Peptide for enhancing expression efficiency of target protein, and fusion protein comprising same

Abstract

The present invention relates to a novel peptide or a partial sequence thereof for enhancing expression efficiency of a target protein, and a fusion protein comprising the same. The novel peptide according to the present invention can enhance expression efficiency of a target protein, and furthermore, the peptide can also be applied to a solubility-enhancing fusion protein in order to enhance solubility of the target protein, so that solubility as well as expression efficiency of the target protein is enhanced, which allows such a peptide to be usefully used for production of a recombinant target protein.

Claims

1. A fusion protein comprising: (a) a peptide for enhancing expression efficiency of a target protein, and (b) a target protein, wherein the peptide is linked to the N-terminus of the target protein and wherein the peptide consists of the amino acid sequence represented by SEQ ID NO: 1.

2. The fusion protein according to claim 1, wherein the amino acid sequence of SEQ ID NO: 1 is derived from urate oxidase.

3. The fusion protein according to claim 1, wherein the peptide contains the amino acid sequence represented by SEQ ID NO: 2.

4. The fusion protein according to claim 1, wherein the target protein is at least one selected from the group consisting of an antigen, an antibody, a cell receptor, an enzyme, a structural protein, a serum protein, and a cellular protein.

5. The fusion protein according to claim 1, further comprising: an RNA interacting domain (RID) as a fusion partner of the fusion protein, wherein the RID contains the amino acid sequence represented by SEQ ID NO: 7.

6. The fusion protein according to claim 5, wherein the fusion protein contains the amino acid sequence represented by SEQ ID NO: 9.

7. The fusion protein according to claim 5, wherein the target protein is norovirus-derived VP1 protein.

8. A method for producing a soluble target protein comprising the fusion protein according to claim 5, the method comprising the steps of: (A) constructing an expression vector that comprises: (a) a polynucleotide encoding the target protein, (b) a polynucleotide linked to the 5′-end of the polynucleotide encoding the target protein and that encodes the peptide that enhances expression efficiency of the target protein, and (c) a polynucleotide encoding the RID that increases solubility of the target protein; (B) introducing the expression vector into a host cell to prepare a transformant; and (C) culturing the transformant so that expression of a recombinant target protein is induced, and obtaining the recombinant target protein.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 illustrates experimental results showing an effect of the peptide (eet1) of the present invention on expression efficiency of EGFP protein (A: SDS-PAGE results showing expression of each fusion protein, B: a graph showing each fusion protein's relative expression level in a case where an expression level of a control (EGFP) treated with 80 μM IPTG is taken as 1, and C: a graph showing each fusion protein's relative activity in a case where protein activity of the control treated with 80 μM IPTG is taken as 1).

(2) FIG. 2 illustrates results obtained by comparing effects of the peptide of the present invention or a part thereof on expression efficiency and activity of EGFP protein (A: SDS-PAGE results showing expression of each fusion protein, B: each fusion protein's relative expression level in a case where an expression level of a control (MSEQHAQ (SEQ ID NO: 5)-EGFP) treated with 80 μM IPTG is taken as 1, C: each fusion protein's relative activity in a case where protein activity of the control treated with 80 μM IPTG is taken as 1, and D: a graph showing protein activity per unit protein).

(3) FIG. 3 illustrates a graph showing an effect of the peptide of the present invention on expression of TruB, an E. coli-derived protein (A: SDS-PAGE results showing expression of each fusion protein, B: a graph showing each fusion protein's relative expression level in a case where an expression level of a control is taken as 1).

(4) FIG. 4 illustrates SDS-PAGE results (A) showing an effect of the peptide of the present invention on hRID, a solubility-enhancing fusion partner, and a graph (B) showing changes in relative expression level.

(5) FIG. 5 illustrates graphs showing effects of an hRID fusion, obtained by addition of the peptide of the present invention, on expression efficiency of a target protein.

(6) FIG. 6 illustrates a schematic diagram showing a structure of a recombinant expression vector for production of soluble norovirus vaccine according to an embodiment of the present invention.

(7) FIG. 7A illustrates results obtained by identifying, with SDS-PAGE, solubility of VP1 protein expressed according to an embodiment of the present invention, in which the left panel shows an expression result of VP1 (70 kDa) recombined with MSAVKAA (SEQ ID NO: 1)-RID and the right panel shows a result of a comparative example (VP1 recombined with MSEQ (SEQ ID NO: 15)-RID; 69 kDa).

