Ph.D.

-12 Phage Display Peptide Library Kit

Rapid Screening of Peptide Ligands with a Phage Display Peptide Library

I n s t r u c t i o n

M a n u a l

New E.coli host strain with tet-selectable F-factor: minimal plates no longer necessary.

Catalog #E8110S Store at 20C

Version 2.7 11/07

Ph.D.-12 Phage Display Peptide Library Kit

Rapid Screening for Peptide Ligands with a Phage Display Peptide Library Table of Contents:
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Media and solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 General M13 methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Panning procedure (immobilized target) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Characterization of binding clones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Alternate panning procedure (solution phase) . . . . . . . . . . . . . . . . . . . . . . . . . 15 Alternate epitope mapping procedure (Protein A/G capture) . . . . . . . . . . . . . . . 16 Appendix: Optimizing peptide binding interactions . . . . . . . . . . . . . . . . . . . . . . 19 Amino acid distribution of library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

The Kit Includes:

(All kit components should be stored at 20C except where noted): Peptide 12-mer Phage Display Library 100 l, 1.5 x 1013 pfu/ml. Supplied in TBS with 50% glycerol. Complexity = 2.7 x 109 transformants. 28 gIII sequencing primer 5- HOGTA TGG GAT TTT GCT AAA CAA C 3, 100 pmol, 1 pmol/l 96 gIII sequencing primer 5- HOCCC TCA TAG TTA GCG TAA CG 3, 100 pmol, 1 pmol/l E. coli ER2738 host strain F lacIq (lacZ)M15 proA+B+ zzf::Tn10(Tet R)/fhuA2 supE thi (lac-proAB) (hsdMS-mcrB)5 (rk mk McrBC). Host strain supplied as 50% glycerol culture; not competent. Store at 70C. Streptavidin, lyophilized 1.5 mg Biotin, 10 mM 100 l
Ph.D. is a trademark of New England Biolabs, Inc. 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 US Patent 5,866,363 please contact the Licensing Ofce, New England Biolabs, Inc. 32 Tozer Road, Beverly, MA 01915. Commercialization of sequences discovered 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, fax 617-225-2501.

Introduction:
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 surface of the 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 identication of peptide ligands for a variety of target molecules (antibodies, enzymes, cell-surface receptors, etc.) by an in vitro selection process called panning (1,2). 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 specically-bound phage (Figure 1). The eluted phage is then amplied and taken through additional binding/amplication cycles to enrich the pool in favor of binding sequences. After 34 rounds, individual clones are characterized by DNA sequencing. Random peptide libraries displayed on phage have been used in a number of applications (3), including epitope mapping (46), mapping protein-protein contacts (7), and identication of peptide mimics of non-peptide ligands (8-11). Bioactive peptides have been identied either by panning against immobilized purifed receptors (12) or against intact cells (13-15). Protease substrates have been identied by attaching an afnity tag upstream from the randomized region, and separating cleaved from uncleaved phage with the appropriate afnity matrix (16). Conversely, larger proteins [antibodies (17), hormones (18), protease inhibitors (19), enzymes (20) and DNA binding proteins (21)] have been displayed on phage, and variants with altered afnity or specicity have been isolated from libraries of random mutants. The Ph.D.-12 Phage Display Peptide Library Kit is based on a combinatorial library of random peptide 12-mers fused to a minor coat protein (pIII) of M13 phage. The displayed peptide 12-mers are expressed at the N-terminus of pIII, i.e., the rst residue of the mature protein is the rst randomized position. The peptide is followed by a short spacer (Gly-Gly-Gly-Ser) and then the wild-type pIII sequence. The library consists of ~ 2.7 x 109 electroporated sequences, amplied once to yield ~55 copies of each sequence in 10 l of the supplied phage. Extensive sequencing of the naive library (see Appendix) has revealed a wide diversity of sequences with no obvious positional biases. Amplifying the supplied library to yield material for additional panning experiments is not recommended, as sequence biases may occur upon reamplication. Experiments at New England Biolabs have identied consensus peptide sequences against streptavidin and monoclonal antibodies (Figure 2). In all cases the library has been demonstrated to be of sufcient complexity to produce multiple DNA sequences encoding the same consensus peptide motifs.

A library of phage, each displaying a different peptide sequence, is exposed to a plate coated with the target.

Unbound phage is washed away. Specically-bound phage is eluted with an excess of a known ligand for the target, or by lowering pH.

+
The eluted pool of phage is amplied, and the process is repeated for a total of 34 rounds.

After 34 rounds, individual clones isolated and sequenced.

