Expression of plastocyanin and cytochrome f of the cyanobacterium Phormidium laminosum in Escherichia coli and Paracoccus denitrificans and the role of leader peptides.
The gene for plastocyanin from the cyanobacterium Phormidium laminosum was successfully expressed in Escherichia coli. Expression of the gene for cytochrome f resulted in the production of holocytochrome f in the periplasmic space of E. coli, but the yield was low. Expression in Paracoccus denitrificans yielded no holoprotein. When the region encoding the cytochrome f leader sequence was replaced with more typical bacterial leader sequences (those from the P. laminosum plastocyanin gene and the Paracoccus versutus cytochrome c-550 gene), much higher yields were consistently obtained in both species. Overexpressed proteins were compared to those isolated from P. laminosum and found to be identical in mass, isoelectric point, redox midpoint potential and (for plastocyanin) 1H-NMR spectrum.
Department of Biochemistry and Cambridge Centre for Molecular Recognition, University of Cambridge, Cambridge CB2 1QW, UK.
B G
BG
Schlarb
M J
MJ
Wagner
E
E
Vijgenboom
M
M
Ubbink
D S
DS
Bendall
C J
CJ
Howe
D020418:Algal Proteins / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D000458:Cyanobacteria / Q000235:genetics; D003574:Cytochrome c Group / Q000235:genetics; D003580:Cytochromes / Q000235:genetics*; D045348:Cytochromes f; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D006090:Gram-Negative Bacteria / Q000235:genetics*; D010231:Paracoccus denitrificans / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D010970:Plastocyanin / Q000235:genetics*; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology*
D020418:Algal Proteins; D001426:Bacterial Proteins; D003574:Cytochrome c Group; D003580:Cytochromes; D021382:Protein Sorting Signals; D010970:Plastocyanin; D045348:Cytochromes f; C031732:cytochrome C-550