Baculovirus production of fully-active phosphoinositide 3-kinase alpha as a p85alpha-p110alpha fusion for X-ray crystallographic analysis with ATP competitive enzyme inhibitors.
Protein Expr Purif
; 73(2): 167-76, 2010 Oct.
Article
em En
| MEDLINE
| ID: mdl-20457255
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 PI3Kalpha 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 p85alpha-p110alpha 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 PI3Kalpha from which X-ray diffracting crystals were grown with inhibitors bound in the active site.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Cristalografia por Raios X
/
Inibidores Enzimáticos
/
Classe II de Fosfatidilinositol 3-Quinases
Tipo de estudo:
Prognostic_studies
Limite:
Animals
Idioma:
En
Revista:
Protein Expr Purif
Ano de publicação:
2010
Tipo de documento:
Article