The vector is available, for research use only, as part of the Ph.D. Peptide Library Cloning System, product #E8101S. In addition to the vector, this product includes an extension primer for second strand synthesis of the randomized library insert and a detailed protocol for construction of random peptide libraries in M13KE.
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Over the last ten years, the Ph.D. libraries from New England Biolabs have become the dominant tools in this field, with hundreds of publications describing applications including epitope mapping/vaccine development (Youn, et al (2004) FEMS Immunol. Med. Microbial. 41, 51-57; Eshaghi et al. (2006) Mol. Immunol.43, 268-278), mapping protein-protein contacts (Carter et al. (2006) J. Mol. Biol. 357, 236-251 ) and identification of peptide mimics of non-peptide ligands (Hou and Gu (2003) J. Immunol.170, 4373-9). Bioactive peptides, which can be used as cell-targeting or gene delivery agents, have been identified either by panning against purified receptors (De et al. (2006) Biochem. Biophys. Res. Commun. 342, 956-62) or against intact cells or tissue samples, both in vitro and in vivo (Kragler (2000) EMBO J.19, 2856-68). It is apparent that applications of the Ph.D. kits have been limited only by the imagination of the scientific community. Please see our applications page for additional references: http://www.neb.com/nebecomm/tech_reference/drug_discovery/phd_application_references.asp
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1. Doorbar, J. and Winter, G. (1994). Isolation of a peptide antagonist to the thrombin receptor using phage display. J. Mol. Biol. 244, 361-369.
2. Goodson, R.J. et al. (1994). High-affinity urokinase receptor antagonists identified with bacteriophage peptide display. Proc. Natl. Aad. Sci. USA 91, 7129-7133.
3. Barry, M.A., Dower, W.J., and Johnston, S.A. (1996). Toward cell-targeting gene therapy vectors: Selection of cell-binding peptides from random peptide-presenting phage libraries. Nature Medicine 2, 299-305.
4. Szardenings, M. et al. (1997) Phage display selection on whole cells yields a peptide specific for melanocortin receptor 1. J. Biol. Chem. 272, 27943-27948.

1. Pasqualini, R. and Ruoslahti, E. (1996). Organ targeting in vivo using phage display peptide libraries. Nature 380, 364-366.
2. Arap, W., Pasqualini. R. and Ruoslahti, E. (1998) Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science 279, 377-380.
3. Chen et al. (2006) Transdermal protein delivery by coadministered peptide identified via phage display. Nat. Biotech.24, 455-460.

The Ph.D.-12 library consists of randomized linear 12-mer peptides, also fused to pIII via the flexible linker Gly-Gly-Gly-Ser. The library contains 1.9 x 10⁹ independent clones, or only a small fraction (less than 1 millionth) of the 20¹² = 4.1 x 10¹⁵ possible 12-mer sequences. The Ph.D.-12 library can be thought of as having the equivalent diversity of a 7-mer library, but spread out over 12 residues. This is useful for targets requiring 7 or fewer defined residues for binding, but which cannot be contained within the 7-residue "window" of the Ph.D.-7 library. For example, the motif ASDXXXTXPY has only six defined positions, but cannot be present in the Ph.D.-7 library. Additionally, 12-mers are long enough to fold into short structural elements, which may be useful when panning against targets that require structured ligands. A caveat is that the increased length of the randomized segment may allow your target to select sequences with multiple weak binding contacts, instead of a few strong contacts.

The Ph.D.-C7C library consists of randomized 7-mer peptides, each flanked by a pair of cysteine residues. In the absence of reducing agents, these cysteines spontaneously form a disulfide bond, resulting in each peptide in the library being constrained in a disulfide loop. The library contains 3.7 x 10⁹ independent clones . Like the other libraries, the library is fused to pIII via the Gly-Gly-Gly-Ser spacer. The Ph.D.-C7C library is useful for targets whose native ligands are in the context of a surface loop, such as antibodies with structural epitopes. Additionally, imposing structural constraint on the unbound ligand results in a less unfavorable binding entropy, improving the overall free energy of binding compared to unstructured ligands. A major disadvantage of the Ph.D. -C7C library is that the disulfide constraint may "freeze out" a conformation required for target binding. The bottom line is that it is impossible to predict in advance which library is suitable for a given target. As a result, we recommend that the Ph.D.-7 library be tried first, regardless of target. Alternatively, all three libraries can be tried simultaneously, as it is very simple to carry out panning experiments in parallel using multiwell plates.
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Glycerol stocks of ER2738 should be stored at -80°C and ideally, upon receipt of the kit, multiple fresh glyercol cultures should made. These, never thawed, will last for many years in storage. Glycerol stocks have a shorter lifetime at -20°C. If colonies do not appear after streaking a plate, we recommend spreading 20-50 µL of ER2738 stock onto an LB/Tet plate. Alternatively, liquid culture can be inoculated and then a plate should be made by streaking turbid culture. If liquid cultures do not grow in LB/Tet, a fresh plate of ER2738 will usually solve the problem. Additional glycerol cultures of ER2738 (#E4104S) can be ordered for the cost of shipping.
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4.4 All or most of the eluted phage plaques are white (colorless) on Xgal/IPTG plates

