2.2 Expression and purification of SdsA1

A His-tagged version of SdsA1 was expressed in BL21 (DE3) E. coli cells from a plasmid that was a kind gift from Prof. Wolf-Dieter Schubert (University of Pretoria) and the Helmholtz Center for Infection Research. Following transformation by electroporation and selection for colonies on lysogeny broth medium plates with chloramphenicol, cells were cultured in the same medium with shaking (50 mL overnight at 37°C followed by inoculation into 0.5 L at an optical density at 600 nm of 0.2) and grown to an optical density of 0.5. Flasks were cooled to room temperature and SdsA1 expression induced by the addition of 0.4 mM IPTG followed by overnight growth at 25°C with shaking.

These cells were lysed by sonication on ice in 50 mM tris-HCl, 10 mM imidazole, pH 7.4, with 2 mM PMSF, which was also the buffer used to equilibrate the Ni-NTA column. Soluble proteins were isolated by centrifugation, loaded onto the Ni-NTA column, and rotated at 4°C for ~72 h. The column was washed three times with two column volumes of 50 mM tris-HCl, 25 mM imidazole, 2 mM PMSF, and 5% glycerol at pH 7.4. SdsA1 was eluted with approximately one column volume of 50 mM tris-HCl, 250 mM imidazole, 2 mM PMSF, and 5% glycerol, pH 7.4, and analyzed by SDS-PAGE and Bradford assay.