• Franziska Noe Deutsche Forschungsanstalt fuer Lebensmittelchemie - Leibniz Institut
  • Tim Frey Deutsche Forschungsanstalt fuer Lebensmittelchemie - Leibniz Institut
  • Julia Fiedler Deutsche Forschungsanstalt fuer Lebensmittelchemie - Leibniz Institut
  • Christiane Geithe Deutsche Forschungsanstalt fuer Lebensmittelchemie - Leibniz Institut
  • Bettina Nowak Deutsche Forschungsanstalt fuer Lebensmittelchemie - Leibniz Institut
  • Dietmar Krautwurst Deutsche Forschungsanstalt fuer Lebensmittelchemie - Leibniz Institut



The assignment of cognate odorant/agonist pairs is a prerequisite for an understanding of odorant coding at the receptor level. However, the identification of new ligands for odorant receptors in cell-based assays has been challenging, due to their individual and rather sub-optimal plasma membrane expression, as compared with other G protein-coupled receptors. Accessory proteins, such as the chaperone RTP1S, or Ric8b, have improved the surface expression of at least a portion of odorant receptors. Typically, recombinant odorant receptors carry N-terminal tags, which proved helpful for their functional membrane expression. The most common tag is the ‘Rho-tag’, representing an N-terminal part of rhodopsin, but also ‘Lucy-‘ or ‘Flag-tag’ extensions have been described. Here, we used a bi-functional N-terminal tag, called ‘IL-6-HaloTag®’, with IL-6 facilitating functional cell surface expression of recombinant odorant receptors, and the HaloTag® protein, serving as a highly specific acceptor for cell-impermeant or cell-permeant, fluorophore-coupled ligands, which enable the quantification of odorant receptor expression by live-cell flow cytometry. Our experiments revealed on average an about four-fold increased surface expression, a four-fold higher signaling amplitude, and a significant higher potency of odorant-induced cAMP signaling of six different human IL-6-HaloTag®-odorant receptors across five different receptor families in NxG 108CC15 cells, as compared to their Rho-tag–HaloTag® constructs. We observed similar results in HEK-293 cells. Moreover, screening an IL-6–HaloTag®-odorant receptor library with allyl phenyl acetate, revealed both known receptors as best responders for this compound. In summary, the IL-6–HaloTag® represents a promising tool for the de-orphaning of odorant receptors.


Buck L, Axel R (1991) A Novel Multigene Family May Encode Odorant Receptors - a Molecular-Basis for Odor Recognition. Cell 65: 175-187.

Lu M, Staszewski L, Echeverri F, Xu H, Moyer BD (2004) Endoplasmic reticulum degradation impedes olfactory G-protein coupled receptor functional expression. BMC Cell Biol 5: 34.

McClintock TS, Sammeta N (2003) Trafficking prerogatives of olfactory receptors. NeuroReport 14: 1547-1552.

Keller A, Zhuang H, Chi Q, Vosshall LB, Matsunami H (2007) Genetic variation in a human odorant receptor alters odour perception. Nature 449: 468-472.

Jaeger SR, McRae JF, Bava CM, Beresford MK, Hunter D, et al. (2013) A mendelian trait for olfactory sensitivity affects odor experience and food selection. Curr Biol 23: 1601-1605.

Geithe C, Noe F, Kreissl J, Krautwurst D (2017) The Broadly Tuned Odorant Receptor OR1A1 is Highly Selective for 3-Methyl-2,4-nonanedione, a Key Food Odorant in Aged Wines, Tea, and Other Foods. Chem Senses 42: 181-193.

Saito H, Kubota M, Roberts RW, Chi Q, Matsunami H (2004) RTP family members induce functional expression of mammalian odorant receptors. Cell 119: 679-691.

Wu L, Pan Y, Chen GQ, Matsunami H, Zhuang H (2012) Receptor-transporting protein 1 short (RTP1S) mediates translocation and activation of odorant receptors by acting through multiple steps. J Biol Chem 287: 22287-22294.

Von Dannecker LE, Mercadante AF, Malnic B (2006) Ric-8B promotes functional expression of odorant receptors. Proc Natl Acad Sci U S A 103: 9310-9314.

Zhuang H, Matsunami H (2007) Synergism of accessory factors in functional expression of mammalian odorant receptors. J Biol Chem 282: 15284-15293.

Yan Q, Bruchez MP (2015) Advances in chemical labeling of proteins in living cells. Cell and Tissue Research 360: 179-194.

