A novel helper phage that improves phage display selection efficiency by preventing the amplification of phages without recombinant protein.
Phage display is a widely used technology for the isolation of peptides and proteins with specific binding properties from large libraries of these molecules. A drawback of the common phagemid/helper phage systems is the high infective background of phages that do not display the protein of interest, but are propagated due to non-specific binding to selection targets. This and the enhanced growth rates of bacteria harboring aberrant phagemids not expressing recombinant proteins leads to a serious decrease in selection efficiency. Here we describe a VCSM13-derived helper phage that circumvents this problem, because it lacks the genetic information for the infectivity domains of phage coat protein pIII. Rescue of a library with this novel CT helper phage yields phages that are only infectious when they contain a phagemid-encoded pIII-fusion protein, since phages without a displayed protein carry truncated pIII only and are lost upon re-infection. Importantly, the CT helper phage can be produced in quantities similar to the VCSM13 helper phage. The superiority of CT over VCSM13 during selection was demonstrated by a higher percentage of positive clones isolated from an antibody library after two selection rounds on a complex cellular target. We conclude that the CT helper phage considerably improves the efficiency of selections using phagemid-based protein libraries.
Kramer
R Arjen
RA
Crucell Holland B.V., PO Box 2048, 2301CA, Leiden, The Netherlands.
Cox
Freek
F
van der Horst
Marieke
M
van der Oudenrijn
Sonja
S
Res
Pieter C M
PC
Bia
Judith
J
Logtenberg
Ton
T
de Kruif
John
J
D001435:Bacteriophages / Q000235:genetics*; D036022:Capsid Proteins; D004268:DNA-Binding Proteins / Q000235:genetics; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics; D009154:Mutation; D021141:Nucleic Acid Amplification Techniques; D019151:Peptide Library; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D020298:U937 Cells; D014760:Viral Fusion Proteins / Q000235:genetics
D036022:Capsid Proteins; D004268:DNA-Binding Proteins; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D014760:Viral Fusion Proteins