The human D1A dopamine receptor: heterologous expression in Saccharomyces cerevisiae and purification of the functional receptor.
Functional human D1A dopamine receptor has been expressed in Saccharomyces cerevisiae. The primary sequence of the receptor was modified to include two affinity tags at the C-terminus of the protein, a FLAG tag (DYKDDDDK), and a His6 tag (HHHHHH). These modifications allowed for purification to near homogeneity using immobilized metal affinity chromatography and immunoaffinity chromatography. Radioligand binding demonstrated that the purified and reconstituted receptor binds the antagonist [3H]SCH23390 with an affinity (KD = 8.0 +/- 3.2 nM) comparable to that of the native receptor.
Andersen
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B
Life Sciences Division, Donner Laboratory, Lawrence Berkeley Laboratory, Berkeley, California 94720, USA.
Stevens
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RC
D000595:Amino Acid Sequence; D001483:Base Sequence; D001667:Binding, Competitive; D002846:Chromatography, Affinity / Q000379:methods; D017931:DNA Primers; D004274:DNA, Recombinant; D006801:Humans; D009842:Oligopeptides; D010455:Peptides / Q000737:chemistry; D010743:Phospholipids / Q000378:metabolism; D017447:Receptors, Dopamine D1 / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014157:Transcription Factors
D017931:DNA Primers; D004274:DNA, Recombinant; D009842:Oligopeptides; D010455:Peptides; D010743:Phospholipids; D017447:Receptors, Dopamine D1; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C098593:dopamine D1A receptor; C085706:FLAG peptide