A new helper phage and phagemid vector system improves viral display of antibody Fab fragments and avoids propagation of insert-less virions.
Phage display technology (PDT) is a powerful method for isolating functional gene products such as antigen-specific monoclonal antibodies (mAbs). To improve the effectiveness of PDT, we sought to optimize display of Fab-g3p (antibody fragment fused with viral gene 3 protein) on phagemid virions and to optimize the yield of such phage. To do so, we constructed a novel helper phage, Phaberge, having a conditional deficiency in g3p production. Unlike most other published g3p-deficient helper phage, Phaberge is produced at high levels, 10(11) PFU/ml. As compared to g3p-sufficient helper phage, Phaberge caused a 5-20-fold increase in display level. Another novel feature is that Phaberge only packages insert-containing, not insert-less, phagemid into infectious virions. This should prove useful in preserving quality of phagemid libraries during propagation. In addition, other parameters were also found to affect production of phagemid virions. In particular, the choice of bacterial host cell, phagemid construct and growth temperature had a substantial impact on display levels, but generally no effect on number of phagemid virions produced. In short, we have established a set of parameters that improve production and quality of phagemid virions which we expect to facilitate the isolation of mAbs or other gene products by PDT.
Soltes
Glenn
G
Cangene Corporation, 3403 American Drive, L4V 1T4, Mississauga, Ontario, Canada.
Barker
Heather
H
Marmai
Kristine
K
Pun
Elaine
E
Yuen
Amy
A
Wiersma
Erik J
EJ
D000911:Antibodies, Monoclonal / Q000235:genetics; D001435:Bacteriophages / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D005814:Genes, Viral; D005822:Genetic Vectors; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics*; D019151:Peptide Library; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014771:Virion / Q000235:genetics; D014780:Viruses / Q000235:genetics*
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D011993:Recombinant Fusion Proteins