Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Overexpression, Purification, and Characterization of $\beta$-Subunit of Group II Chaperonin from Hyperthermophilic Aeropyrum pernix K1

  • Shin, Eun-Jung (Department of Biomaterial Control, Dong-Eui University) ;
  • Lee, Jin-Woo (Department of Biomaterial Control, Dong-Eui University) ;
  • Kim, Jeong-Hwan (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) ;
  • Nam, Soo-Wan (Department of Biomaterial Control, Dong-Eui University)
  • Received : 2009.09.17
  • Accepted : 2009.10.31
  • Published : 2010.03.31
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Abstract

In the present study, overexpression, purification, and characterization of Aeropyrum pernix K1 chaperonin B in E. coli were investigated. The chaperonin $\beta$-subunit gene (ApCpnB, 1,665 bp ORF) from the hyperthermophilic archaeon A. pernix K1 was amplified by PCR and subcloned into vector pET21a. The constructed pET21a-ApCpnB (6.9 kb) was transformed into E. coli BL21 Codonplus (DE3). The transformant cell successfully expressed ApCpnB, and the expression of ApCpnB (61.2 kDa) was identified through analysis of the fractions by SDS-PAGE (14% gel). The recombinant ApCpnB was purified to higher than 94% by using heat-shock treatment at $90^{\circ}C$ for 20 min and fast protein liquid chromatography on a HiTrap Q column step. The purified ApCpnB showed ATPase activity and its activity was dependent on temperature. In the presence of ATP, ApCpnB effectively protected citrate synthase (CS) and alcohol dehydrogenase (ADH) from thermal aggregation and inactivation at $43^{\circ}$ and $50^{\circ}$, respectively. Specifically, the activity of malate dehydrogenase (MDH) at $85^{\circ}$ was greatly stabilized by the addition of ApCpnB and ATP. Coexpression of pro-carboxypeptidase B (pro-CPB) and ApCpnB in E. coli BL21 Codonplus (DE3) had a marked effect on the yield of pro-CPB as a soluble and active form, speculating that ApCpnB facilitates the correct folding of pro-CPB. These results suggest that ApCpnB has both foldase and holdase activities and can be used as a powerful molecular machinery for the production of recombinant proteins as soluble and active forms in E. coli.

