Phage Display Technology

Identification of Peptides as Model Ligands for Affinity Chromatography
  • George K. Ehrlich
  • Pascal Bailon
  • Wolfgang Berthold
Part of the Methods in Molecular Biology book series (MIMB, volume 147)


Protein A has long been the ligand of choice in the affinity purification of immunoglobulin G1 (IgG1) monoclonal antibodies (see Notes 1 and 2). However, current research efforts (1, 2, 3, 4, 5, 6, 7, 8, 9) have been focused on the discovery of small molecules (peptides or peptidomimetics) that share similar binding characteristics with protein A but are more cost effective owing to small size (for ease of synthesis) and stability (for ease of regeneration). The following methods were developed as part of a proof of principle study (1) to determine whether phage display technology could be used to identify peptides as leads in the customization of ligands for affinity chromatography (see Note 3) and (2) to identify a peptide or peptidomimetic for use as a protein A alternative in the affinity purification of monoclonal antibodies. In this study, the constant region (pFc’ fragments; see Note 4) of an IgG1 monoclonal antibody, denoted humanized anti-Tac (HAT), was used as the target for phage display in this study. HAT is a humanized monoclonal antibody against the low-affinity p55 subunit of the interleukin-2 (IL-2) receptor.


Binding Capacity Phage Display Constant Region Phage Display Technology Genetic Computer Group 
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  1. 1.
    Braisted A. C. and Wells J. A. (1996) Minimizing a binding domain from protein A. Proc. Natl. Acad. Sci USA 93, 5688–5692.PubMedCrossRefGoogle Scholar
  2. 2.
    Li R., Dowd V., Stewart D. J., Burton S. J., and Lowe C. R. (1998) Design, synthesis, and application of a protein A mimetic. Nat. Biotechnol. 16, 190–195.PubMedCrossRefGoogle Scholar
  3. 3.
    Fassina G., Verdoliva A., Palombo G., Ruvo M., and Cassini G. (1998) Immunoglobulins specificity of TG1 9318: a novel synthetic ligand for antibody affinity purification. J. Molec. Recogn. 11, 12–133.Google Scholar
  4. 4.
    Guerrier L, Flayeux I., Schwarz A., Fassina G. and Boschetti E. (1998) IRIS: an innovative protein A-peptidomimetic solid phase medium for antibody purification. J. Molec. Recogn. 11, 107–109.CrossRefGoogle Scholar
  5. 5.
    Palombo G., DeFalco S., Tortora G., Cassani G., and Fassina G. (1998) A synthetic ligand for IgA affinity purification. J. Molec. Recogn. 11, 243–246.CrossRefGoogle Scholar
  6. 6.
    Palombo G., Rossi M., Cassani G., and Fassina G. (1998) Affinity purification of mouse monoclonal IgE using a protein A mimetic ligand [TG19318] immobilized on solid supports. J. Molec. Recogn. 11, 247–249.CrossRefGoogle Scholar
  7. 7.
    Palombo G., Verdoliva A., and Fassina G (1998) Affinity purification of immu-noglobulin M using a novel synthetic ligand. J. Chromatogr. B Biomed. Sci. Appl. 715, 137–145.PubMedCrossRefGoogle Scholar
  8. 8.
    Ehrlich G. K. and Bailon P. (1998) Identification of peptides that bind to the constant region of a humanized IgG1 monoclonal antibody using phage display. J. Molec. Recogn. 11, 121–125.CrossRefGoogle Scholar
  9. 9.
    Sengupta J., Sinha P., Mukhopadhyay C., and Ray P. K. (1999) Molecular modeling and experimental approaches toward designing a minimalist protein having Fc-binding activity of Staphylococcal protein A. Biochem. Biophys. Res. Commun. 256, 6–12.PubMedCrossRefGoogle Scholar
  10. 10.
    Queen C., Schneider W. P., Selick H. E., Payne P. W., Landolfi N., Duncan J.F., Avdalovic N. M., Levitt M., Junghans R. P., and Waldman T. A. (1989) A humanized antibody that binds to the interleukin 2 receptor. Proc. Natl. Acad. Sci. USA 86, 10,029–10,033.PubMedCrossRefGoogle Scholar
  11. 11.
    Junghans R. P., Waldman T. A., Landolfi N. F., Avdalovic N. M., Schneider W. P., and Queen C. (1990) Anti-Tac-H, a humanized antibody to the interleukin 2 receptor with new features for immunotherapy in malignant and immune disorders. Cancer Res. 50, 1495–1502.PubMedGoogle Scholar
  12. 12.
    Bailon P., Weber D. V., Keeney R. F., Fredericks J. E., Smith C., Familletti P. C., and Smart J. E. (1987) Receptor-affinity chromatography: A one-step purification for recombinant interleukin-2. Bio/Technology 5, 1195–1198.CrossRefGoogle Scholar
