Applied Biochemistry and Biotechnology

, Volume 67, Issue 3, pp 199–214 | Cite as

Phage presentation and affinity selection of a deletion mutant of human lnterleukin-3

  • Sean Merlin
  • Edwin Rowold
  • Ann Abegg
  • Cathleen Berglund
  • Jon Klover
  • Nick Staten
  • John P. McKearn
  • Stephen C. Lee
Original Articles


A deletion derivative of the cytokine human interleukin-3 (hIL-315–125, comprising amino acids 15–125 of the native protein) was produced as a fusion to the filamentous phage surface protein pIII. The cytokine was detected in association with phage particles by protein immunoblotting. Compared to an equivalent quantity of soluble cytokine, phage-presented hIL-315–125 exhibited reduced biological activity in a hIL-3-dependent cell proliferation assay. The reduction in activity was attributable to presence of phage particles in the assay, rather than directly owing to physical incorporation of the cytokine into the phage particle. Owing to the position of the amber codon in the phagemid vector, the phagemid-produced free hIL-315–125 species (designated hIL-315–125 ε) had 20 amino acids appended to its C-terminus; hIL-315–125 ε did not exhibit reduced bioactivity. hIL-315–125-presenting phage were affinity-selected with either a hIL-3-reactive polyclonal antibody or with cells expressing the heterodimeric hIL-3 receptor. These data are consistent with the use of phage-display technology for the affinity selection of hIL-3 variants with modified biological properties.

Index Entries

Phage display cytokine receptor growth factor proliferation biopanning 



amino acid




acute myelogenous leukemia cell




borate buffered saline


baby ham-ster kidney cell


base pair(s)


bovine serum albumin


bovine somatotropin (growth hormone)


