Expression and characterization of full-length and partial human androgen receptor fusion proteins. Implications for the production and applications of soluble steroid receptors in Escherichia coli.
We have expressed fusion proteins encoding defined segments of the coding segment of the human androgen receptor (hAR) in Escherichia coli using the pGEX-2T expression vector. Large quantities of fusion proteins containing glutathione-S-transferase (GST) linked to the amino or carboxy terminal region of the receptor and a fusion protein containing the complete amino acid sequence of the androgen receptor were produced in soluble form. The GST hAR fusion proteins containing the hormone-binding domain of the androgen receptor exhibit high affinity specific binding for a variety of natural and synthetic androgens. Analysis of the binding properties of the complete and truncated androgen receptor fusion proteins revealed that the amino terminus affects the Kd of the fusion proteins for mibolerone (0.89 vs. 3.43 nM for the truncated and complete fusion proteins, respectively). Despite these differences, both the truncated and complete hAR fusion proteins exhibit a higher affinity for dihydrotestosterone than for testosterone, implying that the preferential affinity for dihydrotestosterone observed in androgen receptor prepared from native sources is a measure of the inherent structure of the hormone-binding domain. Furthermore, the ligand-receptor complex is stable, as the ligand is not easily displaced with unlabelled competitor and is stable to mild heat denaturation. Fusion proteins containing the DNA-binding domain demonstrate specific DNA binding, as evidenced by studies using segments of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) and synthetic glucocorticoid response elements. These studies establish that GST hAR fusion proteins exhibit physical properties similar to those of native androgen receptor. Affinity purification using a glutathione affinity resin and cleavage of the fusion proteins at a thrombin cleavage site permits a marked enrichment using a two-step purification. The use of such methods will facilitate the study of the normal and mutant receptor proteins.
Roehrborn
C G
CG
Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas 75235-8857.
Zoppi
S
S
Gruber
J A
JA
Wilson
C M
CM
McPhaul
M J
MJ
D001483:Base Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D013196:Dihydrotestosterone / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D005982:Glutathione Transferase; D006801:Humans; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000502:physiology; D011944:Receptors, Androgen / Q000302:isolation & purification / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012091:Repetitive Sequences, Nucleic Acid; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism
D015345:Oligonucleotide Probes; D011944:Receptors, Androgen; D011993:Recombinant Fusion Proteins; D013196:Dihydrotestosterone; D013739:Testosterone; D005982:Glutathione Transferase