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Protein Chromatography pp 53–69Cite as

Avoiding Proteolysis During Protein Purification

Part of the Methods in Molecular Biology book series (MIMB,volume 1485)

Abstract

All cells contain proteases which hydrolyze the peptide bonds between amino acids in a protein backbone. Typically, proteases are prevented from nonspecific proteolysis by regulation and by their physical separation into different subcellular compartments; however, this segregation is not retained during cell lysis, which is the initial step in any protein isolation procedure. Prevention of proteolysis during protein purification often takes the form of a two-pronged approach; firstly inhibition of proteolysis in situ, followed by the early separation of the protease from the protein of interest via chromatographical purification. Protease inhibitors are routinely used to limit the effect of the proteases before they are physically separated from the protein of interest via column chromatography. Here, commonly used approaches to reducing or avoiding proteolysis during protein purification and subsequent chromatography are reviewed.

Key words

  • Protease
  • Proteolysis
  • Protease inhibitor buffer
  • Protein purification

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References

  1. O’Fágáin C (1997) Protein stability and its measurement. In: O’Fágáin C (ed) Stabilising protein function. Springer, Berlin, pp 115–125

    Google Scholar 

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

    CAS  CrossRef  PubMed  Google Scholar 

  3. Chung CH, Goldberg AL (1981) The product of the lon (capR) gene in Escherichia coli is the ATP-dependent protease, protease La. Proc Natl Acad Sci U S A 78:4931–4935

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

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

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  5. Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100:57–70

    CAS  CrossRef  PubMed  Google Scholar 

  6. de Souza PM, Bittencourt ML, Caprara CC, de Freitas M, de Almeida RPC, Silveira D, Fonseca YM, Filho EXF, Junior AP, Magalhães PO (2015) A biotechnology perspective of fungal proteases. Braz J Microbiol 46:337–346

    CrossRef  PubMed  PubMed Central  Google Scholar 

  7. Song J, Tan H, Boyd SE, Shen H, Mahmood K, Webb GI, Akutsu T, Whisstock JC, Pike RN (2011) Bioinformatic approaches for predicting substrates of proteases. J Bioinform Comput Biol 9:149–178

    CAS  CrossRef  PubMed  Google Scholar 

  8. Doucet A, Overall CM (2008) Protease proteomics: revealing protease in vivo functions using systems biology approaches. Mol Aspects Med 29:339–358

    CAS  CrossRef  PubMed  Google Scholar 

  9. Deu E, Verdoes M, Bogyo M (2012) New approaches for dissecting protease functions to improve probe development and drug discovery. Nat Struct Mol Biol 19:9–16

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  10. Vanaman TC, Bradshaw RA (1999) Proteases in cellular regulation. J Biol Chem 274:20047

    CAS  CrossRef  PubMed  Google Scholar 

  11. Sandhya C, Sumantha A, Pandey A (2004) Proteases. In: Pandey A, Webb C, Soccol CR, Larroche C (eds) Enzyme technology. Asiatech, New Delhi, India, pp 312–325

    Google Scholar 

  12. Ryan BJ, Henehan GT (2013) Overview of approaches to preventing and avoiding proteolysis during expression and purification of proteins. Curr Protoc Protein Sci 5:5–25

    Google Scholar 

  13. Terpe T (2006) Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial strains. Appl Microbiol Biotechnol 72:211–222

    CAS  CrossRef  PubMed  Google Scholar 

  14. Zeinoddini M, Khajeh K, Hosseinkhani S, Saeedinia AR, Robatjazi SM (2013) Stabilisation of recombinant aequorin by polyols: activity, thermostability and limited proteolysis. Appl Biochem Biotechnol 170:273–280

    CAS  CrossRef  PubMed  Google Scholar 

  15. Chen R (2012) Bacterial expression systems for recombinant protein production: E. coli and beyond. Biotechnol Adv 30:1102–1107

    CAS  CrossRef  PubMed  Google Scholar 

  16. Mattanovich D, Branduardi P, Dato L, Gasser B, Sauer M, Porro D (2012) Recombinant protein production in yeasts. In: Clifton NJ (ed) Methods in molecular biology, vol 824. Humana, Totowa, NJ, pp 329–358

