KR20110140014A - Fusion protein comprising ferritin and gala peptide, cage protein formed thereby, and novel use thereof - Google Patents

Abstract

PURPOSE: A fusion protein of ferritin and GALA peptides and a cage protein containing the same are provided to effectively release drug under a weak acid condition and to be applied as a biosensor. CONSTITUTION: A fusion protein contains a GALA(Glu-Ala-Leu-Ala) motif containing an amino acid sequence of sequence number 13 linked to C-terminal of a polypeptide of a fragment containing ferritin protein, A-helix, B-helix, C-helix, and D-helix or mutant thereof. The ferritin is denoted by amino acid sequence number 1. The fragment is denoted by amino acid sequence 3. The GALA motif contains 4-100 amino acids of amino acid sequence number 13 and is denoted by amino acid sequence number 5 or 7. A cage protein contains the fusion protein.

Description

Fusion protein comprising ferritin and GALA peptide, cage protein formed thereby, and novel use according to the invention.

The present invention relates to a fusion protein of ferritin and GALA peptides, cage proteins constituted by the same, and novel uses thereof, specifically, a fusion protein prepared by introducing a GALA peptide in which structural modifications occur in weak acidity to ferritin protein, Cage proteins constructed thereby and their novel uses.

Cage protein is a protein capable of forming tens to hundreds of macromolecules of monolithic molecular weight due to the precise self-assembly of low molecular weight monoliths. In nature, these include viral capsid protein, ferritin, heat shock protein, and Dps protein, each of which constitutes a cage, which has a very regular and precise interaction with adjacent ones, and the inside of the cage is empty. It is a structure. It has a characteristic that the inside and the outside is sequestered by the same properties as the container (container) of the cage protein, it is frequently used in the medical field as a drug carrier.

On the other hand, the stability of the drug (biostability) in vivo in the drug delivery to the human body of the existing disease treatment methods, the stability and half-life (half-life) can be controlled by the selection of an effective drug carrier. In particular, the method of enclosing the drug in the cage using the cage structure has the advantage that it can perform the function of the effective drug delivery system. However, in order to effectively release the encapsulated drug in the target tissue, the drug structure enclosed in the normal tissue must be maintained and the encapsulated drug must be released while the cage structure is dissociated only in the desired target tissue. Generally, the pH of human blood is 7.4 ± 0.04, but the pH in cancer tissues is known to be 6.0 to 6.5 on average, and this pH difference can be used as an effective drug delivery strategy in drug delivery systems.

However, cage proteins formed by all other cage-forming proteins, including conventional ferritin proteins, retain large cage structures under a wide range of pH conditions but are reversibly dissociated only under strong acid (pH 4.0) conditions such as guanidine hydrochloride. Have the property of becoming. Therefore, the cage protein formed by the ferritin protein or the like maintains the cage structure intact even at pH 6.0 to 6.5, there is a problem that can not effectively release the drug enclosed therein.

Accordingly, the present inventors completed the present invention by confirming that the GALA peptide forms a cage protein with a recombinant ferritin protein fused to ferritin, and that the prepared cage protein is completely dissociated in a weak acid such as pH 6.0 to pH 6.5. .

Accordingly, the present invention provides a carboxyl terminus of a polypeptide selected from the group consisting of (a) a ferritin protein, fragments comprising A-helix, B-helix, C-helix and D-helix, and variants thereof. An object of the present invention is to provide a fusion protein in which a (LA) Glu-Ala-Leu-Ala (MOLA) motif comprising the amino acid sequence of SEQ ID NO: 13 is linked to a (carboxyl terminal).

It is another object of the present invention to provide a cage protein consisting of the fusion protein.

It is another object of the present invention to provide a polynucleotide encoding a fusion protein.

It is another object of the present invention to provide an expression vector into which a polynucleotide is introduced.

It is another object of the present invention to provide a transformant transformed with an expression vector.

In addition, an object of the present invention is to provide a bioactive material carrier, characterized in that the bioactive material is sealed in the cage protein.

In addition, an object of the present invention is to provide a biosensor comprising an indicator that is sealed in the cage protein as an active ingredient.

In order to solve the above problems, the present invention (a) ferritin (ferritin) protein, A-helix, B-helix, C-helix and D-helix containing the protein group consisting of a variant thereof (B) provides a fusion protein to which a GALA (Glu-Ala-Leu-Ala) motif comprising an amino acid sequence of SEQ ID NO: 13 is linked to a carboxyl terminal of a polypeptide selected from.

The present invention also provides a cage protein consisting of the fusion protein.

The present invention also provides a polynucleotide encoding a fusion protein.

The present invention also provides an expression vector into which the polynucleotide is introduced.

The present invention also provides a transformant transformed with the expression vector.

In another aspect, the present invention provides a bioactive material carrier, characterized in that the bioactive material is sealed in the cage protein.

In another aspect, the present invention provides a biosensor comprising a marker is sealed in the cage protein as an active ingredient.

Specific terms used in the present invention will be described below.

Ferritin (ferritin) protein in the present invention can be used without limitation, if each of them is an active protein capable of forming a complex protein of the cage form as a unit, but is not limited thereto, preferably the average molecular weight may be 20kDa to 25kDa More preferably, GenBank accession No: AAA62259.1. (Ferritin light chain, mouse), NCBI accession No: NP_071945.3 (ferritin light chain, rat), NCBI accession No: NP_001108012.1 (ferritin light chain, horse). NCBI accession No: NP_002023.2 (ferritin heavy chain, human). NCBI accession No: NP_034369.1 (ferritin heavy chain, mouse). NCBI accession No: NP_036980.1 (ferritin heavy chain, rat), and NCBI accession No: NP_001093883.1 (ferritin heavy chain, horse) may be one or more selected from the group consisting of, and most preferably a human-derived sequence number It may be a polypeptide represented by the amino acid sequence of 1.

In the present invention, the ferritin protein may be replaced with another protein capable of forming a complex protein in the form of a cage, but is not limited thereto. Preferably, Hepatitis B virus capsid protein (NCBI accession No: NP_647607.1), Tobacco mosaic virus capsid protein (NCBI accession No: NP_597750.1) and CCMV (Cowpea chlorotic mottle virus) capsid protein (NCBI accession No: NP_613277.1).

In the present invention, the fragment of the ferritin protein is not limited as long as the activity capable of forming the complex protein of the cage form is maintained, but may be one containing A-helix, B-helix, C-helix and D-helix in the ferritin protein. Preferably, at least one amino acid residue after the 155th amino acid sequence of SEQ ID NO: 1 may be removed, and more preferably, at least one amino acid residue after the 160th amino acid sequence of SEQ ID NO: 1 may be removed. Most preferably, it may be represented by the amino acid sequence of SEQ ID NO. 3 (see FIG. 3).

