Superose 6 increase 10 300 column

1 mg of total Fab from immunized guinea pigs was incubated overnight with 10–20 μg HIV-1 Env trimers at room temperature. Env-Fab complexes were purified by size exclusion chromatography using a Superose 6 Increase 10/300 column (Cytiva) in a buffer containing 150 mM NaCl and 5 mM HEPES, pH 7.4. The fractions containing the Env-Fab complexes were pooled and concentrated to ∼1 mg/mL.

SpyCatcher003-mi3⁶⁸ (link) displaying SpyTagged SARS-CoV-2 S-6P trimers were prepared as described.^{63 ,68} (link) Briefly, SpyCatcher003-mi3 subunits with N-terminal 6xHis tags were expressed in BL21-CodonPlus (DE3)-RIPL E. coli (Agilent). Bacterial cell pellets were lysed using a cell disruptor in the presence of 2.0 mM PMSF (Sigma). Lysates were centrifuged at 21,000 x g for 30 min, and supernatants were collected and filtered through a 0.2 μm filter. SpyCatcher003-mi3 was purified by Ni-NTA chromatography using a pre-packed HisTrap™ HP column (Cytiva), concentrated in Amicon Ultra-15 centrifugal filters with 30 kDa molecular weight cut-off (Millipore), and purified by SEC on a HiLoad 16/600 Superdex 200 column (GE Healthcare) equilibrated with TBS. S-mi3 nanoparticles were generated by incubating purified SpyCatcher003-mi3 with a 3-fold molar excess of purified SpyTagged S-6P trimer overnight at 4°C in TBS. Conjugated S-mi3 nanoparticles were separated from uncoupled S-6P trimers by SEC using a Superose 6 Increase 10/300 column (Cytiva) equilibrated with PBS (Invitrogen). Fractions corresponding to conjugated S-mi3 were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and pooled.

A total of 100 µL 1 mg/mL FLAG-tagged NLRP3ΔPYD (wild-type or mutant) were applied to a Superose 6 Increase 10/300 column (Cytiva) equilibrated with a buffer containing 20 mM HEPES pH 7.5, 200 mM NaCl, 1 mM MgCl₂, and 2 mM DTT at a flow rate of 0.3 mL/min. The molecular weight was estimated by comparing the elution volume with a set of calibration standards (5 mg/mL Thyreoglobulin (669 kDa), 0.3 mg/mL Ferritin (440 kDa), 4 mg/mL Aldolase (158 kDa), 4 mg/mL Ovalbumin (44 kDa)).

A sequence encoding for an AviTag followed by a 3xFLAG tag was cloned into the C terminus of the endogenous Pre1 gene of a W303 yeast strain. The purification of AviTag-20S core was performed similar to a procedure previously described (56 (link)). Briefly, the yeast strain was grown in yeast extract, peptone, and dextrose at 30°C for 3 days. The cells were pelleted, resuspended in lysis buffer [60 mM Hepes (pH 7.6), 500 mM NaCl, 1 mM EDTA, and 0.2% NP-40], popcorned into liquid nitrogen, and then lysed in a Cryomill 6875D (SPEX SamplePrep). The lysate was allowed to return to room temperature and clarified by centrifugation, and the 20S core particle was purified using anti-Flag M2 affinity resin (Sigma-Aldrich). The elution was concentrated using an Amicon Ultracel-100K, and the concentration was determined by absorbance at 280 nm. After biotinylation through incubation with 25 μM E. coli biotin ligase BirA and 100 μM biotin in the presence of 10 mM ATP and 10 mM MgCl₂ overnight at 4°C, the core was further purified by size exclusion chromatography with a Superose 6 Increase 10/300 column (Cytiva), equilibrated in GF buffer [30 mM Hepes (pH 7.6), 50 mM NaCl, 50 mM KCl, 10 mM MgCl₂, 5% glycerol, and 0.5 mM TCEP]. The extent of biotinylation was determined in a gel shift assay by incubation with NeutrAvidin (Thermo Fisher Scientific).

Expression and purification of SARS-CoV-2 S ectodomains were performed as previously described (Barnes et al., 2020a (link); Barnes et al., 2020b (link)). SARS-CoV-2 S constructs were composed of residues 16–1206 of the early SARS-CoV-2 isolate (GenBank MN985325.1), Alpha variant (GISAID EPI_ISL_601443), Delta variant (GenBank QWK65230.1), or Omicron variant (BA.1) (GISAID EPI_ISL_9845731) with the following stabilizing mutations: 2P (Pallesen et al., 2017 (link)) or 6P (Hsieh et al., 2020 (link)), the furin cleavage site mutated to Ala, a C-terminal TEV protease site (GSG-RENLYFQG), foldon trimerization motif (GGGSG-YIPEAPRDGQAYVRKDGEWVLLSTFL), 8x-His tag (G-HHHHHHHH), and AviTag (GLNDIFEAQKIEWHE). All S constructs were expressed using the Expi293T transient transfections system (GIBCO). S trimers from clarified transfected cell supernatants were purified over HisTrap High Performance columns (Cytiva), followed by size-exclusion chromatography (SEC) using a Superose 6 increase 10 300 columns (Cytiva). Fractions corresponding to S trimers were collected and concentrated in 10% glycerol TBS (20 mM Tris, 150 mM NaCl, pH 8.0) then flash-frozen in liquid nitrogen and stored at –80℃. Downstream purification was completed within 12 hr of the transfected cell harvest to maximize the quality of trimeric, well-folded S trimers.