The gene for North American bullfrog, R. catesbeiana, Sxph (GenBank: U05246.1), including its N-terminal secretory sequence, was codon-optimized and synthesized by GenScript. The Sxph gene was cloned into the BamHI and HindIII multiple cloning sites of pFastBac1 (Invitrogen) in frame with a C-terminal 3C protease cleavage site, followed by green fluorescent protein (GFP) and a His10 tag. Bacmids and baculovirus were generated following the manufacturer’s protocol (Bac-to-Bac, Invitrogen). P2 baculovirus was used for transduction at dilution of 1:40 into Sf9 cells at cell density of 2 × 106 cells ml−1 in ESF921 media (Expression Systems). Cells were grown for 72 hours after transduction, and the expressed Sxph-GFP fusion protein was secreted into the growth media. Cells were removed by centrifugation, and the supernatant was adjusted to pH 8.0 with a final concentration of 50 mM Tris-HCl and treated with 1 mM NiCl2 and 5 mM CaCl2 to precipitate contaminants. Precipitants were removed by centrifugation, and the clarified supernatant was incubated with anti-GFP nanobody-conjugated sepharose beads (55, 56) for 5 hours at room temperature. Beads were washed with 20 column volumes of a buffer containing 300 mM NaCl and 30 mM Tris-HCl (pH 7.4). On-column cleavage of the GFP-His tag was achieved by incubating with 3C protease (0.1 mg ml−1) (57) overnight at 4°C.Cleaved sample was further purified by size exclusion chromatography using a Superdex 200 10/300 GL column in buffer containing 150 mM NaCl and 10 mM HEPES (pH 7.4).

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