Purification of Recombinant LukAB Toxin

A construct to co-purify recombinant LukAB from S. aureus (pOS-1-P_lukAB-sslukA-6His-lukA-lukB) was generated as previously described [24 (link)] and transformed into Newman ΔΔΔΔ [61 (link)] to facilitate purification. This construct was also used to individually express toxin subunits from LukED, PVL, HlgABC [24 (link)]. Toxins were purified from S. aureus as previously described [24 (link)]. Briefly, strains were grown in TSB with 10 μg/ml chloramphenicol for 5 h at 37°C, 180 rpm, to an optical density at 600 nm (OD₆₀₀) of approximately 1.5 (which represents 1 x 10⁹ CFU/ml). The bacteria were then pelleted, and the supernatant was collected and filtered. Nickel-nitrilotriacetic acid (NTA) resin (Qiagen) was incubated with culture supernatant, washed, and eluted with 500 mM imidazole. The protein was dialyzed in 1 × Tris-buffered saline (TBS) plus 10% glycerol at 4°C overnight and then stored at −80°C.