Ni sepharose excel beads

Anti‐Env monoclonal antibodies and isotypic control mGO53 were produced as recombinant human IgG1 monoclonal antibodies by co‐transfection of Freestyle 293‐F suspension cells (Thermo Fisher Scientific) using the PEI precipitation method as previously described^{54 (link)}. Anti-Env antibodies’ variable domains were also cloned into human Fab- and F(ab’)₂-expressing vectors as previously described^{55 (link)} and produced as aforementioned. Recombinant monoclonal IgG1 antibodies, and corresponding Fab and F(ab’)₂ fragments were purified by batch/gravity‐flow affinity chromatography using respectively protein G sepharose 4 fast flow beads (GE Healthcare) and Ni-sepharose Excel beads (GE Healthcare) following the manufacturers’ procedures.

A DNA fragment encoding Ixodes scapularis Salp20 [UniProtKB: Q95WZ1 (residue 22–183)] was amplified by PCR from Salp20 synthetic DNA optimized for mammalian expression (GeneART ThermoFisher), and ligated into BamHI-NotI sites of vector pUPE106.03 (U-Protein Express BV, Utrecht, the Netherlands). The expressed protein contained a cystatin secretion signal peptide, an N-terminal (His6)GlySer-tag and an C-terminal Ala3 cloning artifact due to the NotI restriction site. The construct was transiently expressed in N-acetylglucosaminyltranferase I-deficient (GnTI-) Epstein-Barr virus nuclear antigen I(EBNA1)-expressing HEK293 cells cultured in suspension (U-Protein Express BV, Utrecht, the Netherlands). Secreted Salp20 was captured by incubating culture medium with Ni-Sepharose excel beads (GE Healthcare) at 4°C for 2 h, followed by washing with 25 mM HEPES pH 7.8, 500 mM NaCl, 15 mM imidazole. After elution using the washing buffer supplemented with 250 mM imidazole the sample was further purified by gel-filtration using a Superdex 200 increase 10/300 GL column (GE Healthcare) equilibrated in 25 mM HEPES pH 7.8, 150 mM NaCl. Salp20 was concentrated to 8.4 mg/ml by centrifugation using a 5-kDa cut-off concentrator before plunge freezing in liquid nitrogen and storage at −80°C.

HEK-293F cells (Thermo Fisher) were transiently transfected using PEI 25K (Polysciences) with plasmids pcDNA3.1-mHla-RBD or pcDNA3.1-RBD. After 5 days in shaker culture, media were collected and cleared of debris for 20 min by centrifugation at 6,000 g and filtered using 0.45-mm flasks (Millipore). Proteins in media were loaded onto Ni Sepharose Excel beads (GE Healthcare, Piscataway, NJ, USA), washed with 20 mM phosphate buffer pH 8.0, 300 mM NaCl and 20 mM imidazole, and eluted with 20 mM phosphate buffer pH 8.0, 300 mM NaCl and 300 mM imidazole. Eluates were buffer exchanged with phosphate-buffered saline (PBS) using a HiPrep™ 26/10 desalting column (GE Healthcare, Piscataway, NJ, USA). The peak fractions corresponding to the recombinant proteins were pooled and verified by SDS-PAGE, and the concentration was determined using the BCA method. All purified proteins were stored at -80°C before use.

Protein expression in E. coli BL21 and subsequent purification of PAR1b-N were performed as previously described [19 (link)]. PAR1b-∆spacer was expressed and purified using the same protocol. PAR1b-FL was expressed and purified as previously described [19 (link)], with some modifications to ensure RNA-free preparations without the use of nucleases that may interfere with downstream applications. For the initial lysis, a high salt lysis buffer (PBS, 2% (v/v) Tween 20, 1% (v/v) Triton X-100, 1 mM EDTA, 1 mM DTT, 850 mM NaCl) + 0.3 mg/mL Benzamidine was used. Cleared lysate was mixed with Ni Sepharose excel beads (GE Healthcare, Chicago, IL, USA) and washed thoroughly with wash buffer (high salt lysis buffer + 20 mM imidazole), followed by elution (high salt lysis buffer + 500 mM imidazole). After adjusting the salt concentration to 500 mM NaCl, the subsequent GST purification steps were performed. The final incubation with 28 U/mL PreScission Protease (GE Healthcare, Chicago, IL, USA) was performed in 50 mM Tris-Cl, pH7.5, 500 mM NaCl, 1 mM EGTA, 1 mM DTT at 4 °C overnight.