Production and Characterization of LLO(LT) Conjugates

All peptides used in this study were purchased from Peptide 2.0 Inc., purified by reverse-phase high pressure liquid chromatography, and analyzed by mass-spectroscopy. A pET29b expression vector containing the His-tagged LLO^LT sequence was provided by Dr. Emil Unanue (Washington University, St. Louis, MO)^{36 (link)}. For generation of LLO^LT-N, site-directed mutagenesis was used to change the lysine at position 203 to an asparagine (N). Mutated clones were confirmed by sequencing. LLO^LT and LLO^LT-N were expressed in BL21 (DE3) competent cells (ThermoFisher) and purified as previously described^{36 (link)}. Protein purity was confirmed by SDS-PAGE. For conjugation of NP to LLO^LT and LLO^LT-N, 0.5mg of NP-OSu (LGC Biosearch Technologies) was added to 5mg of either LLO^LT or LLO^LT-N in 10 equal fractions over 20 minutes. The solution was then incubated at room temperature with rotations for 2 hours before being dialyzed into 0.1M NaHCO₃, 145mM NaCl, pH 8.5. NP:LLO^LT(-N) ratio was determined with the following extinction coefficients: LLO^LT(-N) = 75750 M⁻¹cm⁻¹ and NP = 4230 M⁻¹cm⁻¹. Ratios of NP:LLO^LT(-N) ranged from 5:1-10:1, and batches of NP-LLO^LT(-N) were aliquoted and stored at -20°C to allow for continuity across experiments.