 | | | | Ph.D.™-C7C Phage Display Peptide Library Kit |
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Prices are in US dollars and valid only for US orders.
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Description:
Phage display describes a selection technique in which a library of variants of a peptide or protein is expressed on the outside of a phage virion, while the genetic material encoding each variant resides on the inside (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 (Figure 1), 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.
New England Biolabs offers 3 pre-made random peptide libraries, as well as the cloning vector M13KE for construction of custom libraries. The pre-made libraries consist of linear heptapeptide (Ph.D.-7) and dodecapeptide (Ph.D.-12) libraries, as well as a disulfide-constrained heptapeptide (Ph.D.-C7C) library. The randomized segment of the Ph.D.-C7C library is flanked by a pair of cysteine residues, which are oxidized during phage assembly to a disulfide linkage, resulting in the displayed peptides being presented to the target as loops. All of the libraries have complexities in excess of 2 billion independent clones. The randomized peptide sequences in all three libraries are expressed at the N-terminus of the minor coat protein pIII, resulting in a valency of 5 copies of the displayed peptide per virion. In both the Ph.D.-7 and the Ph.D.-12 libraries, the first residue of the peptide-pIII fusion is the first randomized position, while the first randomized position in the Ph.D.-C7C library is preceded by Ala-Cys. All of the libraries contain a short linker sequence (Gly-Gly-Gly-Ser) between the displayed peptide and pIII.
The Ph.D. libraries have been used for myriad applications, including epitope mapping (Figure 2), identification of protein-protein contacts (5) and enzyme inhibitors (6) and discovery of peptide ligands for GroEL (7), HIV (8-11), semiconductor surfaces (12) and small-molecule fluorophores (13) and drugs (14). Bioactive receptor ligands have been identified both by panning against purified receptors (15-18) and against intact cells (19-22). Peptides which target specific cell types have been isolated by in vitro panning and used for cell-specific gene delivery (23-26). Ligands for mold spores (27) and bacterial cells (28) have also been identified using this system, including a peptide that specifically inhibits anthrax toxin, both in vitro and in vivo (29). Finally, 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 (30,31).
Please click the link for Applications of the Ph.D. Phage Display Peptide Libraries.
Figure 1: Panning with a Ph.D. Phage Display Library
Figure 2: Epitope mapping of an anti-β-endorphin monoclonal antibody with the Ph.D.-12 library. The Ph.D.-12 library was panned against anti-β-endorphin antibody 3-E7 in solution (10 nM antibody), followed by affinity capture of the antibody-phage complexes onto Protein A-agarose (rounds 1 and 3) or Protein G-agarose (round 2). Bound phage were eluted with 0.2 M glycine-HCl, pH 2.2. Selected sequences from each round are shown aligned with the first 12 resides of β-endorphin; consensus elements are boxed. The results clearly show that the epitope for this antibody spans the first 7 residues of β-endorphin, and that the bulk of the antibody-antigen binding energy is contributed by the first 4 residues (YGGF), with some flexibility allowed in the third position. Additionally, the conserved position of the selected sequences within the 12 residue window indicates that the free α-amino group of the N-terminal tyrosine is part of the epitope.
Kit Components:
Ph.D.™-C7C Phage Display Peptide Library
-28 glll Sequencing Primer (22-mer)
-96 glll Sequencing Primer (20-mer)
E. coli K12 ER2738
Biotin
Streptavidin, lyophilized
Storage Conditions
Storage Temperature:
-20°C
FAQs
- Where can I find many more detailed FAQs for Phage Display Peptide Libraries?
- Can a different bacterial strain be used with the Ph.D.™ Phage Display?
- No plaques are visible when titering using the Ph.D.™ Phage Display kit.
- I am using Ph.D.™ Phage display and the amplified phage titer is low.
- I am using Ph.D.™ Phage Display and the phage DNA templates do not yield readable sequence.
- I am using Ph.D.™ Phage Display and the sequencing templates do not run where they should on a gel.
- I am using Ph.D.™ Phage Display and after 4 or more rounds of panning all clones are wild-type phage (white plaques).
