Abstract
ErbB (Erythroblastic Leukemia Viral Oncogene Homolog) receptor tyrosine kinases are critical for tissue development and maintenance, and frequently become oncogenic when mutated or overexpressed. In vitro analysis of ErbB receptor kinases can be difficult because of their large size and poor water solubility. Here we report improved production and assembly of the correctly folded full-length EGF receptor (EGFR) into nanolipoprotein particles (NLPs). NLPs are ~10 nm in diameter discoidal cell membrane mimics composed of apolipoproteins surrounding a lipid bilayer. NLPs containing EGFR were synthesized via incubation of baculovirus-produced recombinant EGFR with apolipoprotein and phosphoplipids under conditions that favor self-assembly. The resulting EGFR-NLPs were the correct size, formed dimers and multimers, had intrinsic autophosphorylation activity, and retained the ability to interact with EGFR-targeted ligands and inhibitors consistent with previously-published in vitro binding affinities. We anticipate rapid adoption of EGFR-NLPs for structural studies of full-length receptors and drug screening, as well as for the in vitro characterization of ErbB heterodimers and disease-relevant mutants.
Highlights
The four members of the ErbB family of receptor tyrosine kinases include EGF receptor (EGFR) or ErbB1/ HER1, ErbB2/HER2, ErbB3/HER3, ErbB4/HER4
Insect cells were infected with a multiplicity of infection (MOI) of 10 of control or EGFR baculovirus for 48 hours with and without additional EGF followed by detection of total and phosphorylated EGFR by Western Blot (Fig 1B)
EGFR was isolated from the insect cells after a 48-hour infection with an MOI of 10 of EGFR baculovirus via FLAG-purification from total cell lysates, which resulted in a band whose migration on an SDS-PAGE gel was consistent with full-length EGFR as shown by immunoblotting and Coomassie (Fig 1D and 1E)
Summary
The four members of the ErbB family of receptor tyrosine kinases include EGFR or ErbB1/ HER1 (human epidermal growth factor receptor), ErbB2/HER2, ErbB3/HER3, ErbB4/HER4. Each ErbB receptor consists of a large extracellular ligand-binding domain, a single transmembrane segment, an intracellular juxtamembrane segment, a tyrosine kinase domain, and a carboxy-terminal tail. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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