Overexpression of a partial human androgen receptor in E. coli

Characterization of steroid binding, DNA binding, and immunological properties

Charles Y F Young, Shudong Qiu, James L. Prescott, Donald J. Tindall

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

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-β-D-thiogalactopyranoside induction, a tripartite protein, consisting of β-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 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, ∼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 KCI. 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.

Original languageEnglish (US)
Pages (from-to)1841-1849
Number of pages9
JournalMolecular Endocrinology
Volume4
Issue number12
StatePublished - 1990

Fingerprint

Androgen Receptors
Steroids
Escherichia coli
DNA
Galactosidases
Androgens
Proteins
Testosterone Congeners
Thiogalactosides
Mouse mammary tumor virus
Terminal Repeat Sequences
human AR protein
Response Elements
Collagenases
Antigen-Antibody Complex
Sodium Dodecyl Sulfate
Sucrose
Organism Cloning
Complementary DNA
Western Blotting

ASJC Scopus subject areas

  • Molecular Biology
  • Endocrinology, Diabetes and Metabolism

Cite this

Overexpression of a partial human androgen receptor in E. coli : Characterization of steroid binding, DNA binding, and immunological properties. / Young, Charles Y F; Qiu, Shudong; Prescott, James L.; Tindall, Donald J.

In: Molecular Endocrinology, Vol. 4, No. 12, 1990, p. 1841-1849.

Research output: Contribution to journalArticle

Young, Charles Y F ; Qiu, Shudong ; Prescott, James L. ; Tindall, Donald J. / Overexpression of a partial human androgen receptor in E. coli : Characterization of steroid binding, DNA binding, and immunological properties. In: Molecular Endocrinology. 1990 ; Vol. 4, No. 12. pp. 1841-1849.
@article{2ac7dc817911425782b5a54e77cae71a,
title = "Overexpression of a partial human androgen receptor in E. coli: Characterization of steroid binding, DNA binding, and immunological properties",
abstract = "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-β-D-thiogalactopyranoside induction, a tripartite protein, consisting of β-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 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, ∼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 KCI. 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.",
author = "Young, {Charles Y F} and Shudong Qiu and Prescott, {James L.} and Tindall, {Donald J.}",
year = "1990",
language = "English (US)",
volume = "4",
pages = "1841--1849",
journal = "Molecular Endocrinology",
issn = "0888-8809",
publisher = "The Endocrine Society",
number = "12",

}

TY - JOUR

T1 - Overexpression of a partial human androgen receptor in E. coli

T2 - Characterization of steroid binding, DNA binding, and immunological properties

AU - Young, Charles Y F

AU - Qiu, Shudong

AU - Prescott, James L.

AU - Tindall, Donald J.

PY - 1990

Y1 - 1990

N2 - 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-β-D-thiogalactopyranoside induction, a tripartite protein, consisting of β-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 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, ∼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 KCI. 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.

AB - 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-β-D-thiogalactopyranoside induction, a tripartite protein, consisting of β-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 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, ∼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 KCI. 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.

UR - http://www.scopus.com/inward/record.url?scp=0025613232&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025613232&partnerID=8YFLogxK

M3 - Article

VL - 4

SP - 1841

EP - 1849

JO - Molecular Endocrinology

JF - Molecular Endocrinology

SN - 0888-8809

IS - 12

ER -