In the present invention, the variant is not limited as long as the activity capable of forming a complex protein in cage form is maintained, but may be a protein formed by the addition, deletion, or substitution of some amino acids in the ferritin protein or fragment thereof.

GALA (Glu-Ala-Leu-Ala) motif (motif) in the present invention is not limited to this, if the structure can be formed in a random coil form in neutral conditions or if the pH is changed to weakly acidic, but not limited to this, preferably sequence It may be a polypeptide consisting of 4 to 100 amino acids comprising the amino acid sequence (Glu-Ala-Leu-Ala) of No. 13, more preferably the amino acid sequence of the amino acid sequence of SEQ ID NO: 13 is repeated 1 to 10 times It may be a polypeptide consisting of 4 to 40 amino acids comprising, more preferably a polypeptide represented by the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 7.

The biosensor of the present invention is a sensor capable of detecting a pH change from neutrality of pH 7.0 to 7.4 to weak acidity of pH 6.0 to 6.5, or detecting weakly acidic cells or tissues of pH 6.0 to 6.5 in a cell or tissue, for example For example, weakly acidic cells, preferably cancer (Taillefer, J .; Brasseur, N .; van Lier, JE; Lenaerts, V .; Le Garrec, D .; Leroux, JCJ), osteoarthritis (Pharm. Pharmacol. 2001, 53, 155-166.), Rheumatoid arthritis (Ojugo, AS; McSheehy, PM; McIntyre, DJ; McCoy, C .; Stubbs, M .; Leach, MO), dementia (Judson, IR; Griffiths, JR NMR Biomed. 1999, 12 (8), 495-504), autoimmune diseases (Lee, ES; Shin, HJ; Na, K .; Bae, YHJ Control. Rel. 2003, 90, 363-374.) And stroke related cells ( Kim, GM; Bae, YH; Jo, WH Marcomol. Biosci. 2005, 5, 1118-1124.).

Hereinafter, the present invention will be described in more detail.

In the present invention, the fusion protein is (a) a ferritin protein, a fragment comprising the A-helix, B-helix, C-helix, and D-helix of the protein and a variant thereof. (B) GALA (Glu-Ala-Leu-Ala) motif (motif) comprising the amino acid sequence of SEQ ID NO: 13 is connected to the carboxyl terminal.

The fusion protein of the present invention serves as a unit of the cage (cage) protein, it can form a cage (cage) structure by including the ferritin protein, fragments thereof or variants thereof (a). Particularly, the fragment of the ferritin protein includes A-helix, B-helix, C-helix, and D-helix in the ferritin protein, and may be one in which the E-helix portion of the ferritin protein is removed, in which case a cage-like structure (See <Comparative Example 1>) In addition, the fusion protein of the present invention includes a GALA motif, so that the cage protein formed therefrom may have a property of dissociating into monomers in weak acidity. See>)

The fusion protein of the present invention is most preferably represented by the amino acid sequence of SEQ ID NO: 9 to which the GALA motif represented by the amino acid sequence of SEQ ID NO: 5 is linked to the carboxyl terminus of the fragment of the ferritin protein represented by the amino acid sequence of SEQ ID NO: 3 It may be a protein or a protein represented by the amino acid sequence of SEQ ID NO: 11 to which the GALA motif represented by the amino acid sequence of SEQ ID NO: 7 is linked to the carboxyl terminus of the fragment of the ferritin protein represented by the amino acid sequence of SEQ ID NO: 3.

The fusion protein of the present invention is not limited thereto, but may be preferably inserted into a conventional vector designed to express an external gene so that it can be mass produced by a genetic engineering method. The vector may be appropriately selected or newly produced according to the type and characteristics of the host cell for producing a protein. The method for transforming the vector into a host cell and the method for producing a recombinant protein from the transformant can be easily carried out by conventional methods. Methods such as selection, production, transformation, and expression of recombinant proteins described above can be easily carried out by those skilled in the art, and some modifications are also included in the present invention. (See <Example 1>)

In addition, the cage protein of the present invention is characterized by consisting of the fusion protein of the present invention.

The cage protein of the present invention is not limited thereto, but is preferably a complex in which the fusion protein is regularly arranged as a unit by the self-assembly activity of the ferritin protein, a fragment thereof or a variant thereof included in the fusion protein of the present invention. It may be a protein, and more preferably 24 fusion proteins of the present invention may be formed through a three-dimensional regular arrangement. The cage protein may be, but is not limited to, an average molecular weight of 450 kDa to 550 kDa.

In particular, the cage protein formed by the fusion protein of the present invention is a spherical macromolecule formed by self-assembly of 24 units, the cage protein formed by the ferritin has an outer diameter of about 12 nm and an inner diameter of about 8 nm. It is an empty container structure.

All other cage-forming proteins, including conventional ferritin proteins, maintain large cage structures under a wide range of pH conditions and are reversibly dissociated only in strong acid (pH 4.0) conditions such as guanidine hydrochloride. In other words, cage proteins formed only of ferritin protein retain their container-like cage at neutral pH and are reversibly dissociated under strong acid conditions (eg, pH 4).

However, the cage protein formed by the fusion protein of the present invention contains a fusion protein containing a ferritin protein, a fragment thereof or a variant thereof as a unit, and thus, in a cage form at neutral (preferably pH 7.0 to 7.4). In addition to being present, the fusion protein containing the GALA motif can be included as a unit, so that it can be dissociated into units at weak acidity (preferably pH 6.0 to 6.5) (see <Experimental Example 1>).

Therefore, the cage protein formed by the fusion protein of the present invention has the property that the cage protein formed of the ferritin protein is dissociated in weak acidity such as pH 6.0, whereas the cage structure in which the self-assembled cage is dissociated only in strong acidity below pH 4.0 In addition, the drug can be effectively released in a weakly acidic environment such as cancer tissue, and thus can be effectively used as a drug carrier.In addition, it can be effectively used as a biosensor by sensitively reacting to weakly acidic stimulants and causing structural changes. Can be.

On the other hand, the polynucleotide of the present invention is characterized by encoding the fusion protein.

The polynucleotide of the present invention is not limited thereto, but may preferably be represented by the nucleotide sequence of SEQ ID NO: 10 encoding the fusion protein represented by the amino acid sequence of SEQ ID NO: 9, and also the fusion protein represented by the amino acid sequence of SEQ ID NO: 11 It can be represented by the nucleotide sequence of SEQ ID NO: 12 that encodes.

