Cloning, expression, purification, and characterization of the human Class Ia phosphoinositide 3-kinase isoforms.

Abstract The Class I phosphoinositide 3-kinases (PI3Ks) are lipid kinases that phosphorylate the 3-hydroxyl group of the inositol ring of phosphatidylinositides. Although closely related, experimental evidence suggests that the four Class I PI3Ks may be functionally distinct. To further study their unique biochemical properties, the three human Class Ia PI3K (α, β, and δ) p110 catalytic domains were cloned and co-expressed with the p85α regulatory domain in Sf9 cells. None of the p110 subunits were successfully expressed in the absence of p85α. Successful expression and purification of each p85α/p110 protein required using an excess of the p110 vector over the p85 vector during co-infection of Sf9 cells. Proteins were purified as the p85α/p110 complex by nickel affinity chromatography through an N-terminal His-tag on the p110 subunit using an imidazole gradient. The purification yields were high using the optimized ratio of p85/p110 vector and small culture volumes, with 24 mg/L cell culture media for p85α/p110α, 17.5 mg/L for p85α/p110δ, and 3.5 mg/L for p85α/p110β. The identity of each purified isoform was confirmed by mass spectral analysis and immunoblotting. The activities of the three p85α/p110 proteins and the Class Ib p110γ catalytic domain were investigated using phosphatidylinositol 4,5-bisphosphate (PIP2) as the substrate in a PIP2/phosphatidylserine (PS) liposome. All four enzymes exhibited reaction velocities that were dependent on the surface concentration of PIP2. The surface concentrations that gave maximal activity for each human isoform with 0.5 mM PIP2 were 2.5 mol% PIP2 for p110γ, 7.5 mol% for p85α/p110β, and 10 mol% PIP2 for p85α/p110α and p85α/p110δ. The specific activity of p85α/p110α was three to five times higher than that of the other human isoforms. These kinetic differences may contribute to the unique roles of these isoforms in cells.