Processing and functional display of the 86 kDa heterodimeric penicillin G acylase on the surface of phage fd.
The large heterodimeric penicillin G acylase from Alcaligenes faecalis was displayed on the surface of phage fd. We fused the coding sequence (alpha subunit-internal peptide-beta subunit) to the gene of a phage coat protein. A modified g3p signal sequence was used to direct the polypeptide to the periplasm. Here we show that a heterodimeric enzyme can be expressed as a fusion protein that matures to an active biocatalyst connected to the coat protein of phage fd, resulting in a phage to which the beta-subunit is covalently linked and the alpha-subunit is non-covalently attached. The enzyme can be displayed either fused to the minor coat protein g3p or fused to the major coat protein g8p. In both cases the penicillin G acylase on the phage has the same Michaelis constant as its freely soluble counterpart, indicating a proper folding and catalytic activity of the displayed enzyme. The display of the heterodimer on phage not only allows its further use in protein engineering but also offers the possibility of applying this technology for the excretion of the enzyme into the extracellular medium, facilitating purification of the protein. With the example of penicillin acylase the upper limit for a protein to become functionally displayed by phage fd has been further explored. Polyvalent display was not observed despite the use of genetic constructs designed for this aim. These results are discussed in relation to the pore size being formed by the g4p multimer.
Verhaert
R M
RM
Pharmaceutical Biology, University Centre for Pharmacy, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands. r.m.d.verhaert@farm.rug.nl
Van Duin
J
J
Quax
W J
WJ
D000421:Alcaligenes / Q000201:enzymology / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D002213:Capsid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018388:Codon, Terminator / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D017912:Inovirus / Q000201:enzymology* / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D010399:Penicillin Amidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D018388:Codon, Terminator; D017931:DNA Primers; D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; D010399:Penicillin Amidase