Figure 2.

Characterization of the MnmC recombinant proteins. (A) Coomassie blue staining of SDS–PAGE containing the purified Flag-MnmC, Flag-MnmC(o) and Flag-MnmC(m) proteins used in this work. (B) HPLC analysis of the Flag-MnmC(o) extract (blue line) and markers (red line). FMN: flavin mononucleotide. (C) Half-life of the Flag-MnmC proteins over time as determined by tracking their decline after the addition of glucose to cultures of IC6010 (ΔmnmC) transformed with the plasmids pIC1253, pIC1339 or pIC1340, which express Flag-MnmC, Flag-MnmC(o) and Flag-MnmC(m), respectively. GroEL was used as a loading control. Protein levels were detected by western blotting. (D) Gel filtration analysis of the purified Flag-MnmC proteins [full MnmC protein: green; MnmC(o): blue and MnmC(m): black] and a Flag-MnmC(o)/Flag-MnmC(m) mix (red). The elution positions of the size markers are indicated on the top. Elution fractions a–d from the chromatography of the Flag-MnmC(o)/Flag-MnmC(m) mix were pooled for further analysis. Inset: SDS–PAGE of elution fractions a–d from the Flag-MnmC(o)/Flag-MnmC(m) mix. Fractions (500 μl) were precipitated with trichloroacetic acid before loading. The gel was stained with coomassie blue. The marker molecular masses are indicated on the left. (E) SPR analysis of the MnmC(o)–MnmC(m) interaction. Flag-MnmC(o) was injected into a solution passing over a sensor chip containing the immobilized His-MnmC(m) (600 RU). Representative sensorgrams for various concentrations of Flag-MnmC(o) are shown. (F) Gel filtration analysis of the purified MnmC protein (blue line) and a mix of MnmC with MnmG (black line) or MnmE (red line). The elution positions of the size markers are indicated on the top. Proteins were detected by UV absorbance at 280 nm.