Integrity of Thermus thermophilus cytochrome c(552) synthesized by Escherichia coli cells expressing the host-specific cytochrome c maturation genes,ccmABCDEFGH. Biochemical, spectral, and structural characterization of therecombinant protein

We describe the design of Escherichia coli cells that synthesize a structur ally perfect, recombinant cytochrome c from the Thermus thermophilus cytoch rome c(552) gene. Key features are (1) construction of a plasmid-borne, chi meric cycA gene encoding an Escherichia coli-compatible, N-terminal signal sequence (MetLysIleSerIleTyrAlaThrLeu AlaAlaLeuSerLeuAlaLeuProAlaGlyAla) fo llowed by the amino acid sequence of mature Thermus cytochrome c(552); and (2) coexpression of the chimeric cycA gene with plasmid-borne, host-specifi c cytochrome c maturation genes (ccmABCDEFGH). Approximately 1 mg of purifi ed protein is obtained from 1 L of culture medium. The recombinant protein, cytochrome rsC(552), and native cytochrome c(552) have identical redox pot entials and are equally active as electron transfer substrates toward cytoc hrome ba(3), a Thermus heme-copper oxidase. Native and recombinant cytochro mes c were compared and found to be identical using circular dichroism, opt ical absorption, resonance Raman, and 500 MHz H-1-NMR spectroscopies. The 1 .7 Angstrom 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 generall y useful for expression of alien cytochrome c genes in E. coli.