Expression and purification of active PKB kinase from Escherichia coli
| Autor: | Inbal Linchevski, Karin Assayag, Alexander Levitzki, Anna Itkin, Tamar Geiger, Shoshana Klein, Mario Lebendiker |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
Recombinant Fusion Proteins
DNA Recombinant Gene Expression AKT1 Protein Serine-Threonine Kinases Biology medicine.disease_cause Chromatography Affinity Affinity chromatography FLAG-tag Proto-Oncogene Proteins Escherichia coli medicine Phosphorylation Protein kinase B Base Sequence Kinase Chromatography Ion Exchange Protein Structure Tertiary Pleckstrin homology domain Amino Acid Substitution Biochemistry Genes Bacterial Mutagenesis Site-Directed cardiovascular system Proto-Oncogene Proteins c-akt Biotechnology |
| Zdroj: | Protein Expression and Purification. 41:162-169 |
| ISSN: | 1046-5928 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect