A simple method for displaying recalcitrant proteins on the surface of bacteriophage lambda

Abstract

Bacteriophage lambda (λ) permits the display of many foreign peptides and proteins on the gpD major coat protein. However, some recombinant derivatives of gpD are incompatible with the assembly of stable phage particles. This presents a limitation to current λ display systems. Here we describe a novel, plasmid-based expression system in which gpD deficient λ lysogens can be co-complemented with both wild-type and recombinant forms of gpD. This dual expression system permits the generation of mosaic phage particles that contain otherwise recalcitrant recombinant gpD fusion proteins. Overall, this improved gpD display system is expected to permit the expression of a wide variety of peptides and proteins on the surface of bacteriophage λ and to facilitate the use of modified λ phage vectors in mammalian gene transfer applications.

A tractable method for simultaneous modifications to the head and tail of bacteriophage lambda and its application to enhancing phage-mediated gene delivery

“…We previously designed and evaluated a plasmid-based, trans -complementation system that allowed the incorporation of recalcitrant peptides into the head of phage lambda (17). We reasoned that a similar approach might also permit the display of exogenous peptides on the major tail protein, gpV.…”

“…With this in mind, we set about displaying an ubiquitinylation motif from the hepatitis A virus 3C protease (UBHA) on the surface of lambda phage particles. To do this, the ubiquitinylation motif (LGVKDDWLLV) was fused to the C-terminus of gpD, and then displayed on phage particles (17). …”

“…The plasmids pTrc:gpD and pTrc:gpD-Fusion were derived from pTrcHis (Invitrogen) with the addition of a synthetic, codon-optimized D gene as described (17). The D gene was inserted as an NcoI to BamHI fragment, with subsequent loss of the NcoI site.…”

“…The production of the polyclonal anti-gpD antiserum has been described (17). Polyclonal anti-gpV antiserum was produced by transforming the plasmid pBAD:gpVtrunc into Top10 E. coli (Invitrogen).…”

Progress in phage display: evolution of the technique and its applications

“…Zanghi et al [86] noted that certain proteins were not well tolerated when trying to display them at high copy number as fusions to gpD major coat protein of lambda phage. To this end, they designed a dual plasmid-based expression system (encoding the wild-type and recombinant gpD, respectively) to co-complement lambda lysogens devoid of gD.…”