Human-derived ferritin monomer fragment and fusion polypeptide using same

Abstract

Disclosed are a human-derived ferritin monomer fragment and a fusion polypeptide using the same, and more particularly, to a human-derived ferritin of which a portion of a fourth loop and a fifth helix of the ferritin monomer fragment are removed, and a fusion polypeptide in which a polypeptide or a protein is fused to an N-terminus or a C-terminus of the ferritin monomer fragment.

Claims

1. A fusion polypeptide comprising a human-derived ferritin monomer fragment comprising the amino acid sequence of SEQ ID NO: 2, further comprising a pro-apoptotic polypeptide comprising the amino acid sequence of SEQ ID NO: 3 fused to the N-terminus of SEQ ID NO: 2 and a green fluorescent protein fused to the C-terminus of SEQ ID NO: 2.

2. The fusion polypeptide of claim 1, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 6 or 7.

3. A drug delivery system comprising the fusion polypeptide of claim 1.

4. The drug delivery system of claim 3, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 6 or 7.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 a schematic diagram showing a wild-type ferritin monomer, a short ferritin monomer from which helix V is removed, and a ferritin cage of each of the wild-type ferritin monomer and the short ferritin monomer.

(2) FIG. 2A is a schematic diagram showing a fusion peptide in which GFP is bound to a C-terminus of the short ferritin monomer (sFt-GFP) and a fusion peptide in which GFP is bound to a C-terminus of the wild-type ferritin monomer, and FIG. 2B shows the results of observing whether sFt-GFP and wFt-GFP each form a cage by TEM imaging.

(3) FIG. 3 shows the results of SDS-PAGE analysis of the tendency of a linker to be cleaved after sFt-GFP and wFt-GFP are each cultured with MMP-2 for 1 hour at a temperature of 37 C. (FT: ferritin).

(4) FIG. 4 shows the results of SDS-PAGE analysis of the purified fragments of each of sFt-GFP and wFt-GFP with respect to molecular weight markers (uninduced: whole cell suspension before induction of protein expression by IPTG; induced: whole cell suspension after induction of protein expression; sup: soluble cell lysate (supernatant); ppt: cell lysis precipitant; NTA purified from sup: bound fraction to NTA agarose beads from the soluble cell lysate; and NTA FT from sup: unbound fraction (flow through) to NTA agarose beads from the soluble cell lysate).

(5) FIG. 5 is a schematic diagram showing a fusion peptide (KLAK (SEQ ID NO:16)-sFt-GFP) and a cage formed by the fusion peptide, wherein the fusion peptide includes GFP fused thereto via a linker including a pro-apoptotic peptide at the N-terminus of sFt and a sequence, which can be cleared by MMP-2, at the C-terminus of the sFt.

(6) FIG. 6A shows the results of observing whether the wild-type ferritin, the short ferritin, sFt-GFP fusion peptide, and the KLAK (SEQ ID NO:16)-sFt-GFP fusion peptide each form a cage by TEM imaging, and FIG. 6B shows the results of SEC analysis of the case formed by each of the short ferritin, sFt-GFP fusion peptide, and the KLAK (SEQ ID NO:16)-sFt-GFP fusion peptide.

(7) FIG. 7A is a schematic diagram showing a fusion peptide fused with GFP via a linker including an amino acid sequence, which can be cleaved by MMP-2, at the C-terminus of sFt (type I) and a fusion peptide fused with a pro-apoptotic peptide and GFP via a linker including amino acid sequences, which can be cleaved by MMP-2, at the C-terminus and the N-terminus of sFt (type II), and FIG. 7B shows the results observed after the fusion peptide type I and the fusion peptide type II are each treated with MMP-2 and the cleaved products are loaded on an SDS PAGE gel.

(8) FIG. 8 shows the results observed after sFt-GFP fusion peptide and the KLAK (SEQ ID NO:16)-sFt-GFP fusion peptide are each treated in a cell line so that intracellular uptake of each fusion peptide is determined (A: FACS analysis results of MDA-MB-231 cell line (cells expressing a cancer-targeting peptide receptor (p32), left panel) and HL-60 cell line (control cells not expressing the p32, right panel), B: fluorescence microscopic observation results, C: confocal microscopic Z-stack analysis results).

