Complete his tag purification resin

For His-tag affinity purification, we used 75 μL cOmplete™ His-Tag purification resin (Sigma-Aldrich) to bind 4 mL supernatant and eluted the resin with 75 μL elution buffer. For all protein samples, SDS-PAGE with Coomassie Blue stain was used to verify the proteins.

Coding regions of the CLV1 and TDR extracellular domains (residues 1 to 628 and 1 to 631, respectively) translationally fused with C-terminal 6×His tags were amplified by PCR from Arabidopsis cDNA extracted from 7-day-old wild-type seedlings. DNA fragments were cloned in pPSC8 to generate pPSC8-CLV1 and pPSC8-TDR. Baculovirus-mediated transfection of Sf9 insect cells with pPSC8-CLV1 and pPSC8-TDR, validation of protein expression, and preparation of culture cells was performed by Fujifilm Wako Pure Chemical Industries Ltd. The medium was collected and adjusted to pH 8.0, and then centrifuged at 10,000g for 10 min at 4°C. The supernatant was filtered by a 0.22-μm polyvinylidene difluoride membrane filter at 4°C. The supernatant was incubated with cOmplete His-Tag Purification Resin (Sigma-Aldrich) at 4°C for 1 hour, and the resin was washed twice with wash buffer [50 mM sodium phosphate (pH 8.0), 500 mM NaCl, and 10 mM imidazole]. The protein was then eluted with elution buffer [50 mM sodium phosphate (pH 8.0), 500 mM NaCl, and 400 mM imidazole]. Primers used for the constructs are listed in table S3.

Bi-NAbs in a ScFv format were designed utilizing tetra-glycine-serine (G₄S) peptide linkers^{36 (link), 37 (link)}. C-terminal His-tagged Bi-ScFvs DNA sequences were synthesized (GenScript, Piscataway, NJ) and cloned into the pcDNA3.1(-) vector (Thermo Fisher Scientific) while the Bi-ScFv lacking the C-terminal His-tag was subcloned into an IgG1 Fc vector (InvivoGen) as well as IgG1 Fc vectors carrying knob-into-hole mutations^{32 (link)}. Bi-NAbs were expressed by transient transfection of either Bi-ScFv or Bi-ScFv-IgG1 Fc plasmids in FreeStyle 293 F cells (Thermo Fisher Scientific, Catalog number: R79007). Tri-NAbs were expressed by transient transfection of the Bi-ScFv_{dVRC01-5X-PGT121} IgG1 Fc “knob”, 10E8 HC “hole” and 10E8 LC plasmids in 293 F cells. Supernatants were harvested 5 days post transfection, filtrated, followed by affinity purification. Bi-ScFvs with 6-His-tag were purified by Complete His-tag purification resin (Sigma-Aldrich). Bi-NAb and Tri-NAb with IgG1 Fc were purified by protein A affinity chromatography. Elutes were dialyzed with phosphate-buffered saline (PBS), and concentrated using an Amicon Ultra 10 kDa molecular weight cutoff concentrator (Sigma-Aldrich). Antibody purity was analyzed by SDS–PAGE.