Isolation and Purification of Francisella Capsule

Francisella capsule was isolated by subjecting EtOHp to a modified hot phenol/water extraction followed by water/Triton X-114 partitioning and SDS gel filtration as described previously [4 (link)] (S1A Fig). An alternate protocol to isolate capsular polysaccharides from the nuclease-treated EtOHp (S1B Fig) was developed using deoxycholate (DOC)-based gel sieving [12 (link)]. EtOHp samples were dissolved in 2% DOC in 0.2 M NaCl, 50 mM Tris, 5 mM EDTA and sonicated for 30 min in a bath sonicator. After centrifugation to remove any insoluble debris, the sample was applied to a 16 mm x 30 cm Sephacryl S-200 column on an Akta Purifier FPLC system (GE Healthcare) at a flow rate of 0.5 ml/min. In order to remove any residual DOC that might interfere with subsequent chromatographic analysis, fractions underwent three rounds of ethanol precipitation as described above after addition of purified DNA (150 μg/ml UltraPure™ Salmon Sperm DNA Solution, Invitrogen) to enhance recovery. To control for any effects of the DOC or ethanol washing on subsequent chromatographic analysis, whole EtOHp was also treated in the same way in parallel with isolated capsule or low molecular weight material. Washed samples were raised in water and stored at 4°C until further analysis.