- When performing an experiment using Ph.D.™ Phage Display, the ELISA indicates that background binding to the plate is as high as binding to the target.
- When using the Ph.D.™ Phage Display, panning yielded a consensus sequence, but no ELISA signal.
- I am using Ph.D.™ Phage Display and the streptavidin control experiment did not yield the HPQ consensus sequence.
References
- Sidhu, S.S. et al. (2003) Chembiochem., 4, 14-25.
- Ferrer, M. et al. (1999) J. Pept. Res., 54, 32-42.
- BouHamdan, M. et al. (1998) J. Biol. Chem., 273, 8009-8016.
- Whaley, S.R. et al. (2000) Nature, 405, 665-668.
- Rozinov, M.N. and Nolan, G.P. (1998) Chem. Biol., 5, 713-728.
- Rodi, D.J. et al. (1999) J. Mol. Biol., 285, 197-203.
- Kraft, S. et al. (1999) J. Biol. Chem., 274, 1970-1985.
- Koolpe, M. et al. (2002) J. Biol. Chem., 277, 46974-46979.
- Mummert, M.E. et al. (2000) J. Exp. Med., 192, 769-779.
- Hetian, L. et al. (2002) J. Biol. Chem., 277, 43137-43142.
- White, S.J. et al. (2001) Hypertension, 37, 449-455.
- Azzazy, H.M. and Highsmith, W.E. (2002) Clin. Biochem., 35, 425-445.
- Binetruy-Tournaire, R. et al. (2000) EMBO J., 19, 1525-1533.
- Kragler, F. et al. (2000) EMBO J., 19, 2856-2868.
- Gazouli, M. et al. (2002) J. Pharmacol. Exp. Ther., 303, 627-632.
- Romanczuk, H. et al. (1999) Hum. Gene Ther., 10, 2615-2626.
- Nicklin, S.A. et al. (2000) Circulation, 102, 231-237.
- Jost, P.J. et al. (2001) FEBS Lett., 489, 263-269.
- Rasmussen, U.B. et al. (2002) Cancer Gene Ther., 9, 606-612.
- Tinoco, L.W. et al. (2002) J. Biol. Chem., 277, 36351-36356.
- Stratmann, J. et al. (2002) J. Clin. Microbiol., 40, 4244-4250.
- Mourez, M. et al. (2001) Nat. Biotechnol., 19, 958-961.
- Rodi, D.J. et al. (2002) Curr. Opin. Chem. Biol., 6, 92-96.
- Lee, L. et al. (2002) Arthritis Rheum., 46, 2109-2120.
- Duerr, D.M. et al. (2004) J. Virol. Methods, 116, 177-180.
- Parmley, S.F. and Smith, G.P. (1988) Gene, 73, 305-318.
- Berggard, T. et al. (2002) J. Biol. Chem., 277, 41954-41959.
- Chaudhary, J. et al. (2001) Am. J. Physiol. Cell Physiol., 280, C1027-1030.
- Chen, L. and Sigler, P.B. (1999) Cell, 99, 757-768.
- Biorn, A.C. et al. (2004) Biochemistry, 43, 1928-1938.
- Ferrer, M. and Harrison, S.C. (1999) J. Virol., 73, 5795-5802.
Reagents Sold Separately
-28 glll Sequencing Primer (22-mer)
-96 glll Sequencing Primer (20-mer)
E. coli K12 ER2738
Companion Products
M13KE Phage
Ph.D.™ Peptide Display Cloning System
Ph.D.™-12 Phage Display Peptide Library Kit
Ph.D.™-7 Phage Display Peptide Library Kit
pMAL-pIII Vector
Legal
Licenses/Patents/Disclaimers:
This product is sold for research use only and not for resale in any form. Commercial use of this product may require a license. For license information under U.S. Patent 5,866,363 please contact the Licensing Office, New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938.
Commercialization of sequences covered using 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 the Director of Corporate Development, Dyax Corp., One Kendall Square, Bldg. 600, Cambridge, MA 02139 USA. Fax 617-225-2501.
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