Conceived and designed the experiments: KJL SZ. Performed the experiments: KJL. Analyzed the data: KJL SZ CH. Contributed reagents/materials/analysis tools: KJL SZ CH. Wrote the paper: KJL SZ CH.
The authors have declared that no competing interests exist.
How fishes are able to detect trace molecules in large bodies of water is not understood. It is plausible that they use olfactory receptors to detect water-soluble compounds. How the zebra fish
Membrane proteins play a number of vital roles in all living systems. Approximately 30% of all genes in almost all sequenced genomes code for membrane proteins
There are several bottlenecks in the study of membrane proteins. One of them is the need for an inexpensive method for large-scale production of soluble non-aggregated membrane proteins. This must be accompanied by systematic detergent screens to select detergents that are suitable for long-term stabilization of functional membrane proteins. Only then will it be possible to carry out successful crystallographic screens for structural analyses.
Olfaction is not only common in all animals, insects and microbes, but it is also a fascinating phenomenon of nature. We are interested in understanding how fish detect an extremely scarce food source in a large body of water. It is plausible that fish use olfactory receptors to detect water-soluble compounds. The critical question of how a zebra fish with only 98 functional olfactory receptors (and 35 pseudo-genes)
In order to carry out structural studies, a large amount of receptors is required. To accomplish this, we established a number of mammalian cell lines stably expressing the zebra fish OR131-2 receptor upon induction. We successfully selected a few stable OR131-2 receptor expressing cell lines after clonal selection. We have also optimized the growth and induction conditions of these cell lines for the production of OR131-2 proteins and have worked out a simple method to recover and purify the protein from cell lysates. Our investigations are described in detail below.
The method to produce milligram quantities of human olfactory receptors from HEK293S cells has previously been described
These genes were cloned into a tetracycline inducible vector, pcDNA4/To, which allows external control of protein expression. After transfection of genes into HEK293S cells, we monitored transgene expression in stably transduced cells by immunostaining with a rhodopsin 1D4 (Rho1D4) monoclonal antibody, which recognizes a 9 amino acid TETSQVAPA tag
It has been reported that sodium butyrate, an inhibitor of histone deacetylase, can frequently be added to culture media to enhance recombinant protein expression in mammalian cells
Cell lysates from HEK293S cell clones that have been treated with 1 µg/mL tetracycline and 2.5 mM sodium butyrate (TS), 1 µg/mL tetracycline only (T) or mock treated (M) for 48 hours were spotted onto a nitrocellulose membrane, and stained with a Rho1D4 monoclonal antibody. The Rho1D4 antibody specifically recognizes a nine amino acid TETSQVAPA tag on the carboxyl terminus of recombinant zebra fish OR131-2 protein. Serial two fold dilutions of purified TETSQVAPA tagged human OR17-4 protein (50 ng/ml –0.78 ng/ml) were spotted as positive control standards (Std) to estimate protein yield, and identically prepared cell lysates from HEK293S cells (N) served as negative controls. Altogether, more than 200 HEK293S cell clones expressing the OR131-2 proteins were screened and the higher producing cell clones were selected for further screening to examine the quality of the protein produced.
Fos-Choline14 soluble proteins isolated from harvested cells, were separated on Novex® 4–12% Bis-Tris gels, blotted on Polyvinylidene Fluoride (PVDF) membranes and stained with Rho1D4 monoclonal antibody. A) HEK293s cell clones expressing OR131-2A with protein sequence shown in supplementary
Purification and stabilization of membrane proteins remains one of the most difficult challenges in producing high quality proteins for structural studies. Using immunoaffinity purification in a single purification step, we were able to obtain ∼290 µg purified protein per gram of culture cells, with protein purity of more than 85% and a 90% recovery rate. The Sypro Ruby protein stain in
Purified Fos-Choline 14 solubilized proteins were separated on Novex® 4–12% Bis-Tris gels and either A) stained with Sypro Ruby protein stain or B) blotted on Polyvinylidene Fluoride (PVDF) membrane and stained with Rho1D4 monoclonal antibody. Shown here are the purification of two independent protein clones with protein sequences A and B (see supplementary
We used confocal microscopy to examine the HEK293S cells that expressed OR131-2 receptors in order to verify if the expressed OR131-2 receptor is correctly translocated into the cell plasma membrane.
