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High-Throughput Screening of Dye-Ligands for Chromatography

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

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. The 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 serve as 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 the 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 lie 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.

Key words

  • Dye-ligand chromatography
  • Cibacron blue
  • Biomimetic ligands
  • Protein purification
  • High-throughput screening
  • Dye-ligand library

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Acknowledgements

We thank Ciba Research (India) Pvt. Ltd., for providing the ninety-six novel-affinity resins (CibaFix® Resins CR-001–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.

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Correspondence to Narayan S. Punekar .

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Kumar, S., Punekar, N.S. (2021). High-Throughput Screening of Dye-Ligands for Chromatography. 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_5

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  • DOI: https://doi.org/10.1007/978-1-0716-0775-6_5

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