Protein Engineering vol. 4 no. 8 pp. 955-961, 1991
© 1991 Oxford University Press
RESEARCH-ARTICLE |
Phage-enzymes: expression and affinity chromatography of functional alkaline phosphatase on the surface of bacteriophage
Cambridge Antibody Technology, Daly Research Laboratories, Babraham Hall Babraham, Cambridgeshire CB2 4AT, UK
1To whom correspondence should be addressed
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 (Argl66) and an active site mutant (Ala 166) 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 µM 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.
Keywords: affinity chromatography/alkaline phosphatase/fd/filamentous bacteriophage/gene 3
Received August 12, 1991;
revised October 3, 1991;
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