Browse > Article
http://dx.doi.org/10.5352/JLS.2012.22.4.552

Expression of SARS-3CL Protease in a Cell-Free Protein Synthesis System  

Park, Sun-Joo (Department of Chemistry, Pukyong National University)
Kim, Yong-Tae (Department of Food Science and Technology, Kunsan National University)
Publication Information
Journal of Life Science / v.22, no.4, 2012 , pp. 552-558 More about this Journal
Abstract
Severe acute respiratory syndrome (SARS) is a severe respiratory infectious disease caused by a novel human coronavirus, SARS-CoV. The 3CL protease is a key enzyme in the proteolytic processing of replicase polyprotein precursors, pp1a and pp1ab, which mediate all the functions required for viral genomic replication and transcription. Therefore, this enzyme is a target for the development of chemotherapeutic agents against SARS. A large quantity of active SARS-3CL protease is required for development of anti-SARS agents. Here we have constructed overexpression vector for the production of the SARS-3CL protease. The gene encoding SARS-3CL protease was amplified using polymerase chain reaction and cloned into the pET29a expression vector, resulting in pET29a/SARS-3CLP. Recombinant SARS-3CL protease was successfully synthesized by the dialysis mode of the cell-free protein expression system, and purified by three-step fast protein liquid chromatography using HighQ and MonoP column chromatographies and Sephacryl S-300 gel filtration. In addition, the produced SARS-3CL protease was found to be an active mature form. This study provides efficient methods not only for the development of anti-SARS materials from natural sources, but also for the study of basic properties of the SARS-3CL protease.
Keywords
Cell-free protein synthesis; 3CL protease; coronavirus; protein expression; SARS;
Citations & Related Records
연도 인용수 순위
  • Reference
1 Thiel, V., Ivanov, K. A., Putics, A., Hertzig, T., Schelle, B., Bayer, S., Weissbrich, B., Snijder, E. J., Rabenau, H., Doerr, H. W., Gorbalenya, A. E. and Ziebuhr, J. 2003. Mechanisms and enzymes involved in SARS coronavirus genome expression. J. Gen. Virol. 84, 2305-2315.   DOI   ScienceOn
2 Wu, C. Y., Jan, J. T., Ma, S. H., Kuo, C. J., Juan, H. F., Cheng, Y. S., Hsu, H. H., Huang, H. C., Wu, D., Brik, A., Liang, F. S., Liu, R. S., Fang, J. M., Chen, S. T., Liang, P. H. and Wong, C. H. 2004. Small molecules targeting severe acute respiratory sundrome human coronavirus. Proc. Natl. Acad. Sci. USA 101, 10012-10017.   DOI   ScienceOn
3 Xiong, B., Gui, C. S., Xu, X. Y., Luo, C., Chen, J., Luo, H. B., Chen, L. L., Li, G. W., Sun, T., Yu, C. Y., Yue, L. D., Duan, W. H., Shen, J. K., Qin, L., Shi, T. L., Li, Y. X., Chen, K. X., Luo, X. M., Shen, X., Shen, J. H. and Jiang, H. L. 2003. A 3D model of SARS-CoV 3CL proteinase and 54 inhibitors design by virtual screening. Acta Pharmacol. Sin. 24, 497-504.
4 Zhang, X. W., Yap, Y. L. and Altmeyer, R. M. 2005. Generation of predictive pharmacophore model for SARS-coronavirus main proteinase. Eur. J. Med. Chem. 40, 57-62.   DOI   ScienceOn
5 Ksiazek, T. G., Erdman, D., Goldsmith, C. S., Zaki, S. R., Peret, T., Emery, S., Tong, S., Urbani, C., Comer, J. A., Lim, W., Rollin, P. E., Dowell, S. F., Ling, A. E., Humphrey, C. D., Shieh, W. J., Guarner, J., Paddock, C. D., Rota, P., Fields, B., DeRisi, J., Yang, J. Y., Cox, N., Hughes, J. M., LeDuc, J. W., Bellini, W. J. and Anderson L. J.; SARS Working Group. 2003. A novel coronavirus associated with severe acute respiratory syndrome. N. Engl. J. Med. 348, 1953-1966.   DOI   ScienceOn
6 Lee, N., Hui, D., Wu, A., Chan, P., Cameron, P., Joynt, G. M., Ahuja, A., Yung, M. Y., Leung, C. B., To, L. F., Lui, S. F., Szeto, C. C., Chung, S. and Sung, J. J. 2003. A major outbreak of severe acute respiratory syndrome in Hong Kong. N. Engl. J. Med. 348, 1986-1994.   DOI   ScienceOn
7 Liu, Z., Huang, C., Fan, K., Wei, P., Chen, H., Liu, S., Pei, J., Shi, L., Li, B., Yang, K., Liu, Y. and Lai, L. 2005. Virtual screening of novel noncovalent inhibitors for SARS-CoV 3C-like proteinase. J. Chem. Inf. Model. 45, 10-17.   DOI   ScienceOn
8 Marra, M. A., Jones, S. J., Astell, C. R., Holt, R. A., Brooks-Wilson, A., Butterfield, Y. S., et al. 2003. The genome sequence of the SARS-associated coronavirus. Science 300, 1399-1404.   DOI
9 Myint, S. H. 1995. Human coronavirus infections, pp. 389-401, In Siddell S. G. (eds.), The coronaviridae. Plenum Press.
