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Ph.D. Phage Display Peptide Libraries

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Download: Technical Bulletin (pdf, postscript),
Manuals - Ph.D.-7 (#8100) (pdf, postscript), Ph.D.-12 (#8110) (pdfpostscript), Ph.D.-C7C (#8120) (pdfpostscript)


Frequently Asked Questions


Description

Phage display describes a selection technique in which a peptide or protein is expressed as a fusion with a coat protein of a bacteriophage, resulting in display of the fused protein on the exterior surface of the phage virion, while the DNA encoding the fusion resides within the virion. Phage display has been used to create a physical linkage between a vast library of random peptide sequences to the DNA encoding each sequence, allowing rapid identification of peptide ligands for a variety of target molecules (antibodies, enzymes, cell-surface receptors, etc.) by an in vitro selection process called biopanning (1-3). In its simplest form, biopanning 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. (Alternatively the phage can be reacted with the target in solution, followed by affinity capture of the phage-target complexes onto a plate or bead that specifically binds the target.) The eluted phage is then amplified and taken through additional cycles of biopanning and amplification to successively enrich the pool of phage in favor of the tightest binding sequences. After 3-4 rounds, individual clones are characterized by DNA sequencing and ELISA.

Random peptide libraries displayed on phage have been used in a number of applications (2), including epitope mapping (4-6), mapping protein-protein contacts (7), and identification of peptide mimics of non-peptide ligands (8-11). Bioactive peptides have been identified either by panning against immobilized purifed receptors (12) or against intact cells (13-15).

TWC BioSearch is pleased to offer three kits based on random peptide libraries displayed on bacteriophage M13: The Ph.D.-7 and Ph.D.-12 kits include linear peptide libraries of 7 and 12 residues, respectively, while the Ph.D.-C7C kit features a 7-residue peptide library in which the randomized sequence is flanked by a pair of cysteine residues. Under nonreducing conditions the cysteines will spontaneously form a disulfide cross-link, resulting in phage display of cyclized peptides. Disulfide-constrained peptide libraries (16) have proven useful in identification of structural epitopes (17,18), mirror-image ligands for D-amino acid targets (19), and leads for peptide-based therapeutics (20).

The Ph.D.-7 linear 7-mer library contains 2.0 x 10 9 independent clones, while the Ph.D.-C7C disulfide-constrained library contains 3.7 x 10 9 independent clones. Both libraries are sufficiently complex to contain most if not all of the 20 7 = 1.28 x 109 possible 7-mer sequences. In contrast, the Ph.D.-12 library, with 1.9 x 10 9 independent clones, represents only a very small sampling of the potential sequence space of 2012 = 4.1 x 1015 12-mer sequences. Spreading the equivalent diversity of a 7-mer library over a "window" of 12 residues permits affinity selection of peptide ligands for targets requiring more than 7 ligand residues for tight binding, or that require interaction with fewer residues but spaced too far apart to be contained within a 7-residue peptide. Additionally, the increased length of the displayed peptide allows folding into small structural elements (short helices, b -turns, etc.) that may be necessary for target binding. All three libraries have been amplified once such that each biopanning experiment is carried out using on the order of 100 copies of each sequence in 10 Ál of the supplied phage.

The randomized peptide sequences in all three libraries are expressed between the leader sequence and the mature N-terminus of the minor coat protein pIII, resulting in an average valency of 5 displayed peptides per virion. For the linear Ph.D.-7 and Ph.D.-12 libraries, the first position of the mature displayed fusion protein is the first randomized position. For the Ph.D.-C7C disulfide-constrained library, the randomized sequence is preceded by Ala-Cys at the N-terminus. Each displayed peptide is followed by a short spacer (Gly-Gly-Gly-Ser) and then the wild-type pIII sequence. The display vector used for all three libraries is a derivative of wild-type M13 phage- simple propagation of the library as phage rather than plasmid eliminates the need for antibiotic selection and a separate helper phage superinfection step, saving time and effort. Extensive sequencing of the libraries has revealed a wide diversity of side chains at each position with no obvious positional biases.

All three libraries have been extensively utilized at New England Biolabs to identify consensus peptide ligands for streptavidin, MAP kinase, RNase A, chitin, cell-surface receptors and monoclonal antibodies. In all cases the libraries have been demonstrated to be of sufficient complexity to produce multiple DNA sequences encoding the same consensus peptide motifs.

References

  1. Parmley, S.F. and Smith, G.P. (1988) Gene 73, 305-318.
  2. Reviewed in Cortese, R. et al. (1995) Curr. Opin. Biotechnol. 6, 73-80.
  3. Noren, C.J. (1996) NEB Transcript 8 (1), 1-5.
  4. Scott, J.K. and Smith, G.P. (1990) Science 249, 386-390.
  5. Cwirla, S.E. et al. (1990) Proc. Natl. Acad. Sci. USA 87, 6378-6382.
  6. Felici, F. et al. (1991) J. Mol. Biol. 222, 301-310.
  7. Hong, S.S. and Boulanger, P. (1995) EMBO J. 14, 4714-4727.
  8. Devlin, J.J., Panganiban, L.C. and Devlin, P.E. (1990) Science 249, 404-406.
  9. Oldenburg, K.R. et al. (1992) Proc. Natl. Acad. Sci. USA 89, 5393-5397.
  10. Scott, J.K. et al. (1992) Proc. Natl. Acad. Sci USA 89, 5398-5402.
  11. Hoess, R. et al. (1993) Gene 128, 43-49.
  12. O'Neil, K.T. et al. (1992) Proteins 14, 509-515.
  13. Doorbar, J. and Winter, G. (1994) J. Mol. Biol. 244, 361-369.
  14. Goodson, R.J. et al. (1994) Proc. Natl. Acad. Sci. USA 91, 7129-71331.
  15. Barry, M.A., Dower, W.J. and Johnston, S.A. (1996) Nature Medicine 2, 299-305.
  16. McLafferty, M.A. et al. (1993) Gene 128, 29-36.
  17. Hoess, R.H. et al. (1994) J. Immunol. 153, 724-729.
  18. Luzzago, A. et al. (1993) Gene 128, 51-57.
  19. Schumacher, T.N.M. et al. (1996) Science 271, 1854-1857.
  20. Wrighton, N.C. et al. (1996) Science 271, 458-463.

Examples

The Ph.D. Kits Include:

Ordering Information:

#8100 Ph.D.-7 Peptide 7-mer Library Kit
#8110 Ph.D.-12 Peptide 12-mer Library Kit
#8120 Ph.D.-C7C Disulfide Constrained Peptide Library Kit

Components Sold Separately:

#1258 -28 gIII Sequencing Primer 0.5 A260 units
#1259 -96 gIII Sequencing Primer 0.5 A260 units
#801-N ER2537 Host Strain glycerol culture

The commercial use of these products may require a license from Dyax Corp. under US Patents 5,223,409, 5,403,484 and/or 5,571,698 and associated patent rights. For license information contact TWC BioSearch


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