If not mentioned otherwise, all materials and reagents, such as tetraethyl orthosilicate, FeCl2, FeCl3, (3-aminopropyl)trimethoxysilane, and Ni(OAc)2, were purchased from Sigma-Aldrich (Tokyo, Japan). Ammonium hydroxide was purchased from Wako Pure Chemical Ind. Ltd. (Osaka, Japan).
2.1 Plasmid preparation and expression of SpyCatcher-mCherry-SpyTag in E. coli
The mCherry DNA fragment was subcloned from the plasmid pmCherry (Catalog # 632522, TakaraBio, Japan) using specific primers (mCherryFw: 5’-gtgagcaagggcgaggaggat-3’; mCherryRv: 5’-cttgtacagctcgtccatgcc-3’) into pFastBac-SpyCatcher/SpyTag  and was designated pFB-SC-mCherry-ST. The recombinant plasmid consisted of a His tag, StrepTag II, SpyCatcher, SpyTag, and tobacco etch virus (TEV) protease cleavage sites. The plasmid was used as a template for amplifying the whole gene sequence using primers (Cat-FW: 5'-atgcaccaccaccaccatcaccatcac-3’, pFastBac-RV: 5'-acaaatgtggtatggctgatt-3’) and subsequently subcloned into pET-41a (+) (Novagen, Tokyo, Japan), and the resulting construct was designated pET41-SC-mCherry-ST. The recombinant plasmid was electroporated into Rosetta-gami 2(DE3) E. coli (Novagen, Tokyo, Japan) to express the recombinant mCherry protein.
For the expression of SC-mCherry-ST, E. coli Rosetta-gami™ 2(DE3)/pET41-SC-mCherry-ST were inoculated into 1 L of Luria-Bertani medium (LB broth, Invitrogen, Thermo Fisher Scientific, Tokyo, Japan) containing 25 µg/mL kanamycin and incubated at 37°C until the OD600 reached 0.5. Then, protein expression was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 0.5 mM, followed by incubation at 16°C for 16 h. Subsequently, the cells were collected by centrifugation (6,000 × g, 4°C, 15 min), washed twice with ice-cold phosphate-buffered saline (PBS, pH 7.3), and stored at –80°C before use.
2.2 Recombinant B. mori nucleopolyhedrovirus (BmNPV) bacmid preparation for SpCaVP1 and EDIII
The Norovirus VP1 DNA fragment was subcloned into the pFastBac-SpyCatcher plasmid  from a recombinant Autographa californica nucleopolyhedrovirus (AcMNPV) kindly provided by Dr. Tian-Cheng Li (Department of Virology 2, National Institute of Infectious Diseases, Musashimurayama, Japan). The DNA sequence also encoded poly-tags (His-Strep-TEV-NoroVP1-SpyCatcher) and consisted of a His-tag, a Strep-tag II, a TEV protease cleavage site, and a SpyCatcher. The resulting plasmid for expressing the SpCaVP1 protein was designated pFastBac-HSSc-SpCaVP1. The codon-optimized EDIII DNA fragment was synthesized (Genewiz, Suzhou, China) based on the sequence of Dengue virus 1 (GenBank No. KM204119). The EDIII sequence was then amplified using primers (1DIII-Fw: 5'-agttatgttatgtgcaccgg-3’; 1DIII-Rv: 5'-gcccaaaatagccattcgcc-3’) and further ligated into pFastBac-cSpyTag . The DNA sequence also encoded poly-tags (an EDIII-cSpyTag-TEV-Strep-Flag, SpyTag, TEV protease cleavage site, Strep-Tag, and Flag-Tag). The resulting plasmid for expressing the EDIII protein was designated pFastBac-FSS-EDIII. Both plasmids were constructed and then utilized for the generation of a recombinant BmNPV bacmid. Subsequently, the recombinant baculovirus was generated in cultured Bm5 cells according to our previous reports . The cell culture supernatant was collected and used for serial infections to obtain high-titer virus stocks, which were employed to infect silkworm larvae.
2.3 Expression of recombinant proteins from silkworms
Fifth instar silkworm larvae (Ehime Sansyu, Ehime, Japan) were reared on an artificial diet (Silkmate S2, Nosan, Yokohama, Japan) in a rearing chamber (MLR-351H, Sanyo, Moriguchi, Japan) at 25°C. On the second day, the 5th instar larvae were injected with 10 µl of a PBS solution containing 250 µl/ml recombinant baculovirus stock using a 1 ml syringe (26G × 1/2, 0.45 × 13 mm). Five days postinjection (dpi), the fat body was collected in 5 ml lysis buffer (0.2 mol/l Tris-HCl, pH 7.6, 0.1% IGEPAC, and protease inhibitor) from each silkworm. This solution was sonicated as follows: 10 s at an amplitude of 60–80 with 30 s on ice for each cycle, which was repeated 20 times. The sonicated solution was incubated on ice for 1 h and centrifuged at 4°C and 8000 × g for 15 min. The supernatant was filtered with a 0.2 µm filter and stored at –80°C until use.