Figure 1: Panning with the Ph.D. peptide library. 3

-endorphin:
Y L Y S T T H V N S L N R Y Y Y Y Y Y Y Y Y Y G S Y Y Y Y Y Y Y Y Y Y G G L S P Y P I M Y P N L V G G G G G G G G G G S I G G G G G G G G G G Q G G Q P M S A A K Y K T A G G G G Q P P A S T P F G G G G G A A A P G P F H D T I C M E L A E M D F F F F F F F F F F P N F F F F F F F F L L N M I A A H M P I N G L Y Q T W A F S S F T A L R Q A A E S A G S S G H D T N P T T R N P S P P K F Q M H P G P N A I P M A H H P T A S I S P I V S P A V P P A V T A G T T L G K T K S E D P A Y S K K L L F L N W W T I E H T G A L Y T S T S S A A P S P P P H V E T V W S S L R P Q S P S E P K C V R H C A H N S L M T Y P T P L V R V S N I W T T S F G P K L V D P Q S K V S A R Y P S S K A N T P G V I S P S R T L P P T P N T P P S F G T I P S R K Q M F A D P P H K A P P D L P Y P H L A A A M S F H I F K A P... P Y P M T F P L S S M G M S T L G P F G R L G Q T T R G P A G S A F R

1st round sequences:

2nd round sequences:

3rd round sequences:

Figure 2: Epitope mapping with the Ph.D.-12 library. The library was panned against anti-endorphin monoclonal antibody in solution, followed by afnity capture of the antibody-phage complexes onto Protein A-agarose (rounds 1 and 3) or Protein G-agarose (round 2). Selected sequences from each round are shown aligned with the rst 12 residues of -endorphin; consensus elements are boxed. The results clearly show that the epitope for this antibody spans the rst 4 residues of -endorphin (YGGF), with some exibility allowed in the third position. One clone from round 3 had no insert.

Media and Solutions:

LB Medium: Per liter: 10 g Bacto-Tryptone, 5 g yeast extract, 5 g NaCl. Autoclave, storeat room temperature. LB/IPTG/Xgal Plates: LB medium + 15 g/L agar. Autoclave, cool to < 70C, add 1 ml IPTG/Xgal* and pour. Store plates at4C in the dark. Agarose Top: Per liter: 10 g Bacto-Tryptone, 5 g yeast extract, 5 g NaCl, 1 g MgCl26H2O, 7 g agarose. Autoclave, dispense into 50 ml aliquots. Store solid at room temperature, melt in microwave as needed. Tetracycline Stock: 20 mg/ml in Ethanol. Store at 20C in the dark. Vortex before using. LB-Tet Plates: LB medium + 15 g/l Agar. Autoclave, cool to <70C, add 1 ml Tetracycline stock and pour. Store plates at 4C in the dark. Do not use plates if brown or black. Blocking buffer: 0.1 M NaHCO3 (pH 8.6), 5 mg/ml BSA, 0.02% NaN3. Filter sterilize, storeat4C. TBS: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl. Autoclave, store at room temperature.

PEG/NaCl: 20% (w/v) polyethylene glycol8000, 2.5 M NaCl. Autoclave, store at room temperature. Iodide Buffer: 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 4 M NaI. Store at room temperature in the dark. Streptavidin Stock Solution: Dissolve 1.5 mg Lyophilized Streptavidin (supplied) in 1 ml 10 mM sodium phosphate (pH 7.2), 100 mM NaCI, 0.02% NaN3. Store at 4C or 20C (avoid repeated freezing/thawing). *Note for IPTG/Xgal: Mix 1.25 g IPTG (isopropyl -D-thiogalactoside) and 1g Xgal (5-Bromo-4-chloro-3-indolyl--D-galactoside) in 25 ml Dimethyl formamide. Solution can be stored at 20C in the dark.

General M13 Methods:

M13 is not a lytic phage so plaques are due to diminished cell growth rather than cell lysis and are turbid rather than clear. Strain Maintenance 1. The supplied E. coli host strain ER2738 is a robust F+ strain with a rapid growth rate and is particularly well-suited for M13 propagation. ER2738 is a recA+ strain, but we have never observed spontaneous invivo recombination events with M13 or phagemid vectors. Commercially available F+ strains such as DH5Fand XL1-Blue can probably be substituted for ER2738 but have not been tested with our system. 2. Since M13 is a male-specic coliphage, it is recommended that all cultures for M13 propagation be inoculated from colonies grown on media selective for presence of the F-factor, rather than directly from the supplied glycerol culture. The F-factor of ER2738 contains a mini-transposon which confers tetracycline resistance, so cells harboring the F-factor can be selected by plating and propagating on tetracycline- containing media. Streak out ER2738 from the included glycerol culture onto an LB-Tet plate. Invert and incubate at 37C overnight and store wrapped with paralm at 4C in the dark for a maximum of 1 month. ER2738 cultures for infection can be grown either in LB or LB-Tet media. Loss of F-factor in nonselective media is insignicant as long as cultures are not serially diluted repeatedly.

