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Approaches to Avoid Proteolysis During Protein Expression and Purification

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Protein Chromatography

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2699))

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Abstract

All cells contain proteases, which hydrolyze the peptide bonds between amino acids of 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: first, inhibition of proteolysis in situ, followed by the early separation of the protease from the protein of interest via chromatographic 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. In this chapter, commonly used approaches to reducing or avoiding proteolysis during protein expression and purification are reviewed.

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References

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

    Google Scholar 

  2. Seife C (1997) Blunting Nature's Swiss Army Knife. Science 277:1602–1603

    Article  CAS  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

    Article  CAS  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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  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

    Article  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 Bioinforma Comput Biol 9:149–178

    Article  CAS  Google Scholar 

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

    Article  CAS  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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  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 Publishers Inc., New Delhi, 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. Zhang W, Lu J, Zhang S, Liu L, Pang X, Lv J (2018) Development an effective system to expression recombinant protein in E. coli via comparison and optimization of signal peptides: expression of Pseudomonas fluorescens BJ-10 thermostable lipase as case study. Microb Cell Factories 17(1):50

    Article  Google Scholar 

  14. Hu J, Lu X, Wang H, Wang F, Zhao Y, Shen W, Yang H, Chen X (2019) Enhancing extracellular protein production in Escherichia coli by deleting the d-alanyl-d-alanine carboxypeptidase gene dacC. Eng Life Sci 19(4):270–278

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. 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

    Article  CAS  PubMed  Google Scholar 

  16. 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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  18. 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, pp 329–358

    Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Chapter  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  22. Li F, Wang Y, Li C, Marquez-Lago TT, Leier A, Rawlings ND, Haffari G, Revote J, Akutsu T, Chou K-C, Purcell AW, Pike RN, Webb GI, Ian Smith A, Lithgow T, Daly RJ, Whisstock JC, Song J (2019) Twenty years of bioinformatics research for protease-specific substrate and cleavage site prediction: a comprehensive revisit and benchmarking of existing methods. Brief Bioinform 20(6):2150–2166. https://doi.org/10.1093/bib/bby077

    Article  CAS  PubMed  Google Scholar 

  23. 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

    Article  CAS  PubMed  Google Scholar 

  24. 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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

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

    Google Scholar 

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

  30. 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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  32. 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 

  33. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. 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 

  35. 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 

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

    Article  CAS  Google Scholar 

  37. 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

    Article  CAS  PubMed  Google Scholar 

  38. 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

    Article  Google Scholar 

  39. Hua S, Hu CY, Kim BJ, Totten SM, Myung Jin O, 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

    Article  CAS  PubMed  Google Scholar 

  40. 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

    Article  PubMed  PubMed Central  Google Scholar 

  41. 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

    Article  CAS  PubMed  Google Scholar 

  42. 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

    Article  CAS  PubMed  Google Scholar 

  43. Jia L, Sun Y (2017) Protein asparagine deamidation prediction based on structures with machine learning methods. PLoS One 12(7):e0181347

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. 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

    Article  PubMed  Google Scholar 

  47. 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

    Article  CAS  PubMed  Google Scholar 

  48. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Rawlings ND, Barrett AJ, Bateman A (2011) Asparagine Peptide Lyases; a seventh catalytic type of protteolytic enzymes. J Biol Chem 286:38321–38328

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  Google Scholar 

  52. Wang M, Zhao XM, Tan H et al (2014) Cascleave 2.0, a new approach for predicting caspase and granzyme cleavage targets. Bioinformatics 30:71–80

    Article  PubMed  Google Scholar 

  53. duVerle D, Takigawa I, Ono Y, Sorimachi H, Mamitsuka H (2009) CaMPDB: a resource for Calpain and modulatory proteolysis. Genome Inform 22:202–214

    CAS  Google Scholar 

  54. Li F, Chen J, Leier A, Marquez-Lago T, Liu Q, Wang Y, Jerico Revote A, Smith I, Akutsu T, Webb GI, Kurgan L, Song J (2020) DeepCleave: a deep learning predictor for caspase and matrix metalloprotease substrates and cleavage sites. Bioinformatics 36(4):1057–1065. https://doi.org/10.1093/bioinformatics/btz721

