Homogeneous purification of human recombinant GST-Akt/PKB from Sf9 cells

The plethora of extracellular stimuli modulating the status of a cell resul ts in the engagement of relatively few pathways responsible for transducing signals to the interior of the cell. One such pathway is the activation of phosphatidylinositol 3-kinase (PI-3K), which results in the generation of a membrane-restricted second messenger, polyphosphatidylinositide 3'-phosph ate. Among the enzymes activated by 3'-phosphorylated inositol lipids is Ak t/protein kinase B (PKB), Here we describe a protocol for the expression an d one-step purification of human recombinant GST-PKB in Sf9 cells. This sch eme allows generating large amounts of homogeneously purified GST-PKB with high specific activity to be employed in high-throughput screening or struc tural studies. (C) 1999 Academic Press.