Synthesis and biochemical characterization of EGF receptor in a water-soluble membrane model system

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.

Single-Molecule Fluorescence Detection of the Epidermal Growth Factor Receptor in Membrane Discs

“…Evidence confirming the presence of resonance energy transfer between SS594 on EGFR and Alexa-647 on the Y1068 antibody were obtained in phosphorylation buffer from the decrease in the average fluorescence lifetime of the donor in the presence of the acceptor, which reduced by 4.9 %, 8.3 % and 36.4 % when EGFR: Y1068 antibody molar ratios of 2:1, 1: 2 and 1: 20 were tested, respectively (Figure 4c). The increase in FRET efficiency indicates that antibody binding occurs within the intracellular domain and is consistent with previous work that demonstrates EGFR functionality via ligand interaction when contained within supported membrane mimetics composed of DMPC phospholipids [45,58]. The combination of FCS and FRET measurements are consistent with antibody binding to the intracellular compartment of functional EGFR and thus emphasize the potential of EGFR NLPs as a versatile platform.…”

“…The increase in FRET efficiency indicates that antibody binding occurs within the intracellular domain and is consistent with previous work that demonstrates EGFR functionality via ligand interaction when contained within supported membrane mimetics composed of DMPC phospholipids. 45,57 The combination of FCS and FRET measurements are consistent with antibody binding to the intracellular compartment of functional EGFR and thus emphasize the potential of EGFR NLPs as a versatile platform.…”

“…While in vitro analysis of monomeric EGFR provides a means to overcome complexity, recent evidence points to ligand-induced multimerization of EGFR [62], the assembly of which optimally organizes kinase-active dimers for auto-phosphorylation [63]. In this regard, the utility of the cell-free expression platform presented here may be extended to enable active dimers and multimers of EGFR to be embedded within single NLPs [58] of defined diameter and radius of curvature [64,65]. Relatively low expression yields (< 0.5 ng/μL) do mean conventional activity assays such as Western Blotting are ineffective, but nevertheless, the application of single-molecule techniques are rapidly evolving to enable such systems to be spectroscopically accessed.…”

Cell-Free Co-Translational Approaches for Producing Mammalian Receptors: Expanding the Cell-Free Expression Toolbox Using Nanolipoproteins

“…A bigger challenge is that many other cellular components that associate with ERBB receptors, for example other ERBB family member proteins, are often co-purified with the target protein. Cell-based and insect cell expression systems combined with NLPs have been used to isolate ERBB receptors (Mi et al, 2008; Scharadin et al, 2017). However, overexpression relied on fusion tags and detergent solubilization.…”

Lipid and Protein Transfer between Nanolipoprotein Particles and Supported Lipid Bilayers

“…This is compelling motivation for development of broadly applicable, lipid-based characterization platforms, especially amid growing evidence that lipids not only stabilize, but regulate membrane protein activity. , MP–NLPs offer one modality for examining properties of membrane proteins embedded in lipid bilayers. Several studies using NLPs to examine protein activity, oligomerization, , and diffusion dynamics , have been reported.…”