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Biotechnology and bioprocess engineering v.14 no.5, 2009년, pp.551 - 558   SCI SCIE 피인용횟수: 1
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Overexpression, Purification, and Functional Characterization of the Group II Chaperonin from the Hyperthermophilic Archaeum Pyrococcus horikoshii OT3

Kim, Jeong-Hwan    (Department of Biomaterial Control, Dong-Eui University   ); Shin, Eun-Jung    (Department of Biomaterial Control, Dong-Eui University   ); Jeon, Sung-Jong    (Department of Biomaterial Control, Dong-Eui University   ); Kim, Yeon-Hee    (Department of Biomaterial Control, Dong-Eui University   ); Kim, Pil    (Department of Biotechnology, Catholic University of Korea   ); Lee, Chung-Hwan    (Department of Bioscience and Biotechnology, Konkuk University   ); Nam, Soo-Wan    (Department of Biomaterial Control, Dong-Eui University  );
  • 초록

    Overexpression in Escherichia coli and functional characterization of the group II chaperonin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 were investigated in this study. PhCpn, the chaperonin gene from the P. horikoshii OT3, was amplified by PCR from the P. horikoshii OT3 genomic DNA, subcloned into pET21a vector, and expressed in three E. coli host cells such as BL21, Rosetta, and Codonplus (DE3). Among these host cells, E. coli Rosetta showed the highest expression level of recombinant PhCpn at induction with 1 mM IPTG. The recombinant PhCpn was purified to 91% by heat-shock treatment and anion-exchange chromatography. The ATPase activity of the purified PhCpn increased in a PhCpn concentration-dependent manner. Also, PhCpn protected the inorganic phosphatase from thermal inactivation at 85 and $110^{\circ}C$ , speculating that PhCpn is effective in in vitro holding of the protein. The holding efficiency was enhanced by the addition of $Mg^{2+}$ ion. Through the coexpression of pro-carboxypeptidase B (pro-CPB) and PhCpn in E. coli Rosetta, pro-CPB was produced as a soluble and active form with a marked yield. This result indicated that PhCpn facilitated the in vivo correct folding of pro-CPB and could be used as powerful and novel molecular machinery for the production of recombinant proteins as soluble and active forms in E. coli.


  • 주제어

    chaperonin .   coexpression .   hyperthermophilic archaeum .   protein folding .   Pyrococcus horikoshii.  

  • 참고문헌 (47)

