Recombinant expression and purification of human androgen receptor in a baculovirus system.
A full-length human androgen receptor (hAR) cDNA was used to produce recombinant baculovirus. Spodoptera frugiperda (Sf9) cells infected with this virus expressed protein with an N-terminal hexahistidine tag (His(6)-hAR) in soluble and insoluble forms. The soluble cytosolic His(6)-hAR demonstrated similar association and dissociation half-times for mibolerone, similar binding affinity for mibolerone, and similar steroid specificity as bona fide AR. Under native conditions, the soluble cytosolic His(6)-hAR was purified to apparent homogeneity in the presence of dihydrotestosterone, using metal ion affinity chromatography. The insoluble pellet fraction was solubilized with strong denaturant 6 M guanidine HCl, and His(6)-hAR was purified from it in the presence of 6 M guanidine HCl. Both the solubilized crude pellet fraction and the solubilized/purified His(6)-hAR could be renatured to bind mibolerone. The baculovirus system will therefore provide an efficient means for producing hAR for ligand-binding assays, as well as purifying hAR for detailed molecular analyses.
Zhu
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Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, Ohio State University, Columbus, Ohio 43210, USA.
Bulgakov
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OV
Scott
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SS
Dalton
J T
JT
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D006639:Histidine / Q000235:genetics; D006801:Humans; D007700:Kinetics; D009277:Nandrolone / Q000031:analogs & derivatives / Q000378:metabolism; D011489:Protein Denaturation; D011944:Receptors, Androgen / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018411:Spodoptera / Q000235:genetics; D013256:Steroids / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D045165:Testosterone Congeners / Q000378:metabolism; D014162:Transfection
D011944:Receptors, Androgen; D011993:Recombinant Fusion Proteins; D013256:Steroids; D045165:Testosterone Congeners; D006639:Histidine; D009277:Nandrolone; C100075:mibolerone