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Protein Expr Purif. 2004 Feb;33(2):176-84.

Purification and characterization of the recombinant human dopamine D2S receptor from Pichia pastoris.

Author information

  • 1Department of Bioanalysis and Toxicology, University Centre for Pharmacy, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands. L.A.A.de.Jong@farm.rug.nl

Abstract

The human dopamine D2S receptor was expressed in the methylotrophic yeast Pichia pastoris, where the receptor with a molecular mass of approximately 40kDa exhibited specific and saturable binding properties. The dopamine antagonist [3H]spiperone showed an average dissociation constant K(d) of 0.6+/-0.17 nM for the dopamine D2S receptor. The receptor was solubilized using the non-ionic detergent dodecylmaltoside and purified by affinity chromatography using a Ni(2+) chelate (His-Trap) column or by batch extraction with an anti-FLAG M1 affinity resin. The receptor maintained its biological activity after solubilization and purification from the membrane protein fraction. A 244- or 185-fold enrichment, as judged by an increase in specific binding, was obtained after adsorption to the His-Trap or anti-FLAG materials, respectively.

PMID:
14711504
[PubMed - indexed for MEDLINE]
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