Ni sepharose excel resin

DNA encoding for residues 24–167 of the extracellular portion of human PD-1 (UniProt Q15116) with a C-terminus histidine tag or a corresponding PD-1 N58Q mutant was cloned into pcDNA3.4 by GenScript. Expi293 cells were transiently transfected with plasmid DNA mixed with PEI (Polysciences) and incubated for 5 d. For the expression of non-fucosylated PD-1, Expi293 cells were grown in the presence of 0.6 mM 2FPF. PD-1 proteins were purified by affinity chromatography using Ni Sepharose Excel resin (Cytiva). PD-10 columns (Cytiva) were used to exchange the buffer to PBS (Corning). Protein quality and purity were assessed by SDS–PAGE (Bio-Rad) and size-exclusion chromatography using a Superdex 200 5/150 column (Cytiva) with ACQUITY UPLC (Waters). The protein concentration was determined using a NanoDrop spectrophotometer.

Plasmids encoding Fab were synthesized by GenScript by cloning VL and VH regions into a derivative of the pHL-sec expression vector^{45 (link)} upstream of the human CH, Cκ, or Cλ regions and expressed in Expi293F^TM cells (Thermo Fisher Scientific) as described above. Briefly, the Fabs were purified from cell-free supernatant 4–5 days post-transfection using Ni Sepharose® Excel resin (Cytiva) and size exclusion chromatography (Superdex 200 Increase 10/300 GL; Cytiva) in a buffer containing 20 mM Tris (pH 8.0) and 100 mM NaCl. Purified Fabs were used for Biolayer Interferometry (BLI) experiments and crystallization.

Recombinant immunogens were expressed in expi293F cells, as described for SPEEDesign in vitro screening above. Cell-free supernatant was harvested 4 days after transfection and His-tagged antigens were purified by gravity chromatography using Ni Sepharose excel resin according to manufacturer instructions (Cytiva). Antigens were further purified by size-exclusion chromatography using a Superdex 75 Increase 10/300 GL column equilibrated in 1x PBS. Fractions corresponding to monomeric immunogen were pooled, snap frozen in liquid nitrogen, and stored at −80 °C.
Transfection, expression, and purification of original designs were performed in triplicate on three separate days to calculate immunogen purification yields. Each replicate consisted of a 30 mL culture, and yields were calculated by integrating the area under the monomeric peak on the Abs₂₈₀ chromatogram during size-exclusion chromatography. These yields closely matched yields calculated from pooled fractions. Purification yields for reversion mutants was calculated after nickel purification by measuring the Abs₂₈₀ of eluant. Extinction coefficients were calculated using the ExPASy ProtParam tool^{63 (link)}. Sample purity was assessed by SDS-PAGE (Supplementary Figure 11).

Protein sequences for Pfs25 (PF3D7_1031000) and Pfs28 (PF3D7_1030900) were obtained from PlasmoDB. The wildtype Pfs28 sequence was used, but the Pfs25 construct used for biophysical studies contained three N-linked glycosylation sites at residues 91, 144, and 166 that were mutated from asparagine to glutamine. Single chain variable fragments (scFvs) of mAbs were designed by fusing the V_H region of each mAb to its paired V_L region by a (GGGGS)₄ linker.
All constructs were cloned into the pHLsec plasmid with a C-terminus hexa-histidine tag and transiently expressed in Expi293 cells following manufacturer protocol (Thermo Fisher Scientific, Waltham, MA) as secreted protein then harvested four to five days after transfection. After centrifugation, the supernatant was loaded on Ni Sepharose Excel resin (Cytiva, Marlborough, MA) and washed with 10 column volumes of wash buffer (25 mM Tris pH 7.4, 300 mM NaCl, 30 mM imidazole). Recombinant protein was eluted with five column volumes of elution buffer (25 mM Tris pH 7.4, 300 mM NaCl, 150 mM imidazole) and concentrated using an Amicon Ultra Centrifugal filter with 10 kDa molecular weight cutoff (Millipore Sigma, Burlington, MA). Concentrated eluate was purified by size exclusion chromatography using a Superdex 75 Increase 10/300 GL column (Cytiva, Marlborough, MA) equilibrated in 20 mM Tris pH 8.0, 100 mM NaCl.

SARS-CoV-2 spike receptor-binding domain (RBD) was expressed in mammalian cells and purified as described previously (Wu et al., 2020b ). Briefly, the RBD (residues 319-541) of the SARS-CoV-2 spike (S) protein (GenBank: QHD43416.1) was cloned into a phCMV3 vector and fused with a C-terminal 6xHis tag. The plasmid was transiently transfected into Expi293F cells using ExpiFectamine 293 Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. The supernatant was collected at 7 days post-transfection. The protein was purified with Ni Sepharose excel resin (Cytiva) followed by size exclusion chromatography.