Pierce endotoxin removal columns

Chimeras were purified as described [22 (link)]. Briefly, to express IsdA-CTA₂/B and ClfA-CTA₂/B, ClearColi® (Lucingen, Madison, WI) transformed with pLR001 or pLR003 was grown to an optical density (O.D.600) of 0.9 and induced for 24 h with 0.2 % l-arabinose. Proteins were isolated from the periplasmic extract with 1 mg/mL polymyxin B and purified by affinity chromatography on immobilized d-galactose (Pierce™ D-Galactose Agarose, Thermo Fisher, Waltham, MA). Vaccine proteins were dialyzed into sterile 20% glycerol + 1×PBS and concentrations determined by BCA (Pierce™ BCA, Thermo Fisher). Vaccines were tested to ensure endotoxin levels below 0.05 EU/mL (LAL Endpoint Chromogenic, Lonza, Allendale, NJ), plated for sterility and stored at −80°C until use. For ELISA, IsdA and ClfA were isolated from the cytosol or periplasm of E.coli Top10 (Thermo Fisher) + pCK001/pMAH001 after overnight induction with 1M IPTG. Proteins were purified on cobalt (HisPur™, Thermo Fisher) and dialyzed into sterile 1×PBS. F or use in flow cytometry and OPA, endotoxin was removed (Pierce™ Endotoxin Removal Columns, Thermo Fisher).

To prepare Rapa-loaded FSI, a two-phase encapsulation method was employed. To 10 mL 300 μM FSI in PBS, 3x molar excess Rapa (LC Laboratories, Woburn, MA) in hexane/EtOH mixture (7:3 v/v) was added. After evaporation of the organic phase at 4 °C using a rotary evaporator, the aqueous suspension was centrifuged at 13,000 g to pellet unbound Rapa precipitate. The supernatant was subjected to additional rounds of centrifugation until no pellet was observed. FSI-Rapa was added to a 10 kDa MWCO dialysis bag (Thermo Fischer Scientific, Waltham, MA) and dialyzed against PBS (1:750 sample: dialysate) for 12 hours to remove free Rapa and residual solvent. After mixing FSI-Rapa and ISR in an equimolar ratio, the working formulation was subjected to endotoxin removal by poly-lysine chromatography using Pierce™ endotoxin removal columns (Thermo Fischer Scientific, Waltham, MA) following manufacturer’s protocol. An aliquot of the final material was injected onto a C-18 RP HPLC column (Waters, Milford, MA) and Rapa was quantified at 280 nm using a calibrated standard curve. Concentration of ISR/FSI-Rapa throughout the manuscript refers to Rapa concentration unless otherwise noted.