Functional and Conformational Analysis of TmMutS2 Figure 8. Superimpositions among the TmMutS2-dsDNA/-FWJ-DNA SAXS models and the Smr structure/model. (A) The left structure is the monomeric Smr domain of H. sapiens B3bp (B3bp-Smr), and the right model is the predicted monomeric Smr domain of TmMutS2 (TmS2-Smr). (B) Superimpositions of the TmMutS2-dsDNA/-FWJ-DNA SAXS model to the B3bp-Smr structure and the TmS2-Smr model. (C) Superimpositions of the TmMutS2-dsDNA/-FWJ-DNA SAXS model to the B3bp-exSmr SAXS model (green mesh). The B3bp-Smr structure (red) fits well with the B3bp- exSmr SAXS model. doi:10.1371/journal.pone.0034529.g008 into the bacterial expression vector pET-28aTEV (Novagen, Germany) containing the TEV protease recognition site. In addition, the TmmutS2DSmr and TmmutS2-Smr (only Smr domain of TmMutS2) genes were isolated from pET-28TEV-TmmutS2, and then ligated into pET-28aTEV. The clone sequences were confirmed via automated DNA sequencing. The target proteins were over-expressed in BL21 (DE3) cells (Invitrogen, USA) grown in Luria-Bertani (LB) medium containing 50 mg/mL kanamycin at 37uC. When the UV absorbance of the cells at 600 nm reached 0.6, the protein expression was induced by the addition of 0.5 mM isopropyl-thiogalactopyranoside. The induced cells were then kept at 37uC for an additional 6 h. After the cells were harvested by centrifugation, they were resuspended in a lysis buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.5 mM b-mercaptoethanol, 5% glycerol, 1 mM phenylmethyl-sulfonyl fluoride, 1 mg/mL leupeptin, 1 mg/mL aprotinin, and 1 mg/mL benzamidine), and then disrupted by sonication. The lysate was centrifuged at 13000 rpm for 30 min at 4uC, and the supernatant was used for the further experiments. Prior to chromatography, a heat- activation step performed at 65uC for 1 h was employed for only TmMutS2, TmMutL, and TmRecA to remove the endogenous proteins extracted from the cells. In the case of B3bp-Smr, supernatants were directly applied to a nickel-nitrilotriacetic acid (Ni-NTA, GE Healthcare, Denmark) affinity column pre-equili- brated with buffer I (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.5 mM b-mercaptoethanol, 5% glycerol, and 5 mM imidazole), and then eluted in buffer I with 300 mM imidazole. His-tag fragments of the purified samples were eliminated by adding TEV protease at room temperature (RT) for 8 h. To remove the untreated His-tagged proteins and the cleaved His-tag fragments, the samples were reloaded onto the Ni-NTA affinity column, and the target proteins were obtained via flow-through against buffer I with 30 mM imidazole. For further purification, each protein was individually applied to a Heparin HitrapTM HP (TmMutS2, TmMutL, and TmRecA) and a Mono S (B3bp-Smr) ion exchange column (GE Healthcare, Denmark) equilibrated with buffer II PLoS ONE | www.plosone.org 11 April 2012 | Volume 7 | Issue 4 | e34529