title: Intracellular protein scaffold-mediated display of random peptide libraries for phenotypic screens in mammalian cells.
abstract: BACKGROUND
  Mammalian cell screens of peptide libraries for changes in cellular phenotype may identify novel functional peptides and their cognate binding partners, and allow identification of signal transduction network members or proteins important in disease processes.
  
  
  RESULTS
  Green fluorescent protein (GFP) peptide libraries with different structural biases were tested by retroviral expression in A549 carcinoma cells, HUVEC and other cell types. Three different loop replacement libraries, containing 12 or 18 random residues, were compatible with enhanced GFP (EGFP) folding, as was a C-terminally fused random 20-mer library. Library concentrations in A549 cells ranged from ca. 1 to 54 microM. Replacement of loop 3 with known nuclear localization sequence (NLS) peptides, but not with inactive mutants, directed EGFP to the nucleus. Microscopy-based screens of three different libraries for non-uniform localization revealed novel NLS peptides, novel variants of a peroxisomal localization motif, a variety of partial NLS peptides, peptides localized to the nucleolus, and nuclear-excluded peptides.
  
  
  CONCLUSIONS
  Peptides can be presented by EGFP in conformations that can functionally interact with cellular constituents in mammalian cells. A phenotypic screen resulting in the discovery of novel localization peptides that were not cell type-specific suggests that this methodology may be applied to other screens in cells derived from diseased organisms, and illustrates the use of intracellular combinatorial peptide chemistry in mammalian cells.
authors.affiliation: Rigel Inc., 240 E. Grand Ave., South San Francisco, CA 94080, USA.
authors.forename: B
authors.initials: B
authors.lastname: Peelle
authors.forename: J
authors.initials: J
authors.lastname: Lorens
authors.forename: W
authors.initials: W
authors.lastname: Li
authors.forename: J
authors.initials: J
authors.lastname: Bogenberger
authors.forename: D G
authors.initials: DG
authors.lastname: Payan
authors.forename: D C
authors.initials: DC
authors.lastname: Anderson
mesh_terms: D000818:Animals; D002466:Cell Nucleolus / Q000737:chemistry / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008322:Mammals / Q000378:metabolism; D008403:Mass Screening / Q000379:methods; D016254:Mutagenesis, Insertional / Q000235:genetics; D019913:Nuclear Localization Signals / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019151:Peptide Library; D020675:Peroxisomes / Q000737:chemistry / Q000378:metabolism; D010641:Phenotype; D017510:Protein Folding; D021381:Protein Transport / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured / Q000378:metabolism
chemical_list: D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D019151:Peptide Library; D049452:Green Fluorescent Proteins