Vivaspin filter

The purified His₆-GGT solution was desalted by repeated concentration using a Vivaspin filter (GE Healthcare) followed by dilution with 50 mM HEPES buffer pH 7.0. We re-screened the crystallization conditions of B. subtilis GGT to obtain a new crystal form; the previously obtained crystals had a large unit cell (Wada et al., 2010 ▶ ). The initial crystallization trials were performed using commercially available sparse-matrix screening kits such as Crystal Screen, Crystal Screen 2, Crystal Screen Lite, Crystal Screen Cryo, Natrix, PEG/Ion, PEG/Ion 2 (Hampton Research), Wizard I–III (Emerald BioStructures) and JBScreen 1–6 (Jena Bioscience). The conditions that produced crystals were optimized by varying the concentrations of protein, the precipitants, the buffer system and the pH. All crystallization trials were carried out using the micro-oil batch method at 293 K. Diffraction-quality crystals were produced when the drop was prepared by mixing 0.9 µl protein solution (10 mg ml⁻¹) with 0.9 µl reservoir solution [26%(w/v) PEG 3350, 0.7 M sodium thiocyanate, 6%(v/v) ethylene glycol] layered under 10 µl Al’s oil (Hampton Research). The crystals grew in a week to typical dimensions of 0.1 × 0.1 × 0.3 mm. Acivicin-bound GGT crystals were obtained by soaking the crystals for 2 h in crystallization solution containing 5 mM acivicin.

All constructs were expressed by transient transfection of HEK293F cells (Invitrogen) maintained in Freestyle medium (Life Technologies) at a density of 0.8-1.2 million cells/mL. On the day of transfection, a mix of PEImax (1 μg/μL) with expression plasmids (312.5 μg/L) in a 3:1 ratio in OptiMEM (GIBCO) were added to the cells. Six days post transfection, supernatants were centrifuged for 30 min at 4000 rpm, filtered using 0.22 μm Steritop filters (Merck Millipore), and subjected to affinity purification using Ni-NTA agarose beads. Protein eluates were then concentrated and buffer exchanged to PBS using Vivaspin filters with the appropriate molecular weight cutoff (GE Healthcare). Protein concentrations were determined by the Nanodrop method using the proteins peptidic molecular weight and extinction coefficient as determined by the online ExPASy software (ProtParam).

HEK293F cells (Invitrogen) were transiently transfected with SARS-CoV-2 S-Foldon pPPI4. Cells were grown in Freestyle medium (Life Technologies) and transfected at a density of 0.8–1.2 million cells/mL. A mixture of PEImax (1 µg/mL) and expression plasmid (312.5 µg/mL) in OptiMEM (GIBCO) was made on the day of transfection and added to the cells. After 6 days, cells and growth medium were transferred into centrifuge buckets and spun down at 3000 × g for 30 min. Supernatants were filtered through 0.22-µm Steritop filters (Merck Millipore). After filtration, spikes were purified using Ni-NTA agarose beads. Eluted proteins were concentrated and the buffer was changed to PBS using 100 kDa cutoff Vivaspin filters (GE Healthcare). Next, proteins were applied to a Superose 6 increase 10/300 GL column linked to a NGC chromatography system (BIO-RAD) in PBS. The appropriate size-exclusion fractions were pooled and proteins were stored at −80 °C.

HEK293F cells (0.8-1.2 million cells per mL) were transiently transfected with the coronavirus S constructs. Cells were maintained in Freestyle medium (Life Technologies). A mix of PEImax (937.5 μg/L cells) and expression plasmid (312.5 μg/L cells) was prepared in OptiMEM (Gibco) and added to the cells. Six days after the transfection the supernatants were collected by centrifuging the cell cultures at 3,000 x g for 30 min and were filtered through 0.22 μm Steritop filters (Merck Millipore). Supernatants were subjected to Ni-NTA agarose beads for affinity purification. Proteins were eluted and buffer exchanged to PBS and concentrated using Vivaspin filters (GE Healthcare) with a 100,000 Da cut-off. Additionally, proteins were applied to a Superose 6 increase 10/300 GL column (GE healthcare) in PBS for size exclusion chromatography. Appropriate size fractions were collected and subsequently pooled and concentrated using Vivaspin filters if necessary. Concentrations were measured using the peptidic molecular weight with Nanodrop. Proteins were stored at -80°C. The BtKY72 S protein was acquired from ACRO Biosystems, while influenza A [A/Victoria/2570/2019 (H1N1)pdm09-like virus] hemagglutinin was obtained from The Native Antigen Company. HIV-1 BG505 Env was produced as described elsewhere.⁵⁷

