Creation and biophysical characterization of a high-affinity, monomeric EGF receptor ectodomain using fluorescent proteins

Abstract

X-ray structural studies revealed two conformations of the epidermal growth factor receptor (EGFR) ectodomain (ECD): a compact, tethered conformation in the absence of EGF and an untethered or extended conformation in the presence of EGF. An EGFR-ECD derivative with a monomeric red fluorescent protein (mRFP) at the N-terminus and an enhanced green fluorescent protein (eGFP) at the C-terminus (dual-tag-EGFR-ECD) was created and characterized. The dual-tag-EGFR-ECD construct was shown to have high affinity (nanomolar range) for both EGF and EGFR monoclonal antibody (mAb528). The dual-tag-EGFR-ECD was further characterized by fluorescence-detected analytical ultracentrifugation, lifetime FRET, ..