title: Production of recombinant androgen receptor in a heterologous expression system.
abstract: To facilitate detailed studies of androgen receptor, we have produced a full-length receptor protein and some of its deletion mutants in Spodoptera frugiperda (Sf9) insect cells, using the baculovirus expression system. Recombinant baculovirus DNA-infected Sf9 cells expressed these proteins in very high quantities, which represented as much as 30-40% of total insect cell protein at 72 h after infection. Only < 1% of the recombinant protein was soluble in low-salt buffers; the majority formed electron-dense cytoplasmic aggregates 30-40 nm in diameter. These aggregates could be solubilized in 6 mol/L guanidine HCl, and biologically active receptor was generated by diluting the guanidine HCl preparation 20- to 50-fold. The full-length receptor, expressed either in a soluble or aggregated form, had characteristics typical of a native receptor: it bound steroids with high affinity and specificity, interacted with DNA in a sequence-specific fashion, and was recognized by domain-specific receptor antibodies. Androgen-receptor protein purified to homogeneity in guanidine HCl required the presence of Zn2+ ions during the refolding to reconstitute its DNA-binding form; ZnCl2 was not, however, needed to restore the receptor's steroid-binding activity.
authors.affiliation: Department of Physiology, University of Helsinki, Finland.
authors.forename: O A
authors.initials: OA
authors.lastname: Jänne
authors.forename: J J
authors.initials: JJ
authors.lastname: Palvimo
authors.forename: P
authors.initials: P
authors.lastname: Kallio
authors.forename: M
authors.initials: M
authors.lastname: Mehto
authors.forename: Y B
authors.initials: YB
authors.lastname: Xie
authors.forename: Y P
authors.initials: YP
authors.lastname: Sui
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D009036:Moths; D011944:Receptors, Androgen / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014162:Transfection
chemical_list: D011944:Receptors, Androgen; D011994:Recombinant Proteins