(8) FIG. 7B illustrates results obtained by identifying, with SDS-PAGE, expression in soluble form of the VP1 protein expressed according to an embodiment of the present invention, in which the left panel shows an expression result of VP1 (70 kDa) recombined with MSAVKAA (SEQ ID NO: 1)-RID, the middle panel shows an expression result of VP1 (70 kDa) recombined with MSAV (SEQ ID NO: 2)-RID, and the right panel shows an expression result of a control (MS-RID).

(9) FIG. 8 illustrates a chromatogram result obtained by purifying and identifying, through nickel affinity chromatography, VP1 protein expressed according to an embodiment of the present invention.

(10) FIG. 9 illustrates results obtained by purifying VP1 protein expressed according to an embodiment of the present invention, and then identifying the same with SDS-PAGE, in which the fractions of lanes 18 to 20 are pooled.

(11) FIG. 10 illustrates results obtained by cleaving norovirus VP1 using TEV protease, and then identifying the same with SDS-PAGE.

(12) FIG. 11 illustrates results obtained by performing peptide mapping, and N-terminal and C-terminal amino acid sequence analysis, so as to identify whether the protein obtained through expression and purification consists of the VP1 sequence. FIG. 11A illustrates UPLC peaks obtained by performing analysis after trypsin treatment. FIGS. 11B, 11B-A, 11B-B, and 11B-C illustrate results, identifying that as a result of LC-MS/MS analysis of the peptide fragment (SEQ ID NO: 17) appearing in trypsin treatment, such a peptide fragment exhibits an 83.9% match, in terms of amino acid sequence, with the originally expected sequence. FIG. 11C illustrates results, identifying that the protein's N-terminal sequence (SEQ ID NO: 18) matches norovirus VP1. FIG. 11D illustrates results, identifying that the protein's C-terminal sequence matches norovirus VP1.

(13) FIG. 12A illustrates a chromatogram showing results obtained by performing size exclusion chromatography in order to purify VLP formed after cleavage with TEV protease.

(14) FIG. 12B illustrates results obtained by purifying VLP protein formed after cleavage with TEV protease, and then identifying the same with SDS-PAGE.

(15) FIG. 13 illustrates results obtained by analyzing the purified VLP through dynamic light scattering (DLS) in order to identify its overall diameter.

(16) FIG. 14 illustrates results obtained by identifying, using TEM electron microscopy, whether the purified VP1 proteins have formed VLP [A: E. coli-derived virus-like particle (VLP) identified through electron microscopy, B: recombinant VP1 protein from which a RID tag containing histidine and TEV recognition sequence has not been removed (failure to form VLP structure), C: norovirus VLPs produced using baculovirus-insect cell system, and D: wild-type norovirus virion].

DETAILED DESCRIPTION OF INVENTION

(17) Hereinafter, various examples are presented to help understand the present invention. The following examples are provided only for easier understanding of the present invention, and the scope of protection of the present invention is not limited to the following examples.

(18) <Experimental Methods>

(19) 1. Construction of Protein Expression Vector

(20) pGE-LysRS expression vector was used as a protein expression vector (Choi, S. I. et al., Protein solubility and folding enhancement by interaction with RNA, PLoS ONE (2008), 3:e2677). In pGE-LysRS whose expression is under control of T7 promoter, the LysRS gene is cleaved using Ndel and one of the cleavage sites present in MCS (Kpn1-BamH1-EcoRV-Sal1-Hind3), and EGFP or hRID, or other proteins were inserted into the same location. Here, the amino acid sequence (eet1) of SEQ ID NO: 1 or a part thereof (eet2, SEQ ID NO: 2) was inserted at the N-terminal position of the inserted protein. Then, target proteins to be expressed were inserted using two restriction enzyme sites in the prepared hRID or MCS of hRID vector containing the amino acid sequence of SEQ ID NO: 1.

