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Synthesis and Evaluation of Dye-Ligand Affinity Adsorbents for Protein Purification

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Part of the Methods in Molecular Biology book series (MIMB,volume 2178)

Abstract

Dye-ligand affinity chromatography is a widely used technique in protein purification. The utility of the reactive dyes as affinity ligands results from their unique chemistry, which confers wide specificity toward a large number of proteins. They are commercially available, inexpensive, stable and can easily be immobilized. Significant factors that contribute to the successful operation of a dye-ligand chromatography include matrix type, dye-ligand density, adsorption along with elution conditions and flow rate. The present chapter provides protocols for the synthesis of dye-ligand affinity adsorbents as well as protocols for screening, selection, and optimization of a given dye-ligand purification step. The purification of the glutathione transferases from Phaseolus vulgaris on Cibacron Blue 3GA-Sepharose affinity adsorbent is given as an example.

Key words

  • Affinity chromatography
  • Cibacron Blue 3GA
  • Dye-ligand chromatography
  • Glutathione transferase
  • Triazine dyes

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  • DOI: 10.1007/978-1-0716-0775-6_15
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References

  1. Eisele T, Stressler T, Kranz B, Fischer L (2012) Automated multi-step purification protocol for angiotensin-I-converting-enzyme (ACE). J Chromatogr B Analyt Technol Biomed Life Sci 911:64–70

    CrossRef  CAS  Google Scholar 

  2. Clonis YD, Labrou NE, Kotsira V, Mazitsos C, Melissis S, Gogolas G (2000) Biomimetic dyes as affinity chromatography tools in enzyme purification. J Chromatogr A 891:33–44

    CrossRef  CAS  Google Scholar 

  3. Labrou NE, Mazitsos K, Clonis YD (2005) Dye-ligand and biomimetic affinity chromatography. In: Hage DS (ed) Handbook of affinity chromatography. Marcel Dekker, Inc, New York, NY, pp 231–255

    Google Scholar 

  4. Gallant SR, Koppaka V, Zecherle N (2008) Dye ligand chromatography. In: Zachariou M (ed) Affinity chromatography. Methods in molecular biology™. Humana, Totowa, NJ, pp 61–70

    CrossRef  Google Scholar 

  5. Chronopoulou EG (2018) Column chromatography overview: an evolving method in protein purification. In: Labrou N, Chronopoulou E, Ataya F (eds) Handbook on protein purification: industry challenges and technological developments. Nova Science Publishers, Inc., New York, NY, pp 1–17

    Google Scholar 

  6. Marinou M, Platis D, Ataya FS, Chronopoulou E, Vlachakis D, Labrou NE (2018) Structure-based design and application of a nucleotide coenzyme mimetic ligand: application to the affinity purification of nucleotide dependent enzymes. J Chromatogr A 1535:88–100

    CrossRef  CAS  Google Scholar 

  7. Kumar S, Punekar NS (2014) High-throughput screening of dye-ligands for chromatography. In: Labrou N (ed) Protein downstream processing. Methods in molecular biology (methods and protocols). Humana, Totowa, NJ, pp 53–65

    CrossRef  Google Scholar 

  8. Labrou NE (2014) Protein purification: an overview. In: Labrou N (ed) Protein downstream processing, Methods in molecular biology (methods and protocols), vol 1129. Humana, Totowa, NJ, pp 3–10

    CrossRef  Google Scholar 

  9. Gianazza E, Arnaud P (1982) Chromatography of plasma proteins on immobilized Cibacron Blue F3-GA. Mechanism of the molecular interaction. Biochem J 203:637–641

    CrossRef  CAS  Google Scholar 

  10. Andac M, Galaev I, Denizli A (2012) Dye attached poly(hydroxyethyl methacrylate) cryogel for albumin depletion from human serum. J Sep Sci 35:1173–1182

    CrossRef  CAS  Google Scholar 

  11. Liang-Schenkelberg J, Fieg G, Waluga T (2017) Molecular insight into affinity interaction between cibacron blue and proteins. Ind Eng Chem Res 56:9691–9697

    CrossRef  CAS  Google Scholar 

  12. Wang SSS, Yang SM, Hsin A, Chang YK (2018) Dye-affinity nanofibrous membrane for adsorption of lysozyme: preparation and performance evaluation. Food Technol Biotechnol 56:40–50

    CrossRef  CAS  Google Scholar 

  13. Maltezos A, Platis D, Vlachakis D, Kossida S, Marinou M, Labrou NE (2014) Design, synthesis and application of benzyl-sulfonate biomimetic affinity adsorbents for monoclonal antibody purification from transgenic corn. J Mol Recogn 27:19–31

    CrossRef  CAS  Google Scholar 

  14. Jahanshahi M, Najafpour G (2007) Advanced downstream processing in biotechnology. In: Najafpour GD (ed) Biochemical engineering and biotechnology. Elsevier, Amsterdam, pp 390–415

    CrossRef  Google Scholar 

  15. Burton SJ, Stead CV, Lowe CR (1988) Design and application of biomimetic dyes II: the interaction of C.I. reactive blue 2 analogs bearing terminal ring modifications with horse liver alcohol dehydrogenase. J Chromatogr 455:201–206

    CrossRef  CAS  Google Scholar 

  16. Lindner NM, Jeffcoat R, Lowe CR (1989) Design and application of biomimetic dyes: purification of calf intestinal alkaline phosphatase with immobilized terminal ring analogs of C.I. Reactive Blue 2. J Chromatogr 473:227–240

