Overexpression, characterization, and purification of a recombinant mouse immunophilin FKBP-52 and identification of an associated phosphoprotein.
To gain insight into the structure and function of the immunophilin FKBP-52, a mouse FKBP-52 was overexpressed in Spodoptera frugiperda insect cells (Sf9 cells) with the baculovirus expression system. The purification and characterization of the recombinant FKBP-52 (rFKBP-52) was facilitated by incorporating a histidine 6-mer domain at its N terminus. The rFKBP-52 was highly purified on a N(i)2+ affinity resin with an estimated recovery of 10 mg of pure protein from 1 liter of Sf9 cell culture. Subcellular fractionation revealed that the rFKBP-52 is expressed predominantly in the nuclei of infected Sf9 cells maximally at 48 hr after infection, consistent with the nuclear localization of FKBP-52 in mammalian cells. The rFKBP-52 can be assembled in vitro with the glucocorticoid receptor complex, establishing its functionality and confirming that it is a component of the unactivated glucocorticoid receptor complex. The rFKBP-52 possesses an ATP/GTP binding activity that is stimulated by divalent cations. Furthermore, incubation of purified rFKBP-52 with [gamma-32P]ATP and MgCl2 resulted in the phosphorylation of a 59-kDa nuclear protein. Amino acid sequence analysis of this protein revealed that it is a phosphoprotein or kinase that is associated with the rFKBP-52.
Alnemri
E S
ES
Department of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107.
Fernandes-Alnemri
T
T
Nelki
D S
DS
Dudley
K
K
DuBois
G C
GC
Litwack
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002451:Cell Compartmentation; D002467:Cell Nucleus; D019204:GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D022021:Tacrolimus Binding Proteins
D002352:Carrier Proteins; D006360:Heat-Shock Proteins; D010750:Phosphoproteins; D011965:Receptors, Glucocorticoid; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins; D022021:Tacrolimus Binding Proteins