Superdex 75 or 200 columns

Proteins were expressed in E. coli strain BL21 (DE3) in TB media with 50 μM kanamycin at 17°C overnight after induction with 0.5 mM of IPTG. Selenomethionine labelled protein was obtained by expressing the NTos211 protein construct in E. coli BL21 cells in M9 minimal medium containing 50 mg/l of seleno-methionine (SeMet). The cells were disrupted by sonication on ice for 3 min in lysis buffer (50 mM Tris–HCl pH8, 300 mM NaCl, glycerol 5%, 10 mM imidazole and 2 mM TCEP) with EDTA-free protease inhibitor cocktail (Roche). The protein from the soluble fraction was loaded onto a 5–10 ml chelating sepharose column with nickel, washed with 5 column volumes of lysis buffer containing 10 mM imidazole, 2 column volumes with 40 mM imidazole and eluted with 5 volumes of lysis buffer with 400 mM imidazole. The eluted protein was cleaved with histidine tagged TEV protease overnight at 4°C during dialysis against lysis buffer, yielding an additional glycine before the second amino acid residue of the original sequence. A second nickel column step was performed to remove unwanted material. The resulting untagged proteins were concentrated and purified by size-exclusion chromatography using Superdex 75 or 200 columns (GE Healthcare) with 10 mM HEPES pH 7.6, 150 mM NaCl and 2 mM TCEP.

Expression constructs comprising residues 120–395 of PsrP (BR_120–395), 187–385 (BR_187–385), and 187–378 (BR_187–378) all with a N-terminal poly-His (HHHHHH) tag, were cloned into the pET21d (Novagen) expression vector as described previously20 (link). The mutated expression construct of BR_120–395 (BR*_120–395) with R165S and R168S substitutions was generated following previously described protocols41 (link). All coding sequences of the protein-expression constructs were confirmed by DNA sequencing and are listed in the supplemental information.
Heterologous protein expression in E. coli (T7 express, New England Biolabs) was induced at OD 0.4–0.7 using 400 μM IPTG and performed over night at 25 °C. The poly-Histidine-tagged proteins were purified using immobilized metal affinity (IMAC), cation exchange chromatography (CEC) (HisTrap FF and HiTrap SPFF, GE-Healthcare) and size exclusion chromatography (SEC) on Superdex 75 or 200 columns (GE Healthcare).