Skip to main content
SpringerLink
Log in

High-Throughput Screening of Dye-Ligands for Chromatography

  • Protocol
  • First Online:
Protein Downstream Processing

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1129))

Abstract

Dye-ligand-based chromatography has become popular after Cibacron Blue, the first reactive textile dye, found application for protein purification. Many other textile dyes have since been successfully used to purify a number of proteins and enzymes. While the exact nature of their interaction with target proteins is often unclear, dye-ligands are thought to mimic the structural features of their corresponding substrates, cofactors, etc. The dye-ligand affinity matrices are therefore considered pseudo-affinity matrices. In addition, dye-ligands may simply bind with proteins due to electrostatic, hydrophobic, and hydrogen-bonding interactions. Because of their low cost, ready availability, and structural stability, dye-ligand affinity matrices have gained much popularity. Choice of a large number of dye structures offers a range of matrices to be prepared and tested. When presented in the high-throughput screening mode, these dye-ligand matrices provide a formidable tool for protein purification. One could pick from the list of dye-ligands already available or build a systematic library of such structures for use. A high-throughput screen may be set up to choose best dye-ligand matrix as well as ideal conditions for binding and elution, for a given protein. The mode of operation could be either manual or automated. The technology is available to test the performance of dye-ligand matrices in small volumes in an automated liquid-handling workstation. Screening a systematic library of dye-ligand structures can help establish a structure–activity relationship. While the origins of dye-ligand chromatography lay in exploiting pseudo-affinity, it is now possible to design very specific biomimetic dye structures. High-throughput screening will be of value in this endeavor as well.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 119.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Kopperschlager G, Freyer R, Diezel W et al (1968) Some kinetic and molecular properties of yeast phosphofructokinase. FEBS Lett 1:137–141

    Article  PubMed  Google Scholar 

  2. Haeckel R, Hess B, Lauterborn W et al (1968) Purification and allosteric properties of yeast pyruvate kinase. Hoppe Seylers Z Physiol Chem 349:699–714

    Article  CAS  PubMed  Google Scholar 

  3. Ryan LD, Vestling CS (1974) Rapid purification of lactate dehydrogenase from rat liver and hepatoma: a new approach. Arch Biochem Biophys 160:279–284

    Article  CAS  PubMed  Google Scholar 

  4. Swart AC, Hemker HC (1970) Separation of blood coagulation factors II, VII, IX and X by gel filtration in the presence of dextran blue. Biochim Biophys Acta 222:692–695

    Article  CAS  PubMed  Google Scholar 

  5. Blume KG, Hoffbauer RW, Busch D et al (1971) Purification and properties of pyruvate kinase in normal and in pyruvate kinase deficient human red blood cells. Biochim Biophys Acta 227:364–372

    Article  CAS  PubMed  Google Scholar 

  6. Staal GE, Koster JF, Kamp H et al (1971) Human erythrocyte pyruvate kinase. Its purification and some properties. Biochim Biophys Acta 227:86–96

    Article  CAS  PubMed  Google Scholar 

  7. Bohme HJ, Kopperschlager G, Schulz J et al (1972) Affinity chromatography of phosphofructokinase using Cibacron blue F3G-A. J Chromatogr 69:209–214

    Article  CAS  PubMed  Google Scholar 

  8. Subramanian S (1984) Dye-ligand affinity chromatography: the interaction of Cibacron Blue F3GA with proteins and enzymes. CRC Crit Rev Biochem 16:169–205

    Article  CAS  PubMed  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

    CAS  PubMed Central  PubMed  Google Scholar 

  10. Forde J, Oakey L, Jennings L et al (2005) Fundamental differences in bioaffinity of amino acid dehydrogenases for N6- and S6-linked immobilized cofactors using kinetic-based enzyme-capture strategies. Anal Biochem 338:102–112

    Article  CAS  PubMed  Google Scholar 

  11. Burton SJ (1992) Dye-ligand affinity chromatography. Methods Mol Biol 11:91–103

    CAS  PubMed  Google Scholar 

  12. Lowe CR, Pearson JC (1984) Affinity chromatography on immobilized dyes. In: William BJ (ed) Methods in enzymology, part C: enzyme purification and related techniques. Academic, New York, pp 97–113

    Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  14. Curling J (2004) Affinity chromatography—from textile dyes to synthetic ligands by design, part I. BioPharm Int 17:34–42