2.4 Lysis of recombinant protein expressed in E. coli (mCherry construct)
The E. coli cell pellet was resuspended in 3 ml ice-cold PBS for each 50 ml culture. To a 15 ml solution, 15 µl of 1 µg/ml lysozyme and 15 µl 1 × complete Mini EDTA free Version protease inhibitor (from a 100 × stock solution, Roche, Tokyo, Japan) was added and incubated on ice for 30 min. Sonication was performed on the ice at an amplitude of 70 with a 30-s interval cycle for 20 min. This solution was then centrifuged at 12000 × g for 10 min at 4°C. The supernatant was filtered with a 0.2 µm filter before further use.
2.5 Preparation of Ni-conjugated magnetic nanoparticles (Ni-MNPs)
The superparamagnetic iron oxide nanoparticles (SPIONs) were synthesized by following Massart's method . Five milliliters of ammonium hydroxide were added to 5 mmol of FeCl2 and 10 mmol of FeCl3 in 40 ml of ultrapure water. The mixed solution was vigorously stirred at room temperature for 30 min, and the synthesized MNPs were magnetically separated from the solution. Subsequently, the synthesized MNPs were coated with SiO2 for functionalization and stabilization . The freshly synthesized SPIONs in 120 ml ethanol were sonicated at room temperature for 30 min, and 150 µl of tetraethyl orthosilicate (TEOS) was added to the solution. The [email protected]2 was separated and washed at room temperature after 6 h of stirring.
Amino group conjugation for MNP functionalization was performed by following previously reported protocols . The washed [email protected]2 was dissolved in 100 ml anhydrous toluene, sonicated for 30 min, and then loaded into a three-necked round-bottom flask. (3-Aminopropyl)trimethoxysilane (APTMS) was added slowly and heated at 40°C with vigorous stirring for 24 h. The product was magnetically separated and washed with ethanol several times.
Ni-modified [email protected]2@NH2 was prepared as previously reported . Briefly, 0.32 g of isatoic anhydride was added to the solution, and 0.5 g of amino benzamide (2-AB)-immobilized [email protected]2@NH2 was dissolved in 100 ml of anhydrous toluene and refluxed for 12 h. The prepared [email protected]2@NH2@2-AB was separated by magnetic decantation and washed with ethanol several times. [email protected]2@NH2@2-AB (0.5 g) was suspended in 100 ml of ethanol and ultrasonically dispersed to form a homogeneous solution mixed with 2 mmol Ni(OAc)2·4H2O and refluxed for 12 h. The Magnetically separated [email protected]2@NH2@Ni was washed with ethanol to remove unreacted agents and dried overnight.
Each sample was characterized using transmission electron microscopy (TEM; JEM-2100F, JEOL, Ltd., Tokyo, Japan) with energy dispersive X-ray spectrometry (EDS) for elemental mapping nickel and iron. The zeta potential and hydrodynamic particle size were measured by dynamic light scattering (DLS) using a Zetasizer Nano series (Malvern Inst. Ltd., Malvern, UK).
2.6 Strep-tag affinity chromatography
Purification of mCherry from the E. coli cell lysate was performed using the Strep-Tactin affinity column with a manual peristaltic pump at a low flow rate (0.5 ml/min). The processed lysate was filtered through a 0.8 µm filter before chromatography was performed. The elution fraction, which had a reddish/purple color and strong red fluorescence, was separately collected.
2.7 General MNP purification protocol
Purification with the commercial magnetic bead MagneHis (Promega, Tokyo, Japan) was performed according to the manufacturer's manual, but the amounts used varied depending on the scale. Usually, the Ni particles were vortexed before usage, and then 100–300 µl of particles (2 mg/ml MNP solution) were added to a 1 ml sample. Incubation was performed for 10 min after mixing, but the mixture was stirred for 2 h at 4°C for scaling up. For the magnetic separation a neodymium magnet (TRUSCO Nakayama, Tokyo, Japan) was used. The supernatant was removed, and 500 µl washing buffer (100 mmol/l HEPES and 10 mmol/l imidazole) was added and mixed. After magnetic separation, the previous step was repeated two times. For elution, 200 µl elution buffer (100 mmol/l HEPES and 500 mmol/l imidazole) was used. A different buffer (20 mmol/l Tris-HCl, 0.5 mol/l NaCl and 1 mol/l imidazole, pH 7.5) was applied to ensure complete elution.