In addition, the expression vector of the present invention is characterized in that the polynucleotide is introduced.

Possible expression vectors used in the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like. Suitable expression vectors include membrane targeting or in addition to expression control sequences such as promoters, operators, initiation codons, termination codons, polyadenylation signals, and enhancers. It may be prepared in various ways according to the purpose, including a signal sequence (leader sequence) or a signal sequence for secretion. The promoter of the expression vector may be constitutive or inducible. In addition, the expression vector includes a selection marker for selecting a host cell containing the vector, and in the case of an expression vector capable of replication, includes a replication origin.

The recombinant expression vector of the present invention prepared as described above is not limited thereto but may be preferably represented by a cleavage map of FIG. 9 or 10.

In addition, the transformant of the present invention is transformed with the expression vector may be preferably E. coli.

The transformation may include any method of introducing a nucleic acid into a host cell, and may be performed by transformation techniques known to those skilled in the art. Preferably, microprojectile bombardment, electroporation, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, PEG-mediated fusion, microinjection And liposome-mediated methods and the like.

On the other hand, the bioactive material carrier of the present invention is characterized in that the bioactive material is sealed in the cage protein of the present invention.

The biologically active substance carrier of the present invention maintains a large cage structure in the same way as a cage protein formed of ferritin protein present in nature under neutral conditions, but has a characteristic of releasing the enclosed bioactive substance by dissociation into units in weak acidity. .

The bioactive substance is a substance exhibiting a constant activity when encapsulated in the cage protein of the present invention and administered in vivo, and particularly to weakly acidic cells, preferably cancer, osteoarthritis, rheumatoid arthritis, dementia, autoimmune diseases and stroke-related cells. , Prophylactic or therapeutic activity is not limited to this, but preferably cancer, osteoarthritis, rheumatoid arthritis, dementia, autoimmune diseases and stroke prevention or treatment drugs, more preferably docetaxel, cisplatin (cis- platin, camptothecin, paclitaxel, tamoxifen, anastozole, anasterozole, gleevec, 5-fluorouracil (5-FU), floxuridine, Leuprolide, Flutamide, Zoledronate, Doxorubicin, Vincristine, Gemcitabine, Strep Streptozocin, Carboplatin, Topotecan, Belotecan, Irinotecan, Vinorelbine, Hiroxiurea, Valrubicin, Retinino Retinoic acid series, Methotrexate, Mechlorethamine, Chlorambucil, Busulfan, Doxifluridine, Vinblastin, Mitomycin, Prednisone, Testosterone, Mitoxantron, Aspirin, Salicylates, Ibuprofen, Naproxen, Naproxen, Phenof Fenoprofen, indomethacin, phenyltazone, cyclophosphamide, mechlorethamine, dexamethasone, prednisolone, celecoxib, celecoxib ), Valdecoxib, nimeslied sulide), cortisone (cortisone) and corticosteroids may be selected from the group consisting of.

The method of encapsulating a bioactive substance inside the cage protein of the present invention is a bond using a maleimide reactor to a residue inside a cage, a bond using an iodoacetyl reactor or a pyridyl disulfide. Bonding using a reactor, bonding using a charge-charge interaction, and the like are preferred, but are not limited thereto.

Accordingly, the bioactive substance carrier of the present invention is an active ingredient of a pharmaceutical composition, preferably a pharmaceutical composition for preventing or treating at least one disease selected from the group consisting of cancer, osteoarthritis, rheumatoid arthritis, dementia, autoimmune diseases and stroke. It can be used as. The cancer is squamous cell carcinoma, uterine cancer, cervical cancer, prostate cancer, head and neck cancer, pancreatic cancer, brain tumor, breast cancer, liver cancer, skin cancer, esophageal cancer, testicular cancer, kidney cancer, colon cancer, rectal cancer, stomach cancer, kidney cancer, bladder cancer, ovarian cancer Cancers, including bile duct cancer, gallbladder cancer, and the like are preferred, but are not limited thereto.

The pharmaceutical composition comprises 0.0001 to 50% by weight of the bioactive material carrier as an active ingredient based on the total weight of the composition. The composition of the present invention may further contain one or more active ingredients exhibiting the same or similar functions in addition to the active ingredients.

The pharmaceutical composition may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome and one or more of these components. , A buffer solution, a bacteriostatic agent, and the like may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions, and the like, and may act specifically on target organs. Target organ specific antibodies or other ligands may be used in combination with the carriers so as to be used. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA). have.

The pharmaceutical composition is intravenous, intraperitoneal, intramuscular, subcutaneous, intradermal, nasal, mucosal, inhalation and It can be delivered in vivo by infusion by oral or other route. Dosage varies depending on the subject's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and severity of disease. The daily dosage is about 0.1 to 100 mg / kg for the compound, preferably 0.5 to 10 mg / kg, and more preferably administered once to several times a day.

On the other hand, the biosensor of the present invention is characterized in that the label protein is sealed inside the cage protein.

The biosensor of the present invention is a sensor capable of detecting a pH change from neutrality of pH 7.0 to 7.4 to weak acidity of pH 6.0 to 6.5, or detecting weakly acidic cells or tissues of pH 6.0 to 6.5 in a cell or tissue, for example For example, weakly acidic cells, preferably cancer (Taillefer, J .; Brasseur, N .; van Lier, JE; Lenaerts, V .; Le Garrec, D .; Leroux, JCJ), osteoarthritis (Pharm. Pharmacol. 2001, 53, 155-166.), Rheumatoid arthritis (Ojugo, AS; McSheehy, PM; McIntyre, DJ; McCoy, C .; Stubbs, M .; Leach, MO), dementia (Judson, IR; Griffiths, JR NMR Biomed. 1999, 12 (8), 495-504), autoimmune diseases (Lee, ES; Shin, HJ; Na, K .; Bae, YHJ Control. Rel. 2003, 90, 363-374.) And stroke related cells ( Kim, GM; Bae, YH; Jo, WH Marcomol. Biosci. 2005, 5, 1118-1124.).

The method of encapsulating a marker inside a constant cage protein is preferably, but not limited to, binding using a maleimide reactor to a residue inside the cage, binding using an iodoacetyl reactor or pyridyl disulfide ( Pyridyl disulfide) can be combined with a reactor or a charge-charge interaction.

The marker may be, but is not limited to, an enzyme, a phosphor, a radioisotope, a quencher or a chemical.

Examples of mechanisms by which the biosensor of the present invention can detect a pH change from neutrality of pH 7.0 to 7.4 to weak acidity of pH 6.0 to 6.5, or detect weakly acidic cells or tissues of pH 6.0 to 6.5 in a cell or tissue. For example, it is as follows.

