Domain-level antibody epitope mapping through yeast surface display of epidermal growth factor receptor fragments.
Individual domains from extracellular proteins are potential reagents for biochemical characterization of ligand/receptor interactions and antibody binding sites. Here, we describe an approach for the identification and characterization of stable protein domains with cell surface display in Saccharomyces cerevesiae, using the epidermal growth factor receptor (EGFR) as a model system. Fragments of the EGFR were successfully expressed on the yeast cell surface. The yeast-displayed EGFR fragments were properly folded, as assayed with conformationally specific EGFR antibodies. Heat denaturation of yeast-displayed EGFR proteins distinguished between linear and conformational antibody epitopes. In addition, EGFR-specific antibodies were categorized based on their ability to compete ligand binding, which has been shown to have therapeutic implications. Overlapping EGFR antibody epitopes were determined based on a fluorescent competitive binding assay. Yeast surface display is a useful method for identifying stable folded protein domains from multidomain extracellular receptors, as well as characterizing antibody binding epitopes, without the need for soluble protein expression and purification.
Cochran
Jennifer R
JR
Department of Chemical Engineering and Division of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Kim
Yong-Sung
YS
Olsen
Mark J
MJ
Bhandari
Rashna
R
Wittrup
K Dane
KD
D000818:Animals; D000906:Antibodies / Q000032:analysis*; D000915:Antibody Affinity; D000954:Antigens, Surface / Q000276:immunology; D001667:Binding, Competitive; D018604:Epitope Mapping / Q000379:methods*; D066246:ErbB Receptors / Q000276:immunology*; D006801:Humans; D007118:Immunoassay / Q000379:methods; D010446:Peptide Fragments / Q000276:immunology*; D017434:Protein Structure, Tertiary / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000276:immunology*
D000906:Antibodies; D000954:Antigens, Surface; D010446:Peptide Fragments; D066246:ErbB Receptors