Recombinant Expression and Purification of Human Androgen Receptor in a Baculovirus System

Abstract

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 (His6-hAR) in soluble and insoluble forms. The soluble cytosolic His6-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 His6-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 His6-hAR was purified from it in the presence of 6 M guanidine HCl. Both the solubilized crude pellet fraction and the solubilized/purified His6-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.