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.
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 (MetLysIleSerIleTyrAlaThrLeu AlaAlaLeuSerLeuAlaLeuProAlaGlyAla) 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 A 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.
Fee
J A
JA
Department of Biology, University of California at San Diego, La Jolla 92093, USA. jfee@ucsd.edu
Chen
Y
Y
Todaro
T R
TR
Bren
K L
KL
Patel
K M
KM
Hill
M G
MG
Gomez-Moran
E
E
Loehr
T M
TM
Ai
J
J
Thöny-Meyer
L
L
Williams
P A
PA
Stura
E
E
Sridhar
V
V
McRee
D E
DE
D000595:Amino Acid Sequence; D002455:Cell Division; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D003574:Cytochrome c Group / Q000096:biosynthesis / Q000737:chemistry*; D004579:Electron Transport; D004926:Escherichia coli / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010957:Plasmids / Q000378:metabolism; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D016963:Thermus thermophilus / Q000201:enzymology*; D014466:Ultraviolet Rays
D003574:Cytochrome c Group; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C083951:cytochrome c553; C022302:cytochrome C-552