Purification and functional reconstitution of human olfactory receptor expressed in Escherichia coli

Heehong Yang, Hyun Seok Song, Sae Ryun Ahn, Tai Hyun Park

Research output: Contribution to journalArticlepeer-review

18 Scopus citations

Abstract

Olfactory receptors (ORs), belonging to the Gprotein coupled receptor (GPCR) family, are very difficult to be overexpressed, purified and reconstituted because of their hydrophobicity and complicated structure. These receptors bind to their specific ligands, thus their specificity is very useful for application as a bioelectronic nose. Furthermore, highly purified and well-reconstituted human olfactory receptor (hOR) can be used in various fields, such as in protein-interaction research, drug screening, and analysis of the hOR structure. In this study, human olfactory receptor, hOR2AG1, was produced with high purity and functionally reconstituted in detergent micelles. The hOR2AG1 was overexpressed in Escherichia coli (E. coli) with glutathione S-transferase (GST) and 6xHis-tag as an inclusion body. The hOR2AG1 fusion protein was solubilized in buffer containing sodium dodecyl sulfate (SDS) and purified using Ni-NTA chromatography. The GST domain was removed using proteolytic cleavage before elution from the column. After purification, the hOR2AG1 was successfully reconstituted using nonionic detergents and methyl-ß-cyclodextrin. Finally highly purified and well-reconstituted hOR was obtained, and its biological characteristics were confirmed by using circular dichroism (CD) spectrum and tryptophan fluorescence assay. These results can be applied to develop protein-based sensing systems including a bioelectronic nose and to analyze the native hOR structure using solid-state NMR, X-ray crystallography, or neutron scattering.

Original languageEnglish
Pages (from-to)423-430
Number of pages8
JournalBiotechnology and Bioprocess Engineering
Volume20
Issue number3
DOIs
StatePublished - 25 Jun 2015

Bibliographical note

Publisher Copyright:
© 2015, The Korean Society for Biotechnology and Bioengineering and Springer-Verlag Berlin Heidelberg.

Keywords

  • Escherichia coli
  • human olfactory receptor
  • ligand binding
  • purification
  • reconstitution

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