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 (Kd=40 nM) and an expression level (Bmax 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.
|Journal||European Journal of Biochemistry|
|Publication status||Published - 1995|
|MoE publication type||A1 Journal article-refereed|