Abstract
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.
Copyright 1999 Academic Press.
MeSH terms
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Animals
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Cell Line
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Enzyme Inhibitors / pharmacology
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Enzyme Stability
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Gene Expression
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Glutathione Transferase / genetics
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Glutathione Transferase / isolation & purification
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Glutathione Transferase / metabolism
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Histones / metabolism
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Humans
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Nucleopolyhedroviruses / genetics
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Peptides / metabolism
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Phosphorylation
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Protein Serine-Threonine Kinases*
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Proto-Oncogene Proteins / genetics*
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Proto-Oncogene Proteins / isolation & purification*
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Proto-Oncogene Proteins / metabolism
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Proto-Oncogene Proteins c-akt
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / isolation & purification
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Recombinant Fusion Proteins / metabolism
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Spodoptera
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Staurosporine / pharmacology
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Substrate Specificity
Substances
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Enzyme Inhibitors
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Histones
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Peptides
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Proto-Oncogene Proteins
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Recombinant Fusion Proteins
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Glutathione Transferase
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AKT1 protein, human
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Protein Serine-Threonine Kinases
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Proto-Oncogene Proteins c-akt
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Staurosporine