Superdex 200 increase 10 300

The I53-50A and I53-50B.4.PT1 proteins were expressed as described recently^{45 (link)}. Briefly, Lemo21 cells(DE3) (NEB), which were grown in LB (10 g Tryptone, 5 g Yeast Extract, 10 g NaCl) in 2 L baffled shake flasks or a 10 L BioFlo 320 Fermenter (Eppendorf) were used to express the I53-50A or I53-50B.4PT1 proteins grown. Cells were induced with 1 mM IPTG and maintained shaken for ~16 h at 18 °C. Microfluidization was used to harvest and lyse the cells, using a Microfluidics M110P machine at 18,000 psi in 50 mM Tris, 500 mM NaCl, 30 mM imidazole, 1 mM PMSF, 0.75% CHAPS. Proteins were purified by applying clarified lysates to a 2.6×10 cm Ni Sepharose 6 FF column (Cytiva) on an AKTA Avant150 FPLC system (Cytiva). A linear gradient of 30 mM to 500 mM imidazole in 50 mM Tris, pH 8, 500 mM NaCl, 0.75% CHAPS was used to elute both proteins. Next, the pooled fractions were subjected to size-exclusion chromatography on a Superdex 200 Increase 10/300, or HiLoad S200 pg GL SEC column (Cytiva) in 50 mM Tris pH 8, 500 mM NaCl, 0.75% CHAPS buffer. I53-50A elutes at ∼0.6 column volume (CV) whereas I53-50B.4PT1 elutes at ~0.45 CV. Prior to nanoparticle assembly, protein preparations were tested to confirm low levels of endotoxin.

Murine heavy chain apoferritin plasmid in pET24a vector was received from Masahide Kikkawa (University of Tokyo), expressed and purified using a previously published protocol (Danev, Yanagisawa, and Kikkawa 2019 (link)). Vector was transformed into and expressed in BL21(DE3)pLysS E. coli chemically-competent cells according to the published protocol. Sample concentrated to 10-20 mg/mL was loaded onto a Superdex 200 Increase 10/300 (Cytiva) column equilibrated with 30 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT. The fractions corresponding to the apoferritin peak were pooled and concentrated to 5 mg/mL. For long-term storage, trehalose (5% (v/v) final) was added to concentrated apoferritin prior to aliquoting and flash-freezing in liquid nitrogen for long-term storage at −70 °C.

The I53–50A and I53–50B.4.PT1 proteins were expressed in Lemo21(DE3) (NEB) in LB (10 g Tryptone, 5 g Yeast Extract, 10 g NaCl) grown in 2 L baffled shake flasks or a 10 L BioFlo 320 Fermenter (Eppendorf). Cells were grown at 37°C to an OD600 ~0.8, and then induced with 1 mM IPTG. Expression temperature was reduced to 18°C and the cells shaken for ~16 h. The cells were harvested and lysed by microfluidization using a Microfluidics M110P at 18,000 psi in 50 mM Tris, 500 mM NaCl, 30 mM imidazole, 1 mM PMSF, 0.75% CHAPS. Lysates were clarified by centrifugation at 24,000 g for 30 min and applied to a 2.6×10 cm Ni Sepharose 6 FF column (Cytiva) for purification by IMAC on an AKTA Avant150 FPLC system (Cytiva). Protein of interest was eluted over a linear gradient of 30 mM to 500 mM imidazole in a background of 50 mM Tris pH 8, 500 mM NaCl, 0.75% CHAPS buffer. Peak fractions were pooled, concentrated in 10K MWCO centrifugal filters (Millipore), sterile filtered (0.22 μm) and applied to either a Superdex 200 Increase 10/300, or HiLoad S200 pg GL SEC column (Cytiva) using 50 mM Tris pH 8, 500 mM NaCl, 0.75% CHAPS buffer. I53–50A elutes at ~0.6 column volume (CV). I53–50B.4PT1 elutes at ~0.45 CV. After sizing, bacterial-derived components were tested to confirm low levels of endotoxin before using for nanoparticle assembly.

