A. Kuusinen et al., PURIFICATION OF RECOMBINANT GLUR-D GLUTAMATE-RECEPTOR PRODUCED IN SF21 INSECT CELLS, European journal of biochemistry, 233(3), 1995, pp. 720-726
GluR-D glutamate receptors carrying FLAG and polyHis affinity tags at
the N-terminus and C-terminus, respectively, were expressed in recombi
nant baculovirus-infected Spodoptera frugiperda Sf21 cells. Affinity-t
agged receptors displayed ligand-binding affinity (K-d 40 nM) and an e
xpression level (B-max 10-30 pmol/mg protein) similar to that of insec
t-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-st
ep protocol consisting of immobilized metal-chelation affinity chromat
ography followed by immunoaffinity chromatography. The purified recept
or preparation contained over 2000 pmol high-affinity [H-3]AMPA-bindin
g sites/mg protein, and migrated as a single 110-kDa species in SDS/PA
GE. 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 subtyp
es for biochemical and structural studies.