Method for secretory production of target protein

Abstract

To provide a method for stably and efficiently producing a protein in secretory production of a target protein using BEVS.SOLUTION: The present invention also provides a method for producing a fusion protein, comprising the steps of expressing the fusion protein in cultured insect cells using a recombinant baculovirus comprising a polynucleotide encoding the fusion protein, and recovering the fusion protein from the culture supernatant, wherein the fusion protein is derived from silkworm H1N1 influenza virus hemagglutinin or silkworm osteonectin.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for secreting and producing a target protein using a baculovirus expression vector system. [Background technology]

[0002] The baculovirus expression vector system (BEVS) is a technology for expressing target proteins in insect cells using a recombinant baculovirus vector containing the gene encoding the foreign protein of interest. Unlike protein expression systems using prokaryotes such as E. coli, proteins expressed in insect cells undergo post-translational modifications such as glycosylation, resulting in proteins with the same structure and function as proteins expressed in mammalian cells. Furthermore, baculoviruses do not infect mammals, making them non-pathogenic and easy to handle. For this reason, BEVS is useful as a method for producing biological products such as vaccines, and influenza and human papillomavirus vaccines produced using this method have already been commercialized.

[0003] On the other hand, in the baculovirus-insect cell expression system, if the expressed protein is localized intracellularly, it is necessary to disrupt the cells using ultrasound or detergents to extract the protein. Such procedures can cause the target protein to denature due to the heat generated during ultrasonic disruption, or the detergents can cause changes in the protein's higher-order structure. Furthermore, insect cells have higher protease activity than mammalian cells, so proteins accumulated intracellularly are easily degraded. Therefore, when expressing proteins with low protease resistance, they must be secreted extracellularly. Furthermore, when purifying the produced recombinant protein, secretion into the culture supernatant, which contains fewer contaminating proteins, is more efficient than extraction from within the cells, and therefore is advantageous in that it is expected to improve productivity.

[0004] In general, proteins secreted outside the cell are secreted via the endoplasmic reticulum and Golgi apparatus. These proteins have a secretory signal, called a signal peptide, at their N-terminus. When translation of a protein containing a secretory signal is initiated, the signal recognition particle (SRP) binds to the signal peptide at the protein's N-terminus, and the translated protein is transported into the endoplasmic reticulum via the SRP. After transport into the endoplasmic reticulum, the signal peptide is cleaved by signal peptidase, and the secretory protein is released into the endoplasmic reticulum. Proteins in the endoplasmic reticulum undergo post-translational modifications before being released outside the cell via the Golgi apparatus.

[0005] When a foreign protein is secreted and expressed using BEVS, it is expressed as a fusion protein with a signal peptide. However, it has been reported that secretion efficiency is poor when the signal peptide is derived from an organism other than insect cells, and that secretion efficiency improves when the signal peptide is changed to an insect cell-derived signal peptide (Non-Patent Document 1). Known signal peptides commonly used in BEVS-based expression systems include honeybee-derived Honeybee Melittin (HBM) and baculovirus-derived envelope protein GP64 (Non-Patent Documents 1 and 2). For example, when expressing the hemagglutinin of the A / H5N1 influenza virus subtype in BEVS, it has been reported that secretion levels improve when the signal peptide of the hemagglutinin is replaced with the signal peptide of honeybee-derived HBM (Non-Patent Document 3).

[0006] However, the types of signal peptides reported so far are limited, and secretion efficiency is not necessarily sufficient. Furthermore, after infection of host insect cells, baculoviruses are known to express proteases such as cathepsins and chitinases, and these proteases are localized in the endoplasmic reticulum. Therefore, it is desirable that the expressed proteins be stable against proteases in the endoplasmic reticulum. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Gene. 1991 Feb 15;98(2):177-83. [Non-patent document 2] Methods Mol Biol. 2016;1350:51-71. [Non-patent document 3] Transbound Emerg Dis. 2017 Apr;64(2):432-441. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention relates to providing a method for stably and efficiently producing a protein when secreting and producing a target protein using a BEVS. [Means for solving the problem]

[0009] As a result of investigations conducted in light of the above-mentioned problems, the present inventors have found that when a target protein is fused with and expressed with a specific signal peptide, intracellular degradation of the expressed protein is suppressed, secretion efficiency is improved, and the target protein can be released extracellularly in high yield.