    Google Scholar 

  17. Zhu J (2012) Mammalian cell protein expression for biopharmaceutical production. Biotechnol Adv 30:1158–1170

    CAS  CrossRef  PubMed  Google Scholar 

  18. Beynon RJ, Oliver S (2004) Avoidance of proteolysis in extracts. In: Cutler P (ed) Protein purification protocols, vol 244, Methods in molecular biology. Humana, Totowa, NJ, pp 75–85

    CrossRef  Google Scholar 

  19. Vera A, Arís A, Carrió M, González-Montalbán N, Villaverde A (2005) Lon and ClpP proteases participate in the physiological disintegration of bacterial inclusion bodies. J Biotechnol 119:163–171

    CAS  CrossRef  PubMed  Google Scholar 

  20. Pickering AM, Davies KJ (2012) A simple fluorescence labeling method for studies of protein oxidation, protein modification, and proteolysis. Free Radic Biol Med 52:239–246

    CAS  CrossRef  PubMed  Google Scholar 

  21. Healy N, Greig S, Enahoro H, Roberts H, Drake L, Shaw E, Ashall F (1992) Detection of peptidases in Trypanosoma cruzi epimastigotes using chromogenic and fluorogenic substrates. Parasitology 104:315–322

    CAS  CrossRef  PubMed  Google Scholar 

  22. Vandooren J, Geurts N, Martens E, Van den Steen PE, Opdenakker G (2013) Zymography methods for visualizing hydrolytic enzymes. Nat Methods 10:211–220

    CAS  CrossRef  PubMed  Google Scholar 

  23. Serim S, Haedke U, Verhelst SH (2012) Activity-based probes for the study of proteases: recent advances and developments. ChemMedChem 7:1146–1159

    CAS  CrossRef  PubMed  Google Scholar 

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

  25. Beynon RJ (1998) Prevention of unwanted proteolysis. In: Walker JM (ed) Methods in molecular biology: new protein techniques, vol 3. Humana, Totowa, NJ, pp 1–23

    CrossRef  Google Scholar 

  26. Frank MB (1997) “Notes on Protease Inhibitors” from a Bionet Newsgroup described in Molecular Biology Protocols. http://omrf.ouhsc.edu/~frank/protease.html

  27. Harper JW, Hemmi K, Powers JC (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

    CAS  CrossRef  PubMed  Google Scholar 

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

    CAS  CrossRef  PubMed  Google Scholar 

  29. North MJ, Benyon RJ (1994) Prevention of unwanted proteolysis. In: Beynon RJ, Bond JS (eds) Proteolytic enzymes: a practical approach. Oxford University Press, Oxford, pp 241–249

    Google Scholar 

  30. Sreedharan SK, Verma C, Caves LSD, Brocklehurst SM, Gharbia SE, Shah HN, Brocklehurst KM (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

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  31. Salvensen G, Nagase H (1989) Inhibition of proteolytic enzymes. In: Beynon RJ, Bond JS (eds) Proteolytic enzymes: a practical approach. Oxford University Press, Oxford, pp 83–104

    Google Scholar 

  32. North MJ (1989) Prevention of unwanted proteolysis. In: Beynon RJ, Bond JS (eds) Proteolytic enzymes: a practical approach. IRL Press, Oxford, pp 105–124

    Google Scholar 

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

    CAS  CrossRef  PubMed  Google Scholar 

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

    CAS  CrossRef  PubMed  Google Scholar 

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

    CrossRef  Google Scholar 

  36. Hua S, Hu CY, Kim BJ, Totten SM, Oh MJ, Yun N, Nwosu CC, Yoo JS, Lebrilla CB, An HJ (2013) Glyco-analytical multispecific proteolysis (Glyco-AMP): a simple method for detailed and quantitative glycoproteomic characterization. J Proteome Res 12:4414–4423

    CAS  CrossRef  PubMed  Google Scholar 

  37. Nwosu CC, Huang J, Aldredge DL, Strum JS, Hua S, Seipert RR, Lebrilla CB (2012) In-gel nonspecific proteolysis for elucidating glycoproteins: a method for targeted protein-specific glycosylation analysis in complex protein mixtures. Anal Chem 85:956–963

