A cost-effective protocol for the over-expression and purification of fully-functional and more stable Erwinia chrysanthemi ligand-gated ion channel.

Autor: Elberson BW; Department of Cell Physiology and Molecular Biophysics and Center for Membrane Protein Research, Texas Tech University Health Sciences Center, 3601 4th Street STOP 6551, Lubbock, TX 79430, USA., Whisenant TE; Department of Cell Physiology and Molecular Biophysics and Center for Membrane Protein Research, Texas Tech University Health Sciences Center, 3601 4th Street STOP 6551, Lubbock, TX 79430, USA., Cortes DM; Department of Cell Physiology and Molecular Biophysics and Center for Membrane Protein Research, Texas Tech University Health Sciences Center, 3601 4th Street STOP 6551, Lubbock, TX 79430, USA., Cuello LG; Department of Cell Physiology and Molecular Biophysics and Center for Membrane Protein Research, Texas Tech University Health Sciences Center, 3601 4th Street STOP 6551, Lubbock, TX 79430, USA. Electronic address: luis.cuello@ttuhsc.edu.
Jazyk: angličtina
Zdroj: Protein expression and purification [Protein Expr Purif] 2017 May; Vol. 133, pp. 177-186. Date of Electronic Publication: 2017 Mar 07.
DOI: 10.1016/j.pep.2017.03.006
Abstrakt: The Erwinia chrysanthemi ligand-gated ion channel, ELIC, is considered an excellent structural and functional surrogate for the whole pentameric ligand-gated ion channel family. Despite its simplicity, ELIC is structurally capable of undergoing ligand-dependent activation and a concomitant desensitization process. To determine at the molecular level the structural changes underlying ELIC's function, it is desirable to produce large quantities of protein. This protein should be properly folded, fully-functional and amenable to structural determinations. In the current paper, we report a completely new protocol for the expression and purification of milligram quantities of fully-functional, more stable and crystallizable ELIC. The use of an autoinduction media and inexpensive detergents during ELIC extraction, in addition to the high-quality and large quantity of the purified channel, are the highlights of this improved biochemical protocol.
(Copyright © 2017 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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