Baculovirus production of fully-active phosphoinositide 3-kinase alpha as a p85α–p110α fusion for X-ray crystallographic analysis with ATP competitive enzyme inhibitors☆

Abstract Phosphoinositide 3-kinases have been targeted for therapeutic research because they are key components of a cell signaling cascade controlling proliferation, growth, and survival. Direct activation of the PI3Kα pathway contributes to the development and progression of solid tumors in breast, endometrial, colon, ovarian, and gastric cancers. In the context of a drug discovery effort, the availability of a robust crystallographic system is a means to understand the subtle differences between ATP competitive inhibitor interactions with the active site and their selectivity against other PI3Kinase enzymes. To generate a suitable recombinant design for this purpose, a p85α–p110α fusion system was developed which enabled the expression and purification of a stoichiometrically homogeneous, constitutively active enzyme for structure determination with potent ATP competitive inhibitors (Raha et al., in preparation) [56] . This approach has yielded preparations with activity and inhibition characteristics comparable to those of the full-length PI3Kα from which X-ray diffracting crystals were grown with inhibitors bound in the active site.