Interaction of androgen response elements with the DNA-binding domain of the rat androgen receptor expressed in Escherichia coli

• Published in: The Journal of biological chemistry Vol. 266; no. 6; pp. 3439 - 3443
• Main Authors: De Vos, P, Claessens, F, Winderickx, J, Van Dijck, P, Celis, L, Peeters, B, Rombauts, W, Heyns, W, Verhoeven, G
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 25-02-1991; American Society for Biochemistry and Molecular Biology
• Subjects: Androgens - genetics; Animals; Base Sequence; DNA - genetics; DNA - metabolism; DNA Fingerprinting; Electrophoresis, Polyacrylamide Gel; Escherichia coli - genetics; Gene Expression Regulation, Bacterial; Genes, Bacterial; Genetic Vectors; Molecular Sequence Data; Rats; Receptors, Androgen - genetics; Recombinant Fusion Proteins - analysis; Staphylococcal Protein A - genetics; Staphylococcal Protein A - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)67814-2

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.