Fluorescence resonance (FRET, Fluorescence resonance) occurs when the two fluorophores, for example Cy5.5 and Cy7.5, which generate fluorescence of different wavelengths are connected to the fusion protein of the present invention and form a cage in a neutral pH environment. An energy transfer effect occurs, and it can be used as a biosensor by confirming that the FRET effect disappears by dissociation of the cage in a weakly acidic environment. That is, as the cage structure is dissociated by acidic stimulation, the FRET effect disappears and the pH change from neutrality of pH 7.0 to 7.4 to weak acidity of pH 6.0 to 6.5 is detected in the cell or tissue, or weakly acidic cells of pH 6.0 to 6.5 Or tissue can be detected.

In order to connect the phosphor to the fusion protein, for example, a phosphor capable of causing a FRET effect (preferably an organic phosphor including a Cy series, a Green Fluorescent protein (GFP), a Red Fluorescent protein (RFP) and a Cyan Fluorescent CFP). protein) fluorescent substance selected from the group consisting of proteins) can be combined by using a NHS (N-hydroxysuccinimide) ester functional group as a linker using an amine group of the lysine (lysine) present on the surface of the fusion protein of the present invention.

The fusion protein of the ferritin and GALA peptide of the present invention, the cage protein constituted by the weak acid, such as pH 6.0 to 6.5, compared to the cage structure of the cage protein formed from the conventional ferritin dissociated into units only in a strong acidity of less than pH 4.0 Because of its dissociation property, it can release the drug effectively in weakly acidic environment such as cancer tissue, and can be applied as a biosensor using the characteristic that structural change occurs in response to acid stimulus.

1 is a schematic diagram illustrating the structural dissociation of the GALA motif (coil α-helix) in which the cage protein formed of the fusion protein of the present invention is dissociated into monomers according to pH change (neutral to weak acidity).
2A is a schematic diagram of the tertiary structure of ferritin protein.
Figure 2B is a schematic representation of the cage protein formed by ferritin protein and E-helix portion.
Figure 2C is a diagram showing that when the substitution of the E-helix portion of the ferritin protein with GALA peptides, a collision occurs between GALA peptides due to structural changes in weak acidity (yellow is a random coil, purple is a collision between spiral structures). Means)
Figure 3A is a table showing the dissociation activity of the sequence information of the fusion protein of the present invention and the pH change of the cage protein formed thereby.
Figure 3B is a diagram showing the secondary structure of human ferritin protein and the location where E-helix is removed.
FIG. 3C is an electrophoretic photograph showing that E-helix-free recombinant ferritin protein (ferritin-ΔE) is capable of mass expression in E. coli. (S: soluble fraction, +: IPTC,-: no IPTG)
3D is an electrophoretic photograph showing that fusion proteins having different GALA repeat counts (fGALA ₄ and fGALA ₆ ) can be expressed in E. coli (S: soluble fraction, +: IPTC,-: no IPTG).
4 shows that the cage protein formed from the ferritin protein from which the human ferritin or E-helix portion is removed is maintained in the cage structure at a pH range of 4.0 to 8.0, and both proteins are dissociated into monomers at a pH below 4.0. It is a graph showing that dissociation does not occur in weak acidity.
Figure 5A is a result of size exclusion chromatography and denaturation SDS-PAGE electrophoresis showing the self-assembly and dissociation activity according to the pH of the cage protein formed of the fusion proteins (fGALA ₄ and fGAGA ₆ ) of the present invention.
5B is a profile comparing self-assembly and dissociation degree by overlapping the profiles corresponding to FIG. 5A.
Figure 5C is a graph comparing the degree of dissociation according to the pH change of the cage protein formed of human phosphine or fusion proteins of the present invention (fGALA ₄ and fGAGA ₆ ).
6A is a graph confirming that there is no change in the secondary structure at the neutrality and pH 6.0 of the cage protein formed of human ferritin through CD (Circular Dichroism) analysis.
6B is a graph confirming cage structure changes due to changes in protein folding at pH 6.0 in the case of cage proteins formed of the fusion proteins (fGALA ₄ and fGAGA ₆ ) of the present invention through CD (Circular Dichroism) analysis.
7A is a graph confirming that there is no section in which protein folding due to heat of human ferritin is rapidly changed through thermal stability analysis using 222 nm wavelength of CD spectrum.
7B is a graph confirming that the cage protein formed of the fusion proteins (fGALA ₄ and fGAGA ₆ ) of the present invention has a protein folding conversion point at about 78 ° C. at neutral pH and an additional protein folding conversion point at pH 6.5, which is slightly acidic. .
8 is a result of comparing and analyzing the amino acid sequence of the human ferritin protein and the fusion protein of the present invention (fGAGA ₆ ).
9 is a cleavage map of a vector into which a gene encoding a fusion protein (fGAGA ₄ ) of the present invention is inserted.
10 is a cleavage map of a vector into which a gene encoding a fusion protein (fGAGA ₆ ) of the present invention is inserted.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

< Example  1>

Ferritin and GALA Peptide  Preparation of Fusion Proteins

<1-1> Preparation of Expression Vector

Genetic recombination was used to prepare a fusion protein in which GALA peptide is linked to human ferritin.

Specifically, in order to insert the GALA peptide, the gene corresponding to the E-helix sequence of ferritin was removed, and then the GALA peptide gene was inserted into the ferritin. In this case, pET28a (+), an expression vector of E. coli, was used as an expression vector, and a gene expressing a recombinant protein was inserted using a restriction enzyme.

More specifically, using a forward primer having a nucleotide sequence of SEQ ID NO: 14 and a reverse primer having a nucleotide sequence of SEQ ID NO: 15 (fGALA ₄ ) or SEQ ID NO: 16 (fGALA ₆ ) to which a GALA motif is connected By removing the E-helix moiety having the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 12 and connecting the GALA motif, the gene of the fusion protein of the present invention was PCR (denaturation 95 ℃ (30 sec), annealing temp. 60 ℃ (30 sec) , extention temp., 72 ° C. (1 min), 30 cycles). The PCR amplification product was checked for size through gel agarose gel electrophoresis (agarose ge) electrophoresis (gel extraction). Since Nde I, Xho I restriction enzyme (NEB purchased) was processed for amplified gene for 4 hours at room temperature. The pET28a (+) expression vector (Novagen purchased) was also subjected to the same restriction enzyme treatment and purification, followed by binding to the gene using T4 DNA ligase. Gene insertion was verified using colony-PCR.

The base sequence of SEQ ID NO: 4 was used as a gene encoding a protein (ferritin-ΔE) of the amino acid sequence of SEQ ID NO: 3 in which a portion corresponding to the E-helix sequence was removed from the human ferritin.