3.

4.

Avoiding Phage Contamination The M13 coat protein pIII mediates infectivity by binding to the F-pilus of the recipient bacterium. Display of foreign peptides as N-terminal fusions to pIII appears to attenuate infectivity of the library phage relative to wild-type M13. As a result, there is a strong in vivo selection for any contaminating wild-type phage during the amplication steps between rounds of panning. In the absence of a correspondingly strong in vitro binding selection, even vanishingly small levels of contamination can result in a majority of the phage pool being wild-type phage after three rounds of panning. 1. The potential for contamination with environmental bacteriophage can be minimized by using aerosol-resistant pipet tips for all protocols described in the Manual. Since the library phage are derived from the common cloning vector M13mp19, which carries the lacZ gene, phage plaques appear blue when plated on media containing Xgal and IPTG. Environmental lamentous phage will typically yield white plaques when plated on the same media. These plaques are also larger and fuzzier than the library phage plaques. We strongly recommend plating on LB/IPTG/Xgal plates for all titering steps and, if white plaques are evident, picking ONLY blue plaques for sequencing.

2.

Phage Titering The number of plaques will increase linearly with added phage only when the multiplicity of infection (MOI) is much less than 1 (i.e.,, cells are in excess). For this reason, it is recommended that phage stocks be titered by diluting prior to infection, rather than by diluting cells infected at a high MOI. Plating at low MOI will also ensure that each plaque contains only one DNA sequence. 1. 2. Inoculate 510 ml of LB with a single colony of ER2738 and incubate with shaking until mid-log phase (OD600 ~ 0.5). While cells are growing, melt Agarose Top in microwave and dispense 3 ml into sterile culture tubes, one per expected phage dilution. Equilibrate tubes at 45C until ready for use. Pre-warm 1 LB/IPTG/Xgal plate per expected dilution at 37C until ready for use. Prepare 10-fold serial dilutions of phage in LB. Suggested dilution ranges: for amplied phage culture supernatants, 1081011; for unamplied panning eluates, 101104. Use a fresh pipet tip for each dilution. Aerosol-resistant pipet tips are recommended to prevent cross-contamination. Once culture has reached mid-log phase, dispense 200 l culture into microfuge tubes, 1 for each phage dilution. Add 10 l of each dilution to each tube, vortex quickly, and incubate at room temperature for 15 minutes. One at a time, transfer the infected cells to a culture tube containing 45C Agarose Top, vortex quickly, and IMMEDIATELY pour onto a pre-warmed LB/IPTG/Xgal plate. Spread Agarose Top evenly by tilting plate. Allow plates to cool 5 minutes, invert and incubate overnight at 37C. Inspect plates and count plaques on plates having ~102 plaques. Multiply each number by the dilution factor for that plate to get phage titer in plaque forming units (pfu) per 10 l.

3. 4.

5. 6. 7.

8. 9.

Panning Procedure
The most straightforward method of panning involves directly coating a plastic surface with the target of interest (by nonspecic hydrophobic and electrostatic interaction), washing away the excess, and passing the pool of phage over the target-coated surface. Depending on the target, direct coating can occasionally result in an inaccessible ligand binding site, either due to steric blocking or partial denaturation of the target along the surface. In these cases it is necessary to pre-bind the target with the phage in solution, followed by afnity capture of the phage-target complexes either onto a streptavidin-coated surface (see page 15) or an afnity matrix (page 16). Because of its relative simplicity, we recommend trying the direct coating method described below rst. If no clear consensus binding sequence emerges, then proceed with either of the solution binding procedures. Day One Depending on the available quantity of target molecule and the number of different targets being panned against simultaneously, panning can be carried out in individual sterile polystyrene petri dishes, 12 or 24-well plates, or 96-well microtiter plates. Coat a minimum of 1 plate (or individual well) per targetH Volumes given in the following procedure are for 60 x 15 mm petri dishes, with volumes for microtiter wells given in parentheses. For wells of intermediate size adjust volumes accordingly, but in all cases the number of input phage should remain the same (1.5 x 1011 virions). 1. Prepare a solution of 100 g/ml of the target in 0.1 M NaHCO3 (pH 8.6). Alternative buffers (containing metal ions etc.) of similar ionic strength can be used if necessary for stabilizing the target molecule. Add 1.5 ml of this solution (150 l if using microtiter wells) to each plate (or well) and swirl repeatedly until the surface is completely wet (this may take some effort as the solution may bead up). Incubate overnight at 4C with gentle agitation in a humidied container (e.g., a sealable plastic box lined with damp paper towels). Store plates at 4C in humidied container until needed.