    CAS  Google Scholar 

  15. Lascu I, Porumb H, Porumb T et al (1984) Ion-exchange properties of Cibacron Blue 3G-A Sepharose (Blue Sepharose) and the interaction of proteins with Cibacron Blue 3G-A. J Chromatogr 283:199–210

    Article  CAS  PubMed  Google Scholar 

  16. Baird JK, Sherwood RF, Carr RJ et al (1976) Enzyme purification by substrate elution chromatography from procion dye-polysaccharide matrices. FEBS Lett 70:61–66

    Article  CAS  PubMed  Google Scholar 

  17. Burton SJ, Stead CV, Lowe CR (1990) Design and applications of biomimetic anthraquinone dyes. III. Anthraquinone-immobilised C.I. reactive blue 2 analogues and their interaction with horse liver alcohol dehydrogenase and other adenine nucleotide-binding proteins. J Chromatogr 508:109–125

    Article  CAS  PubMed  Google Scholar 

  18. Ekkundi VS, Punekar NS, Roentgen G et al (2006) Adsorbents comprising anthraquinone dye-ligands for the separation of biological materials. WO/2006/108760, EP20060725379

    Google Scholar 

  19. Kumar S, Dalvi DB, Moorthy M et al (2009) Discriminatory protein binding by a library of 96 new affinity resins: a novel dye-affinity chromatography tool-kit. J Chromatogr B Analyt Technol Biomed Life Sci 877:3610–3618

    Article  CAS  PubMed  Google Scholar 

  20. Garg N, Galaev IY, Mattiasson B (1996) Dye-affinity techniques for bioprocessing: recent developments. J Mol Recognit 9:259–274

    Article  CAS  PubMed  Google Scholar 

  21. Lowe CR, Lowe AR, Gupta G (2001) New developments in affinity chromatography with potential application in the production of biopharmaceuticals. J Biochem Biophys Methods 49:561–574

    Article  CAS  PubMed  Google Scholar 

  22. Labrou NE (2003) Design and selection of ligands for affinity chromatography. J Chromatogr B Analyt Technol Biomed Life Sci 790:67–78

    Article  CAS  PubMed  Google Scholar 

  23. Labrou NE, Eliopoulos E, Clonis YD (1999) Molecular modeling for the design of a biomimetic chimeric ligand. Application to the purification of bovine heart L-lactate dehydrogenase. Biotechnol Bioeng 63:322–332

    Article  CAS  PubMed  Google Scholar 

  24. Clonis YD, Labrou NE, Kotsira VP et al (2000) Biomimetic dyes as affinity chromatography tools in enzyme purification. J Chromatogr A 891:33–44

    Article  CAS  PubMed  Google Scholar 

  25. Lowe CR (2001) Combinatorial approaches to affinity chromatography. Curr Opin Chem Biol 5:248–256

    Article  CAS  PubMed  Google Scholar 

  26. Carlson HA (2002) Protein flexibility and drug design: how to hit a moving target. Curr Opin Chem Biol 6:447–452

    Article  CAS  PubMed  Google Scholar 

  27. Curling J (2004) Affinity Chromatography—from textile dyes to synthetic ligands by design, part II. BioPharm Int 17:60–66

    CAS  Google Scholar 

  28. Wiendahl M, Schulze Wierling P, Nielsen J et al (2008) High throughput screening for the design and optimization of chromatographic processes—miniaturization, automation and parallelization of breakthrough and elution studies. Chem Eng Technol 31:893–903

    Article  CAS  Google Scholar 

  29. Hasnaoui MH, Debbia M, Cochet S et al (1997) Screening of a large number of dyes for the separation of human immunoglobulin G2 from the other immunoglobulin G subclasses immunoglobulin G2 enrichment on immobilized Procion Yellow HE-4R. J Chromatogr A 766:49–60

    Article  CAS  PubMed  Google Scholar 

  30. Raya-Tonetti G, Perotti N (1999) Rapid screening of textile dyes employed as affinity ligands to purify enzymes from yeast. Biotechnol Appl Biochem 29:151–156

    CAS  PubMed  Google Scholar 

  31. Ibrahim-Granet O, Bertrand O (1996) Separation of proteases: old and new approaches. J Chromatogr B Biomed Appl 684:239–263

    Article  CAS  PubMed  Google Scholar 

  32. Mottl H, Keck W (1992) Rapid screening of a large number of immobilized textile dyes for the purification of proteins: use of penicillin-binding protein 4 of Escherichia coli as a model enzyme. Protein Expr Purif 3:403–409