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References

  1. Andra, S., G. Frey, M. Nitsch, W. Baumeister, and K. O. Stetter. 1996. Purification and structural characterization of the thermosome from the hyperthermophilic archaeum Methanopyrus kandleri. FEBS Lett. 379: 127-131. https://doi.org/10.1016/0014-5793(95)01493-4
  2. Andra, S., G. Frey, R. Jaenicke, and K. O. Stetter. 1998. The thermosome from Methanopyrus kandleri possesses an $NH_{4}^{+}$-dependent ATPase activity. Eur. J. Biochem. 255: 93-99. https://doi.org/10.1046/j.1432-1327.1998.2550093.x
  3. Archibald, J. M., J. M. Logsdon, and W. F. Doolittle. 1999. Recurrent paralogy in the evolution of archaeal chaperonins. Curr. Opin. Biotechnol. 9: 1053-1056.
  4. Buchner, J., M. Schmidt, M. Fuchs, R. Jaenicke, R. Rudolph, F. X. Schmid, and T. Kiefhaber. 1991. GroE facilitates refolding of citrate synthase by suppressing aggregation. Biochemistry 30: 1586-1591. https://doi.org/10.1021/bi00220a020
  5. Bult, C. J., O. White, G. J. Olsen, L. Zhou, R. D. Fleischmann, G. G. Sutton, et al. 1996. Complete genome sequence of the methanogenic archaeon Methanococcus jannaschii. Science 273: 1058-1073. https://doi.org/10.1126/science.273.5278.1058
  6. Chang, Z., T. Primm, J. Jakana, I. H. Lee, I. Serysheva, W. Chiu, H. F. Gilbert, and F. A. Cuiocho. 1996. Mycobacterium tuberculosis 16-kDa antigen (Hsp 16.3) functions as an oligomeric structure in vitro to suppress thermal aggregation. J. Biol. Chem. 271: 7218-7223. https://doi.org/10.1074/jbc.271.12.7218
  7. Choi, J. J., J. W. Park, H. K. Shim, S. C. Lee, M. S. Kwon, J. S. Yang, H. O. Hwang, and S. T. Kwon. 2006. Cloning, expression, and characterization of a hyperalkaline phosphatase from the thermophilic bacterium Thermus sp. T351. J. Microbiol. Biotechnol. 16: 272-279.
  8. Ditzel, L., J. Lowe, D. Stock, K. O. Stetter, H. Huber, R. Huber, and S. Steinbacher. 1998. Crystal structure of the thermosome, the archaeal chaperonin and homolog of CCT. Cell 93: 125-138. https://doi.org/10.1016/S0092-8674(00)81152-6
  9. 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. https://doi.org/10.1074/jbc.273.43.28399
  10. Guan, Y. X., H. X. Pan, Y. G. Gao, S. J. Yao, and M. G. Cho. 2005. Refolding and purification of recombinant human interferon-$\gamma$ expressed as inclusion bodies in Escherichia coli using size exclusion chromatography. Biotechnol. Bioprocess Eng. 10: 122-127. https://doi.org/10.1007/BF02932581
  11. 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. https://doi.org/10.1006/jmbi.1999.3008
  12. Hartl, F. U. 1996. Molecular chaperones in cellular protein folding. Nature 381: 571-580. https://doi.org/10.1038/381571a0
  13. Hoffmann, J. H., K. Linke, P. C. Graf, H. Lilie, and U. Jakob. 2004. Identification of a redox-regulated chaperone network. EMBO J. 23: 160-168. https://doi.org/10.1038/sj.emboj.7600016
  14. Isumi, M., S. Fuiwara, 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.
  15. Kawarabayasi, Y., Y. Hino, H. Horikawa, S. Yamazaki, Y. Haikawa, K. Jin-no, et al. 1999. Complete genome sequence of an aerobic hyper-thermophilic crenarchaeon, Aeropyrum pernix K1. DNA Res. 6: 83-101, 145-152. https://doi.org/10.1093/dnares/6.2.83
  16. Kim, H. and I. H. Kim. 2005. Refolding of fusion ferritin by gel filtration chromatography (GFC). Biotechnol. Bioprocess Eng. 10: 500-504. https://doi.org/10.1007/BF02932284
  17. Kim, S., K. R. Willson, and A. L. Horwich. 1994. Cytosolic chaperonin subunits have a conserved ATPase domain but diverged polypeptide-binding domains. Trends Biochem. Sci. 19: 543-548. https://doi.org/10.1016/0968-0004(94)90058-2
  18. Knapp, S., I. Schhmidt-Krey, H. Hebert, T. Bergman, H. Jornvall, and R. Ladenstein. 1994. The molecular chaperonin TF55 from the thermophilic archaeon Sulfolobus solfataricus. A biochemical and structural characterization. J. Mol. Biol. 242: 397-407.
