title: Discovery of improved EGF agonists using a novel in vitro screening platform.
abstract: Directed evolution is a powerful strategy for protein engineering; however, evolution of pharmaceutical proteins has been limited by the reliance of current screens on binding interactions. Here, we present a method that identifies protein mutants with improved overall cellular efficacy, an objective not feasible with previous approaches. Mutated protein libraries were produced in soluble, active form by means of cell-free protein synthesis. The efficacy of each individual protein was determined at a uniform dosage with a high-throughput protein product assay followed by a cell-based functional assay without requiring protein purification. We validated our platform by first screening mock libraries of epidermal growth factor (EGF) for stimulation of cell proliferation. We then demonstrated its effectiveness by identifying EGF mutants with significantly enhanced mitogenic activity at low concentrations compared to that of wild-type EGF. This is the first report of EGF mutants with improved biological efficacy despite much previous effort. Our platform can be extended to engineer a broad range of proteins, offering a general method to evolve proteins for improved biological efficacy.
authors: Department of Bioengineering, Stanford University, Stanford, CA 94305, USA.
authors: Bertrand H
authors: BH
authors: Lui
authors: Jennifer R
authors: JR
authors: Cochran
authors: James R
authors: JR
authors: Swartz
mesh_terms: D000595:Amino Acid Sequence; D049109:Cell Proliferation; D002478:Cells, Cultured; D004815:Epidermal Growth Factor / Q000819:agonists* / Q000235:genetics / Q000378:metabolism*; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D052816:Foreskin / Q000166:cytology / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000494:pharmacology*; D009154:Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D015202:Protein Engineering; D017386:Sequence Homology, Amino Acid
chemical_list: D050505:Mutant Proteins; D004815:Epidermal Growth Factor; C512478:EGFR protein, human; D066246:ErbB Receptors