Figure 2: In vivo solubilization of cyt b₅ using the SIMPLEx strategy.

(a) Western blot analysis of soluble (sol), detergent solubilized (det) and insoluble (ins) fractions prepared from E. coli strain BL21(DE3) expressing either cyt b₅, OspA-cyt b₅, OspA-ApoAI* or OspA-cyt b₅-ApoAI* as indicated. Blot was probed with anti-His antibody. Molecular weight (MW) markers are shown on the left. (b) SEC profiles of Ni²⁺-purified ΔspMBP-cyt b₅-ApoAI*. Protein elutes as an octamer (O) or tetramer (T). (c) Oxidized and reduced spectra of ΔspMBP-cyt b₅-ApoAI* with characteristic peaks at 424 and 409 nm, respectively. (d) Coordination of haeme cofactor by cyt b₅. Cells carrying empty plasmid control or expressing ΔspMBP-cyt b₅-ApoAI* were visually inspected for characteristic red haeme colouring. Purified ΔspMBP-cyt b₅-ApoAI* is also shown. (e) Augmentation of CYP17A1 lyase activity by cyt b₅. CYP17A1 and its substrate 17-hydroxy pregnenolone (17-P5) were incubated with different concentrations (5–25 pmol) of wild-type cyt b₅ (detergent solubilized) or ΔspMBP-cyt b5-ApoAI*, or in the absence of cyt b₅. The percentage of dehydroepiandrosterone (DHEA) product formed was monitored by high-performance liquid chromatography.