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), e ncompassing amino acids (AAs) 647-919, was expressed in Escherichia coli wi th 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 i on affinity chromatography in the presence of 6 M GdnHCl. The isolated prot ein 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 a ntibodies raised against a peptide corresponding to the last 19 AAs (AAs 90 1-919) of hAR revealed that the purified protein contained an immunoreactiv e epitope present within the AR and was of the appropriate size. Further ch aracterization, using matrix-assisted laser desorption/ionization time-of-f light 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, incl uding the last tryptic peptide in the hAR sequence. These data confirm that the purified protein was the intact hAR LBD. AA sequencing of these trypti c peptides, using an HPLC-coupled electrospray ionization ion trap mass spe ctrometer (LC/ESI-ITMS and MS/MS), unambiguously confirmed that the peptide s were from the hAR LBD. The purified His(10)-hAR LBD in 6 M GdnHCl could b e renatured as determined by ligand-binding activity, with a similar equili brium dissociation constant (K-d) for [H-3]-mibolerone and a similar steroi d specificity to the AR isolated from rat ventral prostate.