Fitc 1

Recombinant Tp0751 (V99−P237) was purified as described above except all buffers were prepared without glycerol. Following purification, Tp0751 (V99−P237) was chemically labeled with fluorescein isothiocyanate isomer I (FITC I; SigmaAldrich, Oakville, Ontario, Canada). Five milligrams of recombinant protein were incubated with 0.5 mg of FITC In a final volume of 5 ml of 1 M sodium bicarbonate (pH 9.1) for 1 h at room temperature (RT). Samples were then buffer exchanged into 20 mM HEPES, 150 mM NaCl (pH 7.0) using a 10,000-kDa molecular-weight cutoff (MWCO) Centricon (Millipore, Etobicoke, Ontario, Canada) to remove unconjugated FITC.

Treponemal proteins Tp0327 (I23-S172), Tp0751 (V99-P237), and Tp0751 (E115-P237) were cloned as previously described (Houston et al., 2011 (link), 2014 (link); Parker et al., 2016 (link)). Tp0327 and Tp0751 constructs were purified from Escherichia coli BL21^∗DE3 and subject to nickel affinity chromatography with HisTrap FF columns (GE Healthcare, Mississauga, ON, Canada) and further purified with size exclusion and cation exchange chromatography (HiLoad 16/60 Superdex 75; GE Healthcare, Chicago, IL, United States) on an AKTA Prime Plus FPLC system (GE Healthcare) in a final buffer of 20 mM HEPES, 150 mM NaCl, 1% glycerol, pH 7.0 as previously described (Houston et al., 2011 (link), 2014 (link); Parker et al., 2016 (link)). Labeling of recombinant proteins, Tp0751 (V99-P237) and Tp0327 (I23-S172), with fluorescein isothiocyanate isomer I (FITC I; Sigma-Aldrich, Oakville, Ontario, Canada) was performed as previously described (Lithgow et al., 2020 (link)).