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Definitions

• the present invention relates to a method for producing a recombinant protein in a baculovirus expression system.
• BEVS Baculovirus Expression Vector System
• BEVS is a technology for obtaining a target protein by infecting insect cells with a recombinant baculovirus into which the gene for the target protein has been inserted.
• proteins expressed in BEVS undergo post-translational modifications such as glycosylation, resulting in a structure similar to that of proteins expressed in mammals, and for this reason it is also used as a production technology for biological products such as vaccines.
• Another reason for its commercial use is that BEVS has a higher protein expression level than mammalian cells.
• BEVS is particularly useful for producing vaccines against pathogens that are difficult to cultivate artificially.
• norovirus is difficult to cultivate artificially, but by expressing the VP1 protein in BEVS, virus-like particles (VLPs) are formed, and development of these VP1 VLPs as candidate antigens for norovirus vaccines is progressing (Non-Patent Document 1).
• BEVS is also used in the production of vaccines against human papillomavirus, which is difficult to cultivate artificially like norovirus, and influenza virus, which has a high risk of causing a pandemic due to significant antigenic mutations, as well as other vaccines that have been put into practical use.
• the expressed target protein is generally recovered by physically disrupting the insect cells using a homogenizer or a surfactant, releasing the target protein into the culture medium or extract.
• physical cell disruption methods can affect not only the insect cells but also the three-dimensional structure of the target protein, and there remain issues with its use as a vaccine antigen, where epitopes that recognize the three-dimensional structure may also be important.
• secretion signal such as Honeybee Melittin Signal Peptide, but depending on the properties of the fused target protein, it may remain inside the cell and secretion into the culture supernatant may be difficult. Secretion is particularly difficult for proteins with large molecular size or proteins with high association properties (multimer formation, particle formation).
• the present invention provides a method for efficiently obtaining recombinant proteins expressed using a baculovirus expression vector system.
• the inventors of the present application discovered that in a baculovirus expression vector system, by culturing infected cells in the presence of a proteasome inhibitor to induce cell death, the expressed recombinant protein (target protein) can be efficiently released outside the cells and easily recovered and obtained.
• the present invention relates to the following 1) to 6).
• 1) A method for producing a target protein using a baculovirus expression vector system comprising the steps of infecting insect cells with a recombinant baculovirus and then culturing the infected cells in the presence of a proteasome inhibitor.
• 2) The method according to 1) which comprises a step of removing the cells after the completion of the culture and recovering the target protein.
• the present invention makes it possible to efficiently obtain recombinant proteins expressed using a baculovirus expression vector system.
• CBB staining Evaluation of the extracellular release of target proteins by physiologically active substances with cytotoxic effects. Evaluation of the extracellular release of target proteins by physiologically active substances with cytotoxic effects (Western Blotting). Evaluation of the effect of proteasome inhibitors on the extracellular release of target proteins (CBB staining). Evaluation of the extracellular release of target proteins by proteasome inhibitors (Western Blotting). Evaluation of effective concentrations of proteasome inhibitors. Optimization evaluation of proteasome inhibitor treatment concentration (Western Blotting). Evaluation of proteasome inhibitor treatment time (CBB staining). Evaluation of proteasome inhibitor treatment time (Western Blotting). Evaluation of short-term treatment with proteasome inhibitors (CBB staining). Evaluation of short-term treatment with proteasome inhibitors (Western Blotting). Evaluation of the effect of proteasome inhibitors on extracellular release of target proteins (Sf9 cells).
• the method for producing a protein of the present invention involves producing a protein using a baculovirus expression vector system (BEVS), and comprises a step of infecting insect cells with a recombinant baculovirus and then culturing the cells in the presence of a proteasome inhibitor.
• baculovirus expression vector system refers to an expression system in which a recombinant baculovirus containing a gene for a target protein (also referred to as "target protein”) is infected into insect cells to express the target protein in the cells.
• baculovirus refers to a baculovirus vector in which a gene for a target protein has been incorporated downstream of a polyhedrin promoter or a p10 promoter, which enables gene expression in cultured insect cells.
• the type of baculovirus is not particularly limited as long as it is a virus capable of infecting lepidopteran insects or cultured cells of such insects, but is preferably a nucleopolyhedrosis virus (NPV) or a modified virus thereof, such as BmNPV, HycuNPV, AnpeNPV, AcNPV, etc.