[0010] That is, the present invention relates to the following 1) to 6). 1) A method for producing a target protein, comprising the steps of expressing a fused signal peptide and target protein in cultured insect cells using a recombinant baculovirus containing a polynucleotide encoding the target protein bound to a signal peptide, and recovering the target protein from the culture supernatant, wherein the signal peptide is a signal peptide derived from a silkworm fibroin L chain, a signal peptide derived from the hemagglutinin of influenza A / H1N1 subtype virus, or a signal peptide derived from silkworm osteonectin. 2) The method described in 1), wherein the signal peptide derived from silkworm fibroin L chain is any of the peptides shown in (1) to (3) below, the signal peptide derived from the hemagglutinin of influenza A / H1N1 subtype virus is any of the peptides shown in (4) to (6) below, and the signal peptide derived from silkworm osteonectin is any of the peptides shown in (7) to (9) below. (1) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 (2) A peptide having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NO: 1 and that functions as a signal peptide. (3) A peptide having a function as a signal peptide, which consists of an amino acid sequence in which one or several amino acids are deleted, substituted, or inserted in the amino acid sequence shown in SEQ ID NO: 1. (4) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 (5) A peptide having an amino acid sequence having an identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 2 and having a function as a signal peptide. (6) A peptide having a function as a signal peptide, which consists of an amino acid sequence in which one or several amino acids are deleted, substituted, or inserted in the amino acid sequence shown in SEQ ID NO: 2. (7) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 3 (8) A peptide having an amino acid sequence having an identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 3 and having a function as a signal peptide. (9) A peptide having a function as a signal peptide, which consists of an amino acid sequence in which one or several amino acids are deleted, substituted, or inserted in the amino acid sequence shown in SEQ ID NO: 3. 3) The method according to claim 1) or 2), wherein the fused and expressed protein is a protein in which a signal peptide is bound to the N-terminus of the target protein. 4) A method according to any one of claims 1 to 3), wherein the cultured insect cells are a cell line established from Spodoptera frugiperda. 5) The method according to any one of 1) to 4), wherein the target protein is ferritin protein or nanoparticles of ferritin protein. 6) A recombinant baculovirus comprising a polynucleotide encoding a target protein bound to a signal peptide, wherein the signal peptide is a signal peptide derived from a silkworm fibroin L chain, a signal peptide derived from an A / H1N1 influenza virus hemagglutinin, or a signal peptide derived from silkworm osteonectin. [Effects of the Invention]

[0011] According to the present invention, intracellular degradation of a protein expressed in insect cells is suppressed and secretion efficiency is improved, so that the target protein can be obtained in high yield from the culture supernatant. [Brief explanation of the drawings]

[0012] [Figure 1] Design of constructs used for expression evaluation. [Figure 2] Confirmation of target protein expression by BEVS using SDS-PAGE / CBB staining. [Figure 3] Confirmation of target protein expression by BEVS using Western blot. [Figure 4] Confirmation of ferritin nanoparticle purification through a sucrose cushion using SDS-PAGE / CBB staining. [Figure 5] Confirmation of ferritin nanoparticle purification through a sucrose cushion using Western blot. [Figure 6] Quantification of ferritin nanoparticle amounts by ELISA. DETAILED DESCRIPTION OF THE INVENTION

[0013] Preferred embodiments of the present invention will be described in detail below, but the present invention is not limited to the following embodiments.

