Cytoplasmic and periplasmic expression of a synthetic gene for ferredoxin in Escherichia coli.
A synthetic gene coding for a modified ferredoxin II of Desulfovibrio desulfuricans Norway strain was assembled from 10 oligonucleotides. This gene was cloned into various expression vectors allowing either cytoplasmic expression or export to the periplasmic space. In the latter case, two different constructs were made, each of which contained the OmpA signal peptide: one of these constructs contained 3 additional N-terminal amino acids as compared to the wild-type ferredoxin (56 amino acid residues). The expression of proteins encoded by the 3 constructs was assayed in E coli and the proteins were localized by cell fractionation and immunogold labelling. A low percentage of the periplasmic ferredoxin (approximately 5%) was secreted to the medium in the absence of cell lysis. The recombinant ferredoxin was purified and found to be correctly processed by the leader peptidase. However, due to the high cysteine content intramolecular and intermolecular disulfide bonds were formed and prevented binding of [4Fe-4S] clusters. Reconstitution experiments using these recombinant proteins are in progress.
Bourdineaud
J P
JP
Centre de Biochimie et de Biologie Moléculaire, Marseille, France.
Howard
S P
SP
Pages
J M
JM
Bernadac
A
A
Leroy
G
G
Bruschi
M
M
Lazdunski
C
C
D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D003901:Desulfovibrio / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005288:Ferredoxins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D004269:DNA, Bacterial; D005288:Ferredoxins; D011994:Recombinant Proteins; C026518:apoferredoxin