Biochemical Characterization of Full-Length Oncogenic BRAF together with Molecular Dynamics Simulations Provide Insight into the Activation and Inhibition Mechanisms of RAF Kinases.

The most prevalent BRAF mutation, V600E, occurs frequently in melanoma and other cancers. Although extensive progress has been made toward understanding the biology of RAF kinases, little in vitro characterization of full-length BRAF is available. Herein, we show the successful purification of active, full-length BRAF from mammalian cells for in vitro experiments.

Mechanism of BRAF Activation through Biochemical Characterization of the Recombinant Full-Length Protein.

BRAF kinase plays an important role in mitogen-activated protein kinase (MAPK) signaling and harbors activating mutations in about half of melanomas and in a smaller percentage in many other cancers. Despite its importance, few in vitro studies have been performed to characterize the biochemical properties of full-length BRAF. Herein, a strategy to generate an active, intact form of BRAF protein suitable for in vitro enzyme kinetics is described.

In Vitro Enzyme Kinetics Analysis of EGFR.

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.