(9) FIG. 9A shows the MTT assay results of evaluating the cytotoxicity of the KLAK (SEQ ID NO:16)-sFt-GFP fusion peptide, and FIG. 9B shows the FACS results of evaluating the cytotoxicity of the KLAK (SEQ ID NO:16)-sFt-GFP fusion peptide.

(10) FIG. 10 shows the results of evaluating in vivo cytotoxic activity of the KLAK (SEQ ID NO:16)-sFt-GFP fusion peptide, wherein the results are obtained by histological test using a confocal microscope on a tumor obtained through an animal experiment (Nuclear: blue; apoptotic region: red; and Scale bar=40 m).

BEST MODE

(11) Hereinafter, the present disclosure is described in detail.

(12) Here, Examples below are described herein are illustrative examples of embodiments and are not intended to otherwise limit the scope of embodiments in any way.

EXPERIMENTAL METHOD

(13) 1. Preparation of a Fusion Peptide (KLAK (SEQ ID NO:16)-sFt-GFP) in which CGKRK(KLAKLAK).sub.2 (SEQ ID NO: 3) and GFP are Fused to a Short Ferritin Monomer

(14) A recombinant plasmid expressing Double Chambered Nano Cage (DCNC) was prepared by using a modified pET28 vector (Novagen). For efficient cloning, the modified pET28 vector had KpnI and NheI between NcoI/NdeI and an additional restriction enzymatic site for SpeI between EcoRI/SalI. A gene encoding a short-ferritin (sFt) heavy chain (1 to 161 amino acids) was obtained by PCR using cDNA of a human-derived ferritin heavy chain (Sino Biological Inc.), and then, inserted according to the previously reported method (ACS nano 2013, 7, (9), 7462-7471. etc) to NdeI and BamH1 sites to utilize bacteria-expression.

(15) An oligonucleotide encoding CGKRK(KLAKLAK).sub.2 (SEQ ID NO 3) was synthesized, and then, inserted between KpnI and NheI. A signal enhanced green fluorescent protein (seGFP) gene was prepared by PCR, and then, inserted between SpeI and XhoI. A synthesized and flow linker (GSGGGSG, SEQ ID NO: 17) was inserted between BamHI and EcoRI, and an MMP-2-cleavage sequence (GPLGLAGGGSG, SEQ ID NO: 18) was synthesized and then inserted between EcoRI and SpeI. This sequence finally produced a linker having the GSGGGSGEFGPLGLAGGGSGTS (SEQ ID NO: 5) sequence between GF and the ferritin monomer.

(16) To insert an MMP-2 cleavage site between the N-terminus chamber and the ferritin monomer, the GPLGLAG (SEQ ID NO: 19) sequence was synthesized and then inserted between NheI and NdeI.

(17) An sFt-GFP fusion peptide was prepared according to the same cloning method, except that the CGKRK(KLAKLAK).sub.2 (SEQ ID NO: 3) sequence was inserted.

(18) 2. Expression and Purification of Protein

(19) The protein was over-expressed in cells of E. coli BL21(DE3). Here, the cells were cultured in an LB medium at a temperature of 37 C., and when the OD600 value reached 0.5, the expression of the cells was induced using IPTG 1 mM. Afterwards, the cells were collected through centrifugation, and then, the pellets were disrupted using a lysis buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 1% TritonX-100, 1 mM PMSF, and 0.5 mM DTT) with an ultrasonic processor. The expressed protein obtained from the cell lysates was purified according to the previously reported method using NTA agarose beads (see J. Analytical chemistry 2011, 83, (15), 5834-5843).

(20) 3. Confirmation of KLAK (SEQ ID NO:16)-sFt-GFP DCNC

(21) Following the purification of the protein, the resulting protein was analyzed using the size exclusion chromatography (SEC, Superdex 200 10/300 GL column). Here, the oligomer state was determined by comparing an elusion volume with a standard molecular weight. A protein-elusion profile was observed by measuring the absorbance at 280 nm. TEM images were recorded using FEI Tecnai (Korea Basic Science Institute (KBSI).