    CrossRef  CAS  Google Scholar 

  17. Labrou NE, Eliopoulos E, Clonis YD (1996) Molecular modelling for the design of chimaeric biomimetic dye-ligands and their interaction with bovine heart mitochondrial malate dehydrogenase. Biochem J 315:695–703

    CrossRef  CAS  Google Scholar 

  18. Katsos NE, Labrou NE, Clonis YD (2004) Interaction of L-glutamate oxidase with triazine dyes: selection of ligands for affinity chromatography. J Chromatogr B Analyt Technol Biomed Life Sci 807:277–285

    CrossRef  CAS  Google Scholar 

  19. Labrou NE, Clonis YD (1995) Biomimetic-dye affinity chromatography for the purification of bovine heart lactate dehydrogenase. J Chromatogr 718:35–44

    CrossRef  CAS  Google Scholar 

  20. Labrou NE, Clonis YD (1995) The interaction of Candida boidinii formate dehydrogenase with a new family of chimeric biomimetic dye-ligands. Arch Biochem Biophys 316:169–178

    CrossRef  CAS  Google Scholar 

  21. Burton SJ, McLoughlin SB, Stead V, Lowe SR (1988) Design and application of biomimetic dyes I: synthesis and characterization of terminal ring isomers of C.I. reactive Blue 2. J Chromatogr 435:127–137

    CrossRef  CAS  Google Scholar 

  22. Scopes RK (1986) Strategies for enzyme isolation using dye-ligand and related adsorbents. J Chromatogr 376:131–140

    CrossRef  CAS  Google Scholar 

  23. Kuralay F, Yılmaz E, Uzun L, Denizli A (2013) Cibacron Blue F3GA modified disposable pencil graphite electrode for the investigation of affinity binding to bovine serum albumin. Colloids Surf B Biointerfaces 110C:270–274

    CrossRef  CAS  Google Scholar 

  24. Kirchberger J, Böhme HJ (2000) Dye-ligand affinity chromatography. In: Kastner M (ed) Journal of chromatography library. Elsevier, New York, NY, pp 415–448

    Google Scholar 

  25. Denizli A, Piskin E (2001) Dye-ligand affinity systems. J Biochem Biophys Methods 49:391–416

    CrossRef  CAS  Google Scholar 

  26. Boyer PM, Hsu JT (1992) Effects of ligand concentration on protein adsorption in dye-ligand adsorbents. Chem Eng Sci 47:241–251

    CrossRef  CAS  Google Scholar 

  27. Labrou NE, Clonis YD (1995) Biomimetic-dye affinity chromatography for the purification of L-malate dehydrogenase from bovine heart. J Biotechnol 45:185–194

    CrossRef  Google Scholar 

  28. Berg A, Scouten WH (1990) Dye-ligand centrifugal affinity chromatography. Bioseparation 1:23–31

    CAS  Google Scholar 

  29. Labrou NE (2003) Design and selection of affinity ligands for affinity chromatography. J Chromatogr Β 790:67–78

    CAS  Google Scholar 

  30. Doğan A, Özkara S, Sarı MM, Uzun L, Denizli A (2012) Evaluation of human interferon adsorption performance of Cibacron Blue F3GA attached cryogels and interferon purification by using FPLC system. J Chromatogr B Analyt Technol Biomed Life Sci 893–894:69–76

    CrossRef  CAS  Google Scholar 

  31. Li Z, Cao M, Zhang W, Liu L, Wang J, Ge W, Yuan Y, Yue T, Li R, Yu WW (2014) Affinity adsorption of lysozyme with Reactive Red 120 modified magnetic chitosan microspheres. Food Chem 145:749–755

    CrossRef  CAS  Google Scholar 

  32. Ng IS, Song CP, Ooi CW, Tey BT, Lee YH, Chang YK (2019) Purification of lysozyme from chicken egg white using nanofiber membrane immobilized with Reactive Orange 4 dye. Int J Biol Macromol 134:458–468

    CrossRef  CAS  Google Scholar 

  33. Bradford MM (1976) Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    CrossRef  CAS  Google Scholar 

  34. Chronopoulou E, Madesis P, Asimakopoulou B, Platis D, Tsaftaris A, Labrou NE (2012) Catalytic and structural diversity of the fluazifop-inducible glutathione transferases from Phaseolus vulgaris. Planta 235:1253–1269

    CrossRef  CAS  Google Scholar 

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Acknowledgments

EI acknowledges the State Scholarships Foundation (ΙΚΥ) for financial support. This research is co-financed by Greece and the European Union (European Social Fund- ESF) through the Operational Programme “Human Resources Development, Education and Lifelong Learning” in the context of the project “Strengthening Human Resources Research Potential via Doctorate Research” (MIS-5000432), implemented by the State Scholarships Foundation (ΙΚΥ).

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Correspondence to Nikolaos E. Labrou .

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Chronopoulou, E.G., Premetis, G., Varotsou, C., Georgakis, N., Ioannou, E., Labrou, N.E. (2021). Synthesis and Evaluation of Dye-Ligand Affinity Adsorbents for Protein Purification. In: Labrou, N.E. (eds) Protein Downstream Processing. Methods in Molecular Biology, vol 2178. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0775-6_15

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  • DOI: https://doi.org/10.1007/978-1-0716-0775-6_15

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0774-9

  • Online ISBN: 978-1-0716-0775-6

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