Expression of androgen receptor in insect cells. Purification of the receptor and renaturation of its steroid- and DNA-binding functions

J Biol Chem. 1992 Mar 5;267(7):4939-48.

Abstract

A full-length rat androgen receptor cDNA was used to produce a recombinant baculovirus (AcrAR) by homologous recombination. Spodoptera frugiperda (Sf9) cells infected with this virus expressed a 110-kDa polypeptide that amounted up to about one-third of total cell protein. Studies with AR antibodies confirmed that this protein was indeed rAR. Only a minor portion of the recombinant AR was soluble in buffers without ionic detergents, but its complete solubilization was achieved in 6 M guanidine HCl (GdnHCl). Electron microscopy of cell pellets revealed that AR was localized to electron-dense cytoplasmic aggregates. The soluble cytosolic receptor was biologically active, in that it bound [3H]mibolerone with high affinity and specificity and interacted with an androgen-responsive element. The functions of the GdnHCl-solubilized AR were partially restored by a 20-50-fold dilution. The solubilized receptor was purified to an apparent homogeneity in a single step by gel filtration on a Sephacryl S-400 column in the presence of 6 M GdnHCl. The homogeneous AR protein could be renatured to bind [3H]mibolerone, interact specifically with a DNA element, and be recognized by receptor antibodies. Receptor-DNA interaction was stabilized by an antibody directed against the N-terminal part and abolished by an antibody against the hinge region of the receptor Zn2+ ions were essential for the purified receptor to refold into a specific DNA-binding form during the renaturation, with the optimal ZnCl2 concentration being 50-100 microM depending on the buffer conditions. Cd2+ ions were also capable of restoring the receptor's DNA-binding activity and did so at concentrations 10-fold lower than those of the Zn2+ ions.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Androgens / metabolism*
  • Animals
  • Baculoviridae / genetics
  • Base Sequence
  • Blotting, Western
  • Cations, Divalent
  • Cell Line / ultrastructure
  • Chromatography, Gel
  • DNA / genetics
  • DNA / metabolism*
  • Electrophoresis, Polyacrylamide Gel
  • Gene Expression*
  • Genes, Viral
  • Microscopy, Electron
  • Molecular Sequence Data
  • Moths / cytology
  • Nandrolone / analogs & derivatives
  • Nandrolone / metabolism
  • Protein Denaturation
  • Rats
  • Receptors, Androgen / genetics*
  • Receptors, Androgen / isolation & purification
  • Receptors, Androgen / metabolism
  • Substrate Specificity
  • Testosterone Congeners / metabolism
  • Transfection

Substances

  • Androgens
  • Cations, Divalent
  • Receptors, Androgen
  • Testosterone Congeners
  • Nandrolone
  • DNA
  • mibolerone