[0014] The method for producing a target protein of the present invention includes the steps of expressing a fused signal peptide and target protein in cultured insect cells using a recombinant baculovirus containing a polynucleotide encoding the target protein bound to a signal peptide, and recovering the target protein from the culture supernatant, i.e., a method for secreting and producing a target protein using BEVS, The signal peptide used may be a signal peptide derived from the silkworm fibroin L chain, a signal peptide derived from the hemagglutinin of influenza virus subtype A / H1N1, or a signal peptide derived from silkworm osteonectin.

[0015] In the present invention, a "recombinant baculovirus" is a virus in which a polynucleotide encoding a target protein bound to a signal peptide has been inserted into the viral genome; specifically, a baculovirus virus (baculovirus vector) in which a polynucleotide encoding a target protein bound to a signal peptide has been inserted downstream of a promoter (such as a polyhedrin promoter) that enables gene expression in cultured insect cells. The type of baculovirus is not particularly limited as long as it can infect lepidopteran insects or their cultured cells, but is preferably a nucleopolyhedrovirus (NPV) or a modified version thereof, such as BmNPV, HycuNPV, AnpeNPV, or AcNPV.

[0016] In the present invention, the target protein refers to a heterologous protein that is exogenous to the host insect cell. The target protein is not limited and may be, for example, a protein derived from a microorganism, virus, animal, or plant, or may be a protein with an artificially designed amino acid sequence. Examples of target proteins include physiologically active proteins, receptor proteins, antigenic proteins used as vaccines, enzymes, antibodies, and other biopharmaceuticals, as well as functional proteins used in clinical test reagents, foods, cosmetics, and the like. Furthermore, the target protein may be a monomeric protein, a multimeric protein consisting of two or more subunits, or a protein that has formed a complex (e.g., a particle) such as a nanoparticle (e.g., a nanoparticle of ferritin protein) or a virus-like particle (VLP).

[0017] In the present invention, a signal peptide refers to a peptide that is bound to the N-terminus of a target protein, is expressed as a fusion with the target protein, and has the function of secreting the target protein extracellularly, for example, via the endoplasmic reticulum and Golgi apparatus. Specifically, the signal peptide of the present invention is a signal peptide derived from the silkworm fibroin L chain, a signal peptide derived from the hemagglutinin of influenza virus subtype A / H1N1, or a signal peptide derived from silkworm osteonectin. Such a signal peptide is preferably a peptide having 15 to 20 amino acids, and the proportion of hydrophobic amino acid residues selected from glycine (G), alanine (A), proline (P), valine (V), leucine (L), isoleucine (I), phenylalanine (F), tryptophan (W) and tyrosine (Y) in the H region (helix region) is 80% or more, preferably 90% or more.

[0018] Specifically, the signal peptides derived from the silkworm fibroin L chain include the following peptides (1) to (3). (1) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 (2) A peptide having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NO: 1 and that functions as a signal peptide. (3) A peptide having a function as a signal peptide, which consists of an amino acid sequence in which one or several amino acids are deleted, substituted, or inserted in the amino acid sequence shown in SEQ ID NO: 1.

[0019] Furthermore, examples of signal peptides derived from the hemagglutinin of influenza virus subtype A / H1N1 include the following peptides (4) to (6). (4) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 (5) A peptide having an amino acid sequence having an identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 2 and having a function as a signal peptide. (6) A peptide having a function as a signal peptide, which consists of an amino acid sequence in which one or several amino acids are deleted, substituted, or inserted in the amino acid sequence shown in SEQ ID NO: 2.

[0020] Furthermore, examples of signal peptides derived from silkworm osteonectin include the following peptides (7) to (9). (7) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 3 (8) A peptide having an amino acid sequence having an identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 3 and having a function as a signal peptide. (9) A peptide having a function as a signal peptide, which consists of an amino acid sequence in which one or several amino acids are deleted, substituted, or inserted in the amino acid sequence shown in SEQ ID NO: 3.

[0021] In the above (2), (5), and (8), the identity of the amino acid sequence is more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, and more preferably 99% or more. The amino acid sequence identity is calculated by the Lipman-Pearson method (Lipman, DJ., Pearson, WR.: Science, 227, 1435-1441, 1985). Specifically, it is calculated by performing analysis using the homology analysis (Searchhomology) program in the genetic information processing software Genetyx-Win (Software Development) with Unitsizetocompare (ktup) set to 2.

