Purification of Stm-l.

<p>(A) Size exclusion chromatography using a HiLoad 16/600 Superdex 200 prep grade column. The absorption was measured at 280 nm. The hatched area on the chromatogram (elution volume 55–70 mL) indicates fractions which were combined and applied to the next step of the purification procedure. (B) Ion-exchange chromatography of Stm-l using a MonoQ 5/50 GL column. Fractions from gel filtration step containing Stm-l (ca. 15 mL) were combined and applied to the MonoQ 5/50 GL column. Because of the poor absorption of Stm-l at 280 nm, absorption was measured at 280 and additionally at 220 nm. The arrowheads indicate injection of the sample. Fractions containing purified Stm-l (hatched area) were combined, concentrated and desalted using a HiTrap Desalting column. (C) Commassie Brilliant Blue R 250-stained SDS-PAGE of the samples from the expression and purification of Stm-l. Lanes 1 and 2, the whole bacterial cell extract before and 3 h after the induction of the overexpression of Stm-l with IPTG, respectively; lane 3, the fraction of soluble proteins obtained after cell lysis; lane 4, proteins from a 55–75% ammonium sulfate fraction; lane 5, the combined gel filtration fractions; lane 6, the combined fractions after ion-exchange chromatography; lane 7, purified Stm-l after desalting using a HiTrap Desalting column; lane 8, molecular mass standards. The arrowheads in (C) and (D) mark the positions of Stm-l. (D) Analogical analysis as in (C), but the gel was stained using Stains-all dye <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114308#pone.0114308-Sharma1" target="_blank">[31]</a>.