Fluorescent T7 display phages obtained by translational frameshift.
Lytic phages form a powerful platform for the display of large cDNA libraries and offer the possibility to screen for interactions with almost any substrate. To visualize these interactions directly by fluorescence microscopy, we constructed fluorescent T7 phages by exploiting the flexibility of phages to incorporate modified versions of its capsid protein. By applying translational frameshift sequences, helper plasmids were constructed that expressed a fixed ratio of both wild-type capsid protein (gp10) and capsid protein fused to enhanced yellow fluorescent protein (EYFP). The frameshift sequences were inserted between the 3' end of the capsid gene and the sequence encoding EYFP. Fluorescent fusion proteins are only formed when the ribosome makes a -1 shift in reading frame during translation. Using standard fluorescence microscopy, we could sensitively monitor the enrichment of specific binders in a cDNA library displayed on fluorescent T7 phages. The perspectives of fluorescent display phages in the fast emerging field of single molecule detection and sorting technologies are discussed.
Slootweg
Erik J
EJ
Laboratory of Molecular Recognition and Antibody Technology, Wageningen University Binnenhaven 5, 6709 PD, Wageningen, The Netherlands. erik.slootweg@wur.nl
Keller
Hans J H G
HJ
Hink
Mark A
MA
Borst
Jan Willem
JW
Bakker
Jaap
J
Schots
Arjen
A
D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics*; D017123:Bacteriophage T7 / Q000235:genetics*; D036022:Capsid Proteins / Q000032:analysis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes / Q000032:analysis; D018965:Frameshifting, Ribosomal; D005822:Genetic Vectors; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics*; D008856:Microscopy, Fluorescence; D019151:Peptide Library; D010957:Plasmids / Q000235:genetics; D025941:Protein Interaction Mapping / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D013696:Temperature; D014771:Virion / Q000737:chemistry
D001426:Bacterial Proteins; D036022:Capsid Proteins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; C104967:gene 10 protein, phage T7; C054562:yellow fluorescent protein, Bacteria