNA.

  On the other hand, even when the capsid protein does not constitute a microcapsule, a microcapsule that embeds an enzyme or protein under appropriate conditions can be prepared. For example, the capsid protein is reconstituted by placing the capsid protein in a high-concentration enzyme or protein solution and substituting it with a solution with a low salt condition or high salt concentration by dialysis, etc., thereby embedding the enzyme or protein. Microcapsules can be made.

(11. Purification)
Microcapsules are larger and higher in molecular weight than capsid proteins. Utilizing this difference in size and molecular weight, the microcapsules can be easily purified by removing molecular sieves such as gel chromatography, which is a known purification method, and those that do not form microcapsules by centrifugation and impurities. Can do. For example, the capsid protein of hepatitis B virus can be easily purified by centrifugation using a sucrose density gradient method.

(12. Polysaccharides)
A disaccharide is produced by the glycosidic bond between two monosaccharides, and the polymer compound produced by the continuous glycosidic bond is a polysaccharide. The molecular formula is represented by (C ₆ H ₁₀ O ₅ ) n. Typical polysaccharides include starch and cellulose. The polysaccharide used in the present invention is preferably gum arabic, carrageenan, chitosan or trehalose. Polysaccharides provide a stabilizing effect when mixed with foods and pharmaceuticals, and also have a protective effect on compounds and proteins.

  Among them, trehalose is a non-reducing disaccharide in which two molecules of glucose are bound. In nature, trehalose exists widely in a free state across animals and plants and microorganisms. It is also abundant in yeasts such as baker's yeast and beer yeast, and is known as a carbohydrate that has been eaten by people since ancient times. In recent years, it has attracted attention as a sugar having a high moisturizing effect, and has been found to have a function of protecting cells from a dry environment. Gum arabic is mixed with pharmaceuticals and blended for the purpose of stabilizing and protecting the gum.

(13. Mixed solution of microcapsules and polysaccharides)
Depending on the nature of the encapsulated enzyme or protein, microcapsules can be improved in stability by being prepared with an acidic solution (pH 5) or a solution (Mg) containing a salt. Furthermore, the solution in which the polysaccharide is mixed can further stably maintain the activity of the internal enzyme and protein. For example, by mixing polysaccharides such as gum arabic, carrageenan, trehalose or chitosan with a solution containing microcapsules (1-10% solution) or microcapsules (0.01-10 mg / mL), the internal enzymes and proteins can be stabilized. It becomes possible to hold. In addition, when the microcapsule and polysaccharide solution is naturally dried at room temperature (17 to 37 ° C.), the embedded protein is stably retained.

(14. Application of microcapsules to biochips)
By allowing the solution containing the microcapsules of the present invention and the polysaccharide to coexist, it is possible to produce an adaptable chip even in a dry state. Above all, by using a chip, it can be applied to protein chips and biosensors that detect intermolecular interactions.

  In other words, enzymes and proteins can be artificially placed in a space that does not affect the interaction of the microcapsules formed by the virus capsid protein, and the enzymes and proteins can be stably immobilized on a solid phase such as a glass substrate. Become.

  Further, by adding a surfactant or an alkaline solution to a solution containing microcapsules, it becomes possible to express the embedded protein. That is, the surfactant is SDS represented by an ionic surfactant, and the alkaline solution is preferably a Tris buffer (pH9-11) that does not break the higher-order structure of the embedded protein. Is mentioned.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to this at all. The scope of the present invention should be defined by the contents described in the detailed description of the invention rather than the specific embodiments shown in the Examples.

Reference Example 1 Preparation of capsid protein (HBcAg) and GFP fusion protein Gene preparation
Using HBcAg gene and jellyfish-derived green fluorescent protein GFP gene and expression vector as materials, restriction enzyme and DNA polymerase are used to encode a gene encoding amino acids 1-149 of HBcAg on the 5 ′ side of the multiple cloning site of the expression vector ( SEQ ID NO: 1) was further introduced downstream of GFP. As the GFP gene (SEQ ID NO: 2), a gene manufactured by Clontech was used. As the HBcAg gene, a cloned gene was used using a cDNA library prepared from the serum of a chronic active hepatitis B patient. As an expression vector, pET20b + manufactured by Novagen was used.