2.

3.

Day Two 4. Inoculate 10 ml LB medium with ER2738 (plating culture for titering). If amplifying the eluted phage on the same day, also inoculate 20 ml LB medium in a 250 ml Erlenmeyer ask (do not use a 50 ml conical tube) with ER2738. Incubate both cultures at 37C with vigorous shaking. 5. Pour off the coating solution from each plate and rmly slap it face down onto a clean paper towel to remove residual solution. Fill each plate or well completely with Blocking Buffer. Incubate at least 1 hour at 4C. Discard the blocking solution as in step 5. Wash each plate rapidly 6X with TBST (TBS + 0.1% [v/v] Tween-20). Coat bottom and sides of plate or well by swirling, pour off the solution, and slap the plate face down on a clean

6. 8

paper towel each time. (Alternatively, an automatic plate washer may be used.) Work quickly to avoid drying out the plates. 7. Dilute 4 x 1010 phage (10 l of original library) with 1 ml of TBST (100 l if using microtiter wells). Pipet onto coated plate and rock gently for 1060 minutes at room temperature.

8. Discard nonbinding phage by pouring off and slapping plate face-down onto a clean paper towel. 9. Wash plates 10 times with TBST as in step 6. Use a clean section of paper towel each time to prevent cross-contamination.

10. Elute bound phage with 1 ml (100 l if using microtiter wells) of an appropriate elution buffer for the interaction being studied. Typically this will be a solution of a known ligand for the target (0.11 mM) in TBS, or a solution of the free target (~100 g/ml in TBS) to compete the bound phage away from the immobilized target on the plate. Rock gently for 1060 minutes at room temperature. Pipet eluate into a microcentrifuge tube. 10a. Alternatively, a general buffer for nonspecic disruption of binding interactions is 0.2 M Glycine-HCl (pH2.2), 1 mg/ml BSA. Rock gently for no more than 10 minutes. Pipet eluate into a microcentrifuge tube. Neutralize with 150 l (15 l for microtiter wells) 1MTris-HCl (pH 9.1). 11. Titer a small amount (~1 l) of the eluate as described in General M13 Methods, above. Plaques from the rst or second round eluate titering can be sequenced if desired, see below. (The remaining eluate can be stored overnight at 4C at this point if necessary and amplied the next day. In this case, set up an overnight culture of ER2738 in LB-Tet. The next day dilute the overnight culture 1:100 in 20 ml LB in a 250 ml Erlenmeyer ask and add the unamplied eluate. Incubate at 37C with vigorous shaking for 4.5 hours and proceed to Step 13.) 12. The rest of the eluate should be amplied: Add the eluate to the 20 ml ER2738 culture (should be early-log at this point) and incubate at 37C with vigorous shaking for 4.5 hours. 13. Transfer the culture to a centrifuge tube and spin 10 minutes at 10,000 rpm (Sorvall SS-34, Beckman JA-17 or equivalent) at 4C. Transfer the supernatant to a fresh tube and re-spin. 14. Pipet the upper 80% of the supernatant to a fresh tube and add 1/6 volume of PEG/NaCl. Allow phage to precipitate at 4C for at least 60 minutes, preferably overnight. Day Three 15. Spin PEG precipitation 15 minutes at 10,000 rpm at 4C. Decant supernatant, re-spin briey, and remove residual supernatant with a pipette. 16. Suspend the pellet in 1 ml TBS. Transfer the suspension to a microcentrifuge tube and spin for 5 minutes at 4C to pellet residual cells. 9

