Characterization of human androgen receptor overexpressed in the baculovirus system.
An essential step in the process of understanding the structure and function of the human androgen receptor (hAR) involves the production of large quantities of the hAR. For this purpose, the full-length hAR has been overproduced in insect cells by using a baculovirus genetic expression system. The recombinant hAR is produced in Sf21 insect cells at approximately 7 pmol/mg of protein (1 x 10(6) AR molecules per cell), which is 70-150 times greater than levels detected in androgen target tissues. Androgen can bind to the baculovirus-expressed hAR with high affinity (Kd = 0.46 nM), and the specificity of hormone binding in baculovirus-expressed hAR is essentially identical to that of bona fide hAR. An anti-AR monoclonal antibody can recognize the baculovirus-expressed hAR at approximately 100 kDa upon Western blot analysis. Sucrose gradient analysis shows that baculovirus-expressed hAR complexes sediment at 4 S in a high salt medium and these complexes can interact with anti-AR monoclonal antibody to form complexes that sediment at 8-10 S. Therefore, production of recombinant hAR from the baculovirus expression system will provide an alternative source of biologically active hAR for studies on the molecular mechanisms of androgen action.
Chang
C
C
University of Wisconsin Comprehensive Cancer Center, Madison.
Wang
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C
DeLuca
H F
HF
Ross
T K
TK
Shih
C C
CC
D000728:Androgens / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009036:Moths; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011944:Receptors, Androgen / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D000728:Androgens; D009838:Oligodeoxyribonucleotides; D011944:Receptors, Androgen; D011994:Recombinant Proteins