Eliminating helper phage from phage display

Abstract

Phage display technology involves the display of proteins or peptides, as coat protein fusions, on the surface of a phage or phagemid particles. Using standard technology, helper phage are essential for the replication and assembly of phagemid particles, during library production and biopanning. We have eliminated the need to add helper phage by using 'bacterial packaging cell lines' that provide the same functions. These cell lines contain M13-based helper plasmids that express phage packaging proteins which assemble phagemid particles as efficiently as helper phage, but without helper phage contamination. This results in genetically pure phagemid particle preparations. Furthermore, by using constructs differing in the form of gene 3 that they contain, we have shown that the display, from a single library, can be modulated between monovalent (phagemid-like) and multivalent display (phage-like) without any further engineering. These packaging cells eliminate the use of helper phage from phagemid-based selection protocols; reducing the amount of technical preparation, facilitating automation, optimizing selections by matching display levels to diversity, and effectively using the packaged phagemid particles as means to transfer genetic information at an efficiency approaching 100%.

Fine epitope specificity of antibodies against interleukin-2 explains their paradoxical immunomodulatory effects

“…Taking into account that between 1-10% of the filamentous phage particles display a single copy of the heterologous molecule in similar phagemidbased systems, 16 the concentration of phage-displayed mIL-2 inducing half-maximal cell proliferation could be roughly estimated to be between 0.6 and 6 pmol/L. Control in vitro refolded recombinant mIL-2 induced half-maximal proliferation at 20 pmol/L (Fig.…”

A filamentous phage display system for N‐linked glycoproteins

“…Another possible explanation for the low recovery rate that cannot be entirely ruled out is that a subpopulation of phages produced in pgl cells may not have glycans owing to: (i) inefficiency of PglB-mediated glycosylation of unnatural substrates 14,15 like MBP DQNAT -g3p; and (ii) competition for assembly into phage particles between engineered g3p fusions and wild-type g3p encoded on the helper phage chromosome. 31 Our own recent studies indicate that the efficiency of PglB is not an issue as a fairly high percentage ($40-50%) of MBP with a C-terminal DQNAT glycosylation locus is N-glycosylated (Fisher and DeLisa, unpublished observations). Nonetheless, increasing the efficiency of phage glycosylation could have a net positive effect on our method.…”

“…Alternatively, the ''helperless'' phage system developed by Bradbury and coworkers could offer a similar improvement. 31 A final strategy that may improve phage display of glycoproteins would be to use the cotranslational signal recognition particle (SRP) pathway, which is the primary export route used by eurkaryotic N-glycoproteins. The use of the SRP pathway was also recently demonstrated to enhance the display levels of designed ankyrinrepeat proteins (DARPins) by 700-fold compared to post-translational Sec export.…”

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in <em>E. coli</em>

“…Although M13K07 contains a mutated packaging signal, it can be packaged into phage capsid at low frequency and transferred to infected populations, resulting in cells competent for phage production and the continued spread of phage beyond the initial infection event 25 . Modifications to the helper phage have proven effective at reducing nonspecific packaging, although sometimes at the cost of reduced phage production 26 .…”