Expression of androgen receptor in insect cells. Purification of the receptor and renaturation of its steroid- and DNA-binding functions.
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.
Xie
Y B
YB
Population Council, New York, New York 10021.
Sui
Y P
YP
Shan
L X
LX
Palvimo
J J
JJ
Phillips
D M
DM
Jänne
O A
OA
D000728:Androgens / Q000378:metabolism*; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D002413:Cations, Divalent; D002460:Cell Line / Q000648:ultrastructure; D002850:Chromatography, Gel; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005814:Genes, Viral; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009036:Moths / Q000166:cytology; D009277:Nandrolone / Q000031:analogs & derivatives / Q000378:metabolism; D011489:Protein Denaturation; D051381:Rats; D011944:Receptors, Androgen / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D045165:Testosterone Congeners / Q000378:metabolism; D014162:Transfection
D000728:Androgens; D002413:Cations, Divalent; D011944:Receptors, Androgen; D045165:Testosterone Congeners; D009277:Nandrolone; D004247:DNA; C100075:mibolerone