The OR131-2 protein expressed in HEK293 cells were immunofluorescently labelled with a Rho1D4 monoclonal antibody, followed by a species specific Tetramethylrhodamine (TRITC) labeled secondary antibody (panels A and B) or an Alexa Fluor 488 labeled secondary antibody (panel C). The cell nuclei were counter-stained stained with DAPI (4′,6-diamidino-2-phenylindole).
In order to exert its biological function, the correct folding of a protein is crucial. We wanted to ensure that the purified OR131-2 receptor was correctly folded. Thus, we used circular dichroism (CD) analysis to examine the purified receptor for its α-helical character since almost all GPCRs are known to fold into seven trans-membrane helices
The circular dichroism (CD) spectrum shows the presence of α-helical content in purified OR131-2 protein (0.87 mg/mL). Mean residue ellipticity [θ] has units millidegrees x cm2 x dmol−1.
This study describes the purification of milligram quantities of zebra fish olfactory receptor 131-2 from HEK293S cells. The protein is correctly localized in cell membranes and CD analysis of the purified protein indicates the presence of alpha helical content, affirming our assumption that the purified protein is correctly folded. These results indicate the possibility that the simple GPCRs production and purification protocol described here may be generally applicable to other GPCRs, overcoming one of the first bottlenecks frequently encountered during structural studies of GPCRs.
Although circular dichroism spectroscopy is a useful technique in the determination of protein secondary structures
Olfaction, in particular the mechanism of odorant binding, still remains a vastly unexplored area, although the further neuronal processing of olfaction after odorant receptor activation is a better understood process
Although zebra fish has only 98 olfactory receptors, it can distinguish a wide range of water-soluble compounds, pheromones and other molecules including amino acids, trace amines and decays of scarce food sources in extremely diluted concentrations. Since the zebra fish is an excellent genetic model of vertebrates with few olfactory receptors compared to other species, its mechanism of olfaction should be more easy to study as compared to more complex olfactory systems.
Currently, little is known about how fish olfactory receptors recognize odorants and other molecules in water. In other words, the OR131-2 receptor is still an orphan receptor without known ligands. It is possible that it could recognize more than one compound and perhaps many more compounds in a combinatorial manner as suggested for other olfactory receptors
OR131-2 is probably the first fish olfactory receptor that has been expressed, produced and purified to near-homogeneity for biochemical and structural studies. Our study may stimulate further studies of aqueous olfactory and chemosensory receptors from other amazing marine animals in deep oceans.
Fos-Choline14 (FC14) was purchased from Anatrace (Maumee, OH). All tissue culture media and media additives were purchased from Invitrogen Pte Ltd (Singapore) unless otherwise stated. Restriction enzymes were bought from Fermentas (Ontario, Canada). Sodium butyrate and Dulbecco's Phosphate Buffered Saline (D-PBS) were purchased from Sigma (St. Louis, MO). HEK293s cells stably transduced with pcDNA6/TR were a gift from Brian Cook of Massachusetts Institute of Technology, USA. Rho1D4 monoclonal antibody was purchased from Cell Essentials (Boston, MA, USA). The buffers used for protein purification are as follows: Wash buffer: D-PBS containing 0.2% wt/vol FC14; Elution buffer: Wash buffer containing 500 µM Ac-TETSQVAPA-CONH2 (First Base, Singapore) elution peptide.
HEK293S cells were cultured in Advanced Dulbecco's Modified Eagle Medium/F12 mixture supplemented with non-essential amino acids, 10% fetal bovine serum, 110 mg/mL sodium pyruvate, 15 mM Hepes, 100 units/mL penicillin, 100 µg/mL streptomycin and 5 µg/mL Blasticidin S. Transfection of genes into HEK293s cells was performed using Lipofectamine 2000 (Invitrogen, Singapore) following the manufacturer's instructions. Cell clones stably integrating the transgenes were selected over 3 weeks by supplementing the culture media with 250 µg/mL zeocin.
