Efficient bacterial export of a eukaryotic cytoplasmic cytochrome.
The soluble core domain of cytochrome b5 of liver endoplasmic reticulum was appended at its amino terminus to full-length alkaline phosphatase secretory signal sequence including the ribosomal binding site. The chimeric precursor gene was placed under the transcriptional control of the native pho promoter in a prokaryotic expression vector. Induction of Escherichia coli by growth in a phosphate-limited medium resulted in abundant synthesis of cytochrome b5 as detected spectrophotometrically and by visual transformation of the bacteria to a pink color. The signal-appended cytochrome b5, but not the corresponding signal-deficient derivative, was translocated across the bacterial inner membrane and processed to yield authentic, haem-assembled cytochrome b5 within the periplasm. The eventual processing of the chimeric cytochrome b5 precursor was unusual regarding the known reaction specificity of signal peptidase. The exported, mature haemoprotein was biochemically indistinguishable from its native mammalian counterpart. At peak induction, approximately 6 mg of correctly matured cytochrome b5 per liter of culture was exported. This amount of cytochrome b5 constituted 6% (w/w) of the periplasmic protein. The appearance of the exported apo-cytochrome b5 preceded the formation of holo-protein. Thus the eukaryotic cytoplasmic protein was efficiently exported from E. coli and post-translocationally modified to generate a functional haemoprotein in the periplasm.
Karim
A
A
Department of Biochemistry, School of Life Sciences, University of Wales, Aberystwyth, U.K.
Kaderbhai
N
N
Evans
A
A
Harding
V
V
Kaderbhai
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015786:Cytochromes b5 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D008099:Liver / Q000737:chemistry / Q000648:ultrastructure; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012697:Serine Endopeptidases; D014169:Transformation, Bacterial
D008565:Membrane Proteins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D015786:Cytochromes b5; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase