Overexpression in Escherichia coli of the fdxA gene encoding Rhodobacter capsulatus ferredoxin II.
Different gene expression systems were tested with the aim of overproducing the 7Fe ferredoxin (FdII) from Rhodobacter capsulatus in Escherichia coli. Plasmids bearing the Ptac, ParaB, fdxA gene, encoding FdII, under the control of the PlacUV5, P phi 10 promoters, were compared for their efficiency to promote the synthesis of recombinant ferredoxin. Using a P phi 10-based expression system, recombinant ferredoxin was obtained in a soluble apoform and accumulated up to 20% of the total cell protein. The ferredoxin polypeptide purified from such cells exhibited the correct molecular mass and no detectable heterogeneity when analyzed by mass spectrometry. When other expression systems were used, the ferredoxin was synthesized in holoform but in relatively smaller amounts (0.2 mg/liter of culture). Factors such as promoter strength, efficient translation signals, and stability of the recombinant mRNA were shown to have little effect on the amount of 7Fe ferredoxin produced in E. coli. It is inferred that iron-sulfur clusters insertion may be a rate-limiting factor for synthesis of the 7Fe ferredoxin in the enterobacterium.
Armengaud
J
J
Laboratorie de Biochimie Microbienne (CNRS URA 1130, alliée à l'INSERM), Département de Biologie Moléculaire et Structurale, Centre d'Etudes Nucléaires de Grenoble, France.
Jouanneau
Y
Y
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D004926:Escherichia coli; D005288:Ferredoxins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D005822:Genetic Vectors / Q000235:genetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D016354:Rhodobacter capsulatus / Q000235:genetics*
D001426:Bacterial Proteins; D005288:Ferredoxins; D011993:Recombinant Fusion Proteins