    Article  CAS  PubMed  Google Scholar 

  55. Liu Z, Cao J, Gao X et al (2011) GPS-CCD: a novel computational program for the prediction of calpain cleavage sites. PLoS One 6:e19001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Song J, Wang Y, Li F et al (2018) iProt-Sub: a comprehensive package for accurately mapping and predicting proteasespecific substrates and cleavage sites. Brief Bioinform:bby028

    Google Scholar 

  57. Fan Y-X, Zhang Y, Shen H-B (2013) LabCaS: labeling calpain substrate cleavage sites from amino acid sequence using conditional random fields. Proteins: Structure, Function, and Bioinformatics 81:622–634

    Article  CAS  Google Scholar 

  58. Barkan D, Hostetter D, Mahrus S, Pieper U, Wells J, Craik C, Sali A (2010) Prediction of protease substrates usingsequence and structure features. Bioinformatics 26:1714–1722

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Wilkins MR, Gasteiger E, Bairoch A et al (1999) Protein identification and analysis tools in the ExPASy server. Methods Mol Biol 112:531–552

    CAS  PubMed  Google Scholar 

  60. Boyd SE, de la Garcia Banda M, Pike RN, Whisstock JC, Rudy GB (2004) PoPS: a computational tool for modeling and predicting protease specificity. Proc IEEE Comput Syst Bioinforma Conf:372–381

    Google Scholar 

  61. Li F, Leier A, Liu Q, Wang Y, Xiang D, Akutsu T, Webb GI, Ian Smith A, Marquez-Lago T, Li J, Song J (2020) Procleave: predicting protease-specific substrate cleavage sites by combining sequence and structural information. Genomics Proteomics Bioinformatics 18(1):52–64. https://doi.org/10.1016/j.gpb.2019.08.002

    Article  PubMed  PubMed Central  Google Scholar 

  62. Song J, Tan H, Perry AJ et al (2012) PROSPER: an integrated feature-based tool for predicting protease substrate cleavage sites. PLoS One 7:e50300

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Song J, Li F, Leier A et al (2018) PROSPERous: high-throughput prediction of substrate cleavage sites for 90 proteases with improved accuracy. Bioinformatics 34:684–687

    Article  CAS  PubMed  Google Scholar 

  64. Fu SC, Imai K, Sawasaki T, Tomii K (2014) ScreenCap3: improving prediction of caspase-3 cleavage sites using experimentally verified non-cleavage sites. Proteomics 17–18:2042–2046

    Article  Google Scholar 

  65. Verspurten J, Gevaert K, Declercq W, Vandenabeele P (2009) SitePredicting the cleavage of proteinase substrates. Trends Biochem Sci 34(7):319–323

    Article  CAS  PubMed  Google Scholar 

  66. 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 Parasit 158:1–10

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Google Scholar 

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

    Google Scholar 

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

    Google Scholar 

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

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

    Article  CAS  PubMed  Google Scholar 

  73. 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

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. School of Food Science and Environmental Health, Technological University Dublin, Grangegorman, Dublin, Ireland

    Gary T. Henehan, Barry J. Ryan & Gemma K. Kinsella

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  1. Gary T. Henehan
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  2. Barry J. Ryan
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  3. Gemma K. Kinsella
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Correspondence to Gemma K. Kinsella .

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Editors and Affiliations

  1. Department of Life and Health Sciences, Dundalk Institute of Technology, Dundalk, Louth, Ireland

    Sinéad T Loughran

  2. National Institute for Bioprocessing Research and Training (NIBRT), Blackrock, Dublin, Ireland

    John Joseph Milne

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Henehan, G.T., Ryan, B.J., Kinsella, G.K. (2023). Approaches to Avoid Proteolysis During Protein Expression and Purification. In: Loughran, S.T., Milne, J.J. (eds) Protein Chromatography. Methods in Molecular Biology, vol 2699. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3362-5_6

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  • DOI: https://doi.org/10.1007/978-1-0716-3362-5_6

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