    CrossRef  PubMed  PubMed Central  Google Scholar 

  38. Ghobadi S, Yousefi F, Khademi F, Padidar S, Mostafaie A (2012) An efficient method for purification of nonspecific lipid transfer protein-1 from rice seeds using kiwifruit actinidin proteolysis and ion exchange chromatography. J Sep Sci 35:2827–2833

    CAS  CrossRef  PubMed  Google Scholar 

  39. Yu L, Xiao G, Zhang J, Remmele RL, Eu M, Liu D (2012) Identification and quantification of Fc fusion peptibody degradations by limited proteolysis method. Anal Biochem 428:137–142

    CAS  CrossRef  PubMed  Google Scholar 

  40. Rawlings ND, Morton FR, Kok CY, Kong J, Barrett AJ (2008) MEROPS: the peptidase database. Nucleic Acids Res 36:D320–D325

    CAS  CrossRef  PubMed  Google Scholar 

  41. Rawlings ND, Barrett AJ (1994) Families of serine peptidases. Methods Enzymol 244:19–61

    CAS  CrossRef  PubMed  Google Scholar 

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

    CrossRef  PubMed  Google Scholar 

  43. Dame JB, Reddy GR, Yowell CA, Dunn BM, Kay J, Berry C (1994) Sequence, expression and modelled structure of an aspartic protease from the human malaria parasite Plasmodium falciparum. Mol Biochem Parasitol 64:177–190

    CAS  CrossRef  PubMed  Google Scholar 

  44. Barinka C, Byun Y, Dusich CL, Banerjee SR, Chen Y, Castanares M, Kozikowski AP, Mease RC, Pomper MG, Lubkowski J (2008) Interactions between human glutamate carboxypeptidase II and urea-based inhibitors: structural characterization. J Med Chem 51:7737–7743

    CAS  CrossRef  PubMed  Google Scholar 

  45. Li YY, Bao YL, Song ZB, Sun LG, Wu P, Zhang Y, Fan C, Huang YX, Wu Y, Yu CL, Sun Y, Zheng LH, Wang GN, Li YX (2012) The threonine protease activity of testes-specific protease 50 (TSP50) is essential for its function in cell proliferation. PLoS One 7:e35030

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  46. Rawlings ND, Barrett AJ, Bateman A (2011) Asparagine peptide lyases; a seventh catalytic type of proteolytic enzymes. J Biol Chem 286:38321–38328

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  47. Edwards DR, Handsley MM, Pennington CJ (2008) The ADAM metalloproteases. Mol Aspects Med 29:258–289

    CAS  CrossRef  PubMed  Google Scholar 

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

    CAS  CrossRef  PubMed  Google Scholar 

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

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  50. Jain MK (1982) Handbook of enzyme inhibitors. Wiley, New York, p 222

    Google Scholar 

  51. Jain MK (1982) Handbook of enzyme inhibitors. Wiley, New York, p 334

    Google Scholar 

  52. Jain MK (1982) Handbook of enzyme inhibitors. Wiley, New York, pp 189–190

    Google Scholar 

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

  54. Gordon JA (1991) Use of vanadate as protein-phosphotyrosine phosphatase inhibitor. Methods Enzymol 201:477–482

    CAS  CrossRef  PubMed  Google Scholar 

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

    CAS  CrossRef  Google Scholar 

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Authors and Affiliations

  1. School of Food Science and Environmental Health, Dublin Institute of Technology, Cathal Brugha Street, Dublin 1, Republic of Ireland

    Barry J. Ryan & Gary T. Henehan

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  1. Barry J. Ryan
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  2. Gary T. Henehan
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Correspondence to Barry J. Ryan .

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Editors and Affiliations

  1. School of Biotechnology, Dublin City University, Dublin, Ireland

    Dermot Walls

  2. Department of Applied Sciences, Dundalk Institute of Technology, Dublin, Ireland

    Sinéad T. Loughran

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Ryan, B.J., Henehan, G.T. (2017). Avoiding Proteolysis During Protein Purification. In: Walls, D., Loughran, S. (eds) Protein Chromatography. Methods in Molecular Biology, vol 1485. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6412-3_4

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  • DOI: https://doi.org/10.1007/978-1-4939-6412-3_4

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