Co-transformation of gpD-UBHA and gpV-CD40 expression plasmids into lysogens yields bi-functional phage displaying both peptide fusions

<p><b>Copyright information:</b></p><p>Taken from "A tractable method for simultaneous modifications to the head and tail of bacteriophage lambda and its application to enhancing phage-mediated gene delivery"</p><p></p><p>Nucleic Acids Research 2007;35(8):e59-e59.</p><p>Published online 28 Mar 2007</p><p>PMCID:PMC1885665.</p><p>© 2007 The Author(s)</p> The plasmid pTrc:gpD-UBHA and the plasmid pTrcRSF:gpV-CD40 were co-transformed into lambda lysogens. After lytic induction, phage were purified by CsCl density gradient centrifugation and analyzed. () Phage (5 × 10 p.f.u.) were loaded on a 12% polyacrylamide gel, subjected to SDS-PAGE and transferred to nitrocellulose. Immunoblot analysis was then performed using an anti-gpD rabbit polyclonal antiserum. This revealed full replacement of gpD with gpD-UBHA in phage produced from lysogens carrying the pTrc:gpD-UBHA plasmid. The molecular weight of gpD is ∼12 kDa and of gpD-UBHA is ∼16 kDa. () Phage (5 × 10 p.f.u.) were loaded on a 12% polyacrylamide gel, subjected to SDS-PAGE and transferred to nitrocellulose. Immunoblot analysis was then performed with an anti-gpV polyclonal antiserum. This revealed that the phage expressing the gpV-CD40 fusion protein contained roughly a 1:1 ratio of wild-type gpV and recombinant gpV-CD40. The projected molecular weights are ∼30 kDa for wild type gpV and ∼27 kDa for recombinant gpV-CD40 (Note that the recombinant gpV fusion protein is based on a truncated form of gpV, and is therefore smaller than its wild-type counterpart)