(21) 2. Protein Expression and SDS-PAGE

(22) The prepared protein expression vector was transformed into BL21 (DE3), BL21 (DE3)-pLysS, or BL21 (DE3)-pLysE competent cells, and culture was performed. All transformed E. coli cells were cultured in LB medium containing 50 μg/ml of ampicillin. The E. coli cells transformed with BL21*(DE3)-pLysS or BL21*(DE3)-pLysE were cultured in the medium supplemented with 34 μg/ml of chloramphenicol. Different culture temperature was used for each protein, and culture was performed at a condition of 33° C. to 37° C. When the OD.sub.600 value of E. coli reached 0.5 or higher, IPTG was added at a level of 0 μM to 1 mM to activate T7 promoter, and culture was performed at 33° C. or 37° C. for about 3 hours after addition of IPTG so that a sufficient amount of protein can be produced. Sufficiently cultured E. coli cells were centrifuged and the supernatant was removed. Then, the resulting E. coli harvest was stored. Next, 0.3 ml of PBS was added to the E. coli harvest corresponding to 5 ml of the LB medium, and ultrasonic pulverization was performed to make a lysate. Alternatively, the E. coli harvest corresponding to 1 ml was subjected to treatment with 60 μl of B-PER (Thermo Fisher Scientific), together with DNase and lysozyme at appropriate concentrations, thereby obtaining a lysate. Then, the lysate was analyzed with SDS-PAGE.

(23) 3. Fluorescence Measurement of EGFP Protein

(24) Fluorescence was measured at Ex 485 nm/Em 520 nm using Fluostar Optima (BMG Labtech) to identify activity of the protein expressed in the sample to be analyzed.

EXAMPLES

Example 1

(25) Effect of Peptide of Present Invention on Expression Efficiency and Activity of EGFP Protein

(26) 1-1. SDS-PAGE Analysis

(27) In pGE LysRS plasmid, LysRS was removed using Ndel and Hind3 restriction enzymes, and each of two sequences, EGFP gene sequence and a fusion form (eet1-EGFP) obtained by adding, to the N-terminus of the EGFP gene sequence, gene sequence (SEQ ID NO: 3) encoding the amino acid sequence (eet1) of SEQ ID NO: 1, was inserted into the same location. The vector is in the form of being expressed by T7 promoter and being regulated by lac operator, in which promoter activation is regulated by IPTG. The two recombinant plasmids were respectively transformed into BL21*(DE3)-pLysE competent cells, and protein expression was induced at a condition of 37° C. for 3 hours. Here, treatment with IPTG was performed at four concentrations of 0, 20, 40, and 80 μM.

(28) As a result, as illustrated in FIGS. 1A and 1B, it was found that in a case of the fusion form (eet1-EGFP) of the EGFP gene, its expression is induced better than the control (EGFP).

(29) 1-2. Protein Activity Assay

(30) In order to check whether the expressed EGFP protein is also functional, for the EGFP sample in lysate form, which had been expressed under each condition, its fluorescence value was measured at 485 nm/520 nm using Fluostar Optima.

(31) As a result, as illustrated in FIG. 1C, it was found that in a case of the fusion form to which eet1 has been added, activity as well as expression of the EGFP protein is increased.

Example 2

(32) Effect of Partial Sequence of Peptide of Present Invention

(33) In order to identify an effect exhibited in a case where a partial sequence (eet2, SEQ ID NO: 2) of the peptide (eet1) identified in Example 1 is fused to a target protein, an EGFP fusion protein was expressed in the same manner as in Example 1. As a control, gene (SEQ ID NO: 6) encoding the lysyl tRNA synthetase-derived amino acid sequence (MSEQHAQ) represented by SEQ ID NO: 5 was used. Here, treatment with IPTG was performed at concentrations of 10, 20, 40, and 80 μM.

(34) As a result, as illustrated in FIGS. 2A and 2B, the EGFP proteins (eet1-EGFP, eet2-EGFP) obtained by addition of the novel peptide of the present invention exhibit a lower expression level than the control protein (MSEQHAQ (SEQ ID NO: 5)-EGFP) obtained by being fused with the control peptide. However, as illustrated in FIG. 2C, it was found that at the IPTG concentration of 40 μM or higher, a higher amount of active protein is produced in a case of being fused with eet1 or eet2. From these results, it can be seen that in a case of being fused with the eet2 sequence, lower expression level but better protein activity is exhibited than eet1, and the highest amount of active protein is produced at the IPTG concentration of 80 μM (FIGS. 2A to 2C).

(35) In addition, when this was calculated in terms of activity per unit protein, it was identified that in a case of being fused with the eet1 sequence, a high-quality protein can be expressed in an amount equal to or greater than two times the control and that in a case of being fused with the eet2 sequence, a high-quality protein can be expressed in an amount equal to or greater than three times the control (FIG. 2D). From these results, it can be seen that increasing an expression level of highly active protein is substantially better for improving protein expression efficiency than simply increasing an expression level of protein.

