Characterization of cycP gene expression in Achromobacter xylosoxidans NCIMB 11015 and high-level heterologous synthesis of cytochrome c' in Escherichia coli.
The cycP gene encoding a periplasmic cytochrome c' from the denitrifying beta-proteobacterium Achromobacter xylosoxidans was characterized. The genes flanking cycP encode components of a mobile genetic element characteristic of the beta-proteobacteria, suggesting that cycP has inserted within a transposon or insertion element. The gene therefore does not form part of a denitrification operon or gene cluster. The level of expression of the cycP gene and the level of synthesis of its corresponding gene product were found to increase by maximally 3-fold anaerobically. Expression of cycP appears to occur mainly by non-specific read-through transcription from portions of the insertion element. Conditions were developed for high-level overproduction of cytochrome c' in Escherichia coli, which resulted in signal peptide cleavage concomitant with secretion of the protein into the periplasm. Using a single-step purification, 20-30 mg of pure protein were isolated from a 1-litre culture. Based on UV-visible spectrophotometry the dimeric protein was shown to have a full complement of haem and to be indistinguishable from the native protein purified from A. xylosoxidans. This system provides an excellent platform to facilitate biochemical and structural dissection of the mechanism underlying the novel specificity of NO binding to the proximal face of the haem.
Harris
Roger L
RL
John Innes Centre, Norwich, UK.
Barbieri
Sonia
S
Paraskevopoulos
Kostas
K
Murphy
Loretta M
LM
Eady
Robert R
RR
Hasnain
S Samar
SS
Sawers
R Gary
RG
D042441:Achromobacter denitrificans / Q000201:enzymology* / Q000235:genetics; D045305:Cytochromes c' / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D020869:Gene Expression Profiling; D019897:Periplasm / Q000737:chemistry; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D045305:Cytochromes c'; D004251:DNA Transposable Elements; D021382:Protein Sorting Signals; D011994:Recombinant Proteins