Browse > Article

Expression and Activation of Akt/PKB Protein Kinase using Escherichia coli  

Lee, Jae-Hag (Department of Food and Nutrition, Seoil University)
Publication Information
Microbiology and Biotechnology Letters / v.37, no.2, 2009 , pp. 105-109 More about this Journal
Abstract
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 $27^{\circ}C$ and 0.01-0.09 mM of IPTG for induction of the protein expression. The expressed protein was purified using $Ni^{2+}$-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.
Keywords
Akt/PKB; Escherichia coli expression system; purification; Akt/PKB specific fluorescent peptide; anti-cancer agent;
Citations & Related Records
Times Cited By KSCI : 3  (Citation Analysis)
Times Cited By SCOPUS : 0
연도 인용수 순위
1 Her, J.-H., Y. H. Cheong, J.-H. Kim, S.-K. Sin, C. G. Hyun, J. Chun, S. S. Kang, D.-K. Kang, and S.-K. Hong. 2002. Identification of a protein kinase using a FITC-labelled synthetic peptide in Streptomyces griseus IFO13350. Kor. J. Microbiol Biotechnol. 30(3): 235-240
2 Takeuchi, H., Y. Kondo, K. Fujiwara, T. Kanzawa, H. Aoki, G. B. Mills, and S. Kondo. 2005. Synergistic augmentation of rapamycin-induced autophagy in malignant glioma cells by phosphatidylinositol 3-kinase/protein kinase B inhibitors. Cancer Res. 65: 3336-46   PUBMED   ScienceOn
3 Paz, K., Y.-F. Liu, H. Shorer, R. Hemi, D. LeRoith, M. Quan, H. Kanety, R. Seger, and Y. Zick. 1999. Phosphorylation of insulin receptor substrate-1 (IRS-1) by protein kinase B positively regulates IRS-1 function. J. Biol. Chem. 274: 28816-28822   DOI   PUBMED
4 Kondapaka, S. B., M. J. Zarnowski, D. R. Yver, E. A. Sausville, and S. W. Cushman. 2004. 7-Hydroxystaurosporine (UCN-01) inhibition of Akt t$thr^{308}$ but not $ser^{473}$ phosphorylation: a basis for decreased insulin-stimulated glucose transport. Clinical Cancer Res. 10: 7192-7198   DOI   ScienceOn
5 Yang, H. Y., H.-S. Lee, J.-H. Ko, S.-W. Yeon, T.-Y. Kim, B. Y. Hwang, S. S. Kang, J. Chun, and S.-K. Hong. 2006. Identification of 3'-hydroxymelanetin and liquiritigenin as Akt protein kinase inhibitors. J. Microbiol. Biotechnol. 16: 1384-1391   과학기술학회마을   ScienceOn
6 Kang, S. S., T. Kwon, D. Y. Kwon, and S. I. Do. 1999. Akt protein kinase enhances human telomerase activity through phosphorylation of telomerase reverse transcriptase V subunit. J. Biol. Chem. 274: 13085-13090   DOI   ScienceOn
7 Sharma, S. V., T. Agatsuma, and H. Nakano. 1998. Targeting of the protein chaperone, HSP90, by the transformation suppressing agent, radicicol. Oncogene 16: 2639-2645   DOI   ScienceOn
8 Ko, J. H., S. W. Yeon, H. S. Lee, T. Y. Kim, D. Y. Noh, K. S. Shim, S.-K. Hong, and S. S. Kang. 2004. Inhibitory effects of dithiolo-thione derivative SWU-20009 on Akt activity. J. pharmaceutical society of Korea 48(2): 105-110   과학기술학회마을   ScienceOn
9 Lee, H. Y., S. H. Oh, J. K. Woo, W. Y. Kim, C. S. Van Pelt, R. E. Price, D. Cody, H. Tran, J. M. Pezzuto, R. M. Moriaty, and W. K. Hong. 2005. Chemopreventive effects of deguelin, a novel Akt inhibitor, on tobacco-induced lung tumorigenesis. J. Natl. Cancer Inst. 97: 1695-1699   DOI   ScienceOn
10 Kase, H., K. Iwahashi, Y. Matsuda, M. Takahashi, C. Murakata, A. Sato, and M. Kaneko. 1987. K-252a compounds, novel and potent inhibitors of protein kinase C and cyclic nucleotide-dependent protein kinases. Biochem. Biophys. Res. Commun. 142: 436-440   DOI   ScienceOn
11 Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, U. S. A
12 Bourret, R. B., K. A. Borkovich, and M. I. Simon. 1991. Signal transduction pathways involving protein phosphorylation in prokaryotes. Annu. Rev. Biochem. 60: 401-441   DOI   ScienceOn
13 Fayard, E., L. A. Tintignac, A. Baudry, and B. A. Hemmings. 2005. Protein kinase B/Akt at a glance. J. Cell Sci. 118: 5675-5678   DOI   ScienceOn
14 Hong, S.-K., A. Matsumoto, S. Horinouchi, and T. Beppu. 1993. Effects of protein kinase inhibitors on in vitro protein phosphorylation and cellular differentiation of Streptomyces griseus. Mol. Gen. Genet. 236: 347-354   DOI   ScienceOn
15 Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254   DOI   PUBMED   ScienceOn
16 Uehara, Y. and H. Ogawara. 1991. Use and selectivity of herbimycin A as inhibitor of protein tyrosine kinases. Methods Enzymol. 201: 370-379   DOI   PUBMED
17 Kwon, T., D. Y. Kwon, J. Chun, J. H. Kim, and S. S. Kang. 2000. Akt protein kinase inhibits Rac1-GTP binding through phosphorylation at serine 71 of Rac1. J. Biol. Chem. 275: 423-428   DOI   ScienceOn