    1. Laksanalamai, P., T. A. Whitehead, and F. T. Robb (2004) Minimal protein-folding systems in hyperthermophilic archaea. Nat. Rev. Microbiol. 2: 315-324 
    2. Kim, S., K. R. Willison, and A. L. Horwich (1994) Cystosolic chaperonin subunits have a conserved ATPase domain but diverged polypeptide-binding domains. Trends Biochem. Sci. 19: 543-548 
    3. Phipps, B. M., D. Typke, R. Heger, S. Volker, A. Hoffmann, K. O. Stetter, and W. Baumeister (1993) Structure of a molecular chaperone from a thermophilic archaebacterium. Nature 361: 475-477 
    4. Sigler, P. B., Z. Xu, H. S. Rye, S. G. Burston, W. A. Fenton, and A. L. Horwich (1998) Structure and function in GroEL-mediated protein folding. Annu. Rev. Biochem. 67: 581-608 
    5. Furutani, M., T. Iida, T. Yoshida, and T. Maruyama (1998) Group II chaperonin in a thermophilic methanogen, Methanococcus thermolithotrophicus. Chaperone activity and filament-forming ability. J. Biol. Chem. 273: 28399-28407 
    6. Oh, I. S., T. W. Kim, J. H. Ahn, J. W. Keum, C. Y. Choi, and D. M. Kim (2007) Use of L-buthionine sulfoximine for the efficient expression of disulfide-containing proteins in cell-free extracts of Escherichia coli. Biotechnol. Bioprocess Eng. 5: 574-578     
    7. Thomas, J. G., A. Ayling, and F. Baneyx (1997) Molecular chaperones, folding catalysts, and the recovery of active recombinant proteins from Escherichia coil. Appl. Biochem. Biotechnol. 66: 197-238 
    8. Hartl, F. U. and M. Hayer-Hartl (2002) Molecular chaperones in the cytosol: from nascent chain to folded protein. Science 295: 1852-1858 
    9. Kusmierczyk, A. R. and J. Martin (2003) Nucleotide dependent protein folding in the type II chaperonin from the mesophilic archaeon Methanococcus maripaludis. Biochem. J. 371: 669-673 
    10. Izumi, M., S. Fujiwara, M. Takagi, S. Kanaya, and T. Imanaka (1999) Isolation and characterization of a second subunit of molecular chaperonin from Pyrococcus kodakaraensis KOD1: analysis of an ATPase-deficient mutant enzyme. Appl. Environ. Microbiol. 65: 1801-1805 
    11. Yoshida, T., A. Ideno, R. Suzuki, M. Yohda, and T. Maruyama (2002) Two kinds of archaeal group II chaperonin subunits with different thermostability in Thermococcus strain KS-1. Mol. Microbiol. 44: 761-769 
    12. Fenton, W. A. and A. L. Horwich (1997) GroEL-mediated protein folding. Protein Sci. 6: 743-760 
    13. Chen, H. Y., Z. M. Chu, Y. H. Ma, Y. Zhang, and S. L. Yang (2007) Expression and characterization of the chaperonin molecular machine from the hyperthermophilic archaeon Pyrococcus furiosus. J. Basic Microbiol. 47: 132-137 
    14. Hartl, F. U. (1996) Molecular chaperones in cellular protein folding. Nature 381: 571-579 
    15. Guagliardi, A., L. Cerchia, and M. Rossi (1995) Prevention of in vitro protein thermal aggregation by the Sulfolobus solfataricus chaperonin. Evidence for nonequivalent binding surfaces on the chaperonin molecule. J. Biol. Chem. 270: 28126-28132 
    16. Ranson, N. A., H. E. White, and H. R. Saibil (1998) Chaperonins. Biochem. J. 333: 233-242 
    17. Kubota, H., G. Hynes, and K. Willison (1995) The chaperonin containing t-complex polypeptide 1 (TCP-1). Multisubunit machinery assisting in protein folding and assembly in the eukaryotic cytosol. Eur. J. Biochem. 230: 3-16 
    18. Gutsche, I., L. O. Essen, and W. Baumeister (1999) Group II chaperonins: new TRiC(k)s and turns of a protein folding machine. J. Mol. Biol. 293: 295-312 
    19. Kohda, J., Y. Endo, N. Okumura, Y. Kurokawa, K. Nishihara, H. Yanagi, T. Yura, H. Fukuda, and A. Kondo (2002) Improvement of productivity of active form of glutamate racemase in Escherichia coli by coexpression of folding accessory proteins. Biochem. Eng. J. 10: 39-45 
    20. Yoshida, T., M. Yohda, T. Iida, T. Maruyama, H. Taguchi, K. Yazaki, T. Ohta, M. Odaka, I. Endo, and Y. Kagawa (1997) Structural and functional characterization of homo-oligomeric complexes of alpha and beta chaperonin subunits from the hyperthermophilic archaeum Thermococcus strain KS-1. J. Mol. Biol. 273: 635-645 
    21. Kim, M. J., S. H. Kim, J. H. Lee, J. H. Seo, J. H. Lee, J. H. Kim, Y. H. Kim, and S. W. Nam (2008) High-level secretory expression of human procarboxypeptidase B by fedbatch cultivation of Pichia pastoris and its partial characterization. J. Microbiol. Biotechnol. 18: 1938-1944     
    22. Jeon, S. J. and K. Ishikawa (2005) Characterization of the family I inorganic pyrophosphatase from Pyrococcus horikoshii OT3. Archaea 1: 385-389 
    23. Gething, M. J. and J. Sambrook (1992) Protein folding in the cell. Nature 355: 33-45 
    24. Mogk, A., B. Bukau, and E. Deuerling (2001) Cellular functions of cytosolic E. coli chaperones. pp. 1-34. In: P. Lund (ed.). Molecular chaperones in the cell. Oxford University Press, Oxford, UK 
    25. Shin, E. J., S. L. Park, S. J. Jeon, J. W. Lee, Y. T. Kim, Y. H. Kim, and S. W. Nam (2006) Effect of molecular chaperones on the soluble expression of alginate lyase in E. coli. Biotechnol. Bioprocess Eng. 11: 414-419     
