Homogeneous purification of human recombinant GST-Akt/PKB from Sf9 cells.
The plethora of extracellular stimuli modulating the status of a cell results 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'-phosphate. Among the enzymes activated by 3'-phosphorylated inositol lipids is Akt/protein kinase B (PKB). Here we describe a protocol for the expression and one-step purification of human recombinant GST-PKB in Sf9 cells. This scheme allows generating large amounts of homogeneously purified GST-PKB with high specific activity to be employed in high-throughput screening or structural studies.
Fabbro
D
D
Department of Oncology, Unit Biology Basel, Novartis Pharma AG, Basel, 4057, Switzerland.
Batt
D
D
Rose
P
P
Schacher
B
B
Roberts
T M
TM
Ferrari
S
S
D000818:Animals; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D006801:Humans; D017924:Nucleopolyhedroviruses / Q000235:genetics; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D019311:Staurosporine / Q000494:pharmacology; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D006657:Histones; D010455:Peptides; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D019311:Staurosporine