Purification of recombinant GluR-D glutamate receptor produced in Sf21 insect cells

Arja Kuusinen, Milla Arvola, Christian Oker-Blom, Kari Keinänen (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

39 Citations (Scopus)

Abstract

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.
Original languageEnglish
Pages (from-to)720-726
JournalEuropean Journal of Biochemistry
Volume233
Issue number3
DOIs
Publication statusPublished - 1995
MoE publication typeA1 Journal article-refereed

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Sf9 Cells
Glutamate Receptors
Purification
Insects
Glutamic Acid
Affinity chromatography
Spodoptera
Octoxynol
Chromatography
Affinity Chromatography
Cell culture
Polysaccharides
Polyacrylamide Gel Electrophoresis
Suspensions
Proteins
Cell Culture Techniques
glutamate receptor type D
propionic acid
peptide F

Cite this

Kuusinen, Arja ; Arvola, Milla ; Oker-Blom, Christian ; Keinänen, Kari. / Purification of recombinant GluR-D glutamate receptor produced in Sf21 insect cells. In: European Journal of Biochemistry. 1995 ; Vol. 233, No. 3. pp. 720-726.
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abstract = "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.",
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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 journalArticleScientificpeer-review

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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.

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