title: Directed evolution of the epidermal growth factor receptor extracellular domain for expression in yeast.
abstract: The extracellular domain of epidermal growth factor receptor (EGFR-ECD) has been engineered through directed evolution and yeast surface display using conformationally-specific monoclonal antibodies (mAbs) as screening probes for proper folding and functional expression in Saccharomyces cerevisiae. An EGFR mutant with four amino acid changes exhibited binding to the conformationally-specific mAbs and human epidermal growth factor, and showed increased soluble secretion efficiency compared with wild-type EGFR. Full-length EGFR containing the mutant EGFR-ECD was functional, as assayed by EGF-dependent autophosphorylation and intracellular MAPK signaling in mammalian cells, and was expressed and localized at the plasma membrane in yeast. This approach should enable engineering of other complex mammalian receptor glycoproteins in yeast for genetic, structural, and biophysical studies.
authors.affiliation: Division of Biological Engineering, Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Yong-Sung
authors.initials: YS
authors.lastname: Kim
authors.forename: Rashna
authors.initials: R
authors.lastname: Bhandari
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Cochran
authors.forename: John
authors.initials: J
authors.lastname: Kuriyan
authors.forename: K Dane
authors.initials: KD
authors.lastname: Wittrup
mesh_terms: D019943:Amino Acid Substitution; D000911:Antibodies, Monoclonal; D003001:Cloning, Molecular; D004815:Epidermal Growth Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D016366:Open Reading Frames; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae
chemical_list: D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor