Purification of recombinant GluR-D glutamate receptor produced in Sf21 insect cells

GluR‐D glutamate receptors carrying FLAG and polyHis affinity tags at the N-terminus and C-terminus, respectively, were expressed in recombinant baculovirus-infected Spodoptera frugiperda Sf21 cells. Affinity-tagged receptors displayed ligand‐binding affinity (K_d=40 nM) and an expression level (B_max 10–30 pmol/mg protein) similar to that of insect-cell-expressed wild-type GluR-D, as determined by [³H]-α-amino-5-hydroxy-3-methyl-4-isoxazole propionic acid ([³H]AMPA) binding. The receptor was solubilized in Triton X-100, and purified using a two-step protocol consisting of immobilized metal-chelation affinity chromatography followed by immunoaffinity chromatography. The purified receptor preparation contained over 2000 pmol high-affinity [³H]AMPA-binding sites/mg protein, and migrated as a single 110-kDa species in SDS/PAGE. Peptide: N-glycosidase F treatment reduced the size of GluR-D from 110 kDa to 100 kDa, indicating the presence of N-linked glycans. Up to 100 µg purified GluR-D was obtained from 11 Sf21 suspension culture (2–3×10⁶ cells/ml). High-level expression of affinity-tagged GluRs in insect cells should be an efficient strategy to produce GluR subtypes for biochemical and structural studies.