Example 3

(36) Effect of Peptide According to Present Invention on Expression Efficiency of TruB and TruB-EGFP Protein

(37) In order to examine an effect of the peptide according to the present invention on expression of TruB protein, which is known to be poorly expressed in E. coli, the gene sequence encoding TruB protein and the gene sequence encoding TruB-EGFP protein, and their fusion forms, to each of which the nucleotide sequence of SEQ ID NO: 2 had been added, were respectively inserted into pGE LysRS plasmid using the same method as in Example 1. A total of four recombinant plasmids were transformed into BL21*(DE3)-pLysS competent cells, and protein expression was induced at a condition of 37° C. Here, treatment with IPTG was performed at 1 mM concentration, and over-expression was performed for 3 hours.

(38) As a result, the controls (TruB and TruB-EGFP) were barely expressed to the extent that they are obscured by miscellaneous bands; however, in a case where eet1 of SEQ ID NO: 1 is fused to each of the TruB and TruB-EGFP proteins, it was identified that the respective proteins are expressed. Here, it was found that the expression level of the respective proteins is equal to or greater than three times the controls (FIGS. 3A and 3B).

Example 4

(39) Effect of Peptide According to Present Invention on Expression Efficiency of RID Protein

(40) In order to examine an effect of the peptide according to the present invention on expression of hRID protein (SEQ ID NO: 7), which had been developed as solubility-enhancing fusion partner, in E. coli, its expression level was identified using the same method as in Example 1. Specifically, the gene (SEQ ID NO: 8) encoding hRID protein, and the fusion form (SEQ ID NO: 10), in which an eet1-encoding gene had been linked to the N-terminus of hRID, were respectively inserted into pGE LysRS plasmid. The two recombinant plasmids were transformed into BL21*(DE3)-pLysS competent cells, and protein expression was induced at a condition of 37° C. Here, treatment with IPTG was performed at concentrations of 0, 20, 100, and 1000 μM, and over-expression was performed for 3 hours.

(41) As a result, as illustrated in FIG. 4, it was found that although the control (hRID) is barely expressed, the fusion form (eet1-hRID) obtained by addition of eet1 is well expressed (FIG. 4A). In a case of quantitative analysis of expression level, since the expression level of the control was too low to be used as a baseline, expression level comparison was performed using, as a baseline, the experimental group in which IPTG at 1,000 μM is used (FIG. 4B).

Example 5

(42) Effect of Fusion Protein Containing Peptide and RID According to Present Invention on Expression Efficiency of Three Proteins, CsTA1953, CsTA37, and CsTA422

(43) hRID was used as a fusion partner for increasing expression level and solubility of CsTA1953, CsTA37, and CsTA422 proteins, which are known to be poorly expressed in E. coli or not to be well expressed in a soluble form even in a case of being expressed. In pGE LysRS plasmid, LysRS was cleaved using Ndel and Kpn1, and the gene sequence (SEQ ID NO: 8) encoding hRID or the gene sequence (SEQ ID NO: 10) encoding the eet1-hRID fusion protein was respectively inserted at the same location. Next, CsTA1953 gene, CsTA37 gene, and CsTA422 gene were respectively inserted into the pGE-hRID plasmid and the pGE-eet1-hRID plasmid using BamH1 and Hind3.

(44) A total of six recombinant proteins thus produced were expressed under the same IPTG concentration condition as in Example 4. Among these, for the four recombinant proteins containing CsTA37 and CsTA422, experiments were conducted only at a condition in which treatment with IPTG at 1 mM is performed. These proteins were expressed at a condition of 33° C.

(45) As a result, as illustrated in FIG. 5, it was found that expression efficiency of the CsTA1953, CsTA37, and CsTA422 proteins is improved in a case where the eet1-hRID fusion protein is bound thereto. Also, in this case, all controls (hRID-) were barely expressed to the extent that they are obscured by miscellaneous bands (FIGS. 5A and 5B). In a case of CsTA1953, since the expression level of the controls was too low, quantitative analysis was performed using, as a baseline, the experimental group in which IPTG at 1,000 μM is used (FIG. 5B). It was found that CsTA37 and CsTA422 exhibit at least 7-fold higher expression than the controls (FIG. 5D).

