Purification and characterization of the recombinant human dopamine D2S receptor from Pichia pastoris.
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.
de Jong
Lutea A A
LA
Department of Bioanalysis and Toxicology, University Centre for Pharmacy, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands. L.A.A.de.Jong@farm.rug.nl
Grünewald
Sylvia
S
Franke
Jan Piet
JP
Uges
Donald R A
DR
Bischoff
Rainer
R
D002462:Cell Membrane / Q000378:metabolism; D002793:Cholic Acids / Q000737:chemistry; D003470:Culture Media; D006801:Humans; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D017448:Receptors, Dopamine D2 / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D012995:Solubility
D002793:Cholic Acids; D003470:Culture Media; D017448:Receptors, Dopamine D2; D011993:Recombinant Fusion Proteins; C113776:dopamine D2L receptor; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate