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 (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]-alpha-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 micrograms purified GluR-D was obtained from 1 l Sf21 suspension culture (2-3 x 10(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.
VTT Biotechnology and Food Research, Espoo, Finland.
A
A
Kuusinen
M
M
Arvola
C
C
Oker-Blom
K
K
Keinänen
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D018014:Gene Transfer Techniques; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D017470:Receptors, Glutamate / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D018411:Spodoptera / Q000378:metabolism*
D017470:Receptors, Glutamate; D011994:Recombinant Proteins