한국미생물·생명공학회지 (Microbiology and Biotechnology Letters)

  • 제37권2호
  • /
  • Pages.105-109
  • /
  • 2009
  • /
  • 1598-642X(pISSN)
  • /
  • 2234-7305(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
권호 표지

대장균을 이용한 Akt/PKB Protein Kinase의 발현 및 활성화

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

  • Lee, Jae-Hag (Department of Food and Nutrition, Seoil University)
  • 투고 : 2009.03.13
  • 심사 : 2009.04.16
  • 발행 : 2009.06.28
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

단백질 인산화를 통한 세포내 신호전달기구 중 serine/threonine kinase에 속하는 Akt/PKB는 세포 생존과 사멸, 당대사 등을 조절하는 것으로 알려져 있다. 이러한 이유로, Akt/PKB 단백질은 천연물질들로부터 항암제를 탐색하기 위한 한 가지 target으로 사용되어 왔다. 본 연구에서는 Akt/PKB 단백질을 대량으로 생산하기 위하여 대장균의 단백질 발현 시스템을 이용하여 human Akt/PKB 단백질을 발현시켰다. 대장균에서 대량 발현된 Akt는 일반적인 조건에서는 inclusion body를 형성하였다. 배양온도 $27^{\circ}C$에서 0.01-0.09 mM IPTG로 발현 유도 시 발현된 human Akt/PKB 단백질 상당 부분이 가용화 되었다. 발현된 Akt kinase를 $Ni^{2+}$-NTA agarose column으로 정제하고, anti-Akt antibody를 이용하여 정제된 단백질이 Akt kinase 임을 확인하였다. 정제된 human Akt/PKB 단백질은 세포추출물에 존재하는 인산화 단백질을 이용하여 in vitro에서 인산화 되었으며, 인산화된 활성형 human Akt/PKB protein kinase는 human Akt/PKB protein kinase 특이 형광 peptide를 특이적으로 인산화하였다.

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.

키워드

참고문헌

  1. 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 https://doi.org/10.1146/annurev.bi.60.070191.002153
  2. 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 https://doi.org/10.1016/0003-2697(76)90527-3
  3. 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 https://doi.org/10.1242/jcs.02724
  4. 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
  5. 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 https://doi.org/10.1007/BF00277132
  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 https://doi.org/10.1074/jbc.274.19.13085
  7. 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 https://doi.org/10.1016/0006-291X(87)90293-2
  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
  9. 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 https://doi.org/10.1158/1078-0432.CCR-04-0772
  10. 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 https://doi.org/10.1074/jbc.275.1.423
  11. 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 https://doi.org/10.1093/jnci/dji377
  12. 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
  13. 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 https://doi.org/10.1074/jbc.274.40.28816
  14. 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 https://doi.org/10.1038/sj.onc.1201790
  15. 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
  16. Uehara, Y. and H. Ogawara. 1991. Use and selectivity of herbimycin A as inhibitor of protein tyrosine kinases. Methods Enzymol. 201: 370-379 https://doi.org/10.1016/0076-6879(91)01033-X
  17. 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