[0022] In the above (3), (6), and (9), the term "one or several" used in relation to deletion, substitution, or insertion of amino acids can be, for example, one to three, preferably one to two, and more preferably one. The deletion, substitution, or insertion of one or several amino acids is a conservative mutation that maintains the normal function of the signal peptide. A typical conservative mutation is a conservative substitution, preferably a substitution of a hydrophobic amino acid with another hydrophobic amino acid (substitution between hydrophobic amino acids) in the H region (amino acid positions 3 to 12 in the amino acid sequence shown in SEQ ID NO: 1, amino acid positions 3 to 12 in the amino acid sequence shown in SEQ ID NO: 2, and amino acid positions 3 to 14 in the amino acid sequence shown in SEQ ID NO: 3).

[0023] Recombinant baculoviruses can be produced by methods known in the art. Examples of such methods include those using transfer vectors that can insert a desired gene into baculovirus DNA by homologous recombination in lepidopteran insects. In this method, a transfer vector incorporating a polynucleotide encoding a target protein bound to the signal peptide and baculovirus DNA linearized with a restriction enzyme or the like are co-transfected into cultured lepidopteran insect cells, and recombinant baculoviruses can be obtained by screening the infected cells. Another method involves preparing baculovirus DNA (recombinant bacmid DNA) into which a desired polynucleotide has been inserted by homologous recombination in bacteria. In this method, bacteria containing a baculovirus gene (bacmid) are transformed with a transfer vector incorporating a polynucleotide encoding a target protein bound to a signal peptide to obtain recombinant bacmid DNA. This recombinant bacmid DNA is then transfected into cultured cells of a lepidopteran insect to obtain a recombinant baculovirus. Examples of bacteria used in this method include E. coli strains and their derivatives.

[0024] In the present invention, a polynucleotide encoding a target protein bound to a signal peptide is a polynucleotide in which nucleotides encoding three signal peptides are linked to the 5'-terminus of the nucleotide encoding the target protein. The polynucleotides encoding the target protein and each signal peptide may be the same nucleic acid sequence as in the species of origin, or may be nucleic acid sequences with codons optimized in insect cells.

[0025] As a method for linking a polynucleotide encoding such a signal peptide to the 5' end of a polynucleotide encoding a protein of interest, a polynucleotide encoding the signal peptide may be incorporated into the 5' side of the polynucleotide encoding the protein of interest when constructing a transfer vector, or a polynucleotide encoding the signal peptide may be incorporated into the 5' side of the region in the baculovirus genome backbone into which the polynucleotide encoding the protein of interest is inserted.

[0026] The transfer vector used herein is not particularly limited as long as it has a promoter that enables gene expression in lepidopteran insects or cultured cells of such insects and is vector DNA that allows insertion of a desired gene downstream of the promoter. Such transfer vectors themselves are known in the art, and examples thereof include pM02, pM23, pCPM, pYNG, pBM030, pBM050, pVL1392, pPSC8, and pFastBac. The promoter can be appropriately selected from promoters known in the art, and examples thereof include the polyhedrin promoter, p10 promoter, and silkworm actin promoter.

[0027] Kits for the method using bacmids are commercially available, and known systems include the Bac-to-Bac system (Thermo Fisher Scientific), the BaculoGold system (BD Biosciences), the SuperBAC system (SHEATECH), the BacPAC system (Clontech), the flashBAC system (Oxford Expression), the BacMagic system (Merck), the BestBac system (Expression Systems), and the BacuVance system (GenScript).

[0028] Commercially available baculovirus vectors include, for example, ProFold vectors (AB Vector), ProEasy vectors (AB Vector), FoldHelper vectors (AB Vector), pVL-based vectors (AB Vector), pAc-based vectors (AB Vector), pAB-based vectors (AB Vector), pIEx-based vectors (Merck), pBAC-based vectors (Merck), and pTriEx-based vectors (Merck).

[0029] In the present invention, "insects" refers to insects of the order Lepidoptera that are suitable for expressing recombinant proteins, such as the silkworm (Bombyx mori), the mulberry leaf moth (Spilosoma imparilis), the pernyi moth (Antheraea pernyi), the Spodoptera frugiperda, and the silver looper moth (Trichoplusiani).

[0030] Cultured cells of Lepidoptera insects are not particularly limited as long as they are cell lines established from the above-mentioned Lepidoptera insects, and examples include cell lines established from silkworm cells (BmN cells, BmN4 cells, BoMo cells, etc.), Antheraea persica cells (Anpe cells), Spodoptera frugiperda cells (Sf9 cells, Sf21 cells, etc.), Mulberry webworm cells (SpIm cells), and Oreochromis nigra cells (Tn-5 cells, HIGH FIVE cells, MG1 cells, etc.). Improved insect cell lines derived from these cells by modification are also included.

[0031] Insect cells are inoculated (infected) with the recombinant baculovirus after the insect cells have been cultured and grown in advance. The culture may be either an adherent culture or a suspension culture, but is preferably a suspension culture. Examples of culture media that can be used include commonly used TNM-FH medium (Pharmingen), Express Five SFM (Thermo Fisher Scientific), Sf-900 II SFM (Thermo Fisher Scientific), Sf-900 III (Thermo Fisher Scientific), ExpiSf CD (Thermo Fisher Scientific), IS Sf Insect medium (FUJIFILM Irvine Scientific), 4Cell Insect CD medium (SARTRIUS), HyClone SFM4Insect cell culture media (Cytiva), Insect-XPRESS medium (LONZA), ESF 921 medium (Expression Systems), and ESF SF medium (Expression Systems). Antibiotics such as penicillin, streptomycin, and gentamicin may be added to the culture medium as needed.

[0032] The culture temperature is preferably 25 to 30°C, more preferably 26 to 28°C, and even more preferably 27°C. The cell concentration was 1 × 10 5 ~1×10 7 cells / mL, more preferably 2×10 5 ~5×10 6 cells / mL, more preferably 5 x 10 5 ~3×10 6 cells / mL.

[0033] Infection of the cultured cells with recombinant baculovirus was performed at an insect cell concentration of 1 × 10 5 ~1×10 7 The recombinant baculovirus is inoculated into a culture medium at a concentration of 1000 cells / mL by adding the solution containing the recombinant baculovirus to the culture medium. Here, the virus inoculation amount is, for example, MOI=0.01 to 20, and preferably MOI=0.1 to 1.

[0034] After infection (after inoculation with the virus), the cells are cultured for 1 to 7 days, preferably 2 to 4 days, more preferably 3 days, after inoculation with the virus, whereby the signal peptide and the target protein are fused and expressed within the cells, and the target protein is released outside the cells. During culture, protease inhibitors and nucleases can be added as appropriate.

[0035] After the cultivation is completed, the cells are removed by centrifugation, filtration, etc., and the target protein is recovered from the culture supernatant. The recovered target protein can be purified using methods commonly used for isolating and purifying proteins, such as solvent extraction, salting out, desalting, precipitation with organic solvents, density gradient centrifugation, anion exchange chromatography, cation exchange chromatography, hydrophobic chromatography, ultrafiltration, gel filtration, affinity chromatography, chromatofocusing, electrophoresis, and immunoprecipitation, either alone or in combination. [Example]

[0036] The present invention will be specifically explained below with reference to examples, but the present invention is not limited to these examples.

