Expression of the gene encoding cytochrome c3 from the sulfate-reducing bacterium Desulfovibrio vulgaris in the purple photosynthetic bacterium Rhodobacter sphaeroides.
The gene encoding cytochrome c3 (cyc-gene) from Desulfovibrio vulgaris (Hildenborough) was cloned by G. Voordouw and S. Brenner (1986, Eur. J. Biochem. 159, 347-351). The gene was expressed in Escherichia coli but only the apoprotein was observed (W. Pollock, P. Chemerika, M. Forrest, J. Beatty, and G. Voordouw, 1989, J. Gen. Microbiol. 135, 2319-2328). In this study, the cyc-gene was cloned into the broad host range vector pRK404 and then introduced into the purple photosynthetic bacterium Rhodobacter sphaeroides. Cells grown anaerobically produced a significant amount of recombinant cytochrome c3. The purified protein contains four hemes and the N-terminal protein sequence is identical to the published sequence of the native cytochrome c3. Thus, R. sphaeroides was able to produce the mature cytochrome c3 by combining the four steps of protein synthesis, exporting the protein across the membrane, cleaving the signal peptide, and inserting four hemes. It appears that the D. vulgaris promoter is not very efficiently used by R. sphaeroides. However, replacement of the promoter with a R. sphaeroides promoter should result in cytochrome c3 overproduction.
Cannac
V
V
Department of Biochemistry, University of Arizona, Tucson 85721.
Caffrey
M S
MS
Voordouw
G
G
Cusanovich
M A
MA
D003001:Cloning, Molecular / Q000379:methods; D003574:Cytochrome c Group / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003901:Desulfovibrio / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000235:genetics*; D013053:Spectrophotometry
D003574:Cytochrome c Group; D011994:Recombinant Proteins; C017165:cytochrome c(3)