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

Protein Expr Purif. 1999 Oct;17(1):83-8. doi: 10.1006/prep.1999.1102.

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.

MeSH terms

  • Animals
  • Cell Line
  • Enzyme Inhibitors / pharmacology
  • Enzyme Stability
  • Gene Expression
  • Glutathione Transferase / genetics
  • Glutathione Transferase / isolation & purification
  • Glutathione Transferase / metabolism
  • Histones / metabolism
  • Humans
  • Nucleopolyhedroviruses / genetics
  • Peptides / metabolism
  • Phosphorylation
  • Protein Serine-Threonine Kinases*
  • Proto-Oncogene Proteins / genetics*
  • Proto-Oncogene Proteins / isolation & purification*
  • Proto-Oncogene Proteins / metabolism
  • Proto-Oncogene Proteins c-akt
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Spodoptera
  • Staurosporine / pharmacology
  • Substrate Specificity

Substances

  • Enzyme Inhibitors
  • Histones
  • Peptides
  • Proto-Oncogene Proteins
  • Recombinant Fusion Proteins
  • Glutathione Transferase
  • AKT1 protein, human
  • Protein Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-akt
  • Staurosporine