3-(cyclohexylamino)-1-propane-sulphionic acid




counts per minute


Dulbecco’s modified Eagle’s medium


Dulbecco’s phosphate buffered saline


enzyme-linked immunosorbent assay


fetal bovine serum


human ciliary neurotrophic factor


human granulocyte colony-stimulating factor


human growth hormone


human granulocyte-macrophage colony-stimulating factor


human interleukin-3


receptor for human interleukin-3


human interleukin-5




tail protein of filamentous bacteriophage


DNA polymerase chain reaction


polyethylene glycol


sodium dodecyl sulfate


titerable unit of phage




Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Metcalf, D. (1993),Blood 82, 3515–3523.Google Scholar
  2. 2.
    Biesma, B., Willemse, P. H. B., Mulder, N. H., Sleijfer, D. Th., Gietema, J. A., Mull, R., Limburg, P. C., Bouma, J., Vellenga, E., and de Vries, E. G. E. (1992).Blood 80, 1141–1148.Google Scholar
  3. 3.
    Gianni, A. M., Siena, S., Bregni, M., Di Nicola, M., Peccatori, F., Magni, M., Ravagnani, F., Sklenar, L., and Banadonna, G. (1993).Annals Oncol. 4, 759–766.Google Scholar
  4. 4.
    Zimmerman, T. M., Williams, S. F., Bender, J. G., Lee, W. J., Blake, M., Carreon, J., Swinney, P., Smith, S. J., Schilling, M., Oldham, F., Van Epps, D.,and, Williams, D.E. (1995),J. Hematotherapy 4, 527–529.Google Scholar
  5. 5.
    Purdy, M. H., Hogan, C. J., Hami, L., McNeice, I., Franklin, W., Jones, R. B., Bearman, S. I., Berenson, R. J., Cagnoni, P. J., Heimfeld, S., and Shpall, E. J. (1995),J. Hematotherapy 4, 515–525.Google Scholar
  6. 6.
    Miyajima, A., Mui, A. L.-F., Ogorochi T., and Kazuhiro, S. (1993).Blood 82, 1960–1974.Google Scholar
  7. 7.
    Gram, H., Strittmatter, U., Lorenz, M., Glueck, D., and Zenke, G. (1993),J. Immunol. Meth. 161, 169–176.CrossRefGoogle Scholar
  8. 8.
    Clackson, T. and Wells, J. A. (1994),Tibtech 12, 173–184.Google Scholar
  9. 9.
    Winter, J. (1994),Drug Devel. Res. 33, 71–89.CrossRefGoogle Scholar
  10. 10.
    Thomas, J. W., Baum, C. M., Hood, W. F., Klein, B., Monahan, J. B., Paik, K., Staten, N., Abrams, M., and McKearn, J. P. (1995).Proc. Natl. Acad. Sci. 92, 3779–3783.CrossRefGoogle Scholar
  11. 11.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989).Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.Google Scholar
  12. 12.
    Olins, P. O., Bauer, S. C., Bradford-Goldberg, S., Sterbenz, K., Polazzi, J. O., Caparon, M. H., Klein, B. K., Easton, A. M., Paik, K., Klover, J. A., Thiele, B. R., and McKearn, J. P. (1995),J. Biol. Chem. 270, 23,754–23,760.Google Scholar
  13. 13.
    Parmley, S. F. and Smith, G. P. (1988),Gene 73, 305–318.CrossRefGoogle Scholar
  14. 14.
    Boeke, J. D., Russel, M., and Model, P. (1980).J. Mol. Biol. 144, 103–116.CrossRefGoogle Scholar
  15. 15.
    Laemmli, U. K. (1970),Nature 227, 680–685.CrossRefGoogle Scholar
  16. 16.
    Bachmann, B. J. (1987), in Escherichia coliand Salmonella typhimuriumCellular and Molecular Biology, Neidhardt, F. C., Ingraham, J. L., Low, K. B., Magasanik, B., Schaechter, M., and Umbarger, H. E., eds., American Society for Microbiology, Washington, D. C., pp. 1190–1219.Google Scholar
  17. 17.
    Wood, D. C., Salsgiver, W. J., Kasser, T. R., Lange, G. W., Rowold, E., Violand, B. N., Johnson, A., Leimgruber, R. M., Parr, G. R., Siegel, N. R., Kimack, N. M., Smith, C., Zobel, J. F., Ganguli, S. M., Garbow, J. R., Bild, G., and Krivi, G. G. (1989),J. Biol. Chem. 264, 14,741–14,747.Google Scholar
  18. 18.
    Hippenmeyer, P. J. and Pegg, L. E. (1995),Curr. Opin. Biotech. 6, 548–552.CrossRefGoogle Scholar
  19. 19.
    Vieira, J. and Messing, J. (1987),Meth. Enzymol. 153, 3–11.CrossRefGoogle Scholar
  20. 20.
    Bass, S., Greene, R., and Wells, J. A. (1990),Proteins: Structure, Function and Genetics 8, 309–314.CrossRefGoogle Scholar
  21. 21.
    Saggio, I., Gloaguen, L. and Laufer, R. (1995),Gene 152, 35–39.CrossRefGoogle Scholar
  22. 22.
    Stent, G. S. and Calendar, R. (1978),Molecular Genetics, 2nd ed., Freeman, San Francisco.Google Scholar
  23. 23.
    Roberts, D., Guegler, K., and Winter, J. (1993),Gene 128, 67–69.CrossRefGoogle Scholar
  24. 24.
    Doorbar, J. and Winter, G. (1994),J. Mol. Biol. 244, 361–369.CrossRefGoogle Scholar
  25. 25.
    Barry, M. A., Dower, W. J., and Johnston, S. A. (1996),Nature Medicine 2, 299–305.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 1997

Authors and Affiliations

  • Sean Merlin
    • 1
  • Edwin Rowold
    • 1
  • Ann Abegg
    • 1
  • Cathleen Berglund
    • 1
  • Jon Klover
    • 1
  • Nick Staten
    • 1
  • John P. McKearn
    • 1
  • Stephen C. Lee
    • 1
  1. 1.Searle Research and DevelopmentMonsanto CompanySt. LouisUSA

Personalised recommendations