To generate B cells stably expressing Env-specific BCRs, IgM-negative Ramos B cells^{52 (link)} were essentially subjected to lentiviral transduction. Lentiviruses were produced by co-transfecting a T25 flask of HEK 293 T cells with the plasmids pMDL (1.5 µg), pVSV-g (0.83 µg) and pRSV-Rev (0.6 µg), and the BCR-encoding B-cell specific plasmid pRRL EuB29 gl2-1261 IgGTM.BCR.GFP.WPRE^{53 (link)} (2.4 µg) using lipofectamine 2000 (Invitrogen). Prior to lentiviral production, the gl2-1261 gene was exchanged by Gibson assembly with ordered heavy and light chain genes of either mature PGDM1400, VRC01, PGT121, VRC26.25, and RM19R (Integrated DNA Technologies). Two days post-transfection, supernatants containing lentiviral particles were filtered (0.45 μm) and concentrated to 200 μl using Vivaspin filters using Vivaspin filters with a 100 kDa molecular weight cutoff (GE Healthcare). Subsequently, IgM-negative Ramos B cells^{52 (link)} cultured in RPMI supplemented with 10% fetal calf serum, penicillin (100 U mL−1) and streptomycin (100 µg mL−1) were transduced with the concentrated viral supernatant. Seven days post-transduction, GFP and IgG double-positive cells (i.e. BCR-expressing cells) were sorted on a FACS ARIA-II SORP (BD Biosciences) and cultured.

All 16055 constructs were expressed in transiently transfected HEK293F cells (Invitrogen, cat no. R79009) maintained in Freestyle medium (Life Technologies) using previously described methods^{38 (link),49 (link)}. Briefly, a 3:1 mixture of PEImax (1 µg/µL; 937.5 µg/L cells) and expression plasmids (312.5 µg/L cells) were added to 0.8–1.2 million cells/mL. To ensure optimal furin-mediated cleavage of the Env component, the cells were transfected with (1) a 4:1 ratio of Env plasmid and furin for SOSIP constructs or (2) a 3:1 ratio of Env plasmid and furin for SOSIP-I53-50A constructs. Proteins were purified from vacuum-filtered (0.22 µm filters) transfection supernatants by PGT145 bNAb-affinity chromatography. PGT145-coupled sepharose beads were mixed with the filtered supernatant and after an overnight incubation on a roller, subjected to washing and elution as described earlier^{49 (link)}. Protein eluates were concentrated and buffer exchanged to TN75 (75 mM NaCl, 20 mM Tris HCl pH 8.0) using Vivaspin filters with a 100 kDa molecular weight cutoff (GE Healthcare). Protein concentrations were determined using the Nanodrop method. The required proteins peptidic molecular weight and extinction coefficient were obtained by filling in the protein’s amino acid sequence in the online Expasy software (ProtParam tool).

The nanobody-IgG1 constructs were transiently expressed in HEK-293F cells (Invitrogen). Cells were cultured in Freestyle medium (Life Technologies) at a density of 1×10⁶ cells/mL. At day 1, the cells were transfected using the antibody plasmid and 1 µg/µl PEImax (Polysciences) in a 3:1 ratio in OptiMEM. For the production of the bispecific antibodies, HEK-293F cells were transfected with the J3 IgG1-knob plasmid and either the 2E7-IgG1-hole or 1F10-IgG1-hole plasmid in a 1:1 ratio, with PEImax (1 µg/µl) in a 3:1 ratio in OptiMEM. Supernatant was harvested at day 6, centrifuged (30 minutes, 4000 rpm) and filtered using 0.22 µm Steritop filters (Merck Millipore). Antibodies were purified using protein A/G (Pierce) affinity chromatography. Antibodies were eluted from the column using 0.1 M glycine (pH 2.5) and neutralized using neutralization buffer 1M Tris (pH 8.7). The eluates were concentrated and buffer exchanged to PBS using 50 kDa Vivaspin filters (GE Healthcare). In all assays we correct for protein size using the following calculation: $C=\frac{m}{V}x\frac{1}{MW}$ . Where C is the molar concentration in mol/L or M, m is mass of the protein in grams (g), V is volume of solution in liters (L) and MW is the molecular weight of the protein in g/mol.