Example 6

(46) Effect of Fusion Protein Containing Peptide and RID According to Present Invention on Expression Efficiency of Norovirus VP1 Protein

(47) 6-1. Construction of Recombinant Expression Vector and Expression of VP1 Protein

(48) Norovirus Hu/GII.4/Hiroshima/55/2005/JPN (NCBI access number: AB504310.1)-derived VP1 gene was used for production of norovirus VLP through E. coli, and the VP1 gene in question was obtained through gene synthesis. pGE-LysRS vector was used as an expression vector. This vector is an expression vector made by modifying pGEMEX-1 (Promega) vector. Specifically, the expression vector was cleaved by treatment with Nde I and BamHI restriction enzymes, and a DNA fragment was inserted into the cleaved expression vector, the DNA fragment consisting of the following sequences in a continuous manner: a polynucleotide sequence (SEQ ID NO: 3) encoding MSAVKAA (eet1, SEQ ID NO: 1) or a polynucleotide sequence (SEQ ID NO: 4) encoding MSAV (eet 2, SEQ ID NO: 2); a polynucleotide sequence (SEQ ID NO: 11) encoding 6-histidine tag (Histag); a polynucleotide sequence (SEQ ID NO: 12) encoding TEV recognition sequence (ENLYFQ, SEQ ID NO: 16) from which G has been removed; and a polynucleotide sequence (SEQ ID NO: 14) encoding VP1 (SEQ ID NO: 13). The thus completed recombinant plasmid was transformed into the E. coli host HMS174 (DE3). Initial culture for protein expression was performed as follows: Culture was performed at 37° C. for one day in 15 μg/ml of LB medium supplemented with 50 μg/ml of ampicillin, and then 1 ml of E. coli, which had been cultured on the previous day in 15 ml of LB medium supplemented with the same concentration of ampicillin, was added thereto. Culture was performed at 37° C. until the OD.sub.600 nm reached 0.5 to 0.7. When the appropriate OD value was achieved, overexpression was induced with 1 mM IPTG. After IPTG addition, expression was induced at two different temperatures (37° C., 16° C.). As a comparative example, norovirus VP1 conjugated with RID containing four amino acids, which had been applied in the previous study, was also expressed under the same condition. The expressed protein was collected and checked for solubility through SDS-PAGE.

(49) As a result, as illustrated in FIG. 7A, it was identified that the recombinant VP1 protein is well expressed at 16° C. as well as at 37° C. In addition, as a result of comparison with the comparative example VP1 in terms of expression level, it was found that the recombinant VP1 of the present invention exhibits a markedly increased expression level which is about 2 times or higher than the comparative example VP1 (FIG. 7A). In addition, the (Estimated) norovirus VP1 protein was 59 kDa in size and the VP1 containing RID (8 kDa) was about 70 kDa in size. As a result, it was identified that expression has been induced at an appropriate location.

(50) 6-2. Identification of Effect of Partial Sequence of Peptide of Present Invention

(51) In order to identify an effect exhibited in a case where norovirus VP1 is fused with a partial sequence (eet2, SEQ ID NO: 2) of the peptide (eet1, SEQ ID NO: 1) identified in Example 6-1, the VP1 protein was expressed in the same manner as in Example 6-1. The proteins, which were expressed at 37° C. for 3 hours after addition of 1 mM IPTG, were collected and checked for solubility through SDS-PAGE.

(52) As a result, as illustrated in FIG. 7B, it was identified that the VP1 protein is not expressed in the control in which MS-RID is fused therewith, and a case where VP1 is fused with eet2 shows a similar expression level to eet1.

(53) 6-3. Purification of Norovirus VP1

(54) The proteins, for which solubility had been identified, were purified through nickel (Ni) affinity chromatography. Purification was conducted after E. coli was harvested in an amount of 500 ml, which had been ultimately obtained via 3 ml and 50 ml, using the same culture method as described above. Specifically, equilibrium was first made with A buffer [50 mM Tris-HCl (pH 7.5), 300 mM sodium chloride, 5% glycerol, 0.1 mM 2-mercaptoethanol, and 10 mM imidazole], and equilibrated Ni-NTA column (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK) was used to purify sample proteins. Following the A buffer, B buffer [50 mM Tris-HCl (pH 7.5), 300 mM sodium chloride, 5% glycerol, 0.1 mM 2-mercaptoethanol, and 300 mM imidazole] was used to elute the proteins with linear gradient imidazole in a range of 10 to 300 mM. Target protein-containing fractions were identified through SDS-PAGE. Then, the fractions in question were collected and dialyzed using C buffer (store buffer) [50 mM Tris-HCl (pH 8.5), 10 mM NaCl, 0.1% 2-mercaptoethanol]. The concentration of finally-purified proteins was quantified using BSA (Amresco, Solon, Ohio, USA). The purified VP1 protein was mixed with 20% glycerol at a 1:1 ratio, and then stored at −20° C.

