In vitro enzyme kinetics analysis of EGFR

Abstract

The epidermal growth factor receptor (EGFR) is an essential receptor tyrosine kinase (RTK) that regulates cell proliferation and differentiation, and its abnormal activation contributes to a variety of human cancers. EGFR is activated by the binding of growth factors, such as epidermal growth factor (EGF), resulting in the formation of an asymmetric dimer in which one EGFR molecule allosterically activates the other molecule. This chapter provides a detailed protocol to purify and characterize full-length EGFR bound with EGF. This protocol facilitates the expression and preparation of full-length EGFR in transiently transfected mammalian cell culture. A method is also presented for quantitative evaluation of EGFR enzyme activity.