    26. Yoshida, T., R. Kawaguchi, H. Taguchi, M. Yoshida, T. Yasunaga, T. Wakabayashi, M. Yohda, and T. Maruyama (2002) Archaeal group II chaperonin mediates protein folding in the cis-cavity without a detachable GroES-like co-chaperonin. J. Mol. Biol. 315: 73-85 
    27. Park, S. L., E. J. Shin, S. P. Hong, S. J. Jeon, and S. W. Nam (2005) Production of soluble human granulocyte colony stimulating factor in E. coli by molecular chaperones. J. Microbiol. Biotechnol. 15: 1267-1272     
    28. Kang, C. S., C. W. Park, and I. S. Bang (2008) Production and purification of a cecropin family antibacterial peptide, hinnavin II, in Escherichia coli. Biotechnol. Bioprocess Eng. 13: 377-382 
    29. Okochi, M., K. Kanie, M. Kurimoto, M. Yohda, and H. Honda (2008) Overexpression of prefoldin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 endowed Escherichia coli with organic solvent tolerance. Appl. Biochem. Biotechnol. 79: 443-449 
    30. Valpuesta, J. M., J. Martin-Benito, P. Gomez-Puertas, J. L. Carrascosa, and K. R. Willison (2002) Structure and function of a protein folding machine: the eukaryotic cytosolic chaperonin CCT. FEBS Lett. 529: 11-16 
    31. Guagliardi, A., L. Cerchia, S. Bartolucci, and M. Rossi (1994) The chaperonin from the archaeon Sulfolobus solfataricus promotes correct refolding and prevents thermal denaturation in vitro. Protein Sci. 3: 1436-1443 
    32. Yoshida, T., A. Ideno, S. Hiyamuta, M. Yohda, and T. Maruyama (2001) Natural chaperonin of the hyperthermophilic archaeum, Thermococcus strain KS-1: a heterooligomeric chaperonin with variable subunit composition. Mol. Microbiol. 39: 1406-1413 
    33. Ellis, R. J. (1996) The chaperonins. pp. 2-25. Academic Press, San Diego, USA 
    34. Gribaldo, S., V. Lumia, R. Creti, E. C. de Macario, A. Sanangelantoni, and P. Cammarano (1999) Discontinuous occurrence of the hsp70 (DnaK) gene among archaea and sequence features of HSP70 suggest a novel outlook on phylogenies inferred from this protein. J. Bacteriol. 181: 434-443 
    35. Bukau, B. and A. L. Horwich (1998) The Hsp70 and Hsp60 chaperone machines. Cell 92: 351-366 
    36. Kawarabayasi, Y., M. Sawada, H. Horikawa, Y. Haikawa, Y. Hino, S. Yamamoto, M. Sekine, S. Baba, H. Kosugi, A. Hosoyama, Y. Nagai, M. Sakai, K. Ogura, R. Otsuka, H. Nakazawa, M. Takamiya, Y. Ohfuku, T. Funahashi, T. Tanaka, Y. Kudoh, J. Yamazaki, N. Kushida, A. Oguchi, K. Aoki, and H. Kikuchi (1998) Complete sequence and gene organization of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3 (supplement). DNA Res. 5: 147-155 
    37. Archibald, J. M., J. M. Logsdon, and W. F. Doolittle (1999) Recurrent paralogy in the evolution of archaeal chaperonins. Curr. Biol. 9: 1053-1056 
    38. Okochi, M., H. Matsuzaki, T. Nomura, N. Ishii N, and M. Yohda (2005) Molecular characterization of the group II chaperonin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3. Extremophiles 9: 127-134 
    39. Geladopoulos, T. P., T. G. Sotiroudis, and A. E. Evangelopoulos (1991) A malachite green colorimetric assay for protein phosphatase activity. Anal. Biochem. 192: 112-116 
    40. Hongo, K., H. Hirai, C. Uemura, S. Ono, J. Tsunemi, T. Higurashi, T. Mizobata, and Y. Kawata (2006) A novel ATP/ADP hydrolysis activity of hyperthermostable group II chaperonin in the presence of cobalt or manganese ion. FEBS Lett. 580: 34-40 
    41. Jung, S. M., A. K. Lim, and K. M. Park (2008) Construction of Candida antarctica lipase B expression system in E. coli coexpressing chaperones. Korean J. Biotechnol. Bioeng. 23: 403-407     
    42. Yan, Z., S. Fujiwara, K. Kohda, M. Takagi, and T. Imanaka (1997) In vitro stabilization and in vivo solubilization of foreign proteins by the subunit of a chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1. Appl. Environ. Microbiol. 63: 785-789 
    43. Ellis, R. J. and F. U. Hartl (1996) Protein folding in the cell: competing models of chaperonin function. FASEB J. 10: 20-26 
    44. Kondo, A., J. Kohda, Y. Endo, T. Shiromizu, Y. Kurokawa, K. Nishihara, H. Yanagi, T. Yura, and H. Fukuda (2000) Improvement of productivity of active horseradish peroxidase in Escherichia coli by coexpression of Dsb proteins. J. Biosci. Bioeng. 90: 600-606 
    45. Bukau, B., E. Deuerling, C. Pfund, and E. A. Craig (2000) Getting newly synthesized proteins into shape. Cell 101:119-122 
    46. Kwon, M. J., S. L. Park, S. K. Kim, and S. W. Nam (2002) Overproduction of Bacillus macerans cyclodextrin glucanotransferase in E. coli by coexpression of GroEL/ES chaperone. J. Microbiol. Biotechnol. 12: 1002-1005 
    47. Laksanalamai, P., T. A. Whitehead, and F. T. Robb (2004) Minimal protein-folding systems in hyperthermophilic archaea. Nat. Rev. Microbiol. 2: 315-324 
  • 이 논문을 인용한 문헌 (1)

    1. 2011. "" Journal of microbiology and biotechnology, 21(2): 212~217     

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