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Avoiding Proteolysis During Protein Chromatography

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Protein Chromatography

Part of the book series: Methods in Molecular Biology ((MIMB,volume 681))

Abstract

All cells contain proteases, which effect catalytic hydrolysis of the peptide bond between amino acids in the protein backbone. Typically, proteinases are prevented from nonspecific proteolysis by regulation and physical separation into different subcellular compartments; however, this segregation is not retained during cell lysis to release a protein of interest. Prevention of proteolysis during protein purification often takes the form of a two-pronged approach; first the inhibition of proteolysis in situ, followed by the separation of the protease from the protein of interest via chromatographical purification. Proteinase inhibitors are routinely used to limit the effect of the proteinases before they are physically separated from the protein of interest via column chromatography. Here, commonly used approaches to reducing proteolysis during chromatography are reviewed.

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References

  1. O’Fágáin, C. (1997) Protein stability and its measurement, in Stabilising Protein Function (O’Fágáin, C., ed.), Springer Press, Berlin, pp. 1–14.

    Google Scholar 

  2. Seife, C. (1997) Blunting nature’s Swiss army knife. Science 277, 1602–1603.

    Article  PubMed  CAS  Google Scholar 

  3. Sandhya, C., Sumantha, A., and Pandey, A. (2004) Proteases, in Enzyme Technology (Pandey, A., Webb, C., Soccol, C.R., and Larroche, C., eds.), Asiatech Publishers Inc., New Delhi, India, pp. 312–325.

    Google Scholar 

  4. Rawlings, N.D., Morton, F.R., Kok, C.Y., Kong, J., and Barrett, A.J. (2008) MEROPS: the peptidase database. Nucleic Acids Res. 36, D320–D325.

    Article  PubMed  CAS  Google Scholar 

  5. Rawlings, N.D. and Barrett, A.J. (1994) Families of serine peptidases. Methods Enzymol. 244, 19–61.

    Article  PubMed  CAS  Google Scholar 

  6. Bühling, F., Fengler, A., Brandt, W., Welte, T., Ansorge, S., and Nägler, D.K. (2000) Review: novel cysteine proteases of the papain family. Adv. Exp. Med. Biol. 477, 241–254.

    Article  PubMed  Google Scholar 

  7. Dame, J.B., Reddy, G.R., Yowell, C.A., Dunn, B.M., Kay, J., and Berry, C. (1994) Sequence, expression and modeled structure of an aspartic proteinase from the human malaria parasite Plasmodium falciparum. Mol. Biochem. Parasitol. 64, 177–190.

    Article  PubMed  CAS  Google Scholar 

  8. Edwards, D.R., Handsley, M.M., and Pennington, C.J. (2008) The ADAM metalloproteinases. Mol. Aspects Med. 29, 258–289.

    Article  PubMed  CAS  Google Scholar 

  9. Chung, C.H. and Goldberg, A.L. (1981) The product of the lon (capR) gene in Escherichia coli is the ATP-dependent protease, protease La. Proc. Natl. Acad. Sci. USA 78, 4931–4935.

    Article  PubMed  CAS  Google Scholar 

  10. Hershko, A., Leshinsky, E., Ganoth, D., and Heller, H. (1984) ATP-dependent degradation of ubiquitin-protein conjugates. Proc. Natl. Acad. Sci. USA 81, 1619–1623.

    Article  PubMed  CAS  Google Scholar 

  11. Vanaman, T.C. and Bradshaw, R.A. (1999) Proteases in cellular regulation. J. Biol. Chem. 274, 20047.

    Article  PubMed  CAS  Google Scholar 

  12. Beynon, R.J. and Oliver, S. (2004) Avoidance of proteolysis in extracts, in Protein Purification Protocols, Methods in Molecular Biology (Cutler, P., ed.), Humana, Totowa, NJ, 244, pp. 75–85.

    Chapter  Google Scholar 

  13. http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/learning-center/protease-inhibitors.html.

  14. Beynon, R.J. (1998) Prevention of unwanted proteolysis, in Methods in Molecular Biology: New Protein Techniques (Walker, J.M., ed.), Humana, Totowa, NJ, 3, pp. 1–23.

    Chapter  Google Scholar 

  15. Frank, M.B. (1997) “Notes on Protease Inhibitors” from a Bionet Newsgroup described in Molecular Biology Protocols (http://omrf.ouhsc.edu/∼frank/protease.html).

  16. Harper, J.W., Hemmi, K., and Powers, J.C. (1985) Reaction of serine proteases with substituted isocoumarins: discovery of 3,4- dichloroisocoumarin, a new general mechanism based serine protease inhibitor. Biochemistry 24, 1831–1841.

