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  • Phage Display

    Phage display describes a selection technique in which a library of variants of a peptide or protein are expressed on the outside of phage virions, while the genetic material encoding each variant resides on the inside of the corresponding virion (1-3). This creates a physical linkage between each variant protein sequence and the DNA encoding it, which allows rapid partitioning based on binding affinity to a given target molecule (antibodies, enzymes, cell-surface receptors, etc.) by an in vitro selection process called panning (4). In its simplest form, panning is carried out by incubating a library of phage-displayed peptides with a plate (or bead) coated with the target, washing away the unbound phage, and eluting the specifically bound phage. The eluted phage is then amplified and taken through additional binding/ amplification cycles to enrich the pool in favor of binding sequences. After 3–4 rounds, individual clones are characterized by DNA sequencing and ELISA.

    References

    1. Sidhu, S.S. et al. (2003) Chembiochem. 4, 14–25. PMID: 12512072
    2. Azzazy, H.M. and Highsmith, W.E. (2002) Clin. Biochem.35, 425–445. PMID: 12413604
    3. Parmley, S.F. and Smith, G.P. (1988) Gene 73, 305–318. PMID: 3149606
    4. Rodi, D.J. et al. (2002) Curr. Opin. Chem. Biol. 6, 92–96. PMID: 11827830

    Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects


    Isolating, by biopanning, the phage that binds to bone allows researchers to identify the peptide sequences that stimulate the differentiation of mesenchymal cells and potentiate bone repair.

    Isolating, by biopanning, the phage that binds to bone allows researchers to identify the peptide sequences that stimulate the differentiation of mesenchymal cells and potentiate bone repair.

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    Applications

    • Epitope mapping
    • Identification of protein-protein contacts (1) and enzyme inhibitors (2)
    • Discovery of peptide ligands for GroEL (3), HIV (4-7), semiconductor surfaces (8) and small-molecule fluorophores (9) and drugs (10)
    • Bioactive receptor ligands have been identified both by panning against purified receptors (11-14) and against intact cells (15-18)
    • Peptides which target specific cell types have been isolated by in vitro panning and used for cell-specific gene delivery (19-22)
    • Ligands for mold spores (23) and bacterial cells (24) have also been identified using this system, including a peptide that specifically inhibits anthrax toxin, both in vitro and in vivo (25)
    • Tissue-specific peptides have been isolated by in vivo panning, in which phage is injected into a live animal, the relevant organs harvested and phage isolated from each tissue type (26,27)

    References

    1. Berggard, T. et al. (2002) J. Biol. Chem. 277, 41954–41959. PMID: 12176979
    2. Chaudhary, J. et al. (2001) Am. J. Physiol. Cell Physiol. 280, C1027–1030. PMID: 11245619
    3. Chen, L. and Sigler, P.B. (1999) Cell 99, 757–768. PMID: 10619429
    4. Biorn, A.C. et al. (2004) Biochemistry 43, 1928–1938. PMID: 14967033
    5. Ferrer, M. and Harrison, S.C. (1999) J. Virol. 73, 5795–5802. PMID: 10364331
    6. Ferrer, M. et al. (1999) J. Pept. Res. 54, 32–42. PMID: 10448968
    7. BouHamdan, M. et al. (1998) J. Biol. Chem. 273, 8009–8016. PMID: 9525900
    8. Whaley, S.R. et al. (2000) Nature 405, 665–668. PMID: 10864319
    9. Rozinov, M.N. and Nolan, G.P. (1998) Chem. Biol. 5, 713–728. PMID: 9862799
    10. Rodi, D.J. et al. (1999) J. Mol. Biol. 285, 197–203. PMID: 9878399
    11. Kraft, S. et al. (1999) J. Biol. Chem. 274, 1979–1985. PMID: 9890954
    12. Koolpe, M. et al. (2002) J. Biol. Chem. 277, 46974–46979. PMID: 12351647
    13. Mummert, M.E. et al. (2000) J. Exp. Med. 192, 769–779. PMID: 10993908
    14. Hetian, L. et al. (2002) J. Biol. Chem. 277, 43137–43142. PMID: 12183450
    15. White, S.J. et al. (2001) Hypertension 37, 449–455. PMID: 11230317
    16. Binetruy-Tournaire, R. et al. (2000) EMBO J. 19, 1525–1533. PMID: 10747021
    17. Kragler, F. et al. (2000) EMBO J. 19, 2856–2868. PMID: 10856231
    18. Gazouli, M. et al. (2002) J. Pharmacol. Exp. Ther. 303, 627–632. PMID: 12388644
    19. Romanczuk, H. et al. (1999) Hum. Gene Ther. 10, 2615–2626. PMID: 10566889
    20. Nicklin, S.A. et al. (2000) Circulation 102, 231–237. PMID: 10889136
    21. Jost, P.J. et al. (2001) FEBS Lett. 489, 263–269. PMID: 11165262
    22. Rasmussen, U.B. et al. (2002) Cancer Gene Ther. 9, 606–612. PMID: 12082461
    23. Tinoco, L.W. et al. (2002) J. Biol. Chem. 277, 36351–36356. PMID: 12130641
    24. Stratmann, J. et al. (2002) J. Clin. Microbiol. 40, 4244–4250. PMID: 12409405
    25. Mourez, M. et al. (2001) Nat. Biotechnol. 19, 958–961. PMID: 11581662
    26. Lee, L. et al. (2002) Arthritis Rheum. 46, 2109–2120. PMID: 12209516
    27. Duerr, D.M. et al. (2004) J. Virol. Methods 116, 177–180. PMID: 14738985

    Panning with a Phage Display Peptide Library