Purification and Acylation of LpxA-His6

Strains of interest were inoculated into 500 ml of Luria-Bertani (LB) medium containing 100 μg ml⁻¹ of Ampicillin and were incubated while shaking at 37°C until an OD₆₀₀ of 1.0 was reached. The cultures were induced with 1 mM IPTG followed by incubation at 25°C for four hours. Cells were harvested by centrifugation at 10,000 × g for 10 minutes at 4°C, and processed immediately or frozen at −80°C. Cells were resuspended in 6 ml of 20 mM Hepes, 100 mM NaCl at pH 8.0. Cell suspensions were disrupted by French press at 20,000 psi. Cellular debris was removed by centrifugation at 14,000 × g for 5 minutes at 4°C in a 5424 centrifuge (Eppendorf). Soluble crude cytosol was loaded onto 3 ml of nickel-nitriol-triacetic acid (Ni-NTA) resin (Qiagen). The resin was washed with 10 column volumes of loading buffer containing 500 mM NaCl and then eluted with 20 mM Hepes and 500 mM imidazole (pH 8.0). Purified F. tularensis LpxA-His₆ was desalted using Slide-A-Lyzer Dialysis Cassette (Thermo Scientific) and dialyzed against 20 mM Hepes pH 8.0. Protein concentrations were determined by bicinchoninic acid (BCA) assay. The purification, and acylation of holo-ACP was carried out as described previously, except hydroxypalmitic acid (C16:0) was used in place of hydroxymyrstic acid (C14:0) [22 (link)].