, Volume 60, Issue 7–8, pp 379–383 | Cite as

Affinity Chromatography of Insulin with a Heptapeptide Ligand Selected from Phage Display Library

  • Hai-Qing Yu
  • Xiao-Yan Dong
  • Yan SunEmail author


A heptapeptide phage display library was screened with insulin to find its ligands for affinity chromatography. The peptide was synthesized and coupled to EAH Sepharose 4B (5.4 μmol mL−1 bed). Then, insulin chromatography was carried out with mobile phases of different pH values and by the addition of urea and ethylene glycol. It was found that electrostatic interactions were predominant for the affinity binding, and hydrogen bonding might also contribute somewhat to the affinity. Finally, frontal analysis was performed and the dynamic binding capacity of the affinity column for insulin at 50% breakthrough was estimated at 60.6 mg mL−1 bed, which was about two times higher than the theoretical binding capacity of the monomeric insulin. The result suggests that insulin was bound in dimer state in a stoichiometric relationship with the coupled peptide, indicating the high binding efficiency of the peptide ligand for insulin.


Column liquid chromatography Affinity chromatography Peptide ligand Phage display library Insulin 


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This work was financially supported by the Natural Science Foundation of China (grant 20025617).


  1. Johns K (1991) Protein purification: a new approach to affinity chromatography. In: Subramanian G (ed) Preparative and process-scale liquid chromatography. Ellis Horwood Limited, New York, USA, pp. 236–249Google Scholar
  2. Jones C, Patel A, Griffin S, Martin J,Young P, O’Donnell K, Silverman C, Porter T, Chaiken I (1995) J Chromatogr A 707:3–22CrossRefPubMedGoogle Scholar
  3. Christian SB, Zuckermann RN, Kerr JM, Wang L, Malcolm BA (1992) J Mol Biol 227:711–718PubMedGoogle Scholar
  4. Cwirla SE, Peters EA, Barrett RW, Dower WJ (1990) Proc Natl Acad Sci USA 249:6378–6382Google Scholar
  5. Scott JK, Smith GP (1990) Science 249:386–390PubMedGoogle Scholar
  6. Smith GP, Schultz DA, Ladbury JE (1993) Gene 128:37–42CrossRefPubMedGoogle Scholar
  7. Huang PY, Carbonell RG (1995) Biotechnol Bioeng 47:288–297Google Scholar
  8. Huang PY, Baumbach GA, Dadd CA, Buettner JA, Masecar BL, Hentsch ME, Hammond DJ, Carbonell RG (1996) Bioorg Med Chem 4:699–708CrossRefPubMedGoogle Scholar
  9. Smith RG, Missailidis S, Price MR (2001) J Chromatogr B 766:13–26Google Scholar
  10. Karin A, Roman N, Rainer H, Eva S, Horst S, Djuro J, Alois J (2000) J High Resol Chromatogr 23:47–58CrossRefGoogle Scholar
  11. Boyer PM, Hsu JT (1992) Chem Eng Sci 47:241–251CrossRefGoogle Scholar
  12. Baumbach GA, Hammond DJ (1992)Biopharm May Issue:24–25Google Scholar
  13. Yu HQ, Dong X-Y, Sun Y (2004) Biochem Eng J 18:169–175CrossRefGoogle Scholar
  14. Buettner JA, Dadd CA, Baumbach GA, Masecar BL, Hammond DJ (1996) Int J Peptide Protein Res 47:70–83Google Scholar
  15. Shi Y, Sun Y (2003) Chromatographia 57:29–35Google Scholar
  16. Chen J-L, Bai S, Sun Y (2003) Chromatographia 58:701–706Google Scholar
  17. 17.Kaufman DB, Hentsch ME, Baumbach GA, Buettner JA, Dadd CA, Huang PY, Hammond DJ, Carbonell RG (2002) Biotechnol Bioeng 77:278–289CrossRefPubMedGoogle Scholar
  18. Voet D, Voet J G (1995) Biochemistry, 2nd edition, John Wiley & Sons, Inc, New York, USA, pp. 176–180Google Scholar
  19. Stein EA, Fricher EH (1960) Biochim Biophys Acta 39:287–296CrossRefPubMedGoogle Scholar
  20. Shaltiel S (1984) Hydrophobic chromatography. In: Jakoby W (ed) Enzyme purification and related techniques, Part C. Methods in Enzymology. Vol. 104. Academic Press, London, UK, pp. 69–96Google Scholar
  21. Doty P, Gellert M, Rabinovitch B (1952) J Am Chem Soc 74:2065–2069Google Scholar
  22. Helmerhorst E, Stokes G B (1987) Diabetes 36:261–264PubMedGoogle Scholar
  23. Ma XH, Chen WZ, Zhuang Y, Wang CX, Hou TJ, Qiao XB, Xu XJ (2001) Acta Biophysica Sinica 17:329–336Google Scholar
  24. Lakhiari H, Muller D, Jozefonvicz J (1995) J Chromatogr A 711:93–103CrossRefPubMedGoogle Scholar

Copyright information

© Friedr. Vieweg&Sohn/GWV Fachverlage GmbH 2004

Authors and Affiliations

  1. 1.Department of Biochemical EngineeringSchool of Chemical Engineering and Technology, Tianjin UniversityTianjinChina

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