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KMID : 1234420090370020105
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Korean Journal of Microbiololgy and Biotechnology
2009 Volume.37 No. 2 p.105 ~ p.109
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Expression and Activation of Akt/PKB Protein Kinase using Escherichia coli
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Lee Jae-Hag
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Abstract
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Among signal transduction systems by protein phosphorylation Akt/PKB protein kinase which is one of serine/threonine kinases, is known to regulate the survival and death of the cell and glucose metabolism. Thus, Akt/PKB protein kinase has been used as one of the target proteins to find anti-cancer agents from natural products. In this study, human Akt/PKB protein kinase was expressed in Escherichia coli expression system for the mass production. Human Akt/PKB protein kinase expressed in E. coli formed inclusion body under the general condition. However, most of the expressed protein was solubilized under the culture temperature at 27oC and 0.01-0.09 mM of IPTG for induction of the protein expression. The expressed protein was purified using Ni2+-NTA agarose column and confirmed by using anti-Akt antibody. Subsequently, the purified human Akt/PKB protein kinase was activated by in vitro phosphorylation using cellular extract containing kinases. The activated protein was confirmed to phosphorylate the specific fluorescent peptide specially designed as the artificial substrate for Akt/PKB protein kinase.
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KEYWORD
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Akt/PKB, Escherichia coli expression system, purification, Akt/PKB specific fluorescent peptide, anti-cancer agent
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