(22) 4. Cleavage of KLAK (SEQ ID NO:16)-sFt-GFP DCND by Recombinant MMP-2

(23) Recombinant MMP-2 was purchased from R&D systems (Minneapolis, Minn., USA), and used for a cleavage test according to the manufacturer's instructions. MMP-2 was first activated by incubation with p-aminophenylmercuric acetate (APMA, 1 mM, Sigma, Saint Louis, Mo., USA) at a temperature of 37 C. for 1 hour. Activated MMP-2 (0, 25, 50, 100 ng) was then added to 20 g of DCNC to prepare a TCBN buffer having a final volume of 40 l (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM CaCl.sub.2, and 0.05% Grij-35). After incubation at a temperature of 37 C. for 1 hour, DCNC only or DCNC reacted with MMP-2 was loaded onto 12% SDS-PAGE.

(24) 5. Evaluation of Cell Binding and Intracellular Uptake

(25) A human breast cancer cell line, MDA-MB-231, (ATCC, Manassas, Va.), was cultured in a DMEM (high glucose) medium. To analyze the intracellular uptake of the MDA-MB-231 cells, 210.sup.5 MDA-MB-231 cells were cultured with KLAK-sFt-GFP DCNC or sFt-GFP at a temperature of 37 C. for 1 hour. Then, the cultured MDA-MB-231 cells were washed with PBS and re-suspended, and green fluorescence of the MDA-MB-231 cells was analyzed using FACS Calibur cytometry (BD Biosciences, SanJose, Calif., USA). For microscopic analysis, the MDA-MB-231 cells were seeded into 8 chamber culture slides at a density of 110.sup.5 cells/chamber, and then, cultured overnight for attachment. The MDA-MB-231 cells were cultured with 1.4 M of KLAK (SEQ ID NO:16)-sFt-GFP DCNC or sFt-GFP at a temperature of 37 C. for 1 hour. The nuclei were stained with DAPI, and the slides were analyzed using a fluorescence microscope. To observe the distribution of nanoparticles in the cytoplasm, a confocal microscope (Carl Zeiss, Oberkochen, Germany) was used, and the cells were treated with lectin to stain a cell membrane for z-sectional imaging.

(26) 6. Evaluation of Cytotoxicity of KLAK (SEQ ID NO:16)-sFt-GFP DCNC

(27) The cytotoxicity of KLAK (SEQ ID NO:16)-sFt-GFP DCNC was evaluated using the MDA-MB-231 cell line. The MDA-MB-231 cells were seeded onto a 96-well plate and cultured for 24 hours. Afterwards, the culture medium was replaced with a fresh DMEM medium containing KLAK (SEQ ID NO:16)-sFt-GFP DCNC (0.5 M to 4 M). As a control group, 4 M sFt-GFP was added to a medium. After 48 hours of incubation, the cell viability of the MDA-MB-231 cells was evaluated according to MTT assay.

(28) Whether the apoptosis was induced or not was evaluated according to FACS using Annexin V-Alexa Fluor 647 (Invitrogen) and propidium iodide (PI).

(29) 110.sup.5 MDA-MB-231 cells were cultured in a medium containing 0.35 M, 0.7 M, and 1.4 M of KLAK (SEQ ID NO:16)-sFt-GFP DCNC, respectively, at a temperature of 37 C. for 24 hours. The MDA-MB-231 cells were washed with PBS and binding buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM CaCl.sub.2) each three times. The washed MDA-MB-231 cells were cultured with Annexin V-Alexa Fluor 647 and PI at a temperature of 37 C. for 20 minutes, and then, immediately subjected to FACS analysis. As a control group regarding the apoptosis, etoposide (50 M, Sigma) was treated with cells and then observed.

(30) 7. Effect of KLAK (SEQ ID NO:16)-sFt-GFP DCNC on Apoptosis in Mouse Model

(31) All animal experiments were carried out according to the instructions of the institute and the animal experiment methods approved by the Institutional Animal Care and Use Committee (IACUC) of Kyungpook National University (Authorization number: KNU 2015-0017). Here, the present inventors tried the best to minimize the pain of the animals. Female BALB/c nude mice (4 mice per group, a total of 16 mice) aged 6 to 8 weeks and having a weight of 203 g were used, and the MDA-MB-231 cells (110.sup.6) were infected to the right shoulder of the mice. When 100 mm.sup.3 tumors were observed, 100 L of KLAK (SEQ ID NO:16)-sFt-GFP (30 mol/L) was administered to the mice intravenously three times a week. For comparison, the same amount of a KLAK peptide only, sFt-GFP+KLAK peptide (SEQ ID NO:16), or saline was used. After 4 times of the administration (after 9 days), the animals were euthanized with CO.sub.2. The tumor tissues were removed from the animals, fixed with 4% paraformaldehyde (PFA) overnight, and then, frozen with cryosectioning. A confocal microscope (Zeiss, Germany) was used for the immunohistochemical studies. According to the manufacturer's instructions, the apoptosis was evaluated by a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining method.

Experimental Results (Examples)

Example 1

(32) Preparation and Characteristics of Ferritin (Short Ferritin, sFt) Monomer

(33) It has been verified whether ferritin, which has variously modified surfaces with peptides, chemicals, and proteins, was able to form a cage structure. To expose a ligand to the surface of the cage, two positions were frequently selected, wherein the two positions were each a short loop present between the N-terminus of the ferritin monomer and IV helix and V helix. The V helix which is located inside the cage and is bent protruded out of the case when a large protein fragment was bound to the C-terminus of the ferritin monomer.

(34) To develop a nanoplatform that exhibits dual functionality, the present inventors removed the fifth helix of the ferritin monomer to prepare a short-ferritin (sFt) monomer (see FIG. 1), and the resulting sFT monomer was compared with a wild-type ferritin (wFt) monomer.

(35) That is, to prepare the characteristics of sFt and wFt, GFP which is relatively large peptide was fused to a C-terminus of each of sFt and wFt, thereby preparing a sFt-GFP fusion peptide and a wFt-GFP fusion peptide (see FIG. 2A). Then, whether these two fusion peptides both formed a cage, respectively, was confirmed by transmission electro microscopy (TEM) (see FIG. 2B).

(36) Both sFt-GFP and wFt-GFP were well expressed in E. coli and formed a cage, respectively. However, sFt-GFP was expressed about three times more than wFt-GFP, resulting that the protein expression of sFt-GFP was significantly excellent.

(37) In addition, it was confirmed that sFt-GFP was more easily cleaved by MMP-2 as compared with wFt-GFP (see FIG. 3), and that a His-tag attached to the N-terminus of sFt-GFP was more efficiently separated by NTA-agarose beads as compared with a His-tag attached to the N-terminus of wFt-GFP (see FIG. 4).

(38) According to the results above, it was determined that, since the fusion peptide in which macromolecule GFP was fused to the C-terminus of wFt had a very dense spatial structure, a linker site to be cleaved by MMP-2 was not easily exposed. In this regard, it is deemed that 6 His-tags attached to the N-terminus could be sterically screened. However, in the case of sFT in which V helix was removed from the wFT monomer, even if macromolecule GFP was fused to the C-terminus of sFT, a linker site was easily cleaved by MMP-2 and a His-tag attached to the N-terminus of sFT was easily separated. In this regard, it was confirmed that, as compared with wFt, sFT had a spatial structure that was significantly exposed.

Example 2

(39) Preparation of Pro-Apoptotic Peptide and Fusion Peptide in which GFP was Fused to sFt

(40) According to the experimental results above, it was confirmed that sFt was available as a dual delivery system of a peptide and/or a protein and was easily cleaved by an enzyme so that the release of a fusion peptide was easily facilitated using sFT at a target site. In this regard, the present inventors prepared a fusion peptide (KLAK (SEQ ID NO:16)-sFt-GFP) in which a pro-apoptotic peptide, CGKRK(KLAKLAK).sub.2 (SEQ ID NO: 3), was fused to the N-terminus of sFt and GFP linked with a linker including a sequence that can be cleaved by MMP-2 was fused to the C-terminus of sFt (see FIG. 5).

Example 3

(41) Test on Cage Formation of KLAK (SEQ ID NO:16)-sFt-GFP Fusion Peptide

(42) To determine whether the KLAK (SEQ ID NO:16)-sFt-GFP fusion peptide formed a cage, the KLAK (SEQ ID NO:16)-sFt-GFP fusion peptide was observed by SEC and TEM. In addition, to compare the tendency of cage formation, wFt, sFt, and sFt-GFP fusion peptide were each observed in terms of the cage formation.

