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Purification and Functional Reconstitution of Human Olfactory Receptor Expressed in Escherichia coli

Cited 10 time in Web of Science Cited 10 time in Scopus
Authors
Yang, Heehong; Song, Hyun Seok; Ahn, Sae Ryun; Park, Tai Hyun
Issue Date
2015
Publisher
Korean Society for Biotechnology and Bioengineering
Springer
Citation
Biotechnology and Bioprocess Engineering, vol.20, pp. 423-430
Keywords
human olfactory receptorpurificationreconstitutionEscherichia coliligand binding
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-β-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.
Language
English
URI
http://hdl.handle.net/10371/95453
DOI
https://doi.org/10.1007/s12257-014-0897-4
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College of Engineering/Engineering Practice School (공과대학/대학원)Dept. of Chemical and Biological Engineering (화학생물공학부)Journal Papers (저널논문_화학생물공학부)
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