Quick selection of a chimeric T2 phage that displays active enzyme on the viral capsid.
We designed a bacteriophage T2 system to display proteins fused at the N-terminus of the head protein small outer capsid (SOC) of a T2 phage. To facilitate selection of chimeric phage, a T2 phage encoding the beta-galactosidase gene (betagal) upstream of the soc gene was constructed. The phage, named T2betaGal, produces blue plaques on agar plates containing XGal. Subsequently, a plasmid encoding the target protein upstream of soc was constructed and used to transform E. coli B(E) cells. Transformed cells were infected with T2betaGal and homologous recombination between phage DNA and the plasmid resulted in a chimeric phage that produced transparent plaques due to the excision of the betagal gene. Chitosanase of Bacillus sp. strain K17 (ChoK), consisting of 453 amino acids, was used as a model target protein. Recombinant T2 phage that produced ChoK was named T2ChoK. T2ChoK was produced from T2betaGal at a recombination frequency of about 0.1%. On the other hand, the value for T2betaGal produced from wild-type T2 was 0.001 %. This new system enables us to select recombinant phage very quickly and accurately. The number of molecules of ChoK was calculated at 14.7 per single phage. Latent period and burst size were estimated for the chimeric phages.
Tanji
Yasunori
Y
Department of Bioengineering, Tokyo Institute of Technology, Yokohama 226-8501, Japan. ytanji@bio.titech.ac.jp
Murofushi
Keita
K
Miyanaga
Kazuhiko
K
D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D017122:Bacteriophage T4 / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D001709:Biotechnology; D002213:Capsid / Q000201:enzymology; D002678:Chimera / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005814:Genes, Viral; D006026:Glycoside Hydrolases / Q000235:genetics; D007763:Lac Operon; D019151:Peptide Library; D014170:Transformation, Genetic; D001616:beta-Galactosidase / Q000235:genetics
D004274:DNA, Recombinant; D019151:Peptide Library; D006026:Glycoside Hydrolases; C019868:chitosanase; D001616:beta-Galactosidase