Einhauer A, Jungbauer A (2001) The FLAG? peptide, a versatile fusion tag for the purification of recombinant proteins. Journal of Biochemical and Biophysical Methods 49: 455-465.

Hochuli E, Bannwarth W, Dobeli H, Gentz R, Stuber D (1988) Genetic Approach to Facilitate Purification of Recombinant Proteins with a Novel Metal Chelate Adsorbent. Nat Biotech 6: 1321-1325.

Krautwurst D, Yau KW, Reed RR (1998) Identification of ligands for olfactory receptors by functional expression of a receptor library. Cell 95: 917-926.

Geithe C, Andersen G, Malki A, Krautwurst D (2015) A Butter Aroma Recombinate Activates Human Class-I Odorant Receptors. J Agric Food Chem 63: 9410-9420.

Lossow K, Hubner S, Roudnitzky N, Slack JP, Pollastro F, et al. (2016) Comprehensive Analysis of Mouse Bitter Taste Receptors Reveals Different Molecular Receptive Ranges for Orthologous Receptors in Mice and Humans. J Biol Chem 291: 15358-15377.

Noe F, Polster J, Geithe C, Kotthoff M, Schieberle P, et al. (2017) OR2M3: A Highly Specific and Narrowly Tuned Human Odorant Receptor for the Sensitive Detection of Onion Key Food Odorant 3-Mercapto-2-methylpentan-1-ol. Chem Senses 42: 195-210.

Schmiedeberg K, Shirokova E, Weber HP, Schilling B, Meyerhof W, et al. (2007) Structural determinants of odorant recognition by the human olfactory receptors OR1A1 and OR1A2. J Struct Biol 159: 400-412.

Shepard BD, Natarajan N, Protzko RJ, Acres OW, Pluznick JL (2013) A Cleavable N-Terminal Signal Peptide Promotes Widespread Olfactory Receptor Surface Expression in HEK293T Cells. PLOS ONE 8: e68758.

Los GV, Encell LP, McDougall MG, Hartzell DD, Karassina N, et al. (2008) HaloTag: A Novel Protein Labeling Technology for Cell Imaging and Protein Analysis. ACS Chemical Biology 3: 373-382.

Urh M, Rosenberg M (2012) HaloTag, a Platform Technology for Protein Analysis. Current Chemical Genomics 6: 72-78.

Assier E, Boissier M-C, Dayer J-M (2010) Interleukin-6: From identification of the cytokine to development of targeted treatments. Joint Bone Spine 77: 532-536.

Binkowski B, Fan F, Wood K (2009) Engineered luciferases for molecular sensing in living cells. Curr Opin Biotechnol 20: 14-18.

Jones DT, Reed RR (1989) Golf: an olfactory neuron specific-G protein involved in odorant signal transduction. Science 244: 790-795.

Shirokova E, Schmiedeberg K, Bedner P, Niessen H, Willecke K, et al. (2005) Identification of specific ligands for orphan olfactory receptors. G protein-dependent agonism and antagonism of odorants. J Biol Chem 280: 11807-11815.

Li F, Ponissery-Saidu S, Yee KK, Wang H, Chen ML, et al. (2013) Heterotrimeric G protein subunit Ggamma13 is critical to olfaction. J Neurosci 33: 7975-7984.

Hamprecht B, Glaser T, Reiser G, Bayer E, Propst F (1985) Culture and characteristics of hormone-responsive neuroblastoma X glioma hybrid cells. Methods Enzymol 109: 316-341.

Li S, Ahmed L, Zhang R, Pan Y, Matsunami H, et al. (2016) Smelling Sulfur: Copper and Silver Regulate the Response of Human Odorant Receptor OR2T11 to Low-Molecular-Weight Thiols. J Am Chem Soc.

Touhara K, Sengoku S, Inaki K, Tsuboi A, Hirono J, et al. (1999) Functional identification and reconstitution of an odorant receptor in single olfactory neurons. Proc Natl Acad Sci USA 96: 4040-4045.

Saito H, Chi Q, Zhuang H, Matsunami H, Mainland JD (2009) Odor coding by a Mammalian receptor repertoire. Sci Signal 2: ra9.




How to Cite

Noe F, Frey T, Fiedler J, Geithe C, Nowak B, Krautwurst D. IL-6. J Biol Methods [Internet]. 2017Nov.3 [cited 2021Dec.2];4(4):e81. Available from:




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