Novel helper phage design: intergenic region affects the assembly of bacteriophages and the size of antibody libraries.
Phagemid vectors for display of protein/peptides on the surface of filamentous phage utilize a plasmid genome carrying the phage origin of replication, along with the gene fused to a fragment of gene III. Generation of phage particles displaying the fusion protein also requires superinfection of the host bacterium with a helper virus. We describe here the construction of a new gene III mutant of M13 KO7 bacteriophage and compare its ability to act as helper phage with two mutants derived from Fd tet (fKN 16 and fCA 55). Furthermore, we investigate their capability to act as helper phages in SAP selection, where non-infectious helper phage, expressing antibody fragments but not protein 3, can still infect by first reacting with a soluble antigen-protein 3 fusion protein. Gene III mutants were found to be non-infectious, and high titers of infective particles were obtained only when the helper phage was grown in cells harbouring a gene III-containing plasmid. An amplification of the phage titer of 10(6) x was achieved in M13-derived phages, when used for the selection of specific antibody fragments.
Dueñas
M
M
Department of Immunotechnology, Lund University, Sweden.
Borrebaeck
C A
CA
D000906:Antibodies; D017104:Bacteriophage M13 / Q000235:genetics*; D001435:Bacteriophages; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D017353:Gene Deletion; D005814:Genes, Viral; D005822:Genetic Vectors; D006378:Helper Viruses; D007438:Introns; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis*
D000906:Antibodies; D017931:DNA Primers; D011993:Recombinant Fusion Proteins