  13. 13.
    Bailon P. and Weber D. V. (1988) Receptor-affinity chromatography. Nature (London) 335, 839–840.CrossRefGoogle Scholar
  14. 14.
    Hakimi J., Seals C., Anderson L. E., Podlaski F. J., Lin P., Danho W., Jenson J. S., Perkins A., Donadio P. E., Familletti P. C., Pan Y-C.E., Tsien W.-H., Chizzonite R. A., Casabo L., Nelson D. L., and Cullen B. R. (1987) Biochemical and functional analysis of soluble human interleukin-2 receptor produced in rodent cells. J. Biol. Chem. 262, 17,336–17,341.PubMedGoogle Scholar
  15. 15.
    Weber D. V., Keeney R. F., Familletti P. C., and Bailon P. (1988) Medium-scale ligand-affinity purification of two soluble forms of human interleukin-2 receptor. J. Chrom. Biomed. Appl. 431, 55–63.CrossRefGoogle Scholar
  16. 16.
    Uhlen M., Guss B., Nilsson B., Gatenbeck S., Philipson L., and Lindberg M. (1984) Complete sequence of the Staphylococcal gene encoding protein A. A gene evolved through multiple duplications. J. Biol. Chem. 259, 1695–1702.PubMedGoogle Scholar
  17. 17.
    Nilsson B., Moks T., Jansson B., Abrahmseen L., Elmblad A., Holmgren E., Herichson C., Jones T. A., and Uhlen M. (1987) A synthetic IgG-binding domain based on staphylococcal protein A. Protein Eng. 1, 107–113.PubMedCrossRefGoogle Scholar
  18. 18.
    Maclennan J. (1997) The generation of process suitable, rugged, targeted affinity ligands using phage display technology. Twelfth Sympsium on Affinity Interactions: Fundamentals and Applications of Biomolecular Recognition. Abstract L30.Google Scholar
  19. 19.
    Torigoe H., Shimada I., Waelchli M., Saito A., Sato M., and Arata Y. (1990) 15N nuclear magnetic resonance studies of the B domain of staphylococcal protein A: sequence specific assignments of the imide 15N resonances of the proline residues and the interaction with human immunoglobulin G. FEBS Lett. 269, 174–176.PubMedCrossRefGoogle Scholar
  20. 20.
    Torigoe H., Shimada I., Saito A., Sato M., and Arata Y. (1990) Sequential 1H NMR assignments and secondary structure of the B domain of staphylococcal protein A: structural changes between the free B domain in solution and the Fc-boundB domain in crystal. Biochemistry. 29, 8787–8793.PubMedCrossRefGoogle Scholar
  21. 21.
    Gouda H., Torigoe H., Saito A., Sato M., Arata Y., and Shimada I. (1992) Three-dimensional solution structure of the B domain of staphylococcal protein A: comparisons of the solution and crystal structures. Biochemistry 31, 9665–9672.PubMedCrossRefGoogle Scholar
  22. 22.
    Jendeberg L., Tashiro M., Tejero R., Lyons B. A., Uhlen M., Montelione G. T., and Nilsson B. (1996) The mechanism of binding staphylococcal protein A to immunoglobin G does not involve helix unwinding. Biochemistry 35, 22–31.PubMedCrossRefGoogle Scholar
  23. 23.
    Lyons B. A., Tashiro M., Cedergren L., Nilsson B., and Montelione G. T. (1993) An improved strategy for determining resonance assignments for isotopi-cally enriched proteins and its application to an engineered domain of staphylococcal protein A. Biochemistry 32, 7839–7845.PubMedCrossRefGoogle Scholar
  24. 24.
    Tashiro M. and Montelione G. T. (1995) Structures of bacterial immunoglobu-lin-binding domains and their complexes with immunoglobulins. Curr. Opin. Struct. Biol. 5, 471–481.PubMedCrossRefGoogle Scholar
  25. 25.
    McDowell R. S., Blackburn B. K., Gadek T. R., McGee L. R., Rawson T., Reynolds M. E., Robarge K. D., Somers T. C., Thorsett E. D., Tischler M., Webb R. R., and Venuti M. C. (1994) From peptide to non-peptide. 2. The de novo design of potent, non-peptidal inhibitors of platelet aggregation based on a benzodiazepine scaffold. J. Am. Chem. Soc. 116, 5077–5083.CrossRefGoogle Scholar
  26. 26.
    Ley C. A. (1997) Custom affinity ligands from phage display for large-scale affinity purification. IBC International Conference on Display Technologies. Lake Tahoe, CA.Google Scholar

Copyright information

© Humana Press Inc. 2000

Authors and Affiliations

  • George K. Ehrlich
  • Pascal Bailon
  • Wolfgang Berthold

There are no affiliations available

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