Purification and Characterization of Ssp Protein

E. coli BL21(DE3) harbouring the Ssp-pBAD/Myc-His-B plasmid was grown in LB supplemented 100 µg/mL ampicillin at 37 °C. Expression of Ssp was induced by addition of 0.02% L-arabinose at 30 °C for 4 h. The media was harvested, filtered and concentrated ~40 times using Vivaflow 200 (Sartorius, VF20P2) crossflow cassettes at 4 °C before dialysis in 20 mM Tris, pH 9.0. The secreted protein was purified by two round of anion exchange chromatography, HiTrap Q FF (GE Healthcare, 17515601) and Mono Q 10/100 GL (GE Healthcare, 17516701), with 20 mM Tris, pH 9.0 and elution with 1 M NaCl. Recombinant Ssp was further purified by gel-filtration chromatography using a HiLoad Superdex 200 16/600 (GE Healthcare, 28989335) pre-equilibrated with 25 mM HEPES, 150 mM NaCl, pH 7.0.
SeMet labelled Ssp was expressed in minimal media supplemented with 50 μg/mL of selenomethionine inducing with 0.5% L-arabinose at 20 °C overnight^{69 (link)}. Ssp-SeMet was purified as described above.
Ssp mutants were expressed in E. coli Top10 cells in LB inducing with 0.2% L-arabinose at 20 °C overnight. Media was concentrated using Ultra-0.5 Centrifugal Filter Unit (Amicon, UFC5010BK). Protein was quantified by densitometry using SDS-PAGE with purified Ssp as a standard using Image Lab 6.0 (Bio-Rad). Ssp-ΔE2 was purified in the same manner as Ssp-WT.