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).
Pierce d galactose agarose
Manufactured by Thermo Fisher Scientific
Sourced in United States
Pierce™ D-Galactose Agarose is a chromatography resin designed for the purification of proteins and enzymes that bind to galactose. The resin consists of D-galactose coupled to agarose beads, providing a platform for galactose-binding interactions. This product is intended for laboratory use in research and development applications.
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2 protocols using pierce d galactose agarose
1
Purification and Characterization of Vaccine Proteins
2
Purification and Characterization of Vaccine Chimeras
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Chimeras were purified as previously described [49 (link),51 (link)]. Briefly, to express IsdA-CTA2/B and ClfA-CTA2/B, ClearColi® with pLR001 or pLR003 were grown at 37 °C to an O.D. of 0.9 at 600 nm 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, USA). Vaccine proteins were dialyzed into sterile 5% glycerol + 1X PBS and concentrations were determined by bicinchoninic acid assay (BCA) (Pierce™ BCA, Thermo Fisher, Waltham, MA, USA). Sizes and purities of the vaccine chimeras were confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) prior to mixing at a final protein concentration of 600 µg/5 mL for vaccination (Figure 1 B). Vaccines were tested to ensure endotoxin levels were below 0.05 EU/mL (LAL Endpoint Chromogenic, Lonza, Allendale, NJ, USA), plated for sterility on tryptic soy agar, and stored at −80 °C until use.
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