Akta pure

Genes were heterologously expressed in Escherichia coli BL21(DE3) harboring the plasmid pET28a. The mutants were created using the site-directed mutagenesis kit from Tiangen Biotech. The primer pairs used for site-directed mutagenesis are presented in Supplementary Table S1. E. coli BL21(DE3) was grown in lysogeny broth at 37°C with 200 rpm shaking until the optical density (OD₆₀₀) of the cultures reached 0.8. The cultures were then cooled to 16°C and isopropyl β-d-1-thiogalactopyranoside was added to a finial concentration of 0.5 mM. The cells were further incubated at 16°C with 200 rpm shaking for 20 h. The cultures were centrifuged at 12 000 g for 10 min at 4°C. The cell pellets were resuspended in 50 mM Tris–HCl buffer (pH 8.0, 150 mM NaCl). The suspension was subjected to ultrasonication to disrupt the cells. The cell lysate was then centrifuged at 12 000 g for 20 min at 4°C. The supernatants were filtered through a 0.45-μm membrane (Merck Millipore) and then loaded onto AKTA pure (Cytiva, MA, USA) coupled with a HisTrap HP column (5 ml, Cytiva). The product fraction was eluted with imidazole-containing (50–500 mM) elution buffer (10 mM Tris–HCl, 150 mM NaCl, pH 8.0) at a flow rate of 1 ml/min. The collected product fractions were further subjected to a desalting column (5 ml, Cytiva) with elution buffer to remove imidazole.

Antigen binding fragments were cloned and produced in Expi293F cells as above, except the variable heavy chain was cloned into a pVRC expression vector containing the CH1 domain followed by a HRV 3C cleavate site and a 6X His tag. Fabs were purified by cobalt chromatography (Takara) and further purified over an S200 column on an AKTA pure (Cytiva). To the purified Fabs, 1.2 μL HRV 3C protease (Thermo Scientific, #88947) per 200 μg of Fab was added and incubated overnight at 4°C on a roller. The next day, the cleaved Fab was passed over cobalt resin and purified again over an S200 column in 10 mM Tris HCl, 150 mM NaCl, pH 7.5. The resulting Fabs were concentrated to ~15 mg/mL prior to crystallization.

SEC analyses were performed at room temperature on a system (AKTA Pure, Cytiva), equipped with Superose 6 Increase 10/300 GL column (23 ml, Cytiva) and with an array of detectors, comprised of UV-vis detector (UV-900, Cytiva), a multi-angle light scattering (MALS; miniDAWN TREOS, Wyatt Technology) detector, a dynamic light scattering module (WyattQELS) and with a refractive index detector (Optilab T-rEX, Wyatt Technology). The elution was monitored at 280, 260, and 220 nm (UV–vis detector) and three angles (43.6, 90, and 136.4°) with a 658.9 nm laser beam (MALS detector) (Amartely et al., 2019 (link)). The refractive index of the eluting solvent was determined to be 1.331, the viscosity was 0.8945 cP (typical for the PBS buffer) and the refractive index increment value (d_n/d_c) for ulvan was defined to be 0.127 mL g⁻¹ (Paradossi, Cavalieri & Chiessi, 2002 (link)). The data collection and analyses were performed with ASTRA 6.1 software (Wyatt Technology). A sample (with a typical volume of 0.5 mL) was injected into the column equilibrated with an eluent comprised of MES buffer (10 mM, pH 6.0) and NaCl (50 mM). A typical flow rate was 0.8 mL min⁻¹ and for molecular weight (MW) calibration of this column see Table S1.

HB-79 (producing anti H-2Kd/H-2Dd mouse IgG2a) and HB-27 (producing anti H-2Ld mouse IgG2a) hybridoma cell lines were purchased from ATCC and grown in Iscove medium (Sigma) supplemented with 10% FBS. Hybridoma were then adapted to protein-free PFHM medium (Thermo) for expansion and conditioning. Once cells were dead, the medium containing immunoglobulins was centrifuged and filtered to be run on a MabSelect Sure (ProteinA) column (Cytiva) mounted on Akta Pure (Cytiva). IgGs were then eluted at acid pH and dialyzed against physiologic storage buffer.

Variable heavy and light chains were synthesized as eBlocks (IDT). Full-length, codon-optimized HAs (A/Massachusetts/1/1990 – MA90 [Supplementary file 5], MA90-G189E [Supplementary file 6], and A/Solomon Islands/03/2006 – SI06 [Supplementary file 7]) and full-length human IgG1 heavy and light chains were cloned into a pVRC expression vector containing a C-terminal HRV 3C cleavage site, His tag, FoldOn trimerization domain, and AviTag for HAs and a HRV 3C cleavage site followed by a C-terminal His tag for antibody heavy chains. Recombinant proteins were produced in Expi293F cells (Gibco, #A14527; authenticated by STR profiling and verified mycoplasma-negative by the manufacturer) following the manufacturer’s directions. The trimeric HAs were purified from the supernatant using TALON metal affinity resin (Takara, #635653), washing with PBS, and eluting with PBS containing 200 mM imidazole (pH 7.4). After concentration, proteins were further purified over an S200 column on an AKTA pure (Cytiva). For yeast surface display assays, the HAs were further biotinylated and flash-frozen in liquid nitrogen (see below). For kinetics measurements, the HAs were used within 2 weeks of production and never frozen.