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

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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.

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Authors and Affiliations

  1. School of Chemical and Biological Engineering, Seoul National University, Seoul, 151-742, Korea

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

  2. Harvard-MIT Division of Health Science and Technology (HST), Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA, 02139, USA

    Hyun Seok Song

  3. Advanced Institutes of Convergence Technology, Suwon, 443-270, Korea

    Tai Hyun Park

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  1. Heehong Yang
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Correspondence to Tai Hyun Park.

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Yang, H., Song, H.S., Ahn, S.R. et al. Purification and functional reconstitution of human olfactory receptor expressed in Escherichia coli . Biotechnol Bioproc E 20, 423–430 (2015). https://doi.org/10.1007/s12257-014-0897-4

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  • DOI: https://doi.org/10.1007/s12257-014-0897-4

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