For the MNPs prepared, as described in section 2.5, the volume of sample/buffers/MNPs used varied in each protocol. In most cases, only two elution steps were performed. The basic sequence of the protocols was as follows. The MNPs were sonicated in a water bath for 30 min, and 350 µl of 2 mg/ml solution of MNPs were added to 250 µl of sample and 200 µl of washing buffer (20 mmol/l Tris-HCl, 0.5 mol/l NaCl and 20 mmol/l imidazole, pH 7.5). These were mixed and then incubated on ice for 30 min with occasional gentle mixing. After magnetic separation, the supernatant was removed, 200 µl washing buffer was added, and the mixture was incubated on ice for 10 min. After magnetic separation was performed 2 times, following buffers were used for elution: a weak elution buffer (20 mmol/l Tris-HCl, 0.5 mol/l NaCl and 300 mmol/l imidazole, pH 7.5), a strong elution buffer (20 mmol/l Tris-HCl, 0.5 mol/l NaCl, or 1 mol/l imidazole, pH 7.5). The amount depended on the protocol. The incubation time was 30 min on ice with occasional gentle mixing. After magnetic separation, the previous step was repeated with a strong elution buffer. For the volumes of the individual purification protocols, which are variable or not mentioned here, please see the figure legends which contain the specific information for the respecting figure. For all magnetic separations, the time to attract all MNPs were at least 5 minutes, as the case may also be longer, to ensure that all MNPs settled.
2.8 Optimized MNP3 purification protocol
The MNPs were sonicated in a water bath for 30 min, and 2 ml of 3.75 mg/ml MNPs were added to 1 ml fat body sample and 600 µl washing buffer (20 mmol/l Tris-HCl, 0.5 mol/l NaCl, and 20 mmol/l imidazole, pH 7.5). These were mixed and then incubated on ice for 30 min with occasional additional mixing. After magnetic separation, the supernatant was removed, 200 µl washing buffer was added, and the mixture was incubated for 10 min on ice. After magnetic separation, the washing step was repeated. After magnetic separation was performed during the first elution step, 500 µl of weak elution buffer (20 mmol/l Tris-HCl, 0.5 mol/l NaCl, and 300 mmol/l imidazole, pH 7.5) was used. For the second elution step, 500 µl of strong elution buffer (20 mmol/l Tris-HCl, 0.5 mol/l NaCl, and 1 mol/l imidazole, pH 7.5) was used. As the third elution step, the second elution step was repeated. The incubation was performed for 30 min on ice with occasional additional mixing. For all magnetic separations, the time to attract all MNPs were at least 5 minutes, as the case may also be longer, to ensure that all MNPs settled.
The samples were investigated using 10% polyacrylamide gels. They were diluted with an equal amount of sample buffer (0.125 mol/L Tris-HCl, 4% SDS, 20% glycerol, 0.01% mercaptoethanol, and 0.15 mmol/L bromophenol blue), mixed, and heated at 95°C for 5 min for denaturation. Electrophoresis was carried out with a BioRad SDS-PAGE chamber with a PowerPac Basic (BioRad, Hercules, CA, USA). The constant voltage was set at 90 V for the stacking gel and 120 V for the running gel. The size classification was performed with the PM1700 ExcelBand standard (Smobio, Hsinchu City, Taiwan). The 10% acrylamide gel was stained with Coomassie G-250 for approximately 2 h with a short heating period. Usually, the gels were destained with deionized water for at least 2 h to achieve adequate contrast. For documentation, the gels were scanned with the printer system Apeos Port IV (Fuji Xerox, Tokyo, Japan). The samples were analyzed with a series of dilutions from 8 µl sample to 22 µl sample in sample buffer. Each time, 15 µl of the dilution was loaded in the gel lane.
2.10 Western blotting
For western blotting, the proteins were subjected to SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) (Immobilon-P, Merck, Tokyo, Japan) membranes using the Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad, Tokyo, Japan). Blocking was performed for at least 1 h with 15 ml 5% skim milk in TBS containing 0.1% Tween 20. After washing with TBS and incubation for at least 2 h with mouse anti-Strep-tag antibody (1:10000, QIAGEN, Tokyo, Japan), the blots were rewashed. Incubation with a secondary goat anti-mouse IgG-horseradish peroxidase (HRP) (1:10000, MBL, Nagoya, Japan) was performed for at least 1 h. Immunoreactive bands were visualized using the Immobilon ECL Ultra Western HRP Substrate (Merck K. K., Tokyo, Japan) on the Versa-Doc 4000 MP (BioRad, Hercules, CA, USA).