[0037] Example 1 Confirmation of secretory expression of ferritin nanoparticles using BEVS As shown in Figure 1, genes were created by artificial gene synthesis (Eurofins Genomics) that fused six types of signal peptides with Spytag and ferritin derived from Streptococcus pyogenes. However, when HBM was used as the signal peptide, a His tag and a TEV protease cleavage site were also inserted. The signal peptides used were HBM (HBM) from honeybees (Apis mellifera), GP64 (GP64) from Autographa californica nuclear polyhedrosis virus (AcNPV), cathepsin (Cath) from AcNPV, fibroin L chain (Fibl) from silkworms (Bombyx mori), hemagglutinin (HA) from influenza A / H1N1 subtype, and osteonectin (Ost) from silkworms. The sequences and origins of each are shown in Table 1. The ferritin sequence used was derived from Helicobacter pylori (Accession No.: WP000949190.1).

[0038] [Table 1]

[0039] The constructed artificial gene was subcloned into pfastbac1 (Thermo Fisher Scientific). pfastbac1 with the target gene inserted was transformed into DH10Bac (Thermo Fisher Scientific) to obtain Bacmid. The purified Bacmid was transfected into Sf9 cells (Thermo Fisher Scientific) to obtain recombinant baculovirus. The recombinant baculovirus was cloned by the plaque method, then grown in a 100 mL culture scale and stored at -80°C until use. This frozen recombinant baculovirus was used as a viral seed for protein expression.

[0040] Next, the infectious titer of the prepared virus seeds was measured by the following method. 6 Sf9 cells were seeded at 10 cells / well and allowed to adhere for 1 hour at 27°C. 6 or 10 6.5 , 10 71 mL of diluted recombinant baculovirus was added to each well. After incubation at 27°C for 1 hour, the virus dilution was removed and 2 mL of 1.5% agarose / Grace's Insect medium / 2% FBS was added. After the gel had solidified, the plates were incubated at 27°C for 4 days. 1 mL of 1.5% agarose / Grace's Insect medium / 2% FBS / 62.5 μg / mL Neutral Red was then added. After the gel had solidified, the plates were incubated at 27°C for 3 days. After incubation, the number of plaques was counted and the infectious titer was calculated.

[0041] Expression was compared using the virus seeds expressing ferritin with various signal peptides attached to the N-terminus, as follows. 6 Sf9 cells were seeded at 30 mL culture volume at 1000 cells / mL. The culture medium used was SFM4 (Cytiva) supplemented with penicillin-streptomycin (Thermo Fisher Scientific). Recombinant baculovirus was added to the Sf9 cell culture medium at a multiplicity of infection (MOI) of 0.1 or 1, and the cells were cultured at 27°C and 130 rpm. The cells were harvested after 3 days of culture, with the start of culture considered to be day 0. The harvested cells were centrifuged at 200 × g for 10 minutes. The supernatant and sample buffer (6.4% SDS, 32% glycerol, 0.1 mg / mL bromophenol blue / 200 mM Tris-HCl buffer, pH 6.8) were mixed in equal volumes and heated at 95°C for 5 minutes. The precipitate was suspended in an equal volume of phosphate-buffered saline (PBS), mixed with an equal volume of sample buffer, and heated at 95°C for 5 minutes. The supernatant used for Western blotting (WB) was diluted 5-fold with PBS, mixed with an equal volume of sample buffer, and heated at 95°C for 5 minutes.

[0042] A 12.5% ​​polyacrylamide gel (ATTO) was placed in an electrophoresis apparatus (ATTO) and filled with electrophoresis buffer (0.1% SDS, 192 mM glycine / 25 mM Tris Buffer pH 8.3), and the sample was subjected to SDS-PAGE. After electrophoresis, the gel was stained with Bio-Safe Coomassie Stain (Bio-Rad) for 1 hour, destained with ultrapure water, and then photographed. For WB, the electrophoretic gel was transferred to a PVDF membrane (Merck Millipore) using a semi-dry method. The transferred membrane was then blocked with 10% skim milk / TBS (10 mM Tris-HCl pH 7.5, 500 mM NaCl) and incubated overnight at 4°C with anti-ferritin antibody (MYBioSource) diluted 1:1000 in 1% skim milk / TBS-T (10 mM Tris-HCl pH 7.5, 500 mM NaCl, 0.5% Tween 20). After washing five times with TBS-T, the membrane was incubated with HRP-labeled anti-rabbit IgG antibody (Bethyl) diluted 1:10,000 in 1% skim milk / TBS-T for 1 hour at room temperature, and then immersed in Pierce. TM The cells were developed using ECL Western Blotting Substrate (Thermo Fisher Scientific) and photographed.

[0043] As shown in Figures 2 and 3, intracellular expression was confirmed for all constructs, suggesting that the ferritin sequence was properly inserted into all of the recombinant baculoviruses constructed. Figure 3 indicates that HBM was not secreted into the culture supernatant, but all other constructs secreted ferritin into the culture supernatant. Furthermore, as shown in Figures 2 and 3, the target band intensity was high, likely due to the high secretion amount into the culture supernatant, while the HA and Ost signal peptides produced almost no ferritin degradation product bands in the culture supernatant. This suggests that the use of these signal peptides allows the target protein, ferritin particles, to be secreted into the culture supernatant via a pathway that is less susceptible to degradation or more rapidly than degradation.

[0044] Example 2: Crude purification of ferritin nanoparticles using a sucrose cushion and comparison of secretion amounts To purify only nanoparticles of the target protein, the sucrose cushion method was performed. 5 mL of 25% w / w sucrose / PBS was placed in an ultracentrifuge tube (Hitachi) and 25 mL of the culture medium of Sf9 cells infected with the recombinant baculovirus prepared in Example 1 was layered on top. The tube was centrifuged at 141,000 × g for 6 hours (Hitachi). The supernatant was discarded and the pellet was dissolved in 1 mL of PBS. SDS-PAGE, CBB staining, and WB were performed as in Example 1. As shown in Figures 4 and 5, ferritin bands were confirmed for signal peptides other than HBM after crude purification using the sucrose cushion. Therefore, ferritin carrying these signal peptides was thought to form particles in the culture supernatant. Furthermore, degradation product bands were detected for GP64, Cath, and Fibl, whereas no degradation product bands were detected for HA and Ost. Therefore, it was thought that the use of the HA and Ost signal peptides could secrete ferritin particles into the culture supernatant without degradation.

[0045] Next, ELISA using the SpyTag / Spycatcher system was performed to compare the amount of ferritin forming nanoparticles. 100 μL of Spycatcher3 (Bio-Rad) diluted to 20 μg / mL was added to each well of a 96-well plate and incubated at 4°C overnight. Spycatcher3 forms a covalent bond with Spytag, allowing the fusion protein of Spytag and ferritin expressed in Example 1 to selectively bind. After immobilization of Spycatcher, the plate was washed five times with 300 μL of TBST and then blocked by adding 2.5% skim milk / TBST and incubating at 25°C for 60 minutes. After washing five times with 300 μL of TBST, 100 μL of a sample diluted 25- or 4,000-fold with 0.5% skim milk / TBST was added and incubated at 25°C for 2 hours. 100 μL of 0.5% skim milk / TBST was added as a blank. After washing five times with 300 μL of TBST, 100 μL of 1,000-fold diluted anti-ferritin antibody (MYBioSource) was added as the primary antibody reaction and incubated at 25°C for 60 minutes. After washing five times with 300 μL of TBST, 100 μL of 10,000-fold diluted HRP-labeled anti-rabbit IgG antibody (Bethyl) was added as the secondary antibody reaction and incubated at 25°C for 60 minutes. After washing five times with 300 μL of TBST, 125 μL of TMB (Abcam) was added and incubated at 25°C for 20 minutes. After the reaction, 125 μL of 0.6 N sulfuric acid was added to stop the reaction. The absorbance at 450 nm was measured using a plate reader (PerkinElmer), and the blank value was subtracted to compare the content of ferritin fused to each signal peptide. As shown in Figure 6, in the 25-fold diluted sample, slight color development was observed with HBM, while the signal was saturated with signal peptides other than HBM. This suggests that the amount of ferritin secreted with the HBM-derived signal peptide is smaller than that with other signal peptides. Measurements of the 4000-fold diluted sample showed that Fibl produced the highest amount of ferritin, followed by GP64, HA, and Ost, which all produced similar amounts, and Cath produced the lowest. This demonstrates that the use of a Fibl-derived signal peptide can improve secretion levels compared to GP64. It was also shown that HA and Ost secreted amounts similar to those of GP64.

