Expression in Escherichia coli of c-type cytochrome genes from Rhodopseudomonas viridis.
The genes coding for the photosynthetic reaction center cytochrome c subunit (pufC) and the soluble cytochrome c2 (cycA) from the purple non-sulfur bacterium Rhodopseudomonas viridis were expressed in Escherichia coli. Biosynthesis of the reaction center cytochrome without a signal peptide resulted in the formation of inclusion bodies in the cytoplasm amounting to 14% of the total cellular protein. A series of plasmids coding for the cytochrome subunit with varying N-terminal signal peptides was constructed in attempts to achieve translocation across the E. coli cytoplasmic membrane and heme attachment. However, the two major recombinant proteins with N-termini corresponding to the signal peptide and the cytochrome were synthesized in E. coli as non-specific aggregates without heme incorporation. An increased ratio of precursor as compared to 'processed' apo-cytochrome was obtained when expression was carried out in a proteinase-deficient strain. Cytochrome c2 from R. viridis was synthesized in E. coli as a precursor associated with the cytoplasmic membrane. An expression plasmid was designed encoding the N-terminal part of the 33 kDa precursor protein of the oxygen-evolving complex of Photosystem II from spinach followed by cytochrome c2. Two recombinant proteins without heme were found to aggregate as inclusion bodies with N-termini corresponding to the signal peptide and the mature 33 kDa protein.
Grisshammer
R
R
Max-Planck-Institut für Biophysik, Frankfurt/Main, F.R.G.
Oeckl
C
C
Michel
H
H
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003574:Cytochrome c Group / Q000235:genetics*; D045362:Cytochromes c2; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics*; D011498:Protein Precursors / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D012241:Rhodopseudomonas / Q000235:genetics* / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D003574:Cytochrome c Group; D046911:Macromolecular Substances; D045322:Photosynthetic Reaction Center Complex Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D045362:Cytochromes c2