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. 1993 Jul 15;90(14):6839–6843. doi: 10.1073/pnas.90.14.6839

Overexpression, characterization, and purification of a recombinant mouse immunophilin FKBP-52 and identification of an associated phosphoprotein.

E S Alnemri 1, T Fernandes-Alnemri 1, D S Nelki 1, K Dudley 1, G C DuBois 1, G Litwack 1
PMCID: PMC47028  PMID: 8341706

Abstract

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.

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