Purification of Ultraspiracle from Drosophila melanogaster and its basic biochemical tests

Ultraspiracle (Usp) is a nuclear receptor present in arthropods which participates in control of their molting and metamorphosis. It has been shown that Usp is part of complex engaged in the cross talk between 20E and juvenile hormone (JH) signaling pathways where it interacts with ecdysone receptor (EcR). However Chd64 was also proposed as potential interaction partner for Usp in this cross-talk. Thus, biochemical studies of Usp are important for understanding of its interaction with Chd64. For that reason a new efficient method of Usp purification is presented. Drosophila Usp was cloned into pQE-80L vector. Transformed bacterial BL21(DE3)pLysS cells were grown in LB medium. The first step of purification includes fractionation with solid (NH4)2SO4. Fractions between 0-35% saturation containing Usp were dissolved and then desalted on a PD10 column. Next, Usp was purified using HisTrap column. Target protein was eluted with a buffer containing 250 mM imidazole. Finally Superdex 200 column was used for separation of remaining contaminations. Functionality of the obtained protein was confirmed by electrophoretic mobility shift assay (EMSA) using 32P-labeled hsp27 regulatory element. The previous procedure required heparin column and here it was excluded. Therefore overnight dialysis step can be omitted what reduces loss of Usp and shortens purification making it one-day instead of two-days procedure. EMSA confirmed that obtained protein was functional and it bound to hsp27 in form of monomer and dimer. Our purification method allows to obtain at least 0.6 mg of Usp protein from 1 L culture. Compared with the previous procedure LA2