  19. Kohda, J., H. Kawanishi, K. Suehara, Y. Nakano, and T. Yano. 2006. Stabilization of free and immobilized enzyme using hyperthermophilic chaperonin. J. Biosci. Bioeng. 101: 131-136. https://doi.org/10.1263/jbb.101.131
  20. 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. https://doi.org/10.1016/S1369-703X(01)00154-1
  21. Kubota, H., G. Hynes, and K. Willson. 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. https://doi.org/10.1111/j.1432-1033.1995.tb20527.x
  22. Kwak, Y. H., S. J. Kim, K. Y. Lee, and H. B. Kim. 2000. Stress response of the Escherichia coli groE promoter. J. Microbiol. Biotechnol. 10: 63-68.
  23. Kwon, M. J., S. L. Rark, 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.
  24. Lamark, T., M. Ingebrigtsen, C. Bjornstad, T. Melkko, T. Mollens, and E. Nielsen. 2001. Expression of active human C1 inhibitor serpin domain in Escherichia coli. Prot. Expr. Purif. 22: 349-359. https://doi.org/10.1006/prep.2001.1445
  25. Langer, T., G. Pfeifer, J. Martin, W. Baumeister, and F. U. Hartl. 1992. Chaperonin mediated protein folding: GroES binds to one end of the GroEL cylinder, which accommodates the protein substrate within its central cavity. EMBO J. 11: 4757-4765.
  26. Mayhew, M., A. C. da Silva, J. Martin, H. Erdjument-Bromage, P. Tempst, and F. U. Hartl. 1996. Protein folding in the central cavity of the GroEL-GroES chaperonin complex. Nature 379: 420-426. https://doi.org/10.1038/379420a0
  27. Minuth, T., G. Frey, P. Lindner, R. Rachel, K. O. Stetter, and R. Jaenicke. 1998. Recombinant homo- and hetero-oligomers of an ultrastable chaperonin from the archaeon Pyrodictium occultum show chaperone activity in vitro. Eur. J. Biochem. 258: 837-845. https://doi.org/10.1046/j.1432-1327.1998.2580837.x
  28. Son, H. J., E. J. Shin, S. W. Nam, D. E. Kim, and S. J. Jeon. 2006. Properties of the $\alpha$ subunit of a chaperonin from the hyperthermophilic crenarchaeon Aeropyrum pernix K1. FEMS Microbiol. Lett. 266: 103-109.
  29. Trent, J. D., E. Nimmesgern, J. S. Wall, F. U. Hartl, and A. L. Horwich. 1991. A molecular chaperone from a thermophilic archaebacterium is related to the eukaryotic protein t-complex polypeptide-1. Nature 354: 490-493. https://doi.org/10.1038/354490a0
  30. Waldmann, T., E. Nimmesgern, M. Nitsch, J. Peters, G. Pfeifer, S. Muller, et al. 1995. The thermosome of Thermoplasma acidophilum and its relationship to the eukaryotic chaperonin TRiC. Eur. J. Biochem. 227: 848-856. https://doi.org/10.1111/j.1432-1033.1995.tb20210.x
  31. Wiech, H., J. Buchner, R. Zimmermann, and U. Jakob. 1992. Hsp90 chaperones protein folding in vitro. Nature 358: 169-170. https://doi.org/10.1038/358169a0
  32. Winter, J., K. Linke, A. Jatzek, and U. Jakob. 2005. Severe oxidative stress causes inactivation of DnaK and activation of the redox-regulated chaperone Hsp33. Mol. Cell. 17: 381-392. https://doi.org/10.1016/j.molcel.2004.12.027
  33. Yan, Z., S. Fujiwara, K. Kohda, M. Takagi, and T. Imanaka. 1997. In vitro stabilization and in vivo solubilization of foreign proteins by the beta subunit of a chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1. Appl. Environ. Microbiol. 63: 785-789.
  34. Yoshida, T., M. Yohda, T. Iida, T. Maruyama, H. Taguchi, K. Yazaki, et al. 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. https://doi.org/10.1006/jmbi.1997.1337
  35. Yoshida, T., R. Kawaguchi, H. Taguchi, M. Yoshida, 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. https://doi.org/10.1006/jmbi.2001.5220
  36. Zhi, W., P. Srere, and C. T. Evans. 1991. Conformational stability of pig citrate synthase and some active-site mutants. Biochemistry 30: 9281-9286. https://doi.org/10.1021/bi00102a021
  37. Zhi, W., S. J. Landry, L. M. Gierasch, and P. A. Srere. 1992. Renaturation of citrate synthase: Influence of denaturant and folding assistants. Prot. Sci. 1: 522-529.

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