In addition, two kinds of sequences having different sequence lengths were used as genes encoding the GALA peptide. Specifically, the protein of the amino acid sequence of the GALA peptide is four times that of protein that can be repeated SEQ ID NO: 5 the amino acid sequence of the base sequence of SEQ ID NO: 6 coding for the (GALA ₄₎ and the GALA peptide may be repeated six times SEQ ID NO: 7 A gene having a nucleotide sequence of SEQ ID NO: 8 encoding (GALA ₆ ) was used.

Therefore, the gene encoding the fusion protein of the ferritin and GALA peptide may be represented by the nucleotide sequence of SEQ ID NO: 10 which can be repeated 4 times and the GALA peptide is SEQ ID NO: 12.

This is summarized in Table 1 below.

Kind of gene Sequence Amino acid sequence Human ferritin SEQ ID NO: 2 SEQ ID NO: 1 ferritin-ΔE ^* SEQ ID NO: 4 SEQ ID NO: 3 GALA ₄ SEQ ID NO: 6 SEQ ID NO: 5 GALA ₆ SEQ ID NO: 8 SEQ ID NO: 7 ferritin-ΔE-GALA ₄ (fGALA ₄ ) SEQ ID NO: 10 SEQ ID NO: 9 ferritin-ΔE-GALA ₆ (fGALA ₆ ) SEQ ID NO: 12 SEQ ID NO: 11

^* ferritin-ΔE: A gene in which the portion corresponding to the E-helix sequence has been removed from human ferritin

The schematic diagram of the site where the portion corresponding to the E-helix sequence is removed from the human ferritin is as described in FIG.

<1-2> Expression and Separation Purification

E. coli was transformed with the expression vector prepared in <Example 1-1> to mass produce the fusion protein of the present invention.

Specifically, in <Example 1-1>, the expression vector into which the ferritin-ΔE, ferritin-ΔE-GALA ₄ (fGALA ₄ ) and ferritin-ΔE-GALA ₆ (fGALA ₆ ) genes are inserted into a BL21 (DE3) host cell ( After transforming to Novagen), when grown in LB medium (Sigma-Aldrich) at 37 ℃ and the OD ₆₀₀ value reaches 0.5, expression was induced using 1 mM of IPTG (Isopropyl β-D thiogalactoside). After culturing for 6 more hours, the cells were collected and the cells were lysed using lysis buffer (50mM Tris-HCl, 8.0, 100mM NaCl, 1mM PMSF (phenylmethylsulfanylfluoride)), and only the lysed portion was used for purification.

For purification, the purified protein was purified using affinity chromatography and size exclusion chromatography, and the molecular weight of the purified protein was confirmed by denaturing SDS-PAGE electrophoresis. It was.

More specifically, affinity chromatography was performed using a Ni-NTA column (GE healthcare), and washed with column preparation and protein loading with A buffer (50 mM Tris-HCl, 8.0, 100 mM NaCl). After elution of the protein attached to the column with B buffer (50 mM Tris-HCl, 8.0, 100 mM NaCl, 500 mM Immidazole), the molecular weight was confirmed by electrophoresis (Laemmli, UK Nature 1970, 227, 680-685.).

In addition, size exclusion chromatography was performed using a Superdex 200 10/300 GL column (GE Heathcare), an injection volume of 2 mL, and a buffer of 50 mM Tris-HCl (8.0) and 100 mM NaCl.

As shown in FIG. 3C and FIG. 3D, a gene in which the portion corresponding to the E-helix sequence is removed from human ferritin (ferritin-ΔE) can also be seen in E. coli (FIG. 3C). ), Even in the case of fusion proteins having different repeat counts of GALA peptides, they are expressed in E. coli in large quantities, and the molecular weights of 21.9 (fGALA ₄ ) and 22.7 kDa (fGALA ₆ ) can be seen (FIG. 3D).

< Comparative example  1>

ferritin Formed by -ΔE Cage  Protein pH  Dissociation Activity by Change

Size exclusion chromatography was used to determine whether the ferritin-ΔE protein from which human Eritin sequence corresponding to the E-helix sequence was removed forms a cage protein as a unit. It confirmed and the result is shown in FIG.

Specifically, the samples were tested for each of human ferritin and ferritin-ΔE protein. Wild-type ferritin (molecular weight 440 kDa, NCBI accession No.) purchased from Sigma-Aldrich to produce a standard protein curve using size exclusion chromatography (Superdex 200 10/300 GL column) purchased by GE Healthcare. The elution volumes of the standard protein markers for NP_002023.2), BSA (molecular weight 67 kDa) and Cytochrome C (molecular weight 14 kDa) were 9-10 mL, 15-16 mL, and 20-21 mL, respectively.

The buffer composition used was 50 mM Tris-HCl (8.0), 100 mM NaCl, 1 mM mercaptoethanol, and the injection volume was 2 mL. First, the sample was adjusted to pH through dialysis for pH 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0, and size exclusion chromatography was performed for each pH. At this time the concentration of the sample was 1mg / mL and the injection volume (injection volume) was 2mL. At pH 3.0, the sample had an elution volume of about 20 mL, which was dissociated with no cage structure when compared to the standard protein curve. It was confirmed that the elution volume) represented about 9 mL to form the cage structure (see FIG. 4).

Specifically, as shown in FIG. 4, in the case of ferritin-ΔE protein, as in the conventionally known human ferritin, a cage is formed at a pH of 4.0 to 8.0, but below pH 4.0, the units are separated to form a cage. It can be seen that it is not formed. Therefore, the cage protein can be formed even by removing the E-helix portion of the human ferritin, and it can be seen that the dissociation activity by the pH change inherent in the cage protein is maintained.

< Experimental Example  1>

Of the present invention Cage  Protein pH  Dissociation Activity by Change

Measurement of dissociation activity according to the pH change of the cage protein formed of the fusion protein of the present invention, ie, ferritin-ΔE-GALA ₄ (fGALA ₄ ) and ferritin-ΔE-GALA ₆ (fGALA ₆ ) prepared in Example 1 The results are shown in FIG. 5.

Samples are fGALA ₄ , fGALA ₆ Experiments were performed for each. After adjusting the pH of the sample through dialysis for pH 7.0 and 8.0, the concentration was adjusted to 1 mg / mL and 2 mL was injected into the size exclusion column described in Comparative Example 1 above. The elution volume of the sample was about 9 ml, which was confirmed to form cage structure compared with the standard protein curve of Comparative Example 1, and the elution peak was caused by protein through electrophoresis. (See FIG. 5A).

