Interaction of androgen response elements with the DNA-binding domain of the rat androgen receptor expressed in Escherichia coli.
A fragment of the rat androgen receptor (amino acids 533-637) containing the DNA-binding domain was produced in Escherichia coli as a fusion product with protein A of Staphylococcus aureus. The fusion protein was purified on IgG-Sepharose, a method that does not involve the use of denaturing agents. Approximately 4 mg of fusion protein was obtained from 500 ml of bacterial culture. In gel shift assays, the recombinant DNA-binding domain displays an affinity for a fragment of the long terminal repeat of mouse mammary tumor virus and for an intronic fragment of the gene coding for the C3 component of the androgen-regulated rat prostatic binding protein. In a DNase I footprinting assay, the fusion protein protects a sequence in the C3 fragment that has previously been shown to act as a functional androgen response element. Interestingly, a single base pair mutation in the response element, which abolishes androgen inducibility, also destroys the ability to interact with the recombinant androgen receptor DNA-binding domain.
De Vos
P
P
Laboratory of Experimental Medicine and Endocrinology, Faculty of Medicine, Catholic University of Leuven, Belgium.
Claessens
F
F
Winderickx
J
J
Van Dijck
P
P
Celis
L
L
Peeters
B
B
Rombauts
W
W
Heyns
W
W
Verhoeven
G
G
D000728:Androgens / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D016172:DNA Fingerprinting; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D051381:Rats; D011944:Receptors, Androgen / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000032:analysis; D013205:Staphylococcal Protein A / Q000235:genetics / Q000378:metabolism
D000728:Androgens; D011944:Receptors, Androgen; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D004247:DNA