Gene-dose-dependent expression of soluble mammalian cytochrome b ₅ in Escherichia coli

A synthetic structural gene encoding a mammalian cytochrome b 5, carrying an optimised ribosomal binding sequence, was tandemly polymerised ranging from one (n=1) to six (n=6) gene copies. The gene, placed in pλ-ncyt under the control of the λP L promoter, transcribed mono- to hexahomocistronic mRNA, expressing one to six copies of cytochrome b 5. The expressed levels of cytochrome b 5 in Escherichia coli pλ-ncyt corresponded linearly with the gene dose when up to five copies were present; saturating build-up of the recombinant protein was reached at six gene copies. Cells bearing pλ-6cyt produced 75 μg cytochrome b 5/ml of unit optical density at 600 nm culture, constituting 55% of the soluble bacterial protein. The recombinant protein accumulated predominantly in a haem-deficient, apoform, together with lesser amounts of the halocytochrome b 5. Whereas the overall expressed protein (apo and holo forms) was gene dose dependent, there was an inverse relationship between holocytochrome b 5 production and gene dose. Incubation of the thermally induced bacterial lysates with exogenous haem a converted all of the soluble apocytochrome b 5 into holocytochrome b 5 that was spectrally indistinguishable with its native counterpart. Culture supplementation with the likely metabolic precursors of haem synthesis, 5-aminolevulinic acid, glycine/succinate or glutamate, significantly alleviated the protoporphyrin deficiency during hyperproduction of cytochrome b 5 in E. coli.