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 FK BP-52, a mouse FKBP-52 was overexpresed in Spodoptera frugiperda insec t cells (SF9 cells) with the baculovirus expression system. The purifi cation and characterization of the recombinant FKBP-52 (rFKBP-52) was facilitated by incorporating a histidine 6-mer domain at its N terminu s. The rFKBP-52 was highly purified on a Ni2+ affinity resin with an e stimated recovery of 10 mg of pure protein from 1 liter of Sf9 cell cu lture. Subcellular fractionation revealed that the rFKBP-52 is express ed predominantly in the nuclei of infected Sf9 cells maximally at 48 h r after infection, consistent with the nuclear localization of FKBP-52 in mammalian cells. The rFKBP-52 can be assembled in vitro with the g lucocorticoid receptor complex, establishing its functionality and con firming that it is a component of the unactivated glucocorticoid recep tor complex. The rFKBP-52 possesses an ATP/GTP binding activity that i s stimulated by divalent cations. Furthermore, incubation of purified rFKBP-52 with [gamma-P-32]ATP and MgCl2 resulted in the phosphorylatio n of a 59-kDa nuclear protein. Amino acid sequence analysis of this pr otein revealed that it is a phosphoprotein or kinase that is associate d with the rFKBP-52.