Integrity of Thermus thermophilus cytochrome c₅₅₂ synthesized by Escherichia coli cells expressing the host-specific cytochrome c maturation genes, ccmABCDEFGH: Biochemical, spectral, and structural characterization of the recombinant protein

Abstract

We describe the design of Escherichia coli cells that synthesize a structurally perfect, recombinant cytochrome c from the Thermus thermophilus cytochrome c₅₅₂ gene. Key features are (1) construction of a plasmid-borne, chimeric cycA gene encoding an Escherichia coli-compatible, N-terminal signal sequence (MetLysIleSerIleTyrAlaThrLeuAlaAlaLeuSerLeuAlaLeuProAlaGlyAla) followed by the amino acid sequence of mature Thermus cytochrome c₅₅₂; and (2) coexpression of the chimeric cycA gene with plasmid-borne, host-specific cytochrome c maturation genes (ccmABCDEFGH). Approximately 1 mg of purified protein is obtained from 1 L of culture medium. The recombinant protein, cytochrome rsC₅₅₂, and native cytochrome c₅₅₂ have identical redox potentials and are equally active as electron transfer substrates toward cytochrome ba₃, a Thermus heme-copper oxidase. Native and recombinant cytochromes c were compared and found to be identical using circular dichroism, optical absorption, resonance Raman, and 500 MHz ¹H-NMR spectroscopies. The 1.7 Å resolution X-ray crystallographic structure of the recombinant protein was determined and is indistinguishable from that reported for the native protein (Than, ME, Hof P, Huber R, Bourenkov GP, Bartunik HD, Buse G, Soulimane T, 1997, J Mol Biol 271:629–644). This approach may be generally useful for expression of alien cytochrome c genes in E. coli.