In addition, when the pH was adjusted by dialysis for pH 6.8, 6.5, and 6.0, and the same concentration and volume were added to the same size exclusion column as described in <Comparative Example 1>, the elution peak was I could see it moved. The peak corresponding to about 9 mL shown above pH 7.0 was shifted to about 20 mL in the 6.0 section at pH 6.8, indicating that the cage structure was dissociated according to the standard protein curve of Comparative Example 1. In addition, it was confirmed that the elution peak of the pH 6.8-6.0 by the electrophoresis was due to the protein (see FIG. 5A).

In addition, the elution peak was shifted according to the pH range by overlapping the profile of the size exclusion chromatography performed at each pH, as shown in FIG. 5B.

In addition, since the same concentration of the sample was injected by the same volume, the peak area at each pH was analyzed to determine the percentage of the sample forming the cage structure, and plotted according to the pH value to draw a sigmoidal curve (Sigmoidal curve) as shown in FIG. 5C. It was. 5C shows that about 80% of the samples dissociated in cage structure at pH 6.5.

That is, as shown in FIG. 5, the cage protein into which the GALA peptide is inserted forms a large cage structure under neutral pH conditions, but the self-assembled cage structure in the vicinity of pH 6.0 causes three-dimensional structural change. It can be seen that it dissociates to the smallest unit.

On the other hand, CD (Circular Dichroism) analysis of the degree of protein folding according to the pH change of the cage protein formed by the human ferritin (ferritin) formed of the protein of SEQ ID NO: 1 and the fusion proteins (fGALA ₄ , fGALA ₆ ) of the present invention Comparison was made and the results are shown in FIG. 6.

Specifically, the sample is fGALA ₄ , fGALA ₆ Experiments were performed for each. The concentration of the sample was about 0.075 mg / mL and the buffer composition was 50 mM potassium phosphate, 100 mM NaCl for pH 7.0 and 6.0, respectively. CD scan speed was 10 nm / min in 0.1 cm path length quartz cells and was performed at 25 ° C. Proteins composed only of α-helices had peaks of 208 and 222 nm in the CD spectrum. Human ferritin formed of monomers of the protein of SEQ ID NO: 1 was also composed only of α-helices and used the same characteristic spectrum. CD spectra of human ferritin showed typical peaks characteristic of α-helix at pH 7.0 and 6.0. However, the sample showed peak changes at pH 7.0 and 6.0. A clear decrease occurred at the peak of 208 nm and a shift at the 222 nm peak. This shows the structural change of the monoliths constituting the sample.

That is, as shown in Figure 6, human ferritin (ferritin) formed of the protein of SEQ ID NO: 1 unit has no change in the CD spectrum profile at neutral and pH 6.0 (Fig. 6A), by the fusion protein of the present invention In the cage protein formed, a sharp profile change was observed at a wavelength of 208 and 222 nm at pH 6.0, indicating that the degree of folding of the cage protein of the present invention was changed by weak acidity. (FIG. 6B)

In addition, a thermal stabilization experiment was performed to observe the ellipticity of the CD spectrum at 222 nm for the cage protein formed by human ferritin (ferritin) formed by the protein of SEQ ID NO: 1 and the fusion proteins of the present invention (fGALA ₄ , fGALA ₆ ). The results are shown in FIG. 7.

Specifically, the sample is fGALA ₄ , fGALA ₆ Experiments were performed for each. The thermal stability of the samples was evaluated by observing ellipticity by varying the temperature at 222 nm wavelength. Human ferritin samples formed of the monomer of the protein of SEQ ID NO: 1 were very stable to heat because there was no distinct melting temperature (Tm) at pH 7.0 and 6.0, respectively, when the temperature was changed from 5 ° C to 100 ° C. However, the sample showed a distinct sigmoidal curve at pH 7.0 and the Tm value was 78.0 ± 1.0 ° C. This transition inflection represents unfolding of the scaffold by heat. At pH 6.5 the sample showed two Tm values, 39.0 ± 0.5 ° C and 75.0 ± 1.0 ° C, respectively. This shows that the sample is very unstable in the pH range where the cage structure dissociates. No distinct Tm was found at pH 6.0, indicating that the sample is very unstable. These thermal stability results show the pH sensitivity of the sample with size exclusion chromatography and CD spectra.

That is, as shown in Figure 7, the human ferritin (ferritin) formed as a unit of the protein of SEQ ID NO: 1 shows a stability such that almost no change in protein folding at pH 6.0 (Fig. 7A), The cage protein formed by the fusion protein of the invention showed rapid protein folding conversion near 78 ° C. in neutral and an additional protein folding turning point at pH 6.5 (FIG. 7B).

Therefore, the cage protein formed by the fusion protein of the present invention has a property of dissociating a cage structure in which existing human ferritin is self-assembled only in a strong acidity of less than pH 4.0 into monomers, in weak acidity such as pH 6.0 to 6.5, It can be effectively used as a drug carrier because it can release the drug effectively in a weak acidic environment such as tissues, and can also be effectively used as a biosensor by using a property that reacts to a weakly acidic stimulus to cause structural changes. .