    Article  CAS  PubMed  Google Scholar 

  33. Hondmann DH, Visser J (1990) Screening method for large numbers of dye-adsorbents for enzyme purification. J Chromatogr 510: 155–164

    Article  CAS  PubMed  Google Scholar 

  34. Chapman T (2005) Protein purification: pure but not simple. Nature 434:795–798

    Article  PubMed  Google Scholar 

  35. Uttamchandani M, Walsh DP, Khersonsky SM et al (2004) Microarrays of tagged combinatorial triazine libraries in the discovery of small-molecule ligands of human IgG. J Comb Chem 6:862–868

    Article  CAS  PubMed  Google Scholar 

  36. Stellwagen E (1977) Use of blue dextran as a probe for the nicotinamide adenine dinucleotide domain in proteins. Acc Chem Res 10: 92–98

    Article  CAS  Google Scholar 

  37. Kopperschläger G, Bohme HJ, Hofmann E (1982) Cibacron blue F3G-A and related dyes as ligands in affinity chromatography. In: Fiechter A (ed) Advances in biochemical engineering. Springer, Berlin, pp 101–138

    Google Scholar 

  38. Gallant SR, Koppaka V, Zecherle N (2008) Dye ligand chromatography. Methods Mol Biol 421:61–69

    CAS  PubMed  Google Scholar 

  39. Thompson ST, Cass KH, Stellwagen E (1975) Blue dextran-sepharose: an affinity column for the dinucleotide fold in proteins. Proc Natl Acad Sci U S A 72:669–672

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  40. Small DAP, Atkinson T, Lowe CR (1981) High-performance liquid affinity chromatography of enzymes on silica-immobilised triazine dyes. J Chromatogr A 216:175–190

    Article  CAS  Google Scholar 

  41. Hughes P, Lowe CR, Sherwood RF (1982) Metal ion-promoted binding of proteins to immobilized triazine dye affinity adsorbents. Biochim Biophys Acta 700:90–100

    Article  CAS  PubMed  Google Scholar 

  42. James GT (1978) Inactivation of the protease inhibitor phenylmethylsulfonyl fluoride in buffers. Anal Biochem 86:574–579

    Article  CAS  PubMed  Google Scholar 

  43. Dave K, Ahuja M, Jayashri TN et al (2012) A novel selectable marker based on Aspergillus niger arginase expression. Enzyme Microb Technol 51:53–58

    Article  CAS  PubMed  Google Scholar 

  44. Hey Y, Dean PD (1983) Tandem dye-ligand chromatography and biospecific elution applied to the purification of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. Biochem J 209:363–371

    CAS  PubMed Central  PubMed  Google Scholar 

  45. Morrill PR, Gupta G, Sproule K et al (2002) Rational combinatorial chemistry-based selection, synthesis and evaluation of an affinity adsorbent for recombinant human clotting factor VII. J Chromatogr B Analyt Technol Biomed Life Sci 774:1–15

    Article  CAS  PubMed  Google Scholar 

  46. Scopes RK, Testolin V, Stoter A et al (1985) Simultaneous purification and characterization of glucokinase, fructokinase and glucose-6-phosphate dehydrogenase from Zymomonas mobilis. Biochem J 228:627–634

    CAS  PubMed Central  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Ciba Research (India) Pvt. Ltd. for providing the 96 novel-affinity resins (CibaFix® Resins CR-001 to CR-096). This work was supported by a research fellowship (to Sunil Kumar) from the University Grant Commission, India, and the Board of Research in Nuclear Sciences, Department of Atomic Energy, India.

Author information

Authors and Affiliations

  1. Biotechnology Group, Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, India

    Sunil Kumar

  2. IPB, Univ. Bordeaux, BPRVS, EA 4135, 146 rue LĂ©o Saignat, 33076, Bordeaux, Cedex, France

    Narayan S. Punekar

Authors
  1. Sunil Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Narayan S. Punekar
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Narayan S. Punekar .

Editor information

Editors and Affiliations

  1. Laboratory of Enzyme Technology, Agricultural University of Athens Department of Agr. Biotechnology, Athens, Greece

    Nikolaos E. Labrou

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Kumar, S., Punekar, N.S. (2014). High-Throughput Screening of Dye-Ligands for Chromatography. In: Labrou, N. (eds) Protein Downstream Processing. Methods in Molecular Biology, vol 1129. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-977-2_6

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-977-2_6

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-976-5

  • Online ISBN: 978-1-62703-977-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation