title: Construction and analysis of a genetically tuneable lytic phage display system.
abstract: The Bacteriophage λ capsid protein gpD has been used extensively for fusion polypeptides that can be expressed from plasmids in Escherichia coli and remain soluble. In this study, a genetically controlled dual expression system for the display of enhanced green fluorescent protein (eGFP) was developed and characterized. Wild-type D protein (gpD) expression is encoded by λ Dam15 infecting phage particles, which can only produce a functional gpD protein when translated in amber suppressor strains of E. coli in the absence of complementing gpD from a plasmid. However, the isogenic suppressors vary dramatically in their ability to restore functional packaging to λDam15, imparting the first dimension of decorative control. In combination, the D-fusion protein, gpD::eGFP, was supplied in trans from a multicopy temperature-inducible expression plasmid, influencing D::eGFP expression and hence the availability of gpD::eGFP to complement for the Dam15 mutation and decorate viable phage progeny. Despite being the worst suppressor, maximal incorporation of gpD::eGFP into the λDam15 phage capsid was imparted by the SupD strain, conferring a gpDQ68S substitution, induced for plasmid expression of pD::eGFP. Differences in size, fluorescence and absolute protein decoration between phage preparations could be achieved by varying the temperature of and the suppressor host carrying the pD::eGFP plasmid. The effective preparation with these two variables provides a simple means by which to manage fusion decoration on the surface of phage λ.
authors: Pharmaceutical Sciences School of Pharmacy, University of Waterloo, 200 University Ave, Waterloo, Ontario, N2L3G1, Canada.
authors: Jessica
authors: J
authors: Nicastro
authors: Katlyn
authors: K
authors: Sheldon
authors: Farah A
authors: FA
authors: El-Zarkout
authors: Stanislav
authors: S
authors: Sokolenko
authors: Marc G
authors: MG
authors: Aucoin
authors: Roderick
authors: R
authors: Slavcev
mesh_terms: D010582:Bacteriophage lambda / Q000235:genetics* / Q000378:metabolism; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism / Q000821:virology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D019151:Peptide Library; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D036022:Capsid Proteins; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
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