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Title: 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. Lastly, 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.

Authors:
 [1];  [2];  [3];  [4];  [5];  [4];  [6];  [7]; ORCiD logo [8]
  1. Univ. of California, Sacramento, CA (United States). Dept. of Internal Medicine, Division of Hematology Oncology
  2. Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
  3. Univ. of California, Davis, CA (United States)
  4. Univ. of California, Davis, CA (United States). School of Veterinary Medicine, Molecular Biosciences
  5. Univ. of California, Sacramento, CA (United States). School of Medicine, Biochemistry and Molecular Medicine
  6. Univ. of California, Sacramento, CA (United States). School of Medicine, Biochemistry and Molecular Medicine; Univ. of California, Sacramento, CA (United States). Comprehensive Cancer Center
  7. Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Univ. of California, Sacramento, CA (United States). Comprehensive Cancer Center; Univ. of California, Sacramento, CA (United States). School of Medicine, Dept. of Radiation Oncology
  8. Univ. of California, Sacramento, CA (United States). Dept. of Internal Medicine, Division of Hematology Oncology; Univ. of California, Sacramento, CA (United States). Comprehensive Cancer Center
Publication Date:
Research Org.:
Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23); National Institutes of Health (NIH)
OSTI Identifier:
1361453
Alternate Identifier(s):
OSTI ID: 1395310
Grant/Contract Number:  
AC52-07NA27344
Resource Type:
Journal Article: Published Article
Journal Name:
PLoS ONE
Additional Journal Information:
Journal Volume: 12; Journal Issue: 6; Journal ID: ISSN 1932-6203
Publisher:
Public Library of Science
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; Insects; Cell membranes; Dimers (Chemical physics); Enzyme-linked immunoassays; Immunoblotting; Immunostaining; Phosphorylation; Tyrosine kinases

Citation Formats

Scharadin, Tiffany M., He, Wei, Yiannakou, Yianni, Tomilov, Alexey A., Saldana, Matthew, Cortopassi, Gino A., Carraway, Kermit L., Coleman, Matthew A., and Henderson, Paul T. Synthesis and biochemical characterization of EGF receptor in a water-soluble membrane model system. United States: N. p., 2017. Web. doi:10.1371/journal.pone.0177761.
Scharadin, Tiffany M., He, Wei, Yiannakou, Yianni, Tomilov, Alexey A., Saldana, Matthew, Cortopassi, Gino A., Carraway, Kermit L., Coleman, Matthew A., & Henderson, Paul T. Synthesis and biochemical characterization of EGF receptor in a water-soluble membrane model system. United States. doi:10.1371/journal.pone.0177761.
Scharadin, Tiffany M., He, Wei, Yiannakou, Yianni, Tomilov, Alexey A., Saldana, Matthew, Cortopassi, Gino A., Carraway, Kermit L., Coleman, Matthew A., and Henderson, Paul T. Tue . "Synthesis and biochemical characterization of EGF receptor in a water-soluble membrane model system". United States. doi:10.1371/journal.pone.0177761.
@article{osti_1361453,
title = {Synthesis and biochemical characterization of EGF receptor in a water-soluble membrane model system},
author = {Scharadin, Tiffany M. and He, Wei and Yiannakou, Yianni and Tomilov, Alexey A. and Saldana, Matthew and Cortopassi, Gino A. and Carraway, Kermit L. and Coleman, Matthew A. and Henderson, Paul T.},
abstractNote = {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. Lastly, 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.},
doi = {10.1371/journal.pone.0177761},
journal = {PLoS ONE},
number = 6,
volume = 12,
place = {United States},
year = {Tue Jun 06 00:00:00 EDT 2017},
month = {Tue Jun 06 00:00:00 EDT 2017}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at 10.1371/journal.pone.0177761

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Works referenced in this record:

Different Apolipoproteins Impact Nanolipoprotein Particle Formation
journal, November 2007

  • Chromy, Brett A.; Arroyo, Erin; Blanchette, Craig D.
  • Journal of the American Chemical Society, Vol. 129, Issue 46, p. 14348-14354
  • DOI: 10.1021/ja074753y