Zeocin resistant cell clones were either mock treated or treated with 1 µg/mL tetracycline, 1 µg/mL tetracycline and 2.5 mM sodium butyrate for 48 hours to induce OR131-2 protein expression. Cell lysates was prepared in D-PBS buffer containing protease inhibitor cocktail set III (Merck KGaA, Darmstadt, Germany) and 2% wt/vol FC14. One µL of cell lysate from each treatment condition was spotted onto nitrocellulose membrane, air dried for 30 minutes, incubated with Rho1D4 monoclonal antibody for 1 hour, followed by detection using the ECL Plus kit (GE Healthcare, Singapore) according to manufacturer's instructions.
Proteins in cell lysates were separated via sodium dodecyl sulfate
Immunoaffinity purification was performed using Cyanogen Bromide-activated Sepharose 4B beads (GE Healthcare, Singapore) chemically cross-linked to Rho1D4 monoclonal antibody. To begin purification, cell lysates was incubated at 4°C overnight with the antibody conjugated beads, followed by washing the beads approximately 10 times with 4 bead volumes of wash buffer each time. OR131-2 proteins were eluted with elution buffer containing 500 µM Ac-TETSQVAPA-CONH2 elution peptide. Finally, the protein was concentrated and the elution peptides removed using a centrifugal concentrator with 10,000 molecular weight cut off, Vivaspin 6 (Sartorius Stedim Biotech SA, Aubagne Cedex, France) and buffer exchange was performed 10 times to remove the peptide.
OR131-2 protein expression was induced in zeocin resistant cells cultured on poly L-Lysine (Sigma, St. Louis, MO) treated glass cover-slips by supplementing the culture media with 1 µg/mL tetracycline and 2.5 mM sodium butyrate for 48 hours. The culture media was removed and the cells were fixed and permeablized with ice cold methanol for 2 minutes. Methanol was completely removed by washing with Tris Buffered Saline (TBS) 3 times. To prevent non-specific binding, the cells were incubated in 3% wt/vol Bovine Serum Albumin (PAA Laboratories GmbH, Pasching, Austria) in TBS for 30 minutes. The cells were then stained with Rho1D4 monoclonal antibody diluted 1∶1000 in the above blocking buffer for 1 hour. After washing for 3 times with 0.1% Tween 20 (Sigma, St. Louis, MO) in TBS (TBST), cells were incubated with either a mouse specific Tetramethylrhodamine (TRITC) labeled secondary antibody (Sigma, St. Louis, MO) or an Alexa Fluor 488 labeled secondary antibody (Invitrogen, Singapore) for 1 hour. Following 5 washes with TBST to remove any non-specifically bound antibody, cells were counter-stained with the nucleus specific dye, 4′,6-diamidino-2-phenylindole (DAPI) (Sigma, St Louis, MO) for 5 minutes. Next, cover slips were removed from the wells and mounted on a microscopic glass slide using Hydromount (National Diagnostics, Atlanta, GA). Confocal imaging was performed on a Carl Zeiss LSM 510 META Upright microscope using a 100x oil objective.
CD spectra were measured at 25°C over the wavelength range of 200–250 nm with a step size of 1 nm and an averaging time of 10 sec, using a CD spectrometer (Aviv Associates, Model 410). All spectra were the average of 5 replicate scans. Spectra shown for purified OR131-2 protein were baseline subtracted with wash buffer to remove the effects of the FC14 detergent. The measurements are reported in mean residue ellipticity (millidegrees x cm2 x dmole−1). Analyses of the CD spectra were performed using the CDNN software provided with the Aviv instrument. Protein concentration was determined using the BCA Protein Assay kit (Pierce Biotechnology, Rockford, IL).
A) Three different zebra fish olfactory receptor 131-2 constructs were made (
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Immunofluorescence staining was performed as described (
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Mass spectrometry is performed following standard procedure as described in
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The authors would like to acknowledge IBN Youth Research Program attachment students MaryAnn Zhang, Jiaxin Wu, Adhylakshumie Adhynarayanan, Debra Leong and Furen Zhuang for their excellent technical assistance in the project.