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 [H-3]-alpha-amino-5-hydroxy-3-methyl-4-isoxazole propionic acid ([H-3]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 [H-3]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 mu g purified GluR-D was obtained from 11 Sf21 suspension culture (2-3X10(6) 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.