2. Preparation of microcapsules Using the gene obtained in the above, microcapsules purified with high purity were obtained by expression induction, lysis, centrifugation, ammonium sulfate precipitation, dialysis, sucrose density gradient method and gel filtration chromatography as follows.

That is, an expression vector containing only HBcAg and an expression vector expressing a GFP fusion protein were incorporated into E. coli BL21, cultured for 16 hours, and induced for expression using IPTG (ispporpyl-β-D-thiogalactopyranoside). After further culturing for 3 hours, the cells were collected by centrifugation at 8,000 rpm for 15 minutes. Thus, by expressing a high concentration of the fusion protein in the microbial cells, the fusion protein formed microcapsules. In order to isolate and purify the formed microcapsules, the following operation was further performed. Bacteria were suspended in PBS buffer and disrupted 3 times for 10 seconds with ultrasound. Further, centrifugation was performed at 10,000 rpm for 20 minutes to remove cell debris. Ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) was added to the supernatant after centrifugation to a concentration of 20% to precipitate microcapsules. The pellet (precipitate) was redissolved in PBS buffer and fractionated by the sucrose density gradient method (60% to 5%). At this time, the fraction containing the microcapsules to be collected was confirmed using SDS-PAGE. As a result, it was found that the sucrose concentration was recovered in a concentration of 30 to 50%. Furthermore, it was purified by gel filtration chromatography (High Road Superdex 300 HR26 / 60, Pharmacia) and dialyzed using 5 mM Tris-HCl, 150 mM NaCl solution, and a highly purified microcapsule solution was obtained. .

Examples 1-4, Comparative Examples 1-6 (protein stabilization evaluation)
GFP-embedded microcapsule solution (1 mg / ml) in 10 μL water, natural polysaccharide 5% gum arabic (manufactured by Wako Pure Chemical Industries), 10% gelatin (manufactured by Sigma), chitosan (manufactured by Wako Pure Chemical Industries, Ltd.) Or 10% of 1% carrageenan (manufactured by Wako Pure Chemical Industries, Ltd.) to obtain a microcapsule solution. It apply | coated on the glass substrate and air-dried. Similarly, free GFP (without microcapsules) (Roche) (1 mg / ml) in 10 μL of water, natural polysaccharide 5% gum arabic (manufactured by Wako Pure Chemical Industries), 10% gelatin (manufactured by Sigma), The mixture was mixed with 10 μL of chitosan (manufactured by Wako Pure Chemical Industries, Ltd.) or 1% carrageenan (manufactured by Wako Pure Chemical Industries, Ltd.), coated on a glass substrate, and air dried. The glass substrate was stored at room temperature for 20 days, and the glass substrate was irradiated with UV light at 254 nm to compare the green fluorescence that is the activity of GFP. The results are shown in Table 1.

  Significantly, GFP embedded in microphone capsules retained activity when mixed with gum arabic or carrageenan, which are natural polysaccharides. That is, it is considered that it is possible to immobilize proteins on a glass substrate significantly stably by embedding them in microcapsules rather than GFP alone and adding a preservative.

Examples 5-8, comparative examples 7-11 (expression of protein)
The following treatment was performed on the solution containing the microcapsules obtained in 2 of Reference Example 1.

  Comparative Example 7: Untreated (A).

  Comparative Example 8: Anti-GFP antibody (manufactured by Roche) was added to the immobilized beads (manufactured by Amersham) (immunoprecipitation), and the supernatant was recovered (I).

  Example 5: Dialysis with an alkaline solution (0.2 M Tris-HCl Buffer (pH 10)), and the resulting microcapsule solution was added to beads (Amersham) on which an anti-GFP antibody (Roche) was immobilized ( (Immunoprecipitation) and recovering the supernatant (c).

  Example 6: Dialyzed with an alkaline solution (0.02M Tris-HCl Buffer (pH 10)), and the resulting microcapsule solution was added to beads (Amersham) immobilized with anti-GFP antibody (Roche) ( (Immunoprecipitation) and recovering the supernatant (D).