(55) The results obtained by identifying the purified MSAVKAA (SEQ ID NO: 1)-RID-VP1 fusion protein with nickel affinity chromatography and the results obtained by identifying purification through SDS-PAGE are illustrated in FIGS. 8 and 9. Purification was successfully conducted. After the first purification, the conjugating protein (tag) containing MSAVKAA (SEQ ID NO: 1)-RID was also successfully removed through TEV protease. Subsequently, TEV, cleaved tag, and uncleaved-fusion protein were separated through 2.sup.nd Ni-affinity chromatography to obtain completely purified VP1 (FIG. 10).

(56) 6-4 Amino Acid Sequence Analysis of Purified VP1

(57) In order to check whether the protein obtained through the expression and purification experiments consists of the expected VP1 sequence, peptide mapping, and N-terminal and C-terminal sequencing were conducted by making a request to ISS (www.isslab.co.kr), a specialized analysis organization. First, it was identified that as a result of LC-MS/MS analysis of the peptide fragment appearing in trypsin treatment, such a peptide fragment exhibits an 83.9% match, in terms of amino acid sequence, with the originally expected sequence. It was identified that as a result of comparison of the N-terminal sequence 26-mer and the C-terminal sequence 3-mer, a match with NoV VP1 is observed (FIG. 11).

(58) 6-5. Size Exclusion Chromatography

(59) Biochemical analysis was performed to check whether the dimers of VP1 protein, obtained by cleavage with protease after TEV cleavage, form VLP. Specifically, size exclusion chromatography was performed at 4° C. through a Superdex-200 analytical gel-filtration column.

(60) On the previous day, the fusion protein was cleaved at 16° C. for one day using AcTEV protease. The column was subjected to equilibrium with a buffer [ammonium acetate 250 mM (pH 6.0)]. After completion of the equilibrium, the VP1 sample from which the fusion partner protein had been cleaved was loaded thereon and purification was performed. After purification, calibration was performed using ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (44 kDa), and Blue Dextran 2000 (GE Healthcare), thereby determining the molecular weight of a protein which had appeared as a peak on the chromatogram.

(61) From the viewpoints that the norovirus VLP showed a molecular weight of 10 MDa according to the results of the previous study and that the maximum purification limit of the column used by the present inventors was 800 kDa, it was assumed that VLP would be purified in void in a case of being properly formed. As a result of checking the chromatogram, it was found that in the void, there is a high peak appearing as norovirus VLP (FIG. 12A), and it was found that as a result of checking the purified and harvested fractions with SDS-PAGE, the peak appearing in the void corresponds to norovirus VLP (FIG. 12B). In addition, it was found that hRBD resulting from cleavage with TEV protease has also been purified by chromatography.

(62) 6-6. Dynamic Light Scattering Analysis

(63) In order to identify the overall diameter of purified virus-like particles (VLPs), analysis was performed through dynamic light scattering (DLS).

(64) As a result, it was identified that their overall diameter is observed in a size similar to 30 to 40 nm which the wild-type norovirus shows (FIG. 13).

(65) 6-7. Identification of VLP Formation Through Electron Microscopy

(66) In order to check whether the VP1 proteins purified in the void have formed VLP, observations were made with electron microscopy. Purified norovirus VLPs were first placed on a copper grid for 1 minute and then stained for 15 seconds using 2% uranyl acetate. The pretreated sample was dried at room temperature for 30 minutes and then photographed using transmission electron microscopy (TEM; JEM-1011, JEOL, Japan). The above experiment was carried out at the Research Support Department of Yonsei Biomedical Research Center, Yonsei University College of Medicine.

(67) As a result, as illustrated in FIG. 14, it was found that the purified VP1 proteins form VLP. The thus identified VLP's diameter was 34 nm, which was similar to that of the norovirus VLP produced using the baculovirus-insect cell expression system and the wild-type norovirus. On the other hand, it was found that VP1s, each of which has not been cleaved with TEV protease and with each of which hRBD is fused, do not form VLP and are aggregated (FIG. 14).

(68) As described above, the present invention has been described by way of preferred embodiments. It will be understood by those skilled in the art that various changes and modifications can be made without departing from essential features of the present invention. Therefore, it is to be understood that the above-described examples are illustrative in all aspects and not restrictive. It should be interpreted that the scope of the present invention is shown not in the above-stated description but in the claims, and that all differences falling under the scope equivalent thereto are encompassed by the present invention.