Recombinant expression and purification of human androgen receptor in a baculovirus system

A full-length human androgen receptor (hAR) cDNA was used to produce recomb inant baculovirus. Spodoptera frugiperda (Sf9) cells infected with this vir us expressed protein with an N-terminal hexahistidine tag (His(6)-hAR) in s oluble and insoluble forms. The soluble cytosolic His(6)-hAR demonstrated s imilar association and dissociation half-times for mibolerone, similar bind ing affinity for mibolerone, and similar steroid specificity as bona fide A R. Under native conditions, the soluble cytosolic His(6)-hAR was purified t o 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 mea ns for producing hAR for ligand-binding assays, as well as purifying hAR fo r detailed molecular analyses. (C) 2001 Academic Press.