Expression, solubilization and purification of a human olfactory receptor from Escherichia coli.
Olfactory receptors pertaining to G protein-coupled receptor (GPCR) are integral membrane proteins composed of seven transmembrane spanning domains. It has been reported that these receptor proteins are difficult to overexpress, solubilize, and purify because of their complicated structures and strong hydrophobicity. In this study, full-length human olfactory receptor (hOR) 2AG1 was overexpressed in E. coli as a fusion protein with a glutathione S-transferase (GST) tag mainly as an inclusion body without any mutations or deletions in the gene. This protein was difficult to solubilize with detergents and chaotropic agents, and only N-lauroyl sarcosine was found to be suitable for solubilizing it. In contrast, Triton X-100 was found to solubilize most of the impurity proteins from the insoluble fraction in E. coli. Based on this observation, we applied a simple and efficient column-free method using these two detergents for the purification of the olfactory receptor protein. In this method, the insoluble fraction of the cell lysate was first treated with Triton X-100 to remove impurity proteins. The remaining insoluble fraction was then further treated with N-lauroyl sarcosine to solubilize the olfactory receptor protein. Milligram quantity of the human olfactory receptor was produced. This is the first report to produce full-length of the olfactory receptor from E. coli.
Song
Hyun Seok
HS
School of Chemical and Biological Engineering, Seoul National University, Gwanak-Gu Sillim-Dong San 56-1, Seoul, 151-744, Korea.
Lee
Sang Hun
SH
Oh
Eun Hae
EH
Park
Tai Hyun
TH
D003001:Cloning, Molecular; D003902:Detergents / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D017830:Octoxynol / Q000494:pharmacology; D018035:Receptors, Odorant / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012521:Sarcosine / Q000031:analogs & derivatives / Q000494:pharmacology; D012995:Solubility
D003902:Detergents; D018035:Receptors, Odorant; D011993:Recombinant Fusion Proteins; C025231:sarkosyl; D017830:Octoxynol; D012521:Sarcosine