Construction and exploitation in model experiments of functional selection of a landscape library expressed from a phagemid.
Phage display has evolved during the past 15 years as a powerful technique to select, from libraries of peptides or proteins, binders for various targets or to evolve new functions in proteins. In recent years, the knowledge acquired in phage display technology was exploited to engineer phages as vehicles for receptor-mediated gene delivery. The first vectors generated provided the proof of the concept that development of gene delivery vehicles based on phages was feasible. Results obtained showed that the level of receptor ligand display was an essential factor that determines the efficiency of transduction and suggested that phagemids might be more appropriate than phages for gene delivery. However, due to the limitations of the existing display systems, vectors constructed up to now allowed only relatively low levels of ligand display. The transduction efficiency of these vectors was relatively poor. Here, we describe the construction and optimization of a new phagemid display system that was designed to allow the functional selection of peptides that promote gene delivery from phagemids in a high display format. Peptides are displayed on every copy of the major coat protein pVIII and are expressed from the phagemid itself. The phagemid is rescued as particles by a modified R408 helper phage, deficient in pVIII production. Besides an expression cassette for pVIII, the phagemid also contains the SV40 origin of replication, the GFP gene and the neomycin resistance marker. As a model we constructed a library of octapeptides and showed that the library is amenable to selection on cos-7 cells. Several selection approaches were investigated and a preliminary analysis of the peptides selected was carried out.
Legendre
Daniel
D
Laboratoire de Biochimie Physique et des Biopolymères, Institut des Sciences de la Vie, Université Catholique de Louvain, Place L. Pasteur, 1-1b, 1348, Louvain-la-Neuve, Belgium. legendre@bioc.ucl.ac.be
Fastrez
Jacques
J
D000595:Amino Acid Sequence; D000818:Animals; D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D019556:COS Cells; D003001:Cloning, Molecular; D003673:Defective Viruses / Q000235:genetics; D005819:Genetic Markers; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D006378:Helper Viruses / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D005819:Genetic Markers; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins