Study of two G-protein coupled receptor variants of human trace amine-associated receptor 5

Abstract

Here we report the study of two bioengineered variants of human trace amine-associated receptor 5 (hTAAR5) that were expressed in stable tetracycline-inducible HEK293S cell lines. A systematic detergent screen showed that fos-choline-14 was the optimal detergent to solubilize and subsequently purify the receptors. Milligram quantities of both hTAAR5 variants were purified to near homogeneity using immunoaffinity chromatography followed by gel filtration. Circular dichroism showed that the purified receptors had helical secondary structures, indicating that they were properly folded. The purified receptors are not only suitable for functional analyses, but also for subsequent crystallization trials. To our knowledge, this is the first mammalian TAAR that has been heterologously expressed and purified. Our study will likely stimulate in the development of therapeutic drug targets for TAAR-associated diseases, as well as fabrication of TAAR-based sensing devices.

Figure 1. Construction of stable hTAAR5 and hTAAR5-T4L-inducible HEK293S cell lines using the T-REx system (Invitrogen).

Dot blot analysis was used to analyze the hTAAR5 (a) or hTAAR5-T4L (b) expression level of different clones after selective incubation. The numbers identify each. Three 48-hour induction conditions were tested in parallel for each clone: 1) in plain media (−), 2) in media supplemented with 1μg/ml tetracycline (+), or 3) in media supplemented with 1μg/ml tetracycline plus 2.5mM sodium butyrate (++). The three dots at the bottom of b are positive controls. (c) Western blot analysis using an anti-rho-tag monoclonal antibody was also used to further analyze the expression level of hTAAR5 from clones 2 and 8, or the expression level of hTAAR5-T4L from clones 5 and 8. The symbols “+” and “++” represent the same induction conditions as those in the dot blot analysis.

Figure 2. Optimization of hTAAR5-T4L expression.

hTAAR5-T4L expression was induced with varying the concentrations of both tetracycline and sodium butyrate. After induction, the samples were harvested and analyzed on a dot blot. The results were quantified by spot densitometry and normalized to the range of 0.0 to 1.0 for ease of comparison.

Figure 3. Detergent screening for optimal solubilization of hTAAR5 (a) and hTAAR5-T4L (b) expressed in HEK293S cells.

Expression of hTAAR5 or hTAAR5-T4L was induced with tetracycline (1μg/ml) and sodium butyrate (2.5mM) for 48h, and receptors were solubilized in PBS containing detergents for 1 hour at 4°C. In total, 96 detergents or detergent-mixtures were screened. Each integer from 1 to 96 along the x-axis represents a detergent or detergent mixture (see Supplementary Table S1 online for more information about each detergent or detergent mixture). The ability of each detergent or mixtures to solubilize hTAAR5 or hTAAR5-T4L was been quantified by spot densitometry and normalized to the range from 0.0 to 1.0 for ease of comparison.

Figure 4. Immunoaffinity purification of hTAAR5-T4L. Induced HEK293S cells were harvested and solubilized.

The expressed protein was captured by sepharose beads linked to the anti-rho-tag monoclonal antibody. The captured proteins were washed and then eluted using the rho-tag elution peptide TETSQVAPA. Samples were analyzed on a western blot. Abbreviations: FT, flow through; W, wash; E, elution.

Figure 5. Gel filtration purification of hTAAR5-T4L.

(a) Gel filtration of immunoaffinity-purified hTAAR5-T4L from ∼9g cells (wet weight). Absorbance was recorded at 280nm (black line), 254nm (gray line), and 215nm (dashed line). Peaks 1-6 (indicated by numbers) were pooled and concentrated. Peak 7 contained the rho1D4 elution peptide TETSQVAPA from the immunoaffinity purification. Both Western blotting (b) and silver staining (c) were employed to analyze the peak fractions. Peak numbers refer to those designated in (a). (d) Gel filtration of immunoaffinity-purified hTAAR5 from ∼5g cells (wet weight). Peaks 1−5 (indicated by numbers) were pooled and concentrated. Peak 6 contained the rho1D4 elution peptide TETSQVAPA from the immunoaffinity purification. (e) A Western blot and (f) silver stain were used to analyze the peak fractions.

Figure 6. Circular dichroism spectra of purified hTAAR5 and hTAAR5-T4L monomers.

Mean residue ellipticity [θ] has units of degree × cm² × dmol⁻¹. Each spectrum shown is the average of 3 replicate scans. The spectra show typical α-helical profiles with minima at 208nm and 222nm. The helical contents for hTAAR5 and hTAAR5-T4L are ∼63% and ∼69%, respectively. These results suggest that hTAAR5 and hTAAR5-T4L are folded correctly.