<110> Korea Institute of Science and Technology <120> Fusion protein comprising ferritin and GALA peptide, cage protein          formed thereby, and novel use <130> DPP20102338KR <160> 16 <170> KopatentIn 1.71 <210> 1 <211> 175 <212> PRT <213> Human ferritin <400> 1 Met Ser Ser Gln Ile Arg Gln Asn Tyr Ser Thr Asp Val Glu Ala Ala   1 5 10 15 Val Asn Ser Leu Val Asn Leu Tyr Leu Gln Ala Ser Tyr Thr Tyr Leu              20 25 30 Ser Leu Gly Phe Tyr Phe Asp Arg Asp Asp Val Ala Leu Glu Gly Val          35 40 45 Ser His Phe Phe Arg Glu Leu Ala Glu Glu Lys Arg Glu Gly Tyr Glu      50 55 60 Arg Leu Leu Lys Met Gln Asn Gln Arg Gly Gly Arg Ala Leu Phe Gln  65 70 75 80 Asp Ile Lys Lys Pro Ala Glu Asp Glu Trp Gly Lys Thr Pro Asp Ala                  85 90 95 Met Lys Ala Ala Met Thr Leu Glu Lys Lys Leu Asn Gln Ala Leu Leu             100 105 110 Asp Leu His Ala Leu Gly Ser Ala Arg Thr Asp Pro His Leu Cys Asp         115 120 125 Phe Leu Glu Thr His Phe Leu Asp Glu Glu Val Lys Leu Ile Lys Lys     130 135 140 Met Gly Asp His Leu Thr Asn Leu His Arg Leu Gly Gly Pro Glu Ala 145 150 155 160 Gly Leu Gly Glu Tyr Leu Phe Glu Arg Leu Thr Leu Lys His Asp                 165 170 175 <210> 2 <211> 525 <212> DNA <213> Human ferritin <400> 2 atgagctccc agattcgtca gaattattcc accgacgtgg aggcagccgt caacagcctg 60 gtcaatttgt acctgcaggc ctcctacacc tacctctctc tgggcttcta tttcgaccgc 120 gatgatgtgg ctctggaagg cgtgagccac ttcttccgcg aactggccga ggagaagcgc 180 gagggctacg agcgtctcct gaagatgcaa aaccagcgtg gcggccgcgc tctcttccag 240 gacatcaaga agccagctga agatgagtgg ggtaaaaccc cagacgccat gaaagctgcc 300 atggccctgg agaaaaagct gaaccaggcc cttttggatc ttcatgccct gggttctgcc 360 cgcacggacc cccatctctg tgacttcctg gagactcact tcctagatga ggaagtgaag 420 cttatcaaga agatgggtga ccacctgacc aacctccaca ggctgggtgg cccggaggct 480 gggctgggcg agtatctctt cgaaaggctc actctcaagc acgac 525 <210> 3 <211> 160 <212> PRT <213> Artificial Sequence <220> <223> human ferritin without E helix <400> 3 Met Ser Ser Gln Ile Arg Gln Asn Tyr Ser Thr Asp Val Glu Ala Ala   1 5 10 15 Val Asn Ser Leu Val Asn Leu Tyr Leu Gln Ala Ser Tyr Thr Tyr Leu              20 25 30 Ser Leu Gly Phe Tyr Phe Asp Arg Asp Asp Val Ala Leu Glu Gly Val          35 40 45 Ser His Phe Phe Arg Glu Leu Ala Glu Glu Lys Arg Glu Gly Tyr Glu      50 55 60 Arg Leu Leu Lys Met Gln Asn Gln Arg Gly Gly Arg Ala Leu Phe Gln  65 70 75 80 Asp Ile Lys Lys Pro Ala Glu Asp Glu Trp Gly Lys Thr Pro Asp Ala                  85 90 95 Met Lys Ala Ala Met Ala Leu Glu Lys Lys Leu Asn Gln Ala Leu Leu             100 105 110 Asp Leu His Ala Leu Gly Ser Ala Arg Thr Asp Pro His Leu Cys Asp         115 120 125 Phe Leu Glu Thr His Phe Leu Asp Glu Glu Val Lys Leu Ile Lys Lys     130 135 140 Met Gly Asp His Leu Thr Asn Leu His Arg Leu Gly Gly Pro Glu Ala 145 150 155 160 <210> 4 <211> 480 <212> DNA <213> Artificial Sequence <220> <223> human ferritin without E-helix <400> 4 atgagctccc agattcgtca gaattattcc accgacgtgg aggcagccgt caacagcctg 60 gtcaatttgt acctgcaggc ctcctacacc tacctctctc tgggcttcta tttcgaccgc 120 gatgatgtgg ctctggaagg cgtgagccac ttcttccgcg aactggccga ggagaagcgc 180 gagggctacg agcgtctcct gaagatgcaa aaccagcgtg gcggccgcgc tctcttccag 240 gacatcaaga agccagctga agatgagtgg ggtaaaaccc cagacgccat gaaagctgcc 300 atggccctgg agaaaaagct gaaccaggcc cttttggatc ttcatgccct gggttctgcc 360 cgcacggacc cccatctctg tgacttcctg gagactcact tcctagatga ggaagtgaag 420 cttatcaaga agatgggtga ccacctgacc aacctccaca ggctgggtgg cccggaggct 480                                                                          480 <210> 5 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> GALA4 <400> 5 Leu Ala Glu Ala Leu Ala Glu His Leu Ala Glu Ala Leu Ala Glu   1 5 10 15 <210> 6 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> GALA4 <400> 6 ctggcggaag cgctggcgga acatctggcg gaagcgctgg cggaa 45 <210> 7 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> GALA6 <400> 7 Leu Ala Glu Ala Leu Ala Glu His Leu Ala Glu Ala Leu Ala Glu Ala   1 5 10 15 Leu Ala Glu Ala Leu Ala Glu              20 <210> 8 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> GALA6 <400> 8 ctggcggaag cgctggcgga acatctggcg gaagcgctgg cggaagcgct ggcggaagcg 60 ctggcggaa 69 <210> 9 <211> 175 <212> PRT <213> Artificial Sequence <220> <223> fGALA4 <400> 9 Met Ser Ser Gln Ile Arg Gln Asn Tyr Ser Thr Asp Val Glu Ala Ala   1 5 10 15 Val Asn Ser Leu Val Asn Leu Tyr Leu Gln Ala Ser Tyr Thr Tyr Leu              20 25 30 Ser Leu Gly Phe Tyr Phe Asp Arg Asp Asp Val Ala Leu Glu Gly Val          35 40 45 Ser His Phe Phe Arg Glu Leu Ala Glu Glu Lys Arg Glu Gly Tyr Glu      50 55 60 Arg Leu Leu Lys Met Gln Asn Gln Arg Gly Gly Arg Ala Leu Phe Gln  65 70 75 80 Asp Ile Lys Lys Pro Ala Glu Asp Glu Trp Gly Lys Thr Pro Asp Ala                  85 90 95 Met Lys Ala Ala Met Ala Leu Glu Lys Lys Leu Asn Gln Ala Leu Leu             100 105 110 Asp Leu His Ala Leu Gly Ser Ala Arg Thr Asp Pro His Leu Cys Asp         115 120 125 Phe Leu Glu Thr His Phe Leu Asp Glu Glu Val Lys Leu Ile Lys Lys     130 135 140 Met Gly Asp His Leu Thr Asn Leu His Arg Leu Gly Gly Pro Glu Ala 145 150 155 160 Leu Ala Glu Ala Leu Ala Glu His Leu Ala Glu Ala Leu Ala Glu                 165 170 175 <210> 10 <211> 525 <212> DNA <213> Artificial Sequence <220> <223> fGALA4 <400> 10 atgagctccc agattcgtca gaattattcc accgacgtgg aggcagccgt caacagcctg 60 gtcaatttgt acctgcaggc ctcctacacc tacctctctc tgggcttcta tttcgaccgc 120 gatgatgtgg ctctggaagg cgtgagccac ttcttccgcg aactggccga ggagaagcgc 180 gagggctacg agcgtctcct gaagatgcaa aaccagcgtg gcggccgcgc tctcttccag 240 gacatcaaga agccagctga agatgagtgg ggtaaaaccc cagacgccat gaaagctgcc 300 atggccctgg agaaaaagct gaaccaggcc cttttggatc ttcatgccct gggttctgcc 360 cgcacggacc cccatctctg tgacttcctg gagactcact tcctagatga ggaagtgaag 420 cttatcaaga agatgggtga ccacctgacc aacctccaca ggctgggtgg cccggaggct 480 ctggcggaag cgctggcgga acatctggcg gaagcgctgg cggaa 525 <210> 11 <211> 183 <212> PRT <213> Artificial Sequence <220> <223> fGALA6 <400> 11 Met Ser Ser Gln Ile Arg Gln Asn Tyr Ser Thr Asp Val Glu Ala Ala   1 5 10 15 Val Asn Ser Leu Val Asn Leu Tyr Leu Gln Ala Ser Tyr Thr Tyr Leu              20 25 30 Ser Leu Gly Phe Tyr Phe Asp Arg Asp Asp Val Ala Leu Glu Gly Val          35 40 45 Ser His Phe Phe Arg Glu Leu Ala Glu Glu Lys Arg Glu Gly Tyr Glu      50 55 60 Arg Leu Leu Lys Met Gln Asn Gln Arg Gly Gly Arg Ala Leu Phe Gln  65 70 75 80 Asp Ile Lys Lys Pro Ala Glu Asp Glu Trp Gly Lys Thr Pro Asp Ala                  85 90 95 Met Lys Ala Ala Met Ala Leu Glu Lys Lys Leu Asn Gln Ala Leu Leu             100 105 110 Asp Leu His Ala Leu Gly Ser Ala Arg Thr Asp Pro His Leu Cys Asp         115 120 125 Phe Leu Glu Thr His Phe Leu Asp Glu Glu Val Lys Leu Ile Lys Lys     130 135 140 Met Gly Asp His Leu Thr Asn Leu His Arg Leu Gly Gly Pro Glu Ala 145 150 155 160 Leu Ala Glu Ala Leu Ala Glu His Leu Ala Glu Ala Leu Ala Glu Ala                 165 170 175 Leu Ala Glu Ala Leu Ala Glu             180 <210> 12 <211> 549 <212> DNA <213> Artificial Sequence <220> <223> fGALA6 <400> 12 atgagctccc agattcgtca gaattattcc accgacgtgg aggcagccgt caacagcctg 60 gtcaatttgt acctgcaggc ctcctacacc tacctctctc tgggcttcta tttcgaccgc 120 gatgatgtgg ctctggaagg cgtgagccac ttcttccgcg aactggccga ggagaagcgc 180 gagggctacg agcgtctcct gaagatgcaa aaccagcgtg gcggccgcgc tctcttccag 240 gacatcaaga agccagctga agatgagtgg ggtaaaaccc cagacgccat gaaagctgcc 300 atggccctgg agaaaaagct gaaccaggcc cttttggatc ttcatgccct gggttctgcc 360 cgcacggacc cccatctctg tgacttcctg gagactcact tcctagatga ggaagtgaag 420 cttatcaaga agatgggtga ccacctgacc aacctccaca ggctgggtgg cccggaggct 480 ctggcggaag cgctggcgga acatctggcg gaagcgctgg cggaagcgct ggcggaagcg 540 ctggcggaa 549 <210> 13 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> GALA <400> 13 Glu Ala Leu Ala   One <210> 14 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 14 ggaattccat atgagctccc agattcgtca g 31 <210> 15 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for fGALA4 <400> 15 ccgccgctcg agttattcag ctaaagcttc agctaaatgt tcagctaaag cttcagctaa 60 agcctccggg cc 72 <210> 16 <211> 96 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for fGALA6 <400> 16 ccgccgctcg agttattcag ctaaagcttc agctaaagct tcagctaaag cttcagctaa 60 atgttcagct aaagcttcag ctaaagcctc cgggcc 96

