EXPRESSION OF A BIOLOGICALLY-ACTIVE PLANT CYTOCHROME B(5) IN ESCHERICHIA-COLI

Cytochrome b(5) from tobacco (Nicotiana tabacum) was expressed in Escherichia coli using a T7 polymerase/promoter system as described by Studier, Rosenberg, Dunn and Dubendorff (1990) (Methods Enzymol. 185, 60-89). Transformed cells were red in colour and accumulated cytochrome b(5) 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 b(5), and exhibited a c.d. spectrum resembling that of a mammalian cytochrome b(5). 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 b(5), by the use of gabaculin or succinylacetone, prevented the assembly of the cytochrome b(5) 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 b(5) and can be used in studies of plant microsomal oxidation/reduction reactions.