Extraction and Purification of F1 Capsule Antigen from Y. pestis

Abstract

In this chapter, I describe a simple and efficient strategy to obtain purified F1 antigen from Yersinia pestis. Yersinia pestis strain EV76 is inoculated into brain–heart infusion medium containing sterile glass beads and then incubated at 37 °C for 72 h with shaking at 175 rpm. The supernatant is collected by centrifugation and then precipitated with 40% (w/v) saturated ammonium sulfate. The protein pellet is collected by centrifugation, redissolved in 0.01 M phosphate-buffered saline (pH 7.2), and then precipitated with 25% (w/v) saturated ammonium sulfate. The supernatant is precipitated with 5% saturated ammonium sulfate. Finally, highly purified F1 protein is obtained using a Sephacryl S-200HR column. The F1 protein is quantified based on the area and the intensity of its band in a sodium dodecyl sulfate–polyacrylamide gel stained with Coomassie Brilliant Blue. The F1 protein content is determined using a bicinchoninic acid assay. The purity of the F1 antigen is determined by high-performance liquid chromatography, and Western blotting is used to confirm the specificity of the F1 antigen using a specific monoclonal antibody against the F1 antigen.