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Hsp90 chaperone machinery coexpression increases functional glucocorticoid receptor protein expression, receptor•hsp90 heterocomplex formation, and steroid binding activity
Author(s) -
Biette Kelly M.,
Belyaev Alexander S.,
Murphy Patrick J. M.
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.784.2
Subject(s) - hsp90 , glucocorticoid receptor , chaperone (clinical) , heat shock protein , hsp70 , microbiology and biotechnology , biology , receptor , biochemistry , gene , medicine , pathology
While hsp90 client proteins such as the glucocorticoid receptor (GR) are highly expressed using a baculovirus expression system, the majority of overexpressed protein is misfolded and nonfunctional. Nonaggregated‐GR forms a functional GR•hsp90 heterocomplex with steroid binding activity. We hypothesized baculoviral co‐overexpression of the complete hsp90 chaperone machinery (i.e. hsp90, hsp70, Hop, hsp40, and p23) would decrease overexpressed client protein aggregation and increase functional client protein concentration and activity. A fusion protein containing wild‐type GR ligand binding domain (GST‐LBD) was produced using a baculovirus expression system in the presence or absence of coinfected baculovirus expressing the hsp90 chaperone machinery. Western blot and steroid binding analysis demonstrated baculoviral coexpression of hsp90 chaperone machinery elicited a >;2‐fold increase in client protein expression and a >;7.5‐fold increase in steroid binding activity. Coexpression of the hsp90 chaperone machinery increased formation of functional GST‐LBD•hsp90 heterocomplexes and steroid binding capacity following in vitro reconstitution. These data show baculoviral coinfection with hsp90 chaperone machinery increases overexpressed GR LBD folding and function. We speculate this methodology may be applicable to baculoviral expression of many, if not all, hsp90 client proteins.