Superdex 200 increase 10 300 sec column

SEC-MALS experiments were performed using a Superdex 200 increase 10/300 SEC column (Cytiva), equilibrated in 20 mM HEPES pH 7.0, 150 mM NaCl connected to an HPLC system (Shimadzu Corporation) and DAWN HELEOS-II (Wyatt Technology) light scattering detector and Optilab T-rEX (Wyatt Technology) refractive index detector. 100 µl EphA2 570-976 at a concentration of 3 mg/ml was injected. Data were analyzed in ASTRA software (Version 7.3.1.9, Wyatt Technology).

The nanoparticle components I53-50A and I53-50B.4.PT1,²⁹ (link) and I53_dn5A and I53_dn5B,³⁰ (link) were expressed in Lemo21(DE3) (NEB) in LB (10 g Tryptone, 5 g Yeast Extract, 10 g NaCl) and grown in 2 L baffled shake flasks. 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 were 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, (with 0.75% CHAPS only for I53-50 proteins). 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–500 mM imidazole in a background of 50 mM Tris pH 8, 500 mM NaCl, (with 0.75% CHAPS only for I53-50 proteins) buffer. Peak fractions were pooled, concentrated in 10K MWCO centrifugal filters (Millipore), sterile filtered (0.22 μm) and applied to a Superdex 200 Increase 10/300 SEC column (Cytiva) using 50 mM Tris pH 8, 500 mM NaCl, (with 0.75% CHAPS only for I53-50 proteins) buffer. After sizing, bacterial-derived components were tested to confirm low levels of endotoxin before using for nanoparticle assembly.

MS2-SA and biotinylated S2 or S2_mutS2’ were mixed in a ratio determined using analytical SEC. Mixtures consisting of 5 μg of S2 or S2_mutS2’ and varying amounts of MS2-SA were run through a Superdex 200 increase 10/300 SEC column (Cytiva). The ratio of the mixture with the least amount of MS2-SA that resulted in a chromatogram without a peak corresponding to excess S2 or S2_mutS2’ was the stoichiometric ratio used to generate VLP-S2 and VLP-S2_mutS2’ for characterization and immunization.