A novel strategy for the functional cloning of enzymes using filamentous phage display: the case of nucleotidyl transferases.
In vitro selections for catalytic activity have been designed for the isolation of genes encoding enzymes from libraries of proteins displayed on filamentous phages. The proteins are generally expressed as C-terminal fusions with the N-terminus of the minor coat protein p3 for display on phages. As full-length cDNAs generally contain several stop codons near their 3' end, this approach cannot be used for their expression on the surface of phages. Here we show that in vitro selection for catalytic activity is compatible with a system for expression of proteins as N-terminal fusions on the surface of bacteriophages. It is highlighted for the Stoffel fragment of Taq DNA polymerase I and makes use of (p3-Jun/Fos-Stoffel fragment) fusions. The efficiency of the selection is measured by an enrichment factor found to be about 55 for a phage polymerase versus a phage not expressing a polymerase. This approach could provide a method for the functional cloning of nucleotidyl transferases from cDNA libraries using filamentous phage display.
Brunet
Erika
E
Unité de Chimie Organique, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris 15, France.
Chauvin
Camille
C
Choumet
Valérie
V
Jestin
Jean-Luc
JL
D002213:Capsid / Q000235:genetics; D036022:Capsid Proteins; D003001:Cloning, Molecular / Q000379:methods*; D009713:Nucleotidyltransferases / Q000235:genetics; D019151:Peptide Library; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis; D019914:Taq Polymerase / Q000235:genetics* / Q000378:metabolism
D036022:Capsid Proteins; D019151:Peptide Library; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D011993:Recombinant Fusion Proteins; D009713:Nucleotidyltransferases; D019914:Taq Polymerase