Molecular Biotechnology

, Volume 30, Issue 3, pp 193–205 | Cite as

Overexpression in Escherichia coli and functional reconstitution of anchovy trypsinogen from the bacterial inclusion body

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Abstract

We have synthesized and optimized a high-yielding Escherichia coli expression system to produce trypsinogen from anchovy Engraulis japonicus and have developed conditions for its successful refolding. Recombinant anchovy trypsinogen precipitated in E. coli Rosetta (DE3) placI strain as inclusion bodies was denatured by 6 M guanidine-HCl followed by refolding with drop wise addition to a large excess of a folding buffer containing 0.5 M non-detergent sulfobetaine (NDSB-251) and a redox potential of oxidized and reduced glutathiones. The folded trypsinogen was autocatalytically activated to its mature form, trypsin, and purified with a MonoQ ion-exchange column. NH2-terminal amino acid sequencings revealed that E. coli efficiently processed NH2-terminal methionine residue from the expressed trypsinogen and that trypsinogen was activated at the correct site to generate active trypsin. The recombinant enzyme showed kinetic properties comparable to those of the native enzyme and demonstrated a typical cleavage preference for arginine over lysine residue against a protein substrate. The optimized expression and folding procedures yielded 12 mg of purified, active trypsin from 1 L of bacterial culture or 45 g wet weight cells, which is quite enough for various analytical and semipreparative purposes.

Index Entries

Anchovy trysinogen expression inclusion body nondetergent sulfobetaine refolding 

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References

  1. 1.
    Hedstrom, L., Perona, J. J., and Rutter, W. J. (1994) Converting trypsin to chymotrypsin: residue 172 is a substrate specificity determinant. Biochemistry 33, 8757–8763.PubMedCrossRefGoogle Scholar
  2. 2.
    Ahsan, M. N., Funabara, D., and Watabe, S. (2001) Molecular cloning and characterization of two isoforms of trypsinogen from anchovy pyloric ceca. Mar. Biotechnol. 3, 80–90.PubMedCrossRefGoogle Scholar
  3. 3.
    Ahsan, M. N., Funabara, D., and Watabe, S. (2002) Anchovy trypsin: Purification, cDNA cloning, and molecular modeling of two isoforms. Fish. Sci. 68(Suppl.), 1563–1566Google Scholar
  4. 4.
    Middelberg, A. P. J. (2002) Preparative protein refolding. Trends Biotechnol. 20, 437–443.PubMedCrossRefGoogle Scholar
  5. 5.
    Coligan, J. E., Dunn, B. M., Ploegh, H. L., Speicher, D. W., and Wingfield, P. T. (1995) Current Protocols in Protein Science 2nd ed., John Willey & Sons, NY.Google Scholar
  6. 6.
    Clark, E. D. (2001) Protein refolding for industrial processes. Curr. Opin. Biotechnol. 12, 202–207.PubMedCrossRefGoogle Scholar
  7. 7.
    Fischer, B., Perry, B., Sumner, I., and Goodenough, P. (1993) Isolation, renaturation, and formation of disulfide bonds of eukaryotic proteins expressed in Escherichia coli as inclusion bodies. Biotechnol. Bioeng. 41, 3–13.CrossRefGoogle Scholar
  8. 8.
    Taguchi, S., Ozaki, A., Nonaka, T., Mitsui, Y., and Momose, H. (1999) A cold-adapted protease engineered by experimental evolution system. J. Biochem. (Tokyo) 126, 689–693.Google Scholar
  9. 9.
    Gerday, C., Aittaleb, M., Bentahir, M., Baise, E., Genicot, S., and Gerday, C. (2000) Cold-adapted enzymes: from fundamentals to biotechnology. Trends Biotechnol. 18, 103–107.PubMedCrossRefGoogle Scholar
  10. 10.
    Arnórsdóttir, J., Smáradóttir, R. B., Magnússon, Ó. B., Thorbjarnardóttir, S. H., Eggertsson, G., and Kristjánsson, M. M. (2002) Characterization of a cloned subtilisin-like serine protease from a psychrotrophic Vibrio species. Eur. J. Biochem. 269, 5536–5546.PubMedCrossRefGoogle Scholar
  11. 11.
    Kristjánsdóttir, S. and Gudmundsdóttir, A. (2000) Propeptide dependent activation of the Antarctic krill euphauserase precursor produced in yeast. Eur. J. Biochem. 267, 2632–2639.PubMedCrossRefGoogle Scholar
  12. 12.
    Ahsan, M. N. and Watabe, S. (2001) Kinetic and structural properties of two isoforms of trypsin isolated from the viscera of Japanese anchovy, Engraulis japonicus. J. Prot. Chem. 20, 49–58.CrossRefGoogle Scholar
  13. 13.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  14. 14.
    Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.PubMedCrossRefGoogle Scholar
  15. 15.
    Ueki, N. and Ochiai, Y. (2004) Primary structure and thermostability of bigeye tuna myoglobin in relation to those of other scombridae fish. Fish. Sci. 70, 875–884.CrossRefGoogle Scholar
  16. 16.
    Matsudaira, P. (1987) Sequence from picomole quantities of proteins elctroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem. 262, 10,035–10,038.Google Scholar
  17. 17.
    Craik, C. S., Largman, C., Fletcher, T., et al. (1985) Redesigning trypsin: alteration of substrate specificity. Science 228, 291–297.PubMedCrossRefGoogle Scholar
  18. 18.
    Stroud, R. M., Kay, L. M., and Dickerson, R. E. (1974) The structure of bovine trypsin: electron density maps of the inhibited enzyme at 5Å and at 2.7Å resolution. J. Mol. Biol. 83, 185–208.PubMedCrossRefGoogle Scholar
  19. 19.
    Craik, C. S., Roczniak, S., Largman, C., and Rutter, W. J. (1987) The catalytic role of the active site aspartic acid in serine proteases. Science 237, 909–913.PubMedCrossRefGoogle Scholar
  20. 20.
    Brinkmann, U., Mattes, R. E., and Buckel, P. (1989) High-level expression of recombinant genes in Escherichia coli is dependent on the availability of the dnaY gene product. Gene 85, 109–114.PubMedCrossRefGoogle Scholar
  21. 21.
    Sherman, F., Stewart, J. W., and Tsunasawa, S. (1985) Methionine or not methionine at the beginning of a protein. BioEssays 3, 27–31.PubMedCrossRefGoogle Scholar
  22. 22.
    Wingfield, P. T., Mattaliano, R. J., MacDonald, H. R., et al. (1987) Recombinant-derived interleukin 1α stabilized against specific deamidation. Protein Eng. 1, 413–417.PubMedCrossRefGoogle Scholar
  23. 23.
    Wong, H. H., O’Neill, B. K., and Middelberg, A.P. (1996) Centrifugal processing of cell debris and inclusion bodies from recombinant Escherichia coli. Bioseparation 6, 361–372.PubMedGoogle Scholar
  24. 24.
    Maachupalli-Reddy, J., Kelley, B. D., and De Bernardez, C. E. (1997) Effect of inclusion body contaminants on the oxidative renaturation of hen egg white lysozyme. Biotechnol. Prog. 13, 144–150.PubMedCrossRefGoogle Scholar
  25. 25.
    De Bernardez, C. E., Hevehan, D., Szela, S., and Maachupalli-Reddy, J. (1998) Oxidative renaturation of hen egg-white lysozyme. Folding vs aggregation. Biotechnol. Prog. 14, 47–54.CrossRefGoogle Scholar
  26. 26.
    Tran-Moseman, A., Schauer, N., and De Bernardez, C. E. (1999) Renaturation of Escherichia coli-derived recombinant human macrophage colony-stimulating factor. Protein Expr. Purif. 16, 181–189.PubMedCrossRefGoogle Scholar
  27. 27.
    Vuillard, L., Rabilloud, T., and Golberg, M. E. (1998) Interactions of non-detergent sulfobetaines with early folding intermediates facilitate in vitro protein renaturation. Eur. J. Biochem. 256, 128–135.PubMedCrossRefGoogle Scholar
  28. 28.
    Gu, Z., Su, Z., and Janson, J. (2001) Urea gradient size-exclusion chromatography enhanced the yield of lysozyme refolding. J. Chromatogr. 918, 311–318.CrossRefGoogle Scholar
  29. 29.
    Vuillard, L., Madern, D., Francezi, B., and Rabilloud, T. (1995) Halophilic protein stabilization by the mild solubilizing agents nondetergent sulfobetaines. Anal. Biochem. 1230, 290–294.CrossRefGoogle Scholar
  30. 30.
    Golberg, M. E., Expert-Bezancon, N., Vuillard, L., and Rabilloud, T. (1996) Non-detergent sulphobetaines: a new class of molecules that facilitate in vitro protein renaturation. Fold. Des. 1, 21–27.CrossRefGoogle Scholar
  31. 31.
    Schroeder, D. D., and Shaw, E. (1968) Chromatography of trypsin and its derivatives. Characterization of a new active form of bovine trypsin. J. Biol. Chem. 243, 2943–2949.PubMedGoogle Scholar
  32. 32.
    Rawlings, N. D. (1998) Serine proteases, in Handbook of Proteolytic Enzymes (Barrett, A. J., Rawlings, N. D., and Woessner, J. F., eds.) Academic, London, UK.Google Scholar
  33. 33.
    Walker, J. M. (1994) Basic Protein and Peptide Protocols, Humana, NJ.Google Scholar
  34. 34.
    Gráf, L., Craik, C. S., Patthy, A., Roczniak, S., Fletterick, R. J., and Rutter, W. J. (1987) Selective alteration of substrate specificity by replacement of aspartic acid-189 with lysine in the binding pocket of trypsin. Biochemistry 26, 2616–2623.PubMedCrossRefGoogle Scholar
  35. 35.
    Jónsdóttir, G., Bjarnason, J. B., and Gudmundsdóttir, A. (2004) Recombinant cold-adapted trypsin I from Atlantic cod-expression, purification, and identification. Prot. Exp. Puri. 33, 110–122.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc 2005

Authors and Affiliations

  1. 1.Graduate School of Agricultural and Life SciencesThe University of TokyoTokyoJapan
  2. 2.Fisheries and Marine Resource TechnologyKhulnaBangladesh
  3. 3.Research and Development CentreNichirei Co., Inc.ChibaJapan

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