In:
Protein Science, Wiley, Vol. 9, No. 11 ( 2000-01), p. 2074-2084
Abstract:
We describe the design of Escherichia coli cells that synthesize a structurally perfect, recombinant cytochrome c from the Thermus thermophilus cytochrome c 552 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 c 552 ; 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 rsC 552 , and native cytochrome c 552 have identical redox potentials and are equally active as electron transfer substrates toward cytochrome ba 3 , 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 1 H‐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.
Type of Medium:
Online Resource
ISSN:
0961-8368
,
1469-896X
DOI:
10.1110/ps.9.11.2074
Language:
English
Publisher:
Wiley
Publication Date:
2000
detail.hit.zdb_id:
2000025-X
SSG:
12
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