Phage-enzymes: expression and affinity chromatography of functional alkaline phosphatase on the surface of bacteriophage.
We have demonstrated that an active enzyme can be expressed on the surface of a bacteriophage. The gene encoding alkaline phosphatase from Escherichia coli was cloned upstream of gene 3, which encodes a minor coat protein of the filamentous bacteriophage, fd. A fusion protein of the correct size was detected from viral particles by Western blotting. Ultrafiltration confirmed that the enzyme fusion behaves as part of a larger structure as would be expected of an enzyme fused to a viral particle. Both wild-type alkaline phosphatase (Arg166) and an active site mutant (Ala166) expressed in this way retain catalytic activity and have qualitatively similar kinetic properties to free enzyme. Values were obtained for Km of 72.7 and 1070 microM respectively whilst relative kcat for the mutant was 36% of that for wild-type. Phage particles expressing alkaline phosphatase were bound to an immobilized inhibitor (arsenate-Sepharose) and eluted with product (20 mM inorganic phosphate). In this way, the functional enzyme is co-purified with the DNA encoding it. This may permit a novel approach to enzyme engineering based on affinity chromatography of mutant enzymes expressed on the phage surface.
McCafferty
J
J
Cambridge Antibody Technology, Daly Research Laboratories, Babraham, Cambridgeshire, UK.
Jackson
R H
RH
Chiswell
D J
DJ
D000469:Alkaline Phosphatase / Q000235:genetics*; D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000201:enzymology* / Q000235:genetics / Q000276:immunology / Q000472:pathogenicity; D001483:Base Sequence; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000276:immunology / Q000648:ultrastructure; D014759:Viral Envelope Proteins / Q000032:analysis* / Q000276:immunology / Q000648:ultrastructure
D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; D000469:Alkaline Phosphatase