• NPV nucleopolyhedrosis virus
• Recombinant baculoviruses can be produced by methods known in the art. For example, such methods include a method using a transfer vector that can insert a desired gene into baculovirus DNA by homologous recombination in a lepidopteran insect. In this method, a transfer vector incorporating a gene for a target protein and baculovirus DNA linearized with a restriction enzyme or the like are co-transfected into cultured cells of a lepidopteran insect, and the infected cells are screened to obtain a recombinant baculovirus. Another method is to obtain baculovirus DNA in which a desired gene is inserted by homologous recombination in bacteria.
• This method involves transforming bacteria that already contain baculovirus genes with a transfer vector in which a gene for a target protein has been incorporated, to obtain recombinant DNA.
• This recombinant DNA can be transfected into cultured cells of a lepidopteran insect to obtain a recombinant baculovirus.
• bacteria used in this method include E. coli.
• the transfer vector used in this case is not particularly limited as long as it has a promoter that enables gene expression in lepidopteran insects or cultured cells of these insects and is a 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 above promoter can be appropriately selected from promoters known in the art, and examples thereof include polyhedrin promoter, p10 promoter, and silk actin promoter.
• Bac-to-Bac system Thermo Fisher Scientific
• BaculoGold system BD Biosciences
• SuperBAC system SHEATECH
• BacPAC system BacPAC system
• flashBAC system Oxford Expression
• BacMagic system Merck
• BestBac system Expression Systems
• GenScript BacuVance system
• baculovirus vectors include, for example, ProFold vector (AB Vector), ProEasy vector (AB Vector), FoldHelper vector (AB Vector), pVL vector (AB Vector), pAc vector (AB Vector), pAB vector (AB Vector), pIEx vector (Merck), pBAC vector (Merck), and pTriEx vector (Merck).
• the target protein gene to be incorporated into the recombinant baculovirus is not particularly limited.
• the target protein may be a monomer or may form a complex such as a VLP.
• target proteins include functional proteins used as biopharmaceuticals such as vaccines and antibodies, clinical test reagents, foods, cosmetics, etc.
• insects refers to insects of the order Lepidoptera that are suitable for expressing recombinant proteins, such as the silkworm (Bombyx mori), the mulberry webworm (Spilosoma imparilis), the perennial herb (Antheraea pernyi), the Spodoptera frugiperda, and the nettle looper moth (Trichoplusiani).
• Cultured cells of lepidopteran insects are not particularly limited as long as they are cell lines established from the above-mentioned lepidopteran insects, and examples include 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.). Also included are improved insect cell lines derived by modifying these cells.
• Insect cells are inoculated (infected) with the recombinant baculovirus after the insect cells have been previously cultured and grown.
• the culture may be either adhesion culture or suspension culture, but suspension culture is preferred.
• Culture media 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), Insect-XPRESS medium (LONZA), and ESF 921 medium (Expression Systems), ESF SF medium (Expression Systems), etc. can be used, and antibiotics such as penicillin, streptomycin, and gentamicin may be added to the culture medium as necessary.
• the culture temperature is preferably 25 to 30°C, more preferably 26 to 28°C, and even more preferably 27°C.
• the cell concentration is preferably 1 ⁇ 10 5 to 1 ⁇ 10 7 cells/mL, more preferably 2 ⁇ 10 5 to 5 ⁇ 10 6 cells/mL, and even more preferably 5 ⁇ 10 5 to 2 ⁇ 10 6 cells/mL.
• the cultured cells are infected with the recombinant baculovirus by adding a liquid containing the recombinant baculovirus to a culture medium and inoculating the cells at an insect cell concentration of 1 ⁇ 10 5 to 1 ⁇ 10 7 cells/mL.
• protease inhibitors and nucleases can be added to the medium as appropriate.
• the cells After infection (after inoculation with the virus), the cells are cultured for 1 to 7 days, preferably 2 to 4 days after inoculation with the virus, so that the target protein is produced in the cells.
• the infected cells after infection, are cultured in the presence of a proteasome inhibitor.
• the proteasome is a giant proteolytic enzyme complex that degrades proteins that have completed their role or proteins with structural abnormalities in eukaryotic cells, and is involved in the control of major life phenomena such as cell cycle, signal transduction, immune response, apoptosis, and quality control of newly synthesized proteins.
• proteasome inhibitor refers to a physiologically active substance that has affinity for the 20S core of the proteasome and inhibits the activity of the proteasome.
• examples of such a proteasome inhibitor include peptide aldehyde-type inhibitors such as MG-132, MG-101, and MG-115, peptide boronate-type inhibitors such as MG-262, bortezomib, and ixazomib, peptide epoxyketone-type inhibitors such as carfilzomib and epoxomicin, as well as lactacystin, omuralide, and proteasome inhibitor I.
• MG-132 N-[(phenylmethoxy)carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide
• MG-132 N-[(phenylmethoxy)carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide
• Culturing of infected cells in the presence of such a proteasome inhibitor is carried out by infecting insect cells with a recombinant baculovirus, adding a proteasome inhibitor to the medium within a predetermined period of time, and continuing to culture the cells.
• the proteasome inhibitor is preferably added at a concentration that induces cell death in infected cells, and is preferably added so that the final concentration in the culture medium is 40 ⁇ M or more, preferably 50 ⁇ M or more, more preferably 80 ⁇ M or more.
• the final concentration is more preferably 40 to 1000 ⁇ M, more preferably 50 to 500 ⁇ M, more preferably 50 to 200 ⁇ M, more preferably 50 to 150 ⁇ M, and even more preferably 50 to 100 ⁇ M. Addition within such a range rapidly induces cell death in virus-infected cells and promotes the release of the target protein outside the cells.
• the timing of adding such a proteasome inhibitor is not particularly limited, so long as it is after infection of the cultured cells with the recombinant baculovirus, but it is preferable to add it 24 hours or more, preferably 36 hours or more, and more preferably 48 hours or more, but no later than 72 hours after inoculation of the recombinant baculovirus, and it is effective to add it between 48 and 72 hours after inoculation of the recombinant baculovirus.
• the incubation time (reaction treatment time) in the presence of such a proteasome inhibitor is not particularly limited, but from the viewpoint of improving the yield of the target protein, it is preferably 1 hour or more, more preferably 3 hours or more, more preferably 6 hours or more, and may be 18 hours or more or 24 hours or more, but from the viewpoint of shortening the incubation period, 1 to 24 hours is preferable, and 6 to 24 hours is more preferable.
• the target protein produced in insect cells is efficiently released outside the cells. Therefore, the target protein can be recovered from the supernatant obtained by centrifuging the cell culture medium and removing the cells by filtration or the like, without physically destroying the host cells. Furthermore, if necessary, purified products can be obtained by 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, immunoprecipitation, etc., either alone or in combination.
• DMSO DMSO
• benzonase Merck
• protease inhibitor tablets Thermo Fisher Scientific
• a condition in which no physiologically active substances were added was also performed as a negative control.
• the cell culture medium was collected 72 hours after virus infection, centrifuged at 1,000 ⁇ g for 15 minutes, and the supernatant was collected. The collected supernatant was centrifuged at 11,000 ⁇ g for 60 minutes, and the supernatant was collected.
• the supernatant and sample buffer (8% SDS, 40% glycerol/250 mM Tris-HCl Buffer pH 6.8) were mixed in equal amounts, and then mixed with 1/10 volume of 1 mg/mL BPB. This solution was reacted at 95°C for 5 minutes to prepare an SDS-PAGE sample.
• a 12.5% polyacrylamide gel was placed in an electrophoresis apparatus (ATTO) filled with electrophoresis buffer (0.1% SDS, 192 mM glycine/25 mM Tris Buffer pH 8.3), and the SDS-PAGE sample was electrophoresed. After electrophoresis, the gel was stained with Bio-Safe Coomassie Stain (Bio-Rad) for 1 hour, destained with ultrapure water, and then densitometric analysis was performed using an image analyzer (Cytiva).
• a calibration curve was created using VP1, the concentration of which had been adjusted in advance, as the standard, and the amount of the VP1-equivalent band for each sample was calculated as a relative value to the condition in which no physiologically active substance was added.
• PVDF membrane For Western blotting, electrophoresis was performed as described above, and then a transfer reaction from the gel to a PVDF membrane (Merck) was performed using a semi-dry transfer device (ATTO). After the transfer reaction, the PVDF membrane was immersed in TBS containing 10% skim milk and incubated at room temperature for 2 hours. After washing the PVDF membrane three times with an appropriate amount of TBS, it was immersed in anti-norovirus VP1 protein antibody (manufactured in-house) diluted with TBST containing 1% skim milk and incubated at 4°C for 18 to 20 hours.
• anti-norovirus VP1 protein antibody manufactured in-house
• a calibration curve was created using VP1, the concentration of which had been adjusted in advance, as the standard, and the amount of VP1 band in each sample was calculated as a relative value to the condition in which no physiologically active substance was added.
