Overexpression of a partial human androgen receptor in E. coli: characterization of steroid binding, DNA binding, and immunological properties.
The recent cloning of human androgen receptor (AR) cDNAs in this and other laboratories has provided valuable probes for investigating the structure and function of the AR at the molecular level. We now report the overexpression of a region of the human AR containing both the DNA- and hormone-binding domains in E. coli, which provides a means to produce large amounts of AR for analysis and use in functional studies. Under isopropyl-beta-D-thiogalactopyranoside induction, a tripartite protein, consisting of beta-galactosidase, a collagenase recognition site, and AR polypeptide, was produced in E. coli JM109 using pSS20 a as a vector. About 1 mg of the fused AR could be recovered per liter bacterial culture. The induced protein could readily be detected in a sodium dodecyl sulfate-polyacrylamide gel by Coomassie blue staining. Its identity was confirmed by Western blot analysis using antibodies to both beta-galactosidase and the AR. Scatchard analysis of the androgen-binding activity of the hybrid AR revealed high affinity binding to the synthetic androgen, Mibolerone (Kd, approximately 1.2 nM). Competition studies demonstrated the fusion protein's specificity for androgens. The hybrid receptor formed immune complexes with human anti-AR serum that sedimented at about 19S in 10-50% linear sucrose gradients containing 0.4 M KCl. Gel band shift assays revealed that the hybrid receptor protein forms specific complexes with a synthetic steroid response element derived from the mouse mammary tumor virus long terminal repeat region. These results demonstrate that the recombinant AR expressed in E. coli possesses many of the functional properties characteristic of DNA- and steroid-binding domains of the native AR.
Young
C Y
CY
Department of Urology, Mayo Clinic/Foundation, Rochester, Minnesota 55905.
Qiu
S D
SD
Prescott
J L
JL
Tindall
D J
DJ
D000728:Androgens / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009277:Nandrolone / Q000031:analogs & derivatives / Q000378:metabolism; D010957:Plasmids; D011944:Receptors, Androgen / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013737:Testis / Q000737:chemistry; D045165:Testosterone Congeners / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics
D000728:Androgens; D011944:Receptors, Androgen; D011993:Recombinant Fusion Proteins; D045165:Testosterone Congeners; D009277:Nandrolone; D004247:DNA; C100075:mibolerone; D001616:beta-Galactosidase