Abstract 4275: Production and characterization of ErbB1 (EGFR) associated with nanolipoprotein particles via cell-based and cell-free methods.

Abstract

Abstract The four members of the ErbB family of mammalian receptor tyrosine kinases are essential for the development and maintenance of a variety of tissues. Aberrant activation or over-expression of these receptors leads to the progression of epithelial and brain tumors. An understanding of the mechanistic and structural changes that occur to activate the intracellular receptor tyrosine kinase domain via extracellular ligand binding and receptor dimerization is desirable for identifying specific and efficient methods to inhibit signal transduction. Previous methods to study this process achieved low success due to low protein yield, poor water solubility of the membrane protein, and the large size of receptor. We report progress on overcoming these technical hurdles by forming the ErbB family members in nanolipoprotein particles (NLPs), which are ∼20 nm disc-shaped cell membrane analogs. Here we compare an optimized cell-based method with a novel cell-free method to produce NLPs containing the ErbB1 receptor, also called EGFR. The cell-produced ErbB1-NLPs retain kinase activity compared to cell-free produced material with higher yields. However, the cell-free produced receptors have regions of correct folding and can be produced in less than a day. The cell-free produced constructs can be labeled conveniently with unnatural amino acids to enable characterization of dimerization interactions. These two receptor production systems can be utilized synergistically to enable new studies of conformational changes associated with ligand binding or protein mutations, as well as to identify small molecule inhibitors of these receptors. Citation Format: Tiffany M. Scharadin, Christina Takanishi, Wei He, Matthew Saldano, Kermit L. Carraway, Matthew A. Coleman, Paul T. Henderson. Production and characterization of ErbB1 (EGFR) associated with nanolipoprotein particles via cell-based and cell-free methods. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4275. doi:10.1158/1538-7445.AM2013-4275