Gene-dose-dependent expression of soluble mammalian cytochrome b5 in Escherichia coli

A synthetic structural gene encoding a mammalian cytochrome b5, carrying an optimised ribosomal binding sequence, was tandemly polymerised ranging from one (n = 1) to six (n = 6) gene copies. The gene, placed in p lambda-ncyt under the control of the lambda PL promoter, transcribed mono- to hexahomocistronic mRNA, expressing one to six copies of cytochrome b5. The expressed levels of cytochrome b5 in Escherichia coli p lambda-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 p lambda-6cyt produced 75 micrograms cytochrome b5/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 holocytochrome b5. Whereas the overall expressed protein (apo and holo forms) was gene dose dependent, there was an inverse relationship between holocytochrome b5 production and gene dose. Incubation of the thermally induced bacterial lysates with exogenous haem a converted all of the soluble apocytochrome b5 into holocytochrome b5 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 b5 in E. coli.