(43) The results on this observation are shown in FIG. 6

(44) As shown in FIG. 6A, it was confirmed that the KLAK (SEQ ID NO:16)-sFt-GFP fusion peptide formed with a cage of a certain size together with wFt, sFt, and sFt-GFP fusion peptide. It was also confirmed that KLAK (SEQ ID NO:16)-sFt-GFP fusion peptide and sFt-GFP fusion peptide each formed a cage of a similar size as compared with a cage formed by wFt and sFt.

(45) As shown in FIG. 6B, the SEC analysis results confirm that KLAK (SEQ ID NO:16)-sFt-GFP fusion peptide and sFt-GFP fusion peptide released proteins earlier than proteins released by sFt, resulting that the cages formed by KLAK (SEQ ID NO:16)-sFt-GFP fusion peptide and sFt-GFP fusion peptide were larger than the case formed by sFT.

(46) According to the results above, it was confirmed that, even if a peptide or a protein was fused to the N-terminus and the C-terminus of sFt, there was no problem in the cage formation.

Example 4

(47) Linker Cleavage in KLAK (SEQ ID NO:16)-sFt-GFP Fusion Peptide by Enzyme

(48) It was attempted to determine whether linkers linking peptides fused to KLAK (SEQ ID NO:16)-sFt-GFP fusion peptide could be exposed by an enzyme or whether these linkers could be cleaved by an enzyme. In type I fusion peptide, GFP was fused to the C-terminus of sFt via a linker having a sequence that can be cleaved by MMP-2, and in type II fusion peptide, GFT and pro-apoptotic peptide were fused to the C-terminus and the N-terminus of sFT, respectively, via the same linker (see FIG. 7A). MMP-2 was treated with these two fusion peptides, and then, observed after being loaded onto SDS PAGE gel.

(49) The results of this observation are shown in FIG. 7B.

(50) As shown in FIG. 7B, each linker of type I fusion peptide and type II fusion peptide was cleaved, and products generated by the linker cleavage were observed in a concentration-dependent manner. According to the results above, it was confirmed that, when a linker was used to fuse a target peptide or protein to the C-terminus and/or the N-terminus of sFt, the linker could be exposed and cleaved by an enzyme.

(51) Therefore, by binding a linker having an amino acid sequence that can be decomposed by a specific enzyme to the C-terminus and/or the N-terminus of sFt to fuse a peptide or protein, a resulting fused peptide and protein was able to be released.

Example 5

(52) Evaluation of Binding of KLAK (SEQ ID NO:16)-sFt-GFP Fusion Peptide to Cells and Intracellular Uptake of the Peptide

(53) A p32 receptor, which is a protein over-expressed on a surface of a specific cancer cell, was able to recognize CGKRK peptide (SEQ ID NO: 15) fused to the N-terminus of KLAK (SEQ ID NO:16)-sFt-GFP fusion peptide, and to uptake CGKRK peptide into the cytoplasm. Thus, KLAK (SEQ ID NO:16)-sFt-GFP fusion peptide was observed in consideration of binding thereof to cells and intracellular uptake thereof.

(54) First, MDA-MB-231 cells and HL-60 cells in which p32 is not expressed were treated with KLAK (SEQ ID NO:16)-sFt-GFP fusion peptide or sFt-GFP fusion peptide, and then, cultured at a temperature of 37 C. for 1 hour. Afterwards, FACS was used to analyze whether intracellular fluorescence was expressed.

(55) The results of this analysis are shown in FIG. 8A. As shown in FIG. 8A, in a group of cells treated with KLAK (SEQ ID NO:16)-sFt-GFP fusion peptide including CGKRK peptide at the N-terminus thereof, intracellular fluorescence was observed in a concentration-dependent manner, whereas, in a group of cells treated with sFt-GFP peptide in which GFP was fused to the C-terminus thereof, very low fluorescence was observed due to non-specific pinocytosis that caused transportation of the fusion peptide into cells. In addition, in a group of HL-60 cells, it was confirmed that, regardless of treatment with KLAK (SEQ ID NO:16)-sFt-GFP DCNC, the fusion peptide was non-specifically transported into the cells so that very low fluorescence was observed in the same manner as in a control group and a group of cells treated with sFt-GFP peptide.

