Percentage of functional NTS1.

<p>The total protein content of the NiE, NTFT and NiE fractions was determined by the Amido Black method (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012579#pone-0012579-t003" target="_blank">Table 3</a>). The amount of total NTS1 in the NiE, NTFT and NiE fractions was determined by ImageJ analysis of Coomassie-stained gels. The number of functional NTS1 in pmoles (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012579#pone-0012579-t003" target="_blank">Table 3</a>) was converted into a milligram value using an NTS1 molecular mass of 96.5 kDa. The percent functionality was calculated as [(mg NTS1 by ligand binding analysis)/(mg NTS1 by SDS-PAGE analysis)]*100.</p

Stability of NTS1 in NT70 buffer with and without agonist.

<p>Receptors were incubated with [³H]NT (open squares) or kept in NT70 buffer without agonist (filled squares) at 4°C. [³H]NT binding was determined at the indicated time points. The data shown are from 2 independent experiments.</p

Stability of NTS1 in the absence of agonist.

<p>[³H]NT binding to the NTS1 fusion protein was recorded over time and half-lives were calculated. The amount of functional NTS1 remaining after 6 hrs (NiE and diluted NiE) and after 24 hrs (SN) was estimated from one phase exponential decay fits. Abbreviations: SN, supernatant after ultracentrifugation; NiE, Ni-NTA column eluate.</p

Functional and misfolded NTS1 during purification.

<p>Three different ways (columns A–C) were used to estimate the amounts of functional NTS1, misfolded NTS1 and contaminants in the NiE, NTFT and NTE fractions. All values in columns A–D are given in mg quantities. Abbreviations: NiE, Ni-NTA column eluate; NTFT, flow-through of NT column; NTE, NT column eluate; NA, not applicable.</p>1<p>The amount of functional NTS1 in the NiE, NTFT and NTE fractions was determined by [³H]NT binding (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012579#pone-0012579-t003" target="_blank">Tables 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012579#pone-0012579-t004" target="_blank">4</a>). For example, the NiE contained 3678 pmoles or 0.355 mg functional NTS1.</p>2<p>The total amount of NTS1 protein in the NiE, NTFT and NTE fractions was determined by ImageJ analysis of Coomassie-stained gels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012579#pone-0012579-t004" target="_blank">Table 4</a>).</p>3<p>The amount of functional NTS1 in the NiE and NTE fractions was derived from the respective specific [³H]NT binding values (in pmol/mg, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012579#pone-0012579-t003" target="_blank">Table 3</a>) compared to the theoretical value of 10363 pmol/mg. For example, the NiE fraction has a specific binding value of 3724 pmol/mg and hence 36% (3724/10363) of the total protein in NiE (0.92 mg) is functional NTS1 (0.331 mg).</p>4<p>The amount of functional NTS1 was estimated from data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012579#pone-0012579-t004" target="_blank">Table 4</a>.</p>5<p>The diluted NiE is 91% functional after 6 hrs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012579#pone-0012579-t001" target="_blank">Table 1</a>). This decrease of the amount of functional NTS1 is indicated by a left pointing arrow. The corresponding amount of misfolded NTS1 is listed.</p>6<p>The amount of functional and misfolded NTS1 was calculated from the total NTS1 amount in the respective fractions considering the proportion of functional receptors (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012579#pone-0012579-t004" target="_blank">Table 4</a>). For example, the NiE contained 0.443 mg total NTS1 which is 80% functional⁴ i.e. 0.355 mg functional NTS1 and 0.088 mg misfolded NTS1.</p>7<p>The amount of contaminants was calculated by subtracting the total NTS1 amount from the total protein content of a given fraction (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012579#pone-0012579-t004" target="_blank">Table 4</a>).</p>8<p>The total protein content of the NiE, NTFT and NTE fractions was determined by the Amido Black method.</p

Summary of NTS1 purification.

<p>The percent values refer to the respective protein species in the NiE.</p

Purification of NTS1.

<p>The progress of purification was monitored by SDS-PAGE (NuPAGE 4–12% Bis-Tris gel, Invitrogen, 1× MES SDS buffer) and SimplyBlue staining. Lane M: Novagen Perfect Protein Marker (15–150 kDa); lanes SN, NiE and NTFT: 5 µg of protein per lane; lane NTE: 3 µg of protein. Abbreviations are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012579#pone-0012579-t003" target="_blank">Table 3</a>.</p

Optimization of NTS1 expression under different induction conditions using a stable T-REx-293 cell line.

<p>The data are collected from a selected high-expressing clone. Cells were grown in suspension in CD OptiCHO medium supplemented with 4 mM L-glutamine and 1% certified FBS and were induced in the late exponential growth phase (at a viable cell density of 2 million cells/ml) with tetracycline. The addition of sodium butyrate enhanced expression levels. Intact cells were subjected to [³H]NT binding assay to determine the number of receptors located at the cell-surface. For all conditions, n = 2, error bars indicate SEM (standard error of the mean).</p

[³H]NT saturation binding of NTS1 expressed in (A) T-REx-293 cells and (B) insect cells.

<p>NTS1 was extracted from membranes with the detergent DM/CHS and subjected to radio-ligand binding analysis. Inset: Scatchard transformation. Representative experiments conducted as single data points are shown. (C) Table summarizing the values of the apparent dissociation constants values for [³H]NT binding. These values are not statistically different (P = 0.2, unpaired two-tailed t-test). Data are collected from three repeated experiments.</p

Expression of NTS1 in the transient insect cell system and inducible T-REx-293 system.

<p>(A) Total functional NTS1 numbers were determined by [³H]NT binding assays using detergent solubilized cells (B) Surface localized NTS1 numbers were determined by [³H]NT binding assays using intact cells and combined with data from (A) to calculate percentage of surface localized NTS1 (baculovirus insect cell system: 7 independent expression experiments; T-REx-293 system: 4 independent measurements on one 5L expression experiment). The expression of NTS1 in insect cells was conducted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063679#s2" target="_blank">Materials and Methods</a>. Expression of NTS1 in the T-REx-293 system was induced by the addition of 2 µg/ml tetracycline and 10 mM sodium butyrate, with harvest and analysis 36 hours later.</p

Timeframe for the establishment of the transient baculovirus-insect cells system and stable expression with inducible T-REx-293 system for GPCR expression.

<p>Timeframe for the establishment of the transient baculovirus-insect cells system and stable expression with inducible T-REx-293 system for GPCR expression.</p