Figure 1: In vivo solubilization of EmrE using the SIMPLEx strategy.
From: Making water-soluble integral membrane proteins in vivo using an amphipathic protein fusion strategy
(a) Western blot analysis of soluble (sol), detergent solubilized (det) and insoluble (ins) fractions prepared from E. coli strain BL21(DE3) expressing either EmrE, OspA-EmrE, OspA-ApoAI* or OspA-EmrE-ApoAI* as indicated. Blot was probed with anti-His antibody. Molecular weight (MW) markers are shown on the left. (b) Fluorescence microscopy of BL21(DE3) cells expressing the same constructs in (a) that were each modified with a C-terminal GFP fusion for visualizing protein expression and localization. (c) Ligand-binding activity performed using dimeric, detergent-free OspA-EmrE-ApoAI* or organic-extracted detergent-solubilized EmrE, both of which were purified from BL21(DE3) cells. Assays were performed with EtBr as substrate. Determination of binding constants was based on fluorescence quenching. Data are expressed as the mean of biological quadruplicates and the error, defined as the s.e.m., was <5%.
