Expression and purification of active PKB kinase from Escherichia coli.
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 (DeltaPH-PKB-EEE). DeltaPH-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 DeltaPH-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.
Klein
Shoshana
S
Unit of Cellular Signaling, Department of Biological Chemistry, The Hebrew University of Jerusalem, Safra Campus, Jerusalem 91904, Israel.
Geiger
Tamar
T
Linchevski
Inbal
I
Lebendiker
Mario
M
Itkin
Anna
A
Assayag
Karin
K
Levitzki
Alexander
A
D019943:Amino Acid Substitution; D001483:Base Sequence; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D051057:Proto-Oncogene Proteins c-akt; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D004274:DNA, Recombinant; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt