Functional display of active bovine adrenodoxin on the surface of E. coli by chemical incorporation of the [2Fe-2S] cluster.
The display of heterologous proteins on the surface of living cells bears promising options for a wide variety of biotechnological applications. Up to now, however, cellular surface display was merely restricted to simple polypeptide chains. Here we present for the first time the efficient display of a protein (bovine adrenodoxin) that contains an inorganic, prosthetic group in its active form on the surface of Escherichia coli. For this purpose apo-adrenodoxin was transported to the cell surface and anchored within the outer membrane by the autotransporter pathway. Incorporation of the iron-sulfur cluster was achieved by a single-vial, one-step titration under anaerobic conditions. The biological function of surface-displayed holo-adrenodoxin could be established through adrenodoxin-dependent steroid conversion by two different cytochrome P450 enzymes and the number of functional molecules on the cell surface could be determined to be more than 10(5) per cell. Neither the expression of adrenodoxin nor the incorporation of the chemical iron-sulfur cluster reduced the viability of the bacterial cells.
Jose
J
J
Pharmazeutische und Medizinische Chemie, Naturwissenschaftlich-Technische Fakultät III, Universität des Saarlandes, P.O. Box 151150, 66041 Saarbrücken, Germany. j.jose@rz.uni-sb.de
Bernhardt
R
R
Hannemann
F
F
D000325:Adrenodoxin / Q000737:chemistry* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002384:Catalysis; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D007501:Iron / Q000737:chemistry; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000302:isolation & purification; D013256:Steroids / Q000737:chemistry; D013455:Sulfur / Q000737:chemistry; D013499:Surface Properties
D011993:Recombinant Fusion Proteins; D013256:Steroids; D000325:Adrenodoxin; D013455:Sulfur; D003577:Cytochrome P-450 Enzyme System; D007501:Iron