Laboratory of Applied Stress MicrobiologyLaboratory of Applied Stress Microbiology

content ▼

Research Projects

4. Folding mechanism and functional alteration of microbial proteases by new protein engineering

Conventional protein engineering techniques have so far employed the introduction of mutagenesis in the protease domain to modify the enzymatic properties. A new approach, which we termed ‘pro-sequence engineering’, would be not only an important in investigations of protein folding, but also a promising technology for creating unique proteases with various beneficial properties.

  1. M. Takahashi, T. Sekine, N. Kuba, S. Nakamori, M. Yasuda and H. Takagi: The production of recombinant APRP, an alkaline protease derived from Bacillus pumilus TYO-67, by in vitro-refolding of pro-enzyme fixed on a solid surface. J. Biochem., 136, 549-556 (2004).
  2. H. Takagi and M. Takahashi: A new approach for alteration of protease functions: pro-sequence engineering. Appl. Microbiol. Biotechnol., 63, 1-9 (2003).
  3. H.-C. Mei, Y.-F. Li, C.-C. Hsu, Y.-C. Tsai and H. Takagi: Conversion of the cleavage specificity of subtilisin YaB on oxidized insulin chains to an elastase-like specificity by replacement of Gly124 with Ala. Biosci. Biotech. Biochem., 67, 1601-1604 (2003).
  4. Y. Yabuta, E. Subbian, H. Takagi, U. Shinde and M. Inouye: Folding pathway mediated by an intramolecular chaperone: dissecting conformational changes coincident with autoprocessing and the role of Ca2+ precursor maturation. J. Biochem., 131, 31-37 (2002).
  5. Y. Yabuta, H. Takagi, M. Inouye and U. Shinde: Folding pathway mediated by an intramolecular chaperone. Propeptide-release modulates precise activation of a protease. J. Biol. Chem., 276, 44427-44434 (2001).
  6. H. Takagi, M. Koga, S. Katsurada, Y. Yabuta, U. Shinde, M. Inouye and S. Nakamori: Functional analysis of the propeptides of subtilisin E and aqualysin I as intramolecular chaperones. FEBS Lett., 508, 210-214 (2001).
  7. M. Takahashi, Y. Hasuura, S. Nakamori and H. Takagi: Improved autoprocessing efficiency of mutant subtilisins E with altered specificity by engineering of the pro-region. J. Biochem., 130, 99-106 (2001).
  8. H. Takagi: Engineering specificity of Bacillus subtilis subtilisin E. In Recent Research Developments in Protein Engineering, S.G. Pandalai (ed.), pp. 53-74, Research Signpost, Kerala, India (2001).
  9. H. Takagi, K. Hirai, M. Wada and S. Nakamori: Enhanced thermostability of the single-Cys mutant subtilisin E under oxidizing conditions. J. Biochem., 128, 585-589 (2000).
  10. H. Takagi, K. Hirai, Y. Maeda, H. Matsuzawa and S. Nakamori: Engineering subtilisin E for enhanced stability and activity in polar organic solvents. J. Biochem., 127, 617-625 (2000).
  11. H. Takagi, A. Suzumura, Y. Hasuura, T. Hoshino and S. Nakamori: Efficient selection for thermostable protease in Thermus thermophilus. Biosci. Biotech. Biochem., 64, 899-902 (2000).
  12. H. Takagi, A. Suzumura, T. Hoshino and S. Nakamori: Gene expression of Bacillus subtilis subtilisin E in Thermus thermophilus. J. Ind. Microbiol. Biotechnol., 23, 214-217 (1999).
  13. H. Takagi, M. Yamamoto, I. Ohtsu, and S. Nakamori: Random mutagenesis into conserved Gly154 of subtilisin E: isolation and characterization of the revertant enzymes. Protein Engng., 11, 1205-1210 (1998).
  14. H.-C. Mei, Y.-C. Liaw, Y.-C. Li, D.-C. Wang, H. Takagi, and Y.-C. Tsai: Engineering subtilisin YaB: restriction of substrate specificity by the substitution of Gly124 and Gly151 with Ala. Protein Engng., 11, 109-117 (1998).
  15. H.Takagi, I. Ohtsu, and S. Nakamori: Construction of novel subtilisin E with high specificity, activity and productivity through multiple amino acid substitutions. Protein Engng., 10, 985-989 (1997).
  16. H. Takagi, T. Maeda, I. Ohtsu, Y.-C. Tsai, and S. Nakamori: Restriction of substrate specificity of subtilisin E by introduction of a side chain into a conserved glysine residue. FEBS Letters, 395, 127-132 (1996).
  17. H. Takagi, H. Matsuzawa, T. Ohta, M. Yamasaki and M. Inouye: Studies on the structure and function of subtilisin E by protein engineering. In Subtilisin Enzymes: Practical Protein Engineering, R. Bott and C. Betzel (eds.), pp. 269-275, Prenum Press, New York and London (1996).
  18. H. Takagi: Protein engineering on subtilisin. Int. J. Biochem., 25, 307-312 (1993).
  19. H. Takagi, S. Arafuka, M. Inouye and M. Yamasaki: The effect of amino acid deletion in subtilisin E, based on structural comparison with a microbial alkaline elastase, on its substrate specificity and catalysis. J. Biochem., 111, 584-588 (1992).
  20. H. Takagi, H. Matsuzawa, T. Ohta, M. Yamasaki, and M. Inouye: Studies on the structure and function of subtilisin E by protein engineering. Annals of the New York Academy of Sciences, Vol. 672, 52-59, In C. S. Craik and D. A. Estell (ed.), The New York Academy Sciences, New York (1992).
  21. H. Takagi, T. Takahashi, H. Momose, M. Inouye, Y. Maeda, H. Matsuzawa and T. Ohta: Enhancement of the thermostability of subtilisin E by introduction of a disulfide bond engineered on the basis of structural comparison with a thermophilic serine protease. J. Biol. Chem., 265, 6874-6878 (1990).
  22. H. Takagi, Y. Morinaga, H. Ikemura, and M. Inouye: The role of Pro-239 in the catalysis and heat stability of subtilisin E. J. Biochem., 105, 953-956 (1989).
  23. H. Takagi, Y. Morinaga, H. Ikemura and M. Inouye: Mutant subtilisin E with enhanced protease activity obtained by site-directed mutagenesis. J. Biol. Chem., 263, 19592-19596 (1988).
  24. H. Takagi, Y. Morinaga, M. Tsuchiya, H. Ikemura and M. Inouye: Control of folding of proteins secreted by a high expression secretion vector, pIN-III-ompA: 16-fold increase in production of active subtilisin E in Escherichia coli. Bio/Technology, 6, 948-950 (1988).
  25. H. Ikemura, H. Takagi and M. Inouye: Requirement of pro-sequence for the production of active subtilisin E in Escherichia coli. J. Biol. Chem., 262, 7859-7864 (1987).