10 Peiris, J. S., Yuen, K. Y., Osterhaus, A. D. and Stöhr, K. 2003. The severe acute respiratory syndrome. N. Engl. J. Med. 349, 2431-2441.   DOI   ScienceOn
11 Drosten, C., Gunther, S., Preiser, W., van der Werf, S., Brodt, H. R., Becker, S., Rabenau, H., Panning, M., Kolesnikova, L., Fouchier, R. A., Berger, A., Burguiere, A. M., Cinatl, J., Eickmann, M., Escriou, N., Grywna, K., Kramme, S., Manuguerra, J. C., Müller, S., Rickerts, V., Stürmer, M., Vieth, S., Klenk, H. D., Osterhaus, A. D., Schmitz, H. and Doerr, H. W. 2003. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N. Engl. J. Med. 348, 1967-1976.   DOI   ScienceOn
12 Ruan, Y. J., Wei, C. L., Ee, A. L., Vega, V. B., Thoreau, H., Su, S. T., Chia, J. M., Ng, P., Chiu, K. P., Lim, L., Zhang, T., Peng, C. K., Lin, E. O., Lee, N. M., Yee, S. L., Ng, L. F., Chee, R. E., Stanton, L. W., Long, P. M. and Liu, E. T. 2003. Comparative full-length genome sequence analysis of 14 SARS coronavirus isolates and common mutations associated with putative origins of infection. Lancet 361, 1779-1785.   DOI   ScienceOn
13 Seki, E., Matsuda, N., Yokoyama, S. and Kigawa, T. 2008. Cell-free protein synthesis system from Escherichia coli cells cultured at decreased temperatures improves productivity by decreasing DNA template degradation. Anal. Biochem. 377, 156-161.   DOI   ScienceOn
14 Thiel, V., Herold, J., Schelle, B. and Siddell, S. G. 2001. Viral replicase gene products suffice for coronavirus discontinuous transcription. J. Virol. 75, 6676-6681.   DOI   ScienceOn
15 Fan, K., Wei, P., Feng, Q., Chen, S., Huang, C., Ma, L., Lai, B., Pei, J., Liu, Y., Chen, J. and Lai, L. 2004. Biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3C-like proteinase. J. Biol. Chem. 279, 1637-1642.   DOI
16 Fouchier, R. A., Kuiken, T., Schutten, M., van Amerongen, G., van Doornum, G. J., van den Hoogen, B. G., Peiris, M., Lim, W., Stohr, K. and Osterhaus, A. D. 2003. Aetiology: Koch's postulates fulfilled for SARS virus. Nature 423, 240.   DOI   ScienceOn
17 Kigawa, T., Yabuki, T., Yoshida, Y., Tsutsui, M., Ito, Y., Shibata, T. and Yokoyama, S. 1999. Cell-free production and stable-isotope labeling of milligram quantities of protein. FEBS Lett. 442, 15-19.   DOI   ScienceOn
18 Kao, R. Y., Tsui, W. H., Lee, T. S., Tanner, J. A., Watt, R. M., Huang, J. D., Hu, L., Chen, G., Chen, Z., Zhang, L., He, T., Chan, K. H., Tse, H., To, A. P., Ng, L. W., Wong, B. C., Tsoi, H. W., Yang, D., Ho, D. D. and Yuen, K. Y. 2004. Identification of novel small-molecule inhibitors of severe acute respiratory syndrome-associated coronavirus by chemical genetics. Chem. Biol. 11, 1293-1299.   DOI   ScienceOn
19 Kathryn, V. H. 2003. SARS-associated coronavirus. N. Engl. J. Med. 348, 1948-1951.   DOI   ScienceOn
20 Kigawa, T., Yabuki, T., Matsuda, N., Matsuda, T., Nakajima, R., Tanaka, A. and Yokoyama, S. 2004. Preparation of Escherichia coli cell extract for highly productive cell-free protein expression. J. Struct. Funct. Genomics 5, 63-68.   DOI
21 Krokhin, O., Li, Y., Andonov, A., Feldmann, H., Flick, R., Jones, S., Stroeher, U., Bastien, N., Dasuri, K. V., Cheng, K., Simonsen, J. N., Perreault, H., Wilkins, J., Ens, W., Plummer, F. and Standing, K. G. 2003. Mass spectrometric characterization of proteins from the SARS virus: a preliminary report. Mol. Cell Proteomics 2, 346-356.
22 Donnelly, C. A., Ghani, A. C., Leung, G. M., Hedley, A. J., Fraser, C., Riley, S., Abu-Raddad, L. J., Ho, L. M., Thach, T. Q., Chau, P., Chan, K. P., Lam, T. H., Tse, L. Y., Tsang, T., Liu, S. H., Kong, J. H., Lau, E. M., Ferguson, N. M. and Anderson, R. M. 2003. Epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in Hong Kong. Lancet 361, 1761-1766.   DOI   ScienceOn
23 Anand, K., Ziebuhr, J., Wadhwani, P., Mesters, J. R. and Hilgenfeld, R. 2003. Coronavirus main proteinase (3CLpro) structure: basis for design of anti-SARS drugs. Science 300, 1763-1767.   DOI
24 Blanchard, J. E., Elowe, N. H., Huitema, C., Fortin, P. D., Cechetto, J. D., Eltis, L. D. and Brown, E. D. 2004. High-throughput screening identifies inhibitors of the SARS coronavirus main proteinase. Chem. Biol. 11, 1445-1453.   DOI   ScienceOn
25 Chou, K. C., Wei, D. Q. and Zhong, W. Z. 2003. Binding mechanism of coronavirus main proteinase with ligands and its implication to drug design against SARS. Biochem. Biophys. Res. Commun. 308, 148-151.   DOI   ScienceOn