Analysis of localization of <i>E. coli</i>-produced fusion proteins.

<p>(A) 10% SDS-PAGE analysis of the recombinant proteins from the culture medium (Med), periplasmic fractions (Peri 1 and Peri 2) and cytoplasmic soluble fraction (Cyto) and the insoluble fraction (Insol) from <i>E. coli</i>. The proteins in the gel were visualized by staining with Coomassie Blue R-250. (B) Western blot analysis of the recombinant proteins. Proteins were run on SDS-PAGE and transferred to PVDF membrane. Western blots were probed with an anti-V5-HRP antibody. Approximately 2 µg of each protein was electrophoresed. The molecular mass (kDa) of the molecular weight markers (All Blue, Bio-Rad) are shown in the left margin, and the positions of the fusion proteins (Protein A-AMCase-V5-His) are shown with arrows in the right margin. (C and D) Purification of the recombinant proteins. The fusion proteins were expressed in <i>E. coli</i> and purified from the periplasmic fraction 1 (Peri 1) by IgG Sepharose followed by Ni Sepharose. Proteins separated by SDS-PAGE were stained with Coomassie Blue R-250 (C) or transferred to PVDF membrane (D). Western blots were probed with an Anti-V5-HRP antibody.