ArticleThe Biochemical journal1999
Large-scale production and purification of functional recombinant bovine rhodopsin with the use of the baculovirus expression system.
Article in The Biochemical journal, 1999. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 9 papers.
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Who cites it
9 citing papers in PubMed, 44 citations in OpenAlex.
- Drosophila photoreceptor cells exploited for the production of eukaryotic membrane proteins: receptors, transporters and channels.PloS one · 2011Article
- Expression of neurotransmitter transporters for structural and biochemical studies.Protein expression and purification · 2010Article
- Stability of the neurotensin receptor NTS1 free in detergent solution and immobilized to affinity resin.PloS one · 2010Article
- Autoantibodies to recombinant human CTL2 in autoimmune hearing loss.The Laryngoscope · 2009Article
- Purification of recombinant G-protein-coupled receptors.Methods in enzymology · 2009Review
- Large-scale expression and purification of a G-protein-coupled receptor for structure determination -- an overview.Journal of structural and functional genomics · 2005Article
- Purification and biochemical characterization of the D6 chemokine receptor.The Biochemical journal · 2004Article
- Expression and characterization of recombinant human alpha-3/4-fucosyltransferase III from Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn) cells using the baculovirus expression system.The Biochemical journal · 2001Article
- Expression and purification of recombinant vesicular glutamate transporter VGLUT1 using PC12 cells and High Five insect cells.Biological procedures onlineArticle
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Here we describe a generic procedure for the expression and purification of milligram quantities of functional recombinant eukaryotic integral membrane proteins, exemplified by hexahistidine-tagged bovine rhodopsin. These quantities were obtained with the recombinant baculovirus/Sf9 insect cell-based expression system in large-scale bioreactor cultures with the use of a serum-free and protein-free growth medium. After optimization procedures, expression levels up to 4 mg/l were established. The recombinant rhodopsin could be purified with high overall yield by using immobilized-metal-affinity chromatography on Ni(2+)-agarose. After reconstitution into a native lipid environment, the purified protein was functionally indistinguishable from native rhodopsin with regard to the following parameters: spectral absorbance band, structural changes after photoactivation, and G-protein activation. The procedures developed can be adapted to other membrane proteins. The ability to produce and purify tens of milligrams of functional recombinant eukaryotic membrane protein meets the ever-increasing demand of material necessary to perform detailed biochemical and structural biophysical studies that are essential in unravelling their working mechanism at a molecular level.
Indexed as
Identifiers
10455014PMC1220464W2114873058What OpenQuestion holds
Registered trials
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