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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, Trition 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. ; This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (No. 2009-0080242). This work was also partially supported by the System 2010 program of the Ministry of Knowledge Economy.

Sensing smell is a highly complex biological process, and characterizing and mimicking the interaction between the olfactory receptor (OR) protein and its ligands is extremely challenging. Herein, we report a highly sensitive and selective human nose-like nanobioelectronic nose (nbe-nose), which responds to gaseous odorants sensitively and selectively, has a signal specificity pattern similar to that in the cellular signal transduction pathway, and maintains an antagonistic behavior similar to the human nose. The human olfaction mechanism was mimicked by using carboxylated polypyrrole nanotubes (CPNTs) functionalized with human OR protein. The nbe-nose was able to detect gaseous odorants at a concentration as low as 0.02 parts-per-trillion (ppt), which was comparable to a highly trained, human expert's nose. The nbe-nose can be used scientifically for smell mechanism studies. It can be also applied to various fields that rely on smell monitoring for industrial and public purposes. (C) 2011 Elsevier Ltd. All rights reserved. ; This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Ministry of Education, Science and Technology (MEST) (No. 2011-0000331, No. 2011K000682, No. 2011-0020984). This research was supported by the WCU (World Class University) program through the NRF, funded by the MEST (R31-10013), and funded by an NRF grant through the Korean government (MEST) (No. 2011-0017125). ; OAIID:oai:osos.snu.ac.kr:snu2012-01/102/0000002410/2 ; SEQ:2 ; PERF_CD:SNU2012-01 ; EVAL_ITEM_CD:102 ; USER_ID:0000002410 ; ADJUST_YN:Y ; EMP_ID:A002014 ; DEPT_CD:458 ; CITE_RATE:7.404 ; FILENAME:Mimicking the human smell sensing mechanism with an artificial nose platform.pdf ; DEPT_NM:화학생물공학부 ; EMAIL:thpark@snu.ac.kr ; SCOPUS_YN:Y ; CONFIRM:Y

In this study, we developed a human taste receptor protein, hTAS2R38-functionalized carboxylated polypyrrole nanotube (CPNT)-field effect transistor (PET) as a nanobioelectronic tongue (nbe-tongue) that displayed human-like performance with high sensitivity and selectivity. Taster type (PAV) and nontaster type (AVI) hTAS2R38s were expressed in Escherichia coli (E. coli) at a high level and immobilized on a CPNT-FET sensor platform. Among the various tastants examined, PAV-CPNT-FET exclusively responded to target bitterness compounds, phenylthiocarbamide (PTC) and propylthiouracil (PROP), with high sensitivity at concentrations as low as 1 fM. However, no significant changes were observed in the AVI-CPNT-FET in response to the target bitter tastants. This nbe-tongue exhibited different bitter-taste perception of compounds containing thiourea (N-C=S) moieties such as PTC, PROP, and antithyroid toxin in vegetables, which corresponded to the haplotype of hTAS2R38 immobilized on CPNTs. This correlation with the type of receptor is very similar to the human taste system. Thus, the artificial taste sensor developed in this study allowed for the efficient detection of target tastants in mixture and real food sample with a human-like performance and high sensitivity. Furthermore, our nbe-tongue could be utilized as a substitute for cell-based assays and to better understand the mechanisms of human taste. ; This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Ministry of Education, Science and Technology (MEST) (Grant Nos. 2012-0000144, 2012K001365, 2012-0006564). This research was supported by the WCU (World Class University) program through the NRF, funded by the MEST (R31-10013), and funded by an NRF grant through the Korean government (MEST) (No. 2011-0017125). ; OAIID:oai:osos.snu.ac.kr:snu2013-01/102/0000002410/4 ; SEQ:4 ; PERF_CD:SNU2013-01 ; EVAL_ITEM_CD:102 ; USER_ID:0000002410 ; ADJUST_YN:Y ; EMP_ID:A002014 ; DEPT_CD:458 ; CITE_RATE:13.198 ; FILENAME:human taste receptor-functionalized.pdf ; DEPT_NM:화학생물공학부 ; EMAIL:thpark@snu.ac.kr ; SCOPUS_YN:Y ; CONFIRM:Y