Transient and stable expression of the neurotensin receptor NTS1: a comparison of the baculovirus-insect cell and the T-REx-293 expression systems

Abstract

Nowadays, baculovirus-infected insect cells and tetracycline-inducible mammalian cell lines (T-REx-293) are intensively used for G protein-coupled receptor (GPCR) production for crystallography purposes. Here we constructed a suspension T-REx-293 cell line to stably express an engineered neurotensin receptor 1 (NTS1) mutant and we quantitatively compared this cell line with the transient baculovirus-insect cell system throughout a milligram-scale NTS1 expression and purification process. The two systems were comparable with respect to functional NTS1 expression levels and receptor binding affinity for the agonist [(3)H] neurotensin. However, NTS1 surface display on T-REx-293 cells determined by radio-ligand binding assays was 2.8 fold higher than that on insect cells. This work demonstrates two approaches for preparing milligram quantities of purified NTS1 suitable for structural studies and provides useful input to users in choosing and optimizing an appropriate expression host for other GPCRs.

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).

Figure 3. Purification of NTS1.

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.