Abstract
Protein purification and characterization is required for a full understanding of structure-function relationships. Because proteins have complex structures and can be present at low concentrations, efficient purification protocols are needed. Purification of full-length androgen receptor (AR) is complicated by its low abundance, instability in the absence of androgen, and size and charge similarities with other nuclear proteins. Previous approaches to steroid hormone receptor purification have included traditional chromatography, such as ion exchange, gel filtration, isoelectric focusing chromatography (1,2), and hormone, DNA, and antibody affinity chromatography (3-5), but with low yield and purity. Overexpression of recombinant nuclear receptors or their domains in insect cells (6), Escherichia coli (7), or mammalian cells (8), has facilitated their purification. Purification with histidine (His)-tagged proteins is advantageous, because, unlike protein tags, such as glutathione S-transferase, short His sequences can have minimal effects on protein structure and function, efficiently bind metal-chelating columns mostly independent of protein conformation, and may not require the use of a cleavage step (9,10). This chapter details a procedure for the isolation of nondenatured, recombinant human AR with more than 95% purity using four-step chromatography with milligram yields (see Notes 1-Notes 4). Purified AR may be used in physical and biochemical studies, such as monoclonal antibody development (11), crystallography and nuclear magnetic resonance studies, DNA binding, and solution dimerization (6).
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© 2001 Humana Press Inc.
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Liao, M., Wilson, E.M. (2001). Production and Purification of Histidine-Tagged Dihydrotestosterone-Bound Full-Length Human Androgen Receptor. In: Lieberman, B.A. (eds) Steroid Receptor Methods. Methods in Molecular Biology™, vol 176. Humana Press. https://doi.org/10.1385/1-59259-115-9:67
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DOI: https://doi.org/10.1385/1-59259-115-9:67
Publisher Name: Humana Press
Print ISBN: 978-0-89603-754-0
Online ISBN: 978-1-59259-115-2
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