    Article  PubMed  CAS  Google Scholar 

  17. Hassel, M., Klenk, G., and Frohme, M. (1996) Prevention of unwanted proteolysis during extraction of proteins from protease-rich tissue. Anal. Biochem. 242, 274–275.

    Article  PubMed  CAS  Google Scholar 

  18. North, M.J. and Benyon, R.J. (1994) Prevention of unwanted proteolysis, in Proteolytic Enzymes: A Practical Approach (Beynon, R.J. and Bond, J.S., eds.), Oxford University Press, New York, USA, pp. 241–249.

    Google Scholar 

  19. Sreedharan, S.K., Verma, C., Caves, L.S.D., Brocklehurst, S.M., Gharbia, S.E., Shah, H.N., and Brocklehurst, K.M. (1996) Demonstration that 1-trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane (E-64) is one of the most effective low Mr inhibitors of trypsin-catalysed hydrolysis. Characterization by kinetic analysis and by energy minimization and molecular dynamics simulation of the E-64–b-trypsin complex. Biochem. J. 316, 777–786.

    PubMed  CAS  Google Scholar 

  20. Salvensen, G. and Nagase, H. (1989) Inhibition of proteolytic enzymes, in Proteolytic Enzymes: A Practical Approach (Beynon, R.J. and Bond, J.S., eds.), Oxford University Press, New York, USA, pp. 83–104.

    Google Scholar 

  21. North, M.J. (1989) Prevention of unwanted proteolysis, in Proteolytic Enzymes: A Practical Approach (Beynon, R.J. and Bond, J.S., eds.), IRL Press, Oxford, pp. 105–124.

    Google Scholar 

  22. Barford, D. (1996) Molecular mechanisms of the protein serine/threonine phosphatases. Trends Biochem. Sci. 21, 407.

    Article  PubMed  CAS  Google Scholar 

  23. Castellanos-Serra, L. and Paz-Lago, D. (2002) Inhibition of unwanted proteolysis during sample preparation: evaluation of its efficiency in challenge experiments. Electrophoresis 23, 1745–1753.

    Article  PubMed  CAS  Google Scholar 

  24. Kulakowska-Bodzon, A., Bierczynska-Krzysik, A., Dylag, T., Drabik, A., Suder, P., Noga, M., Jarzebinska, J., and Silberring, J. (2007) Methods for sample preparation in proteomic research. J. Chromatogr. B 849, 1–31.

    Article  Google Scholar 

  25. Pendyala, P.R., Ayong, L., Eatrides, J., Schreiber, M., Pham, C., Chakrabarti, R., Fidock, D., Allen, C.M., and Chakrabarti, D. (2008) Characterization of a PRL protein tyrosine phosphatase from Plasmodium falciparum. Mol. Biochem. Parasitol. 158, 1–10.

    Article  PubMed  CAS  Google Scholar 

  26. Kuwana, T. and Rosalki, S.B. (1991) Measurement of alkaline phosphatase of intestinal origin in plasma by p-bromotetramisole inhibition. J. Clin. Pathol. 44, 236–237.

    Article  PubMed  CAS  Google Scholar 

  27. Jain, M.K. (1982) Handbook of Enzyme Inhibitors, John Wiley and Sons, New York, p. 222.

    Google Scholar 

  28. Jain, M.K. (1982) Handbook of Enzyme Inhibitors, John Wiley and Sons, New York, p. 334.

    Google Scholar 

  29. Jain, M.K. (1982) Handbook of Enzyme Inhibitors, John Wiley and Sons, New York, pp. 189–190.

    Google Scholar 

  30. http://www.emdbiosciences.com/html/cbc/Phosphatase_Inhibitor_Cocktail_Sets.htm

  31. Gordon, J.A. (1991) Use of vanadate as protein-phosphotyrosine phosphatase inhibitor. Methods Enzymol. 201, 477–482.

    Article  PubMed  CAS  Google Scholar 

  32. Bodzon-Kulakowska, A., Bierczynska-Krzysik, A., Dylag, T., Drabik, A., Suder, P., Noga, M., Jarzebinska, J., and Silberring, J. (2007) Methods for samples preparation in proteomic research. J. Chromatogr. B 849, 1–31.

    Article  CAS  Google Scholar 

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Correspondence to Barry J. Ryan .

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Ryan, B.J. (2011). Avoiding Proteolysis During Protein Chromatography. In: Walls, D., Loughran, S. (eds) Protein Chromatography. Methods in Molecular Biology, vol 681. Humana Press. https://doi.org/10.1007/978-1-60761-913-0_4

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  • DOI: https://doi.org/10.1007/978-1-60761-913-0_4

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60761-912-3

  • Online ISBN: 978-1-60761-913-0

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