title: High-yield expression and purification of the Hsp90-associated p23, FKBP52, HOP and SGTα proteins.
abstract: Hsp90 is a ubiquitous molecular chaperone that plays a key role in the malignant development of hormone-dependent pathologies such as cancer. An important role for Hsp90 is to facilitate the stable binding of steroid hormones to their respective receptors enabling the ligand-based signal to be carried to the nucleus and ultimately resulting in the up-regulation of gene expression. Along with Hsp90, this dynamic and transient process also involves the recruitment of additional proteins and co-chaperones that add further stability to the mature receptor-chaperone complex. In the work presented here, we describe four new protocols for the bacterial over-expression and column chromatographic purification of the human p23, FKBP52, HOP and SGTα proteins. Each of these proteins plays a distinct role in the steroid hormone receptor regulatory cycle. Affinity, ion-exchange and size-exclusion techniques were used to produce target yields greater than 50mg/L of cultured media, with each purified sample reaching near absolute sample homogeneity. These results reveal a reliable system for the production of p23, FKBP52, HOP and SGTα substrate proteins for use in the investigation of the Hsp90-associated protein interactions of the steroid hormone receptor cycle.
authors.affiliation: Department of Chemistry, University of Texas at El Paso, 500 W. University Ave., El Paso, TX 79968, USA.
authors.forename: Zacariah L
authors.initials: ZL
authors.lastname: Hildenbrand
authors.forename: Sudheer K
authors.initials: SK
authors.lastname: Molugu
authors.forename: Atanu
authors.initials: A
authors.lastname: Paul
authors.forename: Gustavo A
authors.initials: GA
authors.lastname: Avila
authors.forename: Nadia
authors.initials: N
authors.lastname: Herrera
authors.forename: Chuan
authors.initials: C
authors.lastname: Xiao
authors.forename: Marc B
authors.initials: MB
authors.lastname: Cox
authors.forename: Ricardo A
authors.initials: RA
authors.lastname: Bernal
mesh_terms: D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D004926:Escherichia coli; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry; D006360:Heat-Shock Proteins / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D019746:Intramolecular Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D016014:Linear Models; D018832:Molecular Chaperones; D000071876:Prostaglandin-E Synthases; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D022021:Tacrolimus Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D018841:HSP90 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; C114575:SGTA protein, human; C079373:STIP1 protein, human; D022021:Tacrolimus Binding Proteins; C403485:tacrolimus binding protein 4; D019746:Intramolecular Oxidoreductases; D000071876:Prostaglandin-E Synthases