a) Use Xgal/IPTG plates for all titering steps, and if white plaques are evident, pick only blue plaques for sequencing.

b) Use aerosol-resistant pipet tips and cotton-plugged pipets for all protocols described in the Manual.

c) If contamination problems persist, all of the solutions used for panning should be autoclaved, with the exception of BSA-containing solutions which should be filter sterilized. Solutions used for phage display should not be used for anything else. Pipettors should be disassembled, the barrel autoclaved, and the internal plunger machinery soaked overnight in a detergent solution such as Count-Off™.

d) Since wild-type phage are preferentially amplified during the amplification steps, pick plaques for sequencing directly after the 3rd round elution step. Do not amplify the 3rd round eluate and carry out a 4th round unless the third round sequences show no clear consensus.

e) If all or most of the plaques are white (colorless) after 3 rounds of panning, it is possible that the library simply does not contain any clones that bind tightly to the target. The ideal ligand sequence may not be statistically represented in the library, or the target simply is not capable of binding to a short peptide sequence. In the case of the C7C library, where all the peptides are constrained in a disulfide loop, a ligand sequence where the imposed constraint allows a productive binding conformation will bind more tightly than the same linear sequence due to improved binding entropy. However, if the imposed constraint does not allow a productive binding conformation, than that sequence will likely not bind to the target at all. In this case either of our linear libraries may yield better results.

The sequencing templates prepared by the method in the Manual are single-stranded (approx. 7250 nucleotides), and as a result will not line up with double-stranded markers of the same length. The apparent size will vary depending on the applied voltage, ethidium and agarose concentration in the gel, and whether TBE or TAE is used as running buffer. We strongly recommend using single-stranded M13 DNA (e.g. single-stranded M13mp18, NEB #N4040S, $30 ( USA) for 10 µg) as a marker.
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                              EagI
Fig. 3 sequence: ...GGTGGAGGTTCGGCCGAAACTGTTGAA...
                                   ||||||||||||
wt M13 sequence: ...TATTCTCACTCCGCTGAAACTGTTGAA...

Since the library phage are derived from the common cloning vector M13mp19, which carries the lacZa gene, phage plaques appear blue when plated on media containing Xgal and IPTG, providing an a-complementing strain such as ER2738 is used for plating. Environmental M13-like phage will typically yield white plaques when plated on the same media. These plaques are also slightly larger and "fuzzier" than the library phage plaques. We therefore recommend plating on LB/Xgal/IPTG plates for all titering steps and, if white plaques are evident, picking ONLY blue plaques for sequencing.
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4.9 After 4 or more rounds of panning all clones are wild-type phage (white plaques).

When characterizing phage clones by the ELISA protocol in the Manual, it is difficult to add more than 10¹² virions per 100 µL well. This corresponds to a phage concentration of only 16 nM. At this concentration, an unambiguously positive ELISA signal can only be observed if the binding affinity is in the micromolar range or better. The iterative nature of phage selection permits identification of ligands with a broad range of affinities, from sub-nanomolar to 1 millimolar, so lower affinity ligands will not show a positive ELISA signal. In this case it is necessary to increase the concentration of the selected ligand, either by synthesing a peptide corresponding to the selected sequence (be sure to include the spacer sequence GGGS at the C-terminus, and amidate the C-terminal carboxylate if possible), or by expressing the selected sequence as an N-terminal fusion to a smaller protein (e.g. an MBP fusion constructed with pMal-pIII, NEB #N8101S). Alternatively, a sandwich ELISA can be carried out in which the selected phage is immobilized and an excess of target applied in the liquid phase. This procedure requires an antibody against the target protein, or some other means of detecting bound target protein. Coat the wells overnight with anti-M13 antibody (no HRP), wash, and add serial dilutions of each phage clone (one clone per row). After 1 hour, wash away unbound phage and add an excess of target protein (0.1 - 1 µM) in TBST. Incubate 1-2 hours at RT°, wash away unbound target, and detect bound target with an enzyme-linked antibody.
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