title: A novel strategy for the functional cloning of enzymes using filamentous phage display: the case of nucleotidyl transferases.
abstract: 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.
authors.affiliation: Unité de Chimie Organique, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris 15, France.
authors.forename: Erika
authors.initials: E
authors.lastname: Brunet
authors.forename: Camille
authors.initials: C
authors.lastname: Chauvin
authors.forename: Valérie
authors.initials: V
authors.lastname: Choumet
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Jestin
mesh_terms: 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
chemical_list: 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