Claims (22)

(a) a carboxyl terminal of a polypeptide selected from the group consisting of ferritin protein, fragments comprising A-helix, B-helix, C-helix and D-helix of the protein and variants thereof on
(b) a fusion protein to which a GALA (Glu-Ala-Leu-Ala) motif comprising the amino acid sequence of SEQ ID NO: 13 is linked. The fusion protein of claim 1, wherein the ferritin protein is represented by the amino acid sequence of SEQ ID NO: 1. The fusion protein of claim 1, wherein the fragment is one or more amino acid residues removed after the 155th amino acid sequence of SEQ ID NO: 1. The fusion protein of claim 3, wherein the fragment has one or more amino acid residues removed from the amino acid sequence of SEQ ID 1 after 160. The fusion protein according to claim 4, wherein the fragment is represented by the amino acid sequence of SEQ ID NO. According to claim 1, wherein the GALA motif is a fusion protein consisting of 4 to 100 amino acids comprising the amino acid sequence of SEQ ID NO: 13. The fusion protein of claim 6, wherein the GALA motif comprises an amino acid sequence in which the amino acid sequence of SEQ ID NO: 13 is repeated 1 to 10 times. The fusion protein according to claim 7, wherein the GALA motif is represented by the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 7. The fusion protein of claim 1, wherein the fusion protein is represented by the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 11. Cage protein consisting of the fusion protein of any one of claims 1 to 9 The cage protein of claim 10, wherein the cage protein is a fusion protein in which the protein is regularly arranged as a unit. The cage protein of claim 10, wherein the cage protein has an average molecular weight of 450 kDa to 550 kDa. A polynucleotide encoding the fusion protein of claim 1. The polynucleotide of claim 13, wherein the polynucleotide is represented by a nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 12. An expression vector into which the polynucleotide of claim 13 is introduced. The expression vector of claim 15, wherein the expression vector is represented by a cleavage map of FIG. 9 or FIG. 10. A transformant transformed with the expression vector of claim 16. The transformant of claim 17, wherein the transformant is Escherichia coli. A bioactive substance carrier comprising a bioactive substance enclosed in the cage protein of claim 10. The bioactive substance carrier according to claim 19, wherein the bioactive substance is a drug for preventing or treating a disease selected from the group consisting of cancer, osteoarthritis, rheumatoid arthritis, dementia, autoimmune disease and stroke. A biosensor comprising the labeling agent encapsulated in the cage protein of claim 10 as an active ingredient. The biosensor of claim 21, wherein the biosensor detects a change in pH from pH 7.0 to 7.4 to pH 6.0 to 6.5 in the cell or tissue, or detects a cell or tissue having a pH of 6.0 to 6.5.

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