Mass spectrometric characterization of the human androgen receptor ligand-binding domain expressed in Escherichia coli.
The ligand-binding domain (LBD) of the human androgen receptor (hAR LBD), encompassing amino acids (AAs) 647-919, was expressed in Escherichia coli with an N-terminal polyhistidine tag (His(10)-hAR LBD) from a pET-16b vector. The overexpressed protein was initially insoluble in inclusion bodies, and was subsequently solubilized in 8 M guanidine hydrochloride (GdnHCl). The solubilized His(10)-hAR LBD was purified to apparent homogeneity by metal ion affinity chromatography in the presence of 6 M GdnHCl. The isolated protein migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular mass of 33-34 kDa, as expected from the plasmid construct. Immunoblot analysis with C-terminal antibodies raised against a peptide corresponding to the last 19 AAs (AAs 901-919) of hAR revealed that the purified protein contained an immunoreactive epitope present within the AR and was of the appropriate size. Further characterization, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS), showed a single protein species of average mass 34 580 Da, confirming the size and purity of the purified His(10)-hAR LBD. Detailed tryptic peptide mapping analysis, using MALDI/TOF-MS, identified a total of eight peptides with a 30% coverage of the LBD, including the last tryptic peptide in the hAR sequence. These data confirm that the purified protein was the intact hAR LBD. AA sequencing of these tryptic peptides, using an HPLC-coupled electrospray ionization ion trap mass spectrometer (LC/ESI-ITMS and MS/MS), unambiguously confirmed that the peptides were from the hAR LBD. The purified His(10)-hAR LBD in 6 M GdnHCl could be renatured as determined by ligand-binding activity, with a similar equilibrium dissociation constant (K(d)) for [(3)H]-mibolerone and a similar steroid specificity to the AR isolated from rat ventral prostate.
Zhu
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Department of Pharmaceutical Sciences, The Charles B. Stout Neuroscience Mass Spectrometry Laboratory, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA.
Becklin
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Desiderio
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Dalton
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JT
D000595:Amino Acid Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli; D019791:Guanidine / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008024:Ligands; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009277:Nandrolone / Q000031:analogs & derivatives / Q000493:pharmacokinetics*; D010446:Peptide Fragments / Q000737:chemistry; D011944:Receptors, Androgen / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014316:Tritium; D014357:Trypsin
D008024:Ligands; D010446:Peptide Fragments; D011944:Receptors, Androgen; D011994:Recombinant Proteins; D014316:Tritium; D009277:Nandrolone; C100075:mibolerone; D014357:Trypsin; D019791:Guanidine