17. Transfer the supernatant to a fresh microcentrifuge tube and re-precipitate with 1/6 volume of PEG/NaCl. Incubate on ice 1560 minutes. Microcentrifuge for 10 minutes at 4C. Discard supernatant, re-spin briey, and remove residual supernatant with a micropipet. 18. Suspend the pellet in 200 l TBS, 0.02% NaN3. Microcentrifuge for 1 minute to pellet any remaining insoluble matter. Transfer the supernatant to a fresh tube. This is the amplied eluate. 19. Titer the amplied eluate as described in General M13 Method on LB/IPTG/ Xgal plates. Store at 4C. 20. Coat a plate or well for the second round of panning, Steps 13, page 8. Days Four and Five 21. Count blue plaques and determine phage titer. Use this value to calculate an input volume corresponding to 12 x 1011 pfu. If the titer is too low, succeeding rounds of panning can be carried out with as little as 109 pfu of input phage. 22. Carry out a second round of panning: repeat steps 418 using 12 x 1011 pfu of the rst round amplied eluate as input phage, and raising the Tween concentration in the wash steps to 0.5% (v/v). 23. Titer the resulting second round amplied eluate on LB/IPTG/Xgal plates. 24. Coat a plate or well for the third round of panning, Steps 13, page 8. Day Six 25. Carry out a third round of panning: repeat steps 411 using 12 x 1011 pfu of the second round amplied eluate as input phage, again using 0.5% Tween in the wash steps. 26. Titer the unamplied third round eluate as in step 11 on LB/IPTG/Xgal plates. It is not necessary to amplify the third round eluate unless carrying out a 4th round of panning (optional, see Appendix). Plaques from this titering can be used for sequencing: time the procedure so that plates are incubated no longer than 18 hours, as deletions may occur if plates are grown longer. Store the remaining eluate at 4C. 27. Set up an overnight culture of ER2738 in LB-Tet from a colony, not by diluting the plating culture.

Control Panning Experiment

Follow the above procedure using streptavidin as the target adding 0.1 g/ml streptavidin to the blocking solution to complex to any biotin in the BSA. Elute bound phage with 0.1 mM biotin in TBS for at least 30 minutes. After 3 rounds of enrichment/amplication, the consensus sequence for streptavidin-binding peptides should contain the motif His-Pro-Gln (HPQ) (8).

10

pIII leader sequence Kpn I 5-...TTA TTC GCA ATT CCT TTA GTG GTA CCT TTC TAT TCT CAC TCT 3-...AAT AAG CGT TAA GGA AAT CAC CAT GGA AAG ATA AGA GTG AGA ...Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser His Ser Start of mature 12-mer peptide-gIII fusion NNK NNK NNK NNK NNK NNK NNK NNK NNK NNK NNK NNK GGT GGA GGT NNM NNM NNM NNM NNM NNM NNM NNM NNM NNM NNM NNM CCA CCT CCA Xxx Xxx Xxx Xxx Xxx Xxx Xxx Xxx Xxx Xxx Xxx Xxx Gly Gly Gly Eag I TCG GCC GAA ACT GTT GAA AGT AGC CGG CTT TGA CAA CTT TCA Ser Ala Glu Thr Val Glu Ser TGT TTA GCA AAA TCC CAT ACA GAA ACA AAT CGT TTT AGG GTA TGT CTT Cys Leu Ala Lys Ser His Thr Glu 28 sequencing primer

AAT TCA TTT ACT AAC GTC TGG AAA GAC GAC AAA ACT TTA GAT TTA AGT AAA TGA TTG CAG ACC TTT CTG CTG TTT TGA AAT CTA Asn Ser Phe Thr Asn Val Trp Lys Asp Asp Lys Thr Leu Asp CGT GCA Arg TAC GCT AAC TAT GAG GGC...-3 ATG CGA TTG ATA CTC CCG...-5 Tyr Ala Asn Tyr Glu Gly... 96 sequencing primer

K = G or T; M = A or C

Figure 3: N-terminal sequence of random 12-mer peptide-gIII fusion. The fusion is expressed with a leader sequence that is removed upon secretion at the position indicated by the arrow, resulting in the peptide positioned directly at the N-terminus of the mature protein. The hybridization positions of the 28 and 96 primers are indicated.

T
First Position

Second Position

G
Cys (C) Trp (W) Arg (R) Arg (R) Ser (S) Arg (R) Gly (G) Gly (G) T G T G T G T G

T C A G

Phe (F) Leu (L) Leu (L) Leu (L) Ile (I) Met (M) Val (V) Val (V)

Ser (S) Ser (S) Pro (P) Pro (P) Thr (T) Thr (T) Ala (A) Ala (A)

Tyr (Y) Gln*(Q) His (H) Gln (Q) Asn (N) Lys (K) Asp (D) Glu (E)

Figure 4: Reduced genetic code. The randomized region of the library encodes all 20 amino acids with only 32 codons. This increases the relative frequency of residues with a single codon, as well as removing 2 of the 3 stop codons. The amber stop codon TAG (*) is suppressed by Gln in the strain used to construct the library.

Third Position 11

Characterization of Binding Clones

Plaque amplication 1. Dilute the ER2738 overnight culture 1:100 