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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
O’Fágáin C (1997) Protein stability and its measurement. In: O’Fágáin C (ed) Stabilising protein function. Springer Press, Berlin, pp 115–125
Seife C (1997) Blunting Nature's Swiss Army Knife. Science 277:1602–1603
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
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
Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100:57–70
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
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
Doucet A, Overall CM (2008) Protease proteomics: revealing protease in vivo functions using systems biology approaches. Mol Asp Med 29:339–358
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
Vanaman TC, Bradshaw RA (1999) Proteases in cellular regulation. J Biol Chem 274:20047
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
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
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
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
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
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
Chen R (2012) Bacterial expression systems for recombinant protein production: E. coli and beyond. Biotechnol Adv 30:1102–1107
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
Zhu J (2012) Mammalian cell protein expression for biopharmaceutical production. Biotechnol Adv 30:1158–1170
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
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
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
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
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
Vandooren J, Geurts N, Martens E, Van den Steen PE, Opdenakker G (2013) Zymography methods for visualizing hydrolytic enzymes. Nat Methods 10:211–220
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
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
Frank, M. B. (1997) “Notes on Protease Inhibitors” from a Bionet Newsgroup described in Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/protease.html)
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
Hassel M, Klenk G, Frohme M (1996) Prevention of unwanted proteolysis during extraction of proteins from protease-rich tissue. Anal Biochem 242:274–275
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
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
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
North MJ (1989) Prevention of unwanted proteolysis. In: Beynon RJ, Bond JS (eds) Proteolytic enzymes: a practical approach. IRL Press, Oxford, pp 105–124
Barford D (1996) Molecular mechanisms of the protein serine/threonine phosphatases. Trends Bioch Sci 21:407
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
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
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
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
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
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
Jia L, Sun Y (2017) Protein asparagine deamidation prediction based on structures with machine learning methods. PLoS One 12(7):e0181347
Rawlings ND, Morton FR, Kok CY, Kong J, Barrett AJ (2008) MEROPS: the peptidase database. Nucleic Acids Res 36:D320–D325
Rawlings ND, Barrett AJ (1994) Families of serine peptidases. Methods Enzymol 244:19–61
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
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
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
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
Rawlings ND, Barrett AJ, Bateman A (2011) Asparagine Peptide Lyases; a seventh catalytic type of protteolytic enzymes. J Biol Chem 286:38321–38328
Edwards DR, Handsley MM, Pennington CJ (2008) The ADAM metalloproteases. Mol Asp Med 29:258–289
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
duVerle D, Takigawa I, Ono Y, Sorimachi H, Mamitsuka H (2009) CaMPDB: a resource for Calpain and modulatory proteolysis. Genome Inform 22:202–214
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
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
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
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
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
Wilkins MR, Gasteiger E, Bairoch A et al (1999) Protein identification and analysis tools in the ExPASy server. Methods Mol Biol 112:531–552
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
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
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
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
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
Verspurten J, Gevaert K, Declercq W, Vandenabeele P (2009) SitePredicting the cleavage of proteinase substrates. Trends Biochem Sci 34(7):319–323
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
Kuwana T, Rosalki SB (1991) Measurement of alkaline phosphatase of intestinal origin in plasma by p-bromotetramisole inhibition. J Clin Pathol 44:236–237
Jain MK (1982) Handbook of enzyme inhibitors. Wiley, New York, p 222
Jain MK (1982) Handbook of enzyme inhibitors. Wiley, New York, p 334
Jain MK (1982) Handbook of enzyme inhibitors. Wiley, New York, pp 189–190
http://www.emdbiosciences.com/html/cbc/Phosphatase_Inhibitor_Cocktail_Sets.htm
Gordon JA (1991) Use of vanadate as protein-phosphotyrosine phosphatase inhibitor. Methods Enzymol 201:477–482
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
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
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
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3362-5_6
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3361-8
Online ISBN: 978-1-0716-3362-5
eBook Packages: Springer Protocols