Figure 1.
[3H]NT saturation binding of NTS1 expressed in (A) T-REx-293 cells and (B) insect cells.
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.
Figure 2.
Optimization of NTS1 expression under different induction conditions using a stable T-REx-293 cell line.
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).
Table 1.
Purification of NTS1 from different host cells.
Figure 3.
The progress of purification was monitored by SDS-PAGE (NuPAGE 4–12% Bis-Tris gel, Invitrogen, 1× MES SDS buffer) and SimplyBlue staining. The arrow indicate the NTS1 band. Lane 1: Novagen Perfect Protein Marker (15–150 kDa); lane 2: Talon eluate of NTS1 produced in T-REx-293 cells (3.5 µg); lane 3: Talon eluate of NTS1 produced in insect cells (6 µg); Western blot analysis of total cell extract was performed using the HisProbe-HRP reagent recognizing the histidine tag. Lane 4: NTS1 produced in T-REx-293 cells (122,000 lyzed cells with 113 ng functional NTS1); lane 5: NTS1 produced in insect cells (110,500 lyzed cells with 107 ng functional NTS1).
Figure 4.
Expression of NTS1 in the transient insect cell system and inducible T-REx-293 system.
(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 Materials and Methods. 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.
Figure 5.
Timeframe for the establishment of the transient baculovirus-insect cells system and stable expression with inducible T-REx-293 system for GPCR expression.