  Example 7: After adding 2 times the amount of a surfactant solution (10% SDS) and treating at room temperature for 1 hour, beads obtained by immobilizing an anti-GFP antibody (Roche) on the resulting microcapsule solution (Amersham) ) (Immunoprecipitation) and recover the supernatant (e).

  Example 8: After adding a double amount of a surfactant solution (1% SDS) and treating at room temperature for 1 hour, beads obtained by immobilizing an anti-GFP antibody (Roche) on the resulting microcapsule solution (Amersham) ) (Immunoprecipitation) and recover the supernatant (f).

  Comparative Example 9: Free GFP (Roche) (1 mg / ml) was added to an anti-GFP antibody (Roche) -immobilized beads (Amersham) (immunoprecipitation), and the supernatant was recovered ( G).

  Comparative Example 10: Free GFP (Roche) (1 mg / ml) was dialyzed with an alkaline solution (0.02M Tris-HCl Buffer (pH 10)), and the resulting microcapsule solution was anti-GFP antibody (Roche) Is added to the immobilized beads (manufactured by Amersham) (immunoprecipitation), and the supernatant is recovered (ku).

  Comparative Example 11: Free GFP (Roche) (1 mg / ml) was added twice with a surfactant solution (1% SDS) and treated at room temperature for 1 hour, and the resulting microcapsule solution was treated with anti-GFP antibody. (Roche) was added to the immobilized beads (Amersham) (immunoprecipitation), and the supernatant was recovered (K).

  The nine types of samples were electrophoresed (SDS PAGE). SDS PAGE conditions were as follows: 12.5% acrylamide gel was used. The sample was dissolved in the presence of SDS and energized for 70 minutes at 1000 V and 20 mA. The obtained gel and PVDF membrane (manufactured by ATTO) were installed in a blotting apparatus (manufactured by ATTO) and energized for 25 minutes at 150 mA. The resulting membrane was blocked overnight with 10% Block Ace (Dainippon Pharmaceutical Co., Ltd.), treated with anti-GFP antibody (Roche) for 3 hours, and then washed with a washing solution containing 0.01% Tween20. Then, it was treated with anti-mouse antibody-HRP conjugate (Amersham) for 2 hours. The membrane was thoroughly washed with a washing solution and developed with a Western blot detection system (Amersham).

  As a result, the reaction was confirmed only for GFP (molecular weight of about 66000) in the microcapsule to which the surfactant or the alkaline solution was added (FIG. 2-U to K). In other words, the control a is broken by SDS in the SDS PAGE and reacts with the antibody. However, because it has a microcapsule structure, it does not react with the GFP antibody immobilized on the beads. There is no GFP in Kiyomizu. Therefore, it was not detected. It is considered that the microcapsules have a higher order structure broken under alkaline conditions or in the presence of a surfactant, and the embedded enzymes and proteins are exposed. In addition, GFP (molecular weight 26000) and anti-GFP antibody reacted from Ki's experiment, and the reaction between GFP (molecular weight 26000) and anti-GFP antibody immobilized on beads was inhibited by alkaline solution or SDS. Obviously not.

Reference Example 2 Preparation of fusion protein of capsid protein (HBcAg) and lysozyme Gene preparation
Using HBcAg gene and human lysozyme protein Lys gene and expression vector as materials, restriction enzyme and DNA polymerase are used as a gene 1 (sequence) encoding amino acids 1-149 of HBcAg on the 5 ′ side of the multiple cloning site of the expression vector. Number 1) was further introduced downstream of human lysozyme. The Lys gene (SEQ ID NO: 3) was obtained by gene cloning from a human placenta cDNA library manufactured by Takara Shuzo. As the HBcAg gene, a cloned gene was used using a cDNA library prepared from the serum of a chronic active hepatitis B patient. As an expression vector, pET20b + manufactured by Novagen was used.

2. Preparation of microcapsules Using the gene obtained in the above, microcapsules purified with high purity were obtained by expression induction, lysis, centrifugation, ammonium sulfate precipitation, dialysis, sucrose density gradient method and gel filtration chromatography as follows.

