Purification of DksA and Associated Proteins

Genes encoding wild-type or dksA point mutants were cloned into the GST fusion plasmid pGEX-6P-1 (GE) (10 (link)). Full-length or truncated dnaJ, tig, and rpoC genes were directionally cloned as C-terminal 6His fusions into the NdeI and XhoI sites of the pET-22b(+) plasmid (Novagen). All constructs were confirmed by sequence analysis. Plasmids were expressed in E. coli BL21 (DE3) (Invitrogen) or E. coli Origami B (DE3) pLysS (Novagen) (SI Appendix, Table S1). Cells grown in LB broth at 37 °C to an OD₆₀₀ of 0.5–0.7 were then treated with 0.1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). After 3 h, the cells were harvested, disrupted by sonication, and centrifuged to obtain cell-free supernatants. GST and 6His-tagged fusion proteins were purified using Glutathione-Sepharose 4B (bioWORLD) and TALON metal-affinity chromatography (Clontech), respectively. To remove the GST tag from recombinant GST-DksA protein, PreScission protease (PSP), prepared in PBS buffer supplemented 10 mM DTT, was added. After an overnight incubation at 4 °C, protein was eluted and further purified by size-exclusion chromatography on Superdex 75 (GE Healthcare Life Sciences). Purified DksA proteins were aliquoted inside a BACTRON anaerobic chamber (Shel Lab). The purity and mass of the recombinant proteins were assessed by SDS/PAGE.