(56) In addition, MDA-MB-231 cells were treated with KLAK (SEQ ID NO:16)-sFt-GFP fusion peptide or sFt-GFP fusion peptide, and then, cultured at a temperature of 37 C. for 1 hour. Afterwards, a fluorescent microscope was used to observe the intracellular uptake of the fusion peptide.

(57) The results of this observation are shown in FIG. 8B. As shown in FIG. 8B, green fluorescence along with nuclei (blue) was observed in a group of cells treated with KLAK (SEQ ID NO:16)-sFt-GFP fusion peptide, resulting that the fusion peptide was uptake into the cytoplasm. However, in a group of cells treated with sFt-GFP fusion peptide, only nuclei of the cells were observed without green fluorescence in the cytoplasm.

(58) However, as a result of the confocal microscopic Z-stack analysis on the cells treated with KLAK (SEQ ID NO:16)-sFt-GFP fusion peptide, as shown in FIG. 8C, green fluorescent color was located within the cell membrane shown in red color, confirming again that the fusion peptide was uptake in the cells.

Example 6

(59) Evaluation of Cytotoxic of KLAK (SEQ ID NO:16)-sFt-GFP Fusion Peptide

(60) MDA-MB-231 cell line was treated with KLAK (SEQ ID NO:16)-sFt-GFP fusion peptide, culture for 48 hours, and subjected to MTT assay to analyze cell viability of the cells.

(61) The results of the MTT assay are shown in FIG. 9A.

(62) As shown in FIG. 9A, it was confirmed that the treatment with KLAK (SEQ ID NO:16)-sFt-GFP fusion peptide reduced cell viability in a concentration-dependent manner. However, since the cell viability was not significantly inhibited due to excessive cell division during incubation for 48 hours, an additional experiment was carried out.

(63) That is, after KLAK (SEQ ID NO:16)-sFt-GFP fusion peptide was treated with cells, the cells were stained with Annexin V and PI, and the resulting cells were subjected to evaluation of cell viability thereof using FACS.

(64) The results of the evaluation are shown in FIG. 9B. After KLAK (SEQ ID NO:16)-sFt-GFP fusion peptide was treated with the cells for 24 hours, the survival cells were reduced to 70%. That is, most of the dead cells were stained only with Annexin V, meaning that apoptosis was induced early. However, the extent of the apoptosis induced by KLAK (SEQ ID NO:16)-sFt-GFP fusion peptide was similar with the apoptosis induced by 50 M of etoposide which is used as a positive control group.

(65) In addition, a mouse heterogeneous model was used for an animal experiment carried out to confirm the apoptosis effect of KLAK (SEQ ID NO:16)-sFt-GFP DCNC. As follows, saline, KLAK peptide (SEQ ID NO:16), sFt-GFP+KLAK fusion peptide (SEQ ID NO:16), and KLAK (SEQ ID NO:16)-sFt-GFP (30 mol/L) were intravenously administered to each of four groups.

(66) The results of the administration are shown in FIG. 10.

(67) As compared with other groups, a group treated with KLAK (SEQ ID NO:16)-sFt-GFP did not seem to have any influence in tumor growth. As shown in FIG. 10, a site of apoptosis increased within tumors. In addition, even if the amount of KLAK (SEQ ID NO:16)-sFt-GFP DCNC administered to the mouse was not enough to inhibit tumor growth, it was confirmed that KLAK (SEQ ID NO:16)-sFt-GFP DCNC stimulated apoptosis in tumors.

(68) In conclusion, it was confirmed that the fusion peptide in which the pro-apoptotic peptide was fused according to the present disclosure caused the apoptosis after being uptaken into the cells.

INDUSTRIAL APPLICABILITY

(69) The fusion polypeptide in which the polypeptide or the protein is fused to the N-terminus and/or the C-terminus of the human-derived ferritin monomer fragment having the amino acid sequence represented by SEQ ID NO: 2 has very excellent production efficiency, forms a protein cage by self-assembly, thereby enabling to encapsulate active ingredients in the protein cage. In addition, due to low steric screening effect with a property of a three-dimensional structure of the fusion polypeptide, the physiological activity of the polypeptide or the protein fused to the N-terminus or the C-terminus is also excellent. In this regard, the fusion polypeptide is highly effective in terms of industrial applicability as being useful in diagnosing disease or developing a therapeutic agent.