That is, an expression vector for expressing only HBcAg and an expression vector for expressing a fusion protein containing human lysozyme were incorporated into E. coli BL21, cultured for 16 hours, and induced for expression using IPTG (ispporpyl-β-D-thiogalactopyranoside). After further culturing for 3 hours, the cells were collected by centrifugation at 8,000 rpm for 15 minutes. Thus, by expressing a high concentration of the fusion protein in the microbial cells, the fusion protein formed microcapsules. In order to isolate and purify the formed microcapsules, the following operation was further performed. Bacteria were suspended in PBS buffer and disrupted 3 times for 10 seconds with ultrasound. Further, centrifugation was performed at 10,000 rpm for 20 minutes to remove cell debris. Ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) was added to the supernatant after centrifugation to a concentration of 20% to precipitate microcapsules. The pellet (precipitate) was redissolved in PBS buffer and fractionated by the sucrose density gradient method (60% to 5%). At this time, the fraction containing the microcapsules to be collected was confirmed using SDS-PAGE. As a result, it was found that the sucrose concentration was recovered in a concentration of 30 to 50%. Furthermore, it was purified by gel filtration chromatography (High Road Superdex 300 HR26 / 60, Pharmacia) and dialyzed using 5 mM Tris-HCl, 150 mM NaCl solution, and a highly purified microcapsule solution was obtained. .

Example 9, Comparative Example 12 (protein stabilization and expression evaluation)
A microcapsule solution (1 mg / ml) embedded with human lysozyme and 10 μL of 5% gum arabic (manufactured by Wako Pure Chemical Industries, Ltd.), a natural polysaccharide, were mixed to obtain a microcapsule solution. In the same way, mix human free lysozyme (without microcapsules) (Funakoshi Co., Ltd.) (1 mg / ml) with 10 μL of water and natural polysaccharide 5% gum arabic (Wako Pure Chemical Industries, Ltd.) at room temperature. Stored for 20 days. Next, the microcapsule solution and free human lysozyme solution (without microcapsules) were added with equal amounts of 0.2 MTris-HCl (pH 10) solution, which was an alkaline solution, and treated at room temperature for 1 hour. Next, 2.9 mL of a Micrococcus lysodeikticus culture solution was prepared, 0.1 mL of the above solution or 0.1 mL of lysozyme standard solution (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the mixture was allowed to stand at room temperature for 5 minutes. Next, when the absorbance OD450 of the culture solution alone or the microcapsule solution added to the culture solution was measured to measure the biological activity (RBA) of the lysozyme, the microcapsule solution was RBA 55%, free human lysozyme. 30% and 59% with lysozyme standard solution addition. That is, it is presumed that the lysozyme in the microcapsule was stably maintained, and was exposed to the outside by adding an alkaline solution to express protease activity.

  The present invention can be applied not only to enzyme stabilization methods but also to enzyme detergents, protein chips, biosensor applications, and the like, but the application range is not limited thereto.

FIG. 3 is a cross-sectional view of an HBcAgT4 aggregate. It is a figure which shows the immunoblotting result of Examples 5-8 and Comparative Examples 7-11.

Explanation of symbols

A: Inner radius (11.2 nm)
B: Inner surface area (1576 nm ² )

Claims (8)

A microcapsule composition comprising a protein having a particle-forming ability and a polysaccharide. The microcapsule composition according to claim 1, wherein an enzyme or protein is embedded in the microcapsule. The microcapsule composition according to claim 2, wherein a protein having particle-forming ability and an enzyme or protein are fused. The microcapsule composition according to any one of claims 1 to 3, wherein the polysaccharide is at least one selected from gum arabic, carrageenan, trehalose and chitosan. The microcapsule composition according to any one of claims 1 to 4, wherein the protein having particle-forming ability is a viral capsid protein or a mutant protein thereof. 6. The microcapsule composition according to claim 5, wherein the capsid protein is a capsid protein of Hepadnaviridae virus. The microcapsule composition according to claim 6, wherein the hepadnavirus is hepatitis B virus. The enzyme or protein embedded in the microcapsule is expressed by adding a surfactant or an alkaline solution to the microcapsule composition according to any one of claims 2 to 7. Enzyme or protein expression method.

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