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Directed evolution of the epidermal growth factor receptor extracellular domain for expression in yeast
Author(s) -
Kim YongSung,
Bhandari Rashna,
Cochran Jennifer R.,
Kuriyan John,
Wittrup K. Dane
Publication year - 2005
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.20618
Subject(s) - autophosphorylation , epidermal growth factor receptor , epidermal growth factor , yeast , saccharomyces cerevisiae , mutant , extracellular , glycoprotein , microbiology and biotechnology , erbb3 , biology , intracellular , receptor , monoclonal antibody , biochemistry , cell surface receptor , antibody , kinase , genetics , gene , protein kinase a
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. Proteins 2006. © 2005 Wiley‐Liss, Inc.