The I53-50A and I53-50B.4.PT1 proteins were expressed in Lemo21(DE3) (NEB) in LB (10 g Tryptone, 5 g Yeast Extract, 10 g NaCl) grown in 2 L baffled shake flasks or a 10 L BioFlo 320 Fermenter (Eppendorf). Cells were grown at 37°C to an OD600 ∼0.8, and then induced with 1 mM IPTG. Expression temperature was reduced to 18°C and the cells shaken for ∼16 h. The cells were harvested and lysed by microfluidization using a Microfluidics M110P at 18,000 psi in 50 mM Tris, 500 mM NaCl, 30 mM imidazole, 1 mM PMSF, 0.75% CHAPS. Lysates were clarified by centrifugation at 24,000 g for 30 min and applied to a 2.6 × 10 cm Ni Sepharose 6 FF column (Cytiva) for purification by IMAC on an AKTA Avant150 FPLC system (Cytiva). Protein of interest was eluted over a linear gradient of 30 mM to 500 mM imidazole in a background of 50 mM Tris pH 8, 500 mM NaCl, 0.75% CHAPS buffer. Peak fractions were pooled, concentrated in 10K MWCO centrifugal filters (Millipore), sterile filtered (0.22 μm) and applied to either a Superdex 200 Increase 10/300, or HiLoad S200 pg GL SEC column (Cytiva) using 50 mM Tris pH 8, 500 mM NaCl, 0.75% CHAPS buffer. I53-50A elutes at ∼0.6 column volume (CV). I53-50B.4PT1 elutes at ∼0.45 CV. After sizing, bacterial-derived components were tested to confirm low levels of endotoxin before using for nanoparticle assembly.

The oligomerization state of the rRBD protein was evaluated by SEC. Elution fractions from IMAC containing purified rRBD were collected and concentrated, and the buffer was exchanged and loaded on a Superdex 200 increase 10/300 (Cytiva) equilibrated in 100 mM sodium phosphate buffer pH 7.4 and 150 mM NaCl. The elution peaks were collected, and the rRBD was developed by SDS-PAGE and western blot, as described previously.

Cells pellets were resuspended into lysis buffer LB2 (50 mM Tris-HCl pH 8, 500 mM NaCl, 5% glycerol, 0.1% Triton X-100, 10 mM imidazole, 0.5 mM PMSF, cOmplete EDTA-free Protease Inhibitor Cocktail and 70 U/mL of benzonase) and sonicated at 4 °C. Lysates were clarified by centrifugation at 5 °C at 18500 rpm for 30 min and loaded to gravity flow Ni-NTA agarose resin (QIAGEN) previously equilibrated with wash buffer WB2_1 (50 mM Tris-HCl pH 8, 500 mM NaCl, 10 mM imidazole). Resin was then washed with 10 column volumes of wash buffer WB2_1 followed by 10 column volumes of wash buffer WB2_2 (50 mM Tris-HCl pH 8, 1 M NaCl, 10 mM imidazole). Pcf2-6His was eluted with 5 column volumes of the elution buffer EB2 (50 mM Tris-HCl pH 8, 500 mM NaCl, 250 mM imidazole, 0.5 mM TCEP, and 1 X cOmplete EDTA-free Protease Inhibitor Cocktail). The eluate was then concentrated to 300 µL (with Amicon Ultra-15 30 kDa filter concentrators). 1 mM DTT, 1 mM MgCl and ≈2000 U of benzonase were added to the concentrated eluate and the sample was incubated 2 hr at 4 °C and injected into a Superdex 200 increase 10/300 (Cytiva) previously equilibrated with the final buffer FB2 (50 mM Tris-HCl pH 8, 500 mM NaCl and 1 mM DTT). Pcf2-6His containing fractions were pooled and directly used for CAF-1 reconstitution or stored at –70 °C with cOmplete EDTA-free Protease Inhibitor Cocktail, 0.5 mM TCEP and 30% glycerol.