Abstract
| Original language | English |
|---|---|
| Pages (from-to) | 720-726 |
| Journal | European Journal of Biochemistry |
| Volume | 233 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - 1995 |
| MoE publication type | A1 Journal article-refereed |
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Purification of recombinant GluR-D glutamate receptor produced in Sf21 insect cells. / Kuusinen, Arja; Arvola, Milla; Oker-Blom, Christian; Keinänen, Kari (Corresponding Author).
In: European Journal of Biochemistry, Vol. 233, No. 3, 1995, p. 720-726.Research output: Contribution to journal › Article › Scientific › peer-review
TY - JOUR
T1 - Purification of recombinant GluR-D glutamate receptor produced in Sf21 insect cells
AU - Kuusinen, Arja
AU - Arvola, Milla
AU - Oker-Blom, Christian
AU - Keinänen, Kari
N1 - Project code: BEL2031
PY - 1995
Y1 - 1995
N2 - 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.
AB - 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.
U2 - 10.1111/j.1432-1033.1995.720_3.x
DO - 10.1111/j.1432-1033.1995.720_3.x
M3 - Article
VL - 233
SP - 720
EP - 726
JO - FEBS Journal
JF - FEBS Journal
SN - 1742-464X
IS - 3
ER -