Expression and purification of active PKB kinase from Escherichia coli

Shoshana Klein, Tamar Geiger, Inbal Linchevski, Mario Lebendiker, Anna Itkin, Karin Assayag, Alexander Levitzki

Research output: Contribution to journalArticlepeer-review

Abstract

PKB/Akt is a protein involved in control of apoptosis, proliferation and cellular metabolism, and it has been found to be activated in many cancers. Activation of PKB involves recruitment of the enzyme by its PH domain to the cell membrane, and phosphorylation at two residues, T308 and S473. To produce active PKB kinase from Escherichia coli, we constructed a derivative of PKB lacking the PH domain and mutated to glutamate at residues S124, T450 and the activating residue S473 (ΔPH-PKB-EEE). ΔPH-PKB-EEE was expressed in E. coli together with PDK1, the kinase responsible for phosphorylating PKB at T308, which was expressed as a GST-fusion. Full-length ΔPH-PKB-EEE was obtained by using a double tag strategy: His6 at the N-terminus and FLAG at the C-terminus. The protein was purified by nickel affinity chromatography, followed by passage over an anti-FLAG column. The final purification step, anion exchange over a monoQ column, separated phosphorylated from unphosphorylated protein. Active recombinant PKB kinase was thus produced from E. coli, by a simple, reproducible procedure.

Original languageEnglish
Pages (from-to)162-169
Number of pages8
JournalProtein Expression and Purification
Volume41
Issue number1
DOIs
StatePublished - May 2005
Externally publishedYes

Keywords

  • Co-expression
  • E. coli
  • FLAG-tag
  • His-tag
  • Kinase
  • PKB
  • Recombinant protein

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