Intracellular protein scaffold-mediated display of random peptide libraries for phenotypic screens in mammalian cells

Background: Mammalian cell screens of peptide libraries for changes in cell ular phenotype may identify novel Functional peptides and their cognate bin ding partners, and allow identification of signal transduction network memb ers or proteins important in disease processes. Results: Green fluorescent protein (GFP) peptide libraries with different s tructural biases were tested by retroviral expression in A549 carcinoma cel ls, HUVEC and other cell types. Three different loop replacement libraries, containing 12 or 18 random residues, were compatible with enhanced GFP (EG FP) folding, as was a C-terminally fused random 20-mer library. Library con centrations in A549 cells ranged from ca. 1 to 54 muM. Replacement of loop 3 with known nuclear localization sequence (NLS) peptides. but not with ina ctive mutants, directed EGFP to the nucleus. Microscopy-based screens of th ree different libraries for non-uniform localization revealed novel NLS pep tides, novel variants of a peroxisomal localization motif. a variety of par tial NLS peptides, peptides localized to the nucleolus, and nuclear-exclude d peptides. Conclusions: Peptides can be presented by EGFP in conformations that can fu nctionally interact with cellular constituents in mammalian cells. A phenot ypic screen resulting in the discovery of novel localization peptides that were not cell type-specific suggests that this methodology may be applied t o other screens in cells derived from diseased organisms, and illustrates t he use of intracellular combinatorial peptide chemistry in mammalian cells. (C) 2001 Elsevier Science Ltd. Ali rights reserved.