Molecular Cloning and Purification

The coding region of murine full-length (1-223) σ1R (AF004927), and the C-terminal region of the glutamate receptor NMDAR1 (NM_008169) (residues 834-938), were all amplified by RT-PCR using total RNA isolated from mouse brains as the template. Specific primers containing an upstream Sgf I and a downstream Pme I restriction site were used, as described previously [36 (link)]. The PCR products were cloned downstream of the GST coding sequence and the TEV protease site. The sequenced proteins were identical to the GenBank™ sequences. The vector was introduced into E. coli BL21 (KRX #L3002, Promega, Madrid, Spain), and clones were selected on solid medium containing ampicillin. After 3 h induction at room temperature (1 mM IPTG and 0.1% Rhamnose), the cells were collected by centrifugation, and the pellets were maintained at –80° C.
The purification of GST fusion proteins was done under native conditions on GStrap FF columns (GE#17-5130-01, Healthcare, Barcelona, Spain) and when necessary the fusion proteins retained were cleaved on the column with ProTEV protease (Promega, #V605A) and further purification was achieved by high-resolution ion exchange (Enrich Q, BioRad #780-0001) or electroelution of the corresponding gel band (GE 200, Hoefer Scientific Instruments, San Francisco, CA, USA). The sequences were confirmed through automated capillary sequencing.