Browse > Article

Expression of MEK1 Fusion Protein in Yeast for Developing Cell Based Assay System, a Major Substrate of LeTx  

Hwang, Hye-Hyun (Department of Chemistry, College of Natural Sciences, Hanyang University)
Kim, Joung-Mok (Department of Chemistry, College of Natural Sciences, Hanyang University)
Choi, Kyoung-Jae (Department of Chemistry, College of Natural Sciences, Hanyang University)
Park, Hae-Chul (Department of Chemistry, College of Natural Sciences, Hanyang University)
Han, Sung-Hwan (Department of Chemistry, College of Natural Sciences, Hanyang University)
Chung, Hoe-Il (Department of Chemistry, College of Natural Sciences, Hanyang University)
Koo, Bon-Sung (Microbial Function Team, National Institute of Agricultural Biotechnology, RDA)
Park, Joon-Shik (NANO Mechatronics Research Center, Korea Electronics Technology Institute)
Yoon, Moon-Young (Department of Chemistry, College of Natural Sciences, Hanyang University)
Publication Information
Korean Journal of Microbiology / v.42, no.3, 2006 , pp. 195-198 More about this Journal
Abstract
Lethal toxin is a critical virulence factor of anthrax. It is composed two protein: protective antigen (PA) and lethal factor (LF). PA binds to specific cell surface receptors and, forms a membrane channel that mediates entry of LF into the cell. LF is a zinc-dependent metalloprotease, which cleaves MKKs [MAPK (mitogen-activated protein kinase) kinases] at peptide bonds very close to their N-termini. In this study, we suggest application of cell-based assays in the early phase of drug discovery, with a particular focus on the use of yeast cells. We constructed MEK1 expression system in yeast to determine LF activity and approached cell-based assay system to screen inhibitors, in which the results covering the construction of LF-substrate in yeast expression vector, expression, and LF-mediated proteolysis of substrate were described. These results could provided the basic steps in design of cell-based assay system with the high efficiency, rapidly and easy way to screening of inhibitors.
Keywords
Bacillus anthracis; cell-based assay system; lethal factor; yeast expression system;
Citations & Related Records
Times Cited By KSCI : 1  (Citation Analysis)
연도 인용수 순위
1 Xu, B., S. Stippec, F.L. Rovinson, and M.H. Cobb. 2001. Hydrophobic as well as charged residues in both MEK1 and ERK2 are important for their proper docking. J. Biol. Chem. 276, 26509- 26515   DOI   ScienceOn
2 Agrawal, A., J. Lingappa, S.H. Leppla, S. Agrawal, A. Jabbar, C. Quinn, and B. Pulendran. 2003. Impairment of dentritic cells and adaptive immunity by anthrax lethal toxin. Nature 424, 329-334   DOI   ScienceOn
3 Dixon, T.C., M. Meselson, J. Guillemin, and P.C. Hanna. 1999. Anthrax. Engl. J. Med. 341, 815-826   DOI   ScienceOn
4 Drum, C.L., S.Z. Yan, J. Bard, Y.Q. Shen, D. Lu, S. Soelaiman, Z. Grabarek, A. Bohm, and W.J. Tang. 2002. Structural basis for the activation of anthrax adenylyl cyclase exotoxin by calmodulin. Nature 415, 396-402   DOI   ScienceOn
5 Duesery, N.S., C.P. Webb, S.H. Leppla, V.M. Gordon, K.R. Klimpel, T.D. Copeland, N.G. Ahn, M.K. Oskarsson, K. Fukasawa, K.D. Paull, and C.F. Bande Woude. 1998. Protolytic inactivation of MAP-Kinase-kinase by anthrax lethal factor. Science 280, 734-737   DOI
6 Leppla, S.H. 1988. Production and purification of anthrax toxin, in: Methods in Enzymology. Academic press. 165, 103-116
7 Mock, M. and A. Fouet. 2001. Anthrax. Annu. Rev. Microbiol. 55, 647-671   DOI   ScienceOn
8 Vitale, G., L. Bernardi, G. Napolitani, M. Mock, and C. Montecucco. 2000. Susceptibility of mitogen-activated protein kinase kinase family members to proteolysis by anthrax lethal factor. Biochem. J. 352, 739-745   DOI
9 Klimpel, K.R., S.S. Molloy, G. Thomas, and S.H. Leppla. 1992. Anthrax toxin protective antigen is activated by a cell surface protease with the sequence specificity and catalytic properties of furin. Proc. Natl. Acad. Sci. USA. 89, 10277-10281   DOI   ScienceOn
10 황혜현, 김정목, 최경재, 정회일, 한성환, 구본성, 윤문영. 2005. Yeast 내에서 탄저병 원인균인 Bacillus anthracis의 치사독소인 Lethal Factor 단백질 발현. 미생물학회지 41, 275-280   과학기술학회마을
11 Vitale, G., R. Pellizzari, C. Recchi, G. Napolitani, M. Mock, and C. Montecucco. 1998. Anthrax lethal factor cleaves the N-terminus of MAPKKs and induces tyrosine/threonine phosphorylation of MAPKs in cultured macrophages. Biochem. Biophys. Res. Commun. 38, 706-711
12 Bardwell, A.J., L.J. Flatauer, K. Matsukuma, J. Thormer, and L. Bardwell. 2001. A conserved docking site in MEKs mediates high-affinity binding to MAP kinases and cooperates with a scaffold protein to enhance signal transmission. J. Biol. Chem. 276, 10374-10386   DOI   ScienceOn
13 Morton, N. and M.D. Swartz. 2001. New Engl. J. Med. 345, 1621- 1626   DOI   ScienceOn
14 Gietz, D., A.St. Jean, T.A. Woods, and T.H. Schiestl. 1992. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 20, 1425   DOI   ScienceOn
15 Pellizzari, R., C. Guidi-Rontani, G. Vitale, M. Mock, and C. Montecucco. 1999. Anthrax lethal factor cleaves MKK3 in macrophages and inhibit the LPS/IFN$\gamma$- induced release of NO and TNF$\alpha$.FEBS Lett. 462, 199-204   DOI   ScienceOn
16 Collier, R.J. and J.A. Young. 2003. Anthrax toxin. Annu. Rev. Cell Dev. Biol. 19, 45-70   DOI   ScienceOn
17 Mogridge, J., K. Cunningham, and R.J. Collier. 2002. Stoichiometry of anthrax toxin complexes. Biochemistry. 41, 1079-1082   DOI   ScienceOn
18 Dong, C., R.J. Kavis, and R.A. Flavell. 2002. MAP kinases in the immune response. Annu. Rev. Immunol. 20, 55-72   DOI   ScienceOn
19 Ezzell, J.W. and T.G. Abshire. 1988. Immunological analysis of cell associated antigens of Bacillus anthracis. Infect. Immun. 56, 349-356
20 Grossier, F. and M. Mock. 2001. Toxins of Bacillus anthracis. Toxicon. 39, 1747-1755   DOI   ScienceOn
21 Beauregard, K.E., R.J. Collier, and J.A. Swanson. 2000. Proteolytic activation of receptor-bound anthrax protective antigen on macrophages promotes its internalization. Cell Microiol. 2, 251-258   DOI   ScienceOn
22 Burke, D., D. Dawson, and T. Stearns. 2000. Methods in yeast genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y