[0046] The above results indicate that by fusing Fibl, HA, and Ost as signal peptides to the N-terminus of the target protein and expressing it, it becomes possible to secrete it into the culture supernatant in BEVS, and the amount secreted is equal to or greater than that of the commonly used signal peptides of HBM and GP64. In particular, Fibl allows for high secretion levels, while HA and Ost allow for secretory expression with little or no degradation, and all three signal peptides are thought to be useful in the production of recombinant proteins.

[0047] Non-Patent Document 3 discloses that when A / H5N1 influenza virus subtype hemagglutinin is expressed in BEVS, the secretion amount can be improved by substituting the signal peptide of the hemagglutinin with a signal peptide derived from HBM. However, as described above, when ferritin is expressed as a fusion protein with an HBM-derived signal peptide, ferritin is hardly secreted into the culture supernatant. In contrast, secretion into the culture supernatant has been confirmed with the signal peptide (HA) derived from A / H1N1 influenza virus hemagglutinin. This indicates that secretion performance differs even among influenza virus hemagglutinin signal peptides. It is also believed that the signal peptides of the present invention are superior in secreting proteins that form large complexes, such as ferritin.

Claims

1. A method for producing a target protein, comprising the steps of expressing a fused signal peptide and target protein in cultured insect cells using a recombinant baculovirus containing a polynucleotide encoding the target protein bound to a signal peptide, and recovering the target protein from the culture supernatant, wherein the signal peptide is a signal peptide derived from a silkworm fibroin L chain, a signal peptide derived from the hemagglutinin of influenza virus A / H1N1 subtype, or a signal peptide derived from silkworm osteonectin.

2. The method according to claim 1, wherein the signal peptide derived from the silkworm fibroin L chain is any one of the peptides shown in (1) to (3) below, the signal peptide derived from the hemagglutinin of influenza A / H1N1 subtype virus is any one of the peptides shown in (4) to (6) below, and the signal peptide derived from silkworm osteonectin is any one of the peptides shown in (7) to (9) below. (1) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 (2) A peptide having an amino acid sequence having an identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 1 and having a function as a signal peptide. (3) A peptide having a function as a signal peptide, which consists of an amino acid sequence in which one or several amino acids are deleted, substituted, or inserted in the amino acid sequence shown in SEQ ID NO:

1. (4) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 (5) A peptide having an amino acid sequence having an identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 2 and having a function as a signal peptide. (6) A peptide having a function as a signal peptide, which consists of an amino acid sequence in which one or several amino acids are deleted, substituted, or inserted in the amino acid sequence shown in SEQ ID NO:

2. (7) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 3 (8) A peptide having an amino acid sequence having an identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 3 and having a function as a signal peptide. (9) A peptide having a function as a signal peptide, which consists of an amino acid sequence in which one or several amino acids are deleted, substituted, or inserted in the amino acid sequence shown in SEQ ID NO:

3.

3. The method according to claim 1 or 2, wherein the fused and expressed protein is a protein in which a signal peptide is bound to the N-terminus of the target protein.

4. 3. The method according to claim 1 or 2, wherein the cultured insect cells are a cell line established from Spodoptera frugiperda.

5. The method according to claim 1 or 2, wherein the target protein is ferritin protein or nanoparticles of ferritin protein.

6. A recombinant baculovirus comprising a polynucleotide encoding a target protein bound to a signal peptide, wherein the signal peptide is a signal peptide derived from a silkworm fibroin L chain, a signal peptide derived from the hemagglutinin of influenza virus A / H1N1 subtype, or a signal peptide derived from silkworm osteonectin.

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