Talon beads

Recombinant soluble Fab antibodies were expressed in TG1 E. coli bacteria and purified by immobilized metal affinity chromatography (IMAC) using Talon beads (Takara Bio USA, Inc., Mountain View, CA, USA) as described previously [27 (link)]. In vivo biotinylation was performed as described previously [28 (link)]. To generate Fab-ETA′ immunotoxins, the biotinylation sequence AviTag was replaced by the sequence of a truncated version of the pseudomonas exotoxin A (ETA′). For better intracellular transport and efficacy, the 5 C-terminal amino acids of ETA′ were exchanged for the KDEL motif by PCR [29 (link)]. Expression of FAB-ETA′ immunotoxins was performed in E. coli strain TG1.

α-Toxin (AT) plasmid was generously provided by Yeongjin Hong at the Chonnam National University, Korea (Shin et al., 2006 (link)). BL21 cells (Fisher NC9122855) expressing α-toxin are pelleted and resuspended in 0.5× RIPA (75 mm NaCl, 0.5% P-40, 0.25% NaDOC, 0.05% SDS, and 12.5 mm Tris) before being sonicated. Cell extract is filtered and incubated with washed TALON beads (Takara 635501) at 4°C for 30 min. After washing, AT-coated TALON beads are resuspended as 1:1 slurry in wash buffer (20 mm Tris, 500 mm NaCl, pH7.9), and 60 μl of slurry is added to 900 μl of media samples and incubated for 1 h at 25°C. Beads are pelleted by centrifugation, supernatant is collected, beads are washed with wash buffer before being resuspended as a 1:1 slurry in wash buffer and used in Nluc assays.

The proteins were expressed in suspension in Expi293F cells (Gibco) transfected with ExpiFectamine 293 (Gibco) at a density of 3 million cells per mL using either 1 mg of spike DNA or 0.5 mg of each heavy and light chain of the Fab per litre of culture. The cells were maintained in FreeStyle medium in a humidified, 8% CO₂ atmosphere, at 37 °C, shaking at 125 rpm. The supernatants were harvested either twice, after 3–4 and 6–7 days, for the spike or once, after 6 days, for the CR3022 Fab.
The harvested, clarified supernatants were incubated overnight with TALON beads (Takara), washed briefly, eluted with imidazole, and loaded on a Superdex 200 Increase 10/300 GL column (GE Life Sciences) in a pH 7.4 phosphate buffer containing 70 mM NaCl. The Fab used in binding assays was then cleaved with recombinant TEV overnight and the protease, cleaved-off tags, and uncleaved proteins removed by incubation with TALON beads. The purity of all samples was confirmed with SDS-PAGE and the integrity of the spike trimers confirmed by cryoEM (described elsewhere¹² ).

Protein expression was performed as described previously.^{24 (link),29 (link),30 (link)} The open reading frame of virF was cloned into pET15b using primers virFpet15 FP/RP and pET15 FP/RP listed in Table 2, by Gibson assembly. Clones were confirmed by PCR using primers pET15bchk FP/RP and DNA sequencing. Plasmids were electroporated into E. coli BL21 DE3 for expression. Cultures were grown to OD₆₀₀ of 0.6 and induced with 5 mM IPTG (Isopropyl-β-D-1-thiogalactopyranoside) and grown for four hours aerobically at 37°C. Cells were centrifuged and aliquots were run on 12% SDS-PAGE to confirm induction of protein expression. Post confirmation, pellets were dissolved in Xtractor buffer followed by sonication to lyse the cells. Talon beads (TaKaRa) were used to extract His-tagged VirF by affinity purification following Takara’s protocol. Elution of VirF was confirmed by SDS-PAGE. Eluted proteins were dialyzed overnight in dialysis buffer (50 mM sodium phosphate, 300 mM NaCl, 10% glycerol). Size and purity of the protein were confirmed by running aliquots on 12% SDS-PAGE. Protein concentrations were calculated in NanoDrop (Thermo Scientific).

SARS-CoV-2 PLPro and SARS-CoV PLpro were purified from Escherichia coli as described before (19 (link)). Briefly, BL21(DE3) E. coli competent cells (NEB) were transformed with plasmids and grown in LB medium to an OD600 of 0.6–0.8 at 37 °C. Protein production was induced by addition of 0.5 mM isopropyl-D-thiogalactopyranoside (IPTG) overnight at 18 °C. Lysates were incubated with TALON beads (Takara) pre-equilibrated with lysis buffer and nonspecific proteins were cleared with washing. Proteins were eluted with elution buffer (50 mM Tris-HCl, 150 mM NaCl, 250 mM imidazole, 2 mM DTT, pH 8.5). Eluted proteins were buffer exchanged to storage buffer (20 mM Tris-HCl, 100 mM NaCl, 2 mM DTT, pH 8.5) and used for in vitro experiments.
The expression and purification of 3CLpro protein and protein substrate protein were performed as described previously (29 (link), 30 (link)). The sequence of SARS-CoV-2 3CLpro was obtained from GenBank (accession number: YP_009725301), codon-optimized, and ordered from GenScript. A C-terminal poly-histidine-maltose binding protein (His6-MBP) tag with two in-between Factor Xa digestion sites was inserted. The recombinant protein substrate His6 – CFP – TSAVLQ↓SGFRKM – YFP (where ↓ represents the cleavage site) was constructed, expressed, and purified as described (31 , 32 (link)).