• Example 2 Evaluation of extracellular release of target protein by proteasome inhibitors
• High Five cells (Thermo Fisher Scientific Express Five (trademark) SFM) were seeded at a seeding concentration of 1 x 10 6 cells/mL and a culture volume of 30 mL.
• various proteasome inhibitors dissolved in DMSO (Table 5), benzonase (Merck) to a final concentration of 50 U/mL, and 30 mL of protease inhibitor tablets (Thermo Fisher Scientific) were added, and cell culture was continued.
• a condition in which no proteasome inhibitor was added was also performed as a negative control.
• the cell culture medium was collected 72 hours after virus infection, centrifuged at 1,000 ⁇ g for 15 minutes, and the supernatant was collected. The collected supernatant was centrifuged at 11,000 ⁇ g for 60 minutes, and the supernatant was collected.
• the supernatant and sample buffer (8% SDS, 40% glycerol/250 mM Tris-HCl Buffer pH 6.8) were mixed in equal amounts, and then mixed with 1/10 volume of 1 mg/mL BPB. This solution was reacted at 95°C for 5 minutes to prepare an SDS-PAGE sample.
• Reference Example 1 Concentration of physiologically active substance inducing cell death High Five cells (Thermo Fisher Scientific Express FiveTM SFM) were seeded at a seeding concentration of 1 ⁇ 10 6 cells/mL and a culture volume of 30 mL.
• Various physiologically active substances (Table 8) having cytotoxicity dissolved in DMSO were added to this culture solution, and the culture was continued.
• a condition in which no physiologically active substance was added was also performed as a negative control.
• the culture solution was collected 24 hours after the addition, and stained with a staining solution obtained by diluting 0.4% trypan blue (Thermo Fisher Scientific) 2-fold with DPBS (Fujifilm Wako Pure Chemical).
• the stained cells were poured into a modified Neubawell cell counter plate (WATSON), and live and dead cells were counted using an optical microscope, and the live cell concentration was calculated from the counting results.
• WATSON modified Neubawell cell counter plate
• the viable cell concentrations 24 hours after the addition of various physiologically active substances are shown in Table 8.
• the viable cell concentrations were reduced to 6 to 46% compared to the control condition A, suggesting that these treatments were able to induce cell death.
• the final concentrations were the same as those under the treatment conditions in Example 1, suggesting that cell death was able to be induced under the conditions in Example 1.
• both conditions B and C were treated with MG-132, but at 40 ⁇ M the viable cell concentration was reduced to 46% compared to the control condition A, and at 100 ⁇ M the viable cell concentration was reduced to 35% compared to condition A.
• the induction of cell death was determined to be a viable cell concentration of 50% or less compared to when no MG-132 was added.
• the concentration is up to 10 to 20 ⁇ M (Oncol Lett. 2018 Nov; 16 (5): 5900-5906., Cancer Res. 2019 Feb 1; 79 (3): 534-545.), and it is thought that cell death is strongly induced in the concentration range exceeding this.
• Example 1 when the results of Example 1 and this Reference Example are considered together, the amount of protein released into the culture supernatant does not change only depending on the strength of cell death induction, suggesting that the efficiency of intracellular protein release to the outside of the cells differs depending on the pattern of cell death induced. In other words, it was thought that cell death induced by a proteasome inhibitor is suitable for promoting the release of the target protein to the outside of the cells in BEVS.
• Example 3 Evaluation of Effective Concentration of Proteasome Inhibitors
• High Five cells (Thermo Fisher Scientific Express Five (trademark) SFM) were seeded at a seeding concentration of 1 x 10 6 cells/mL and a culture volume of 30 mL.
• MG-132 MedChemexpress
• benzonase Merck
• protease inhibitor tablets Thermo Fisher Scientific
• 72 hours after virus infection the cell culture solution was collected, centrifuged at 1,000 ⁇ g for 15 minutes, and the supernatant was collected. The collected supernatant was centrifuged at 11,000 ⁇ g for 60 minutes, and the supernatant was further collected.
• the supernatant and sample buffer (8% SDS, 40% glycerol/250 mM Tris-HCl Buffer pH 6.8) were mixed in equal amounts, and 1/10 volume of 1 mg/mL BPB was further mixed. This solution was reacted at 95°C for 5 minutes to prepare an SDS-PAGE sample.
• Example 4 Optimization of proteasome inhibitor treatment concentration High Five cells (Thermo Fisher Scientific, Express FiveTM SFM) were seeded at a seeding concentration of 1 ⁇ 10 6 cells/mL and a culture volume of 30 mL in the same manner as in Example 1. This culture solution was infected with a recombinant baculovirus incorporating the gene sequence of norovirus VP1 protein at an MOI of 0.1.
• MG-132 MedChemexpress
• Benzonase Merck
• protease inhibitor tablets Thermo Fisher Scientific
• 72 hours after virus infection the cell culture medium was collected, centrifuged at 1,000 ⁇ g for 15 minutes, and the supernatant was collected. The collected supernatant was centrifuged at 11,000 ⁇ g for 60 minutes, and the supernatant was further collected.
• sample buffer 8% SDS, 40% glycerol/250 mM Tris-HCl buffer pH 6.8
• 1/10 volume of 1 mg/mL BPB was mixed in. This solution was reacted at 95° C. for 5 minutes to prepare a sample for Western blotting.
• High Five cells Thermo Fisher Scientific Express Five (trademark) SFM
• a condition in which no physiologically active substance was added was also performed.
• the cell culture medium was collected 18 and 24 hours after the drug was added, and the supernatant was collected after centrifugation at 1,000 ⁇ g for 15 minutes.
• the collected supernatant was centrifuged at 11,000 ⁇ g for 60 minutes, and the supernatant was collected again.
• the supernatant and sample buffer (8% SDS, 40% glycerol/250 mM Tris-HCl Buffer pH 6.8) were mixed in equal amounts, and 1/10 volume of 1 mg/mL BPB was further mixed. This solution was reacted at 95°C for 5 minutes to prepare an SDS-PAGE sample.
• Example 2 SDS-PAGE and densitometry analysis were performed as in Example 1, and the relative values of the VP1-corresponding bands for each drug treatment time compared to non-addition conditions were calculated.
• PVDF membrane For Western blotting, electrophoresis was performed as described above, and then a transfer reaction from the gel to a PVDF membrane (Merck) was performed using a semi-dry transfer device (ATTO). After the transfer reaction, the PVDF membrane was immersed in TBS containing 10% skim milk and incubated at room temperature for 2 hours. After washing the PVDF membrane three times with an appropriate amount of TBS, it was immersed in anti-norovirus VP1 protein antibody (manufactured in-house) diluted with TBST containing 1% skim milk and incubated at 4°C for 18 to 20 hours.
• anti-norovirus VP1 protein antibody manufactured in-house
• Example 6 Evaluation of short-term treatment with proteasome inhibitors
• High Five cells (Thermo Fisher Scientific Express FiveTM SFM) were seeded at a seeding concentration of 1 ⁇ 10 6 cells/mL and a culture volume of 30 mL.
• MG-132 MedChemexpress
• a condition in which no physiologically active substance was added was also performed as a negative control.
• the cell culture medium was recovered 72 hours after virus infection, and centrifuged at 1,000 ⁇ g for 15 minutes, and the supernatant was recovered. The collected supernatant was centrifuged at 11,000 ⁇ g for 60 minutes, and the supernatant was collected. The supernatant was mixed with an equal amount of sample buffer (8% SDS, 40% glycerol/250 mM Tris-HCl Buffer pH 6.8), and then mixed with 1/10 volume of 1 mg/mL BPB. This solution was reacted at 95° C. for 5 minutes to prepare an SDS-PAGE sample.
• sample buffer 8% SDS, 40% glycerol/250 mM Tris-HCl Buffer pH 6.8
• Example 2 Similar to Example 1, SDS-PAGE and densitometry analysis were performed to calculate the relative value of the VP1-corresponding band. In addition, similar to Example 5, Western blotting was performed to obtain the VP1 band image.
• Example 7 Evaluation of the extracellular release of a target protein by a proteasome inhibitor (Sf9 cells)
• MG-132 MedChemexpress
• benzonase Merck
• a protease inhibitor tablet Thermo Fisher Scientific
• 24 hours after the drug addition the cell culture solution was collected, centrifuged at 1,000 ⁇ g for 15 minutes, and the supernatant was collected. The collected supernatant was centrifuged at 11,000 ⁇ g for 60 minutes, and the supernatant was further collected.
• the supernatant and sample buffer (8% SDS, 40% glycerol/250 mM Tris-HCl Buffer pH 6.8) were mixed in equal amounts, and 1/10 volume of 1 mg/mL BPB was further mixed. This solution was reacted at 95°C for 5 minutes to prepare an SDS-PAGE sample.

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