High-yield expression and purification of the Hsp90-associated p23, FKBP52, HOP and SGTα proteins.
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.
Hildenbrand
Zacariah L
ZL
Department of Chemistry, University of Texas at El Paso, 500 W. University Ave., El Paso, TX 79968, USA.
Molugu
Sudheer K
SK
Paul
Atanu
A
Avila
Gustavo A
GA
Herrera
Nadia
N
Xiao
Chuan
C
Cox
Marc B
MB
Bernal
Ricardo A
RA
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
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