Expression of a biologically active plant cytochrome b₅ in Escherichia coli

Cytochrome b₅ from tobacco (Nicotiana tabacum) was expressed in Escherichia coli using a T7 polymerase/promoter system as described by Studier, Rosenberg, Dunn and Dubendorff. Transformed cells were red in colour and accumulated cytochrome b₅ to a level of around 30% of the total cell protein. The purified cytochrome had oxidized, reduced and low-temperature absorbance spectra characteristic of plant microsomal cytochrome b₅, and exhibited a c.d. spectrum resembling that of a mammalian cytochrome b₅. The recombinant protein appeared to be correctly assembled and biologically active, being reduced by NADH in the presence of microsomal membranes prepared from the developing seeds of sunflower (Helianthus annuus). Inhibition of haem synthesis in the transformed E. coli cells expressing cytochrome b₅, by the use of gabaculin or succinylacetone, prevented the assembly of the cytochrome b₅ holoprotein but had little effect on the accumulation of cytochrome apoprotein. The recombinant protein expressed in E. coli therefore has the biochemical features of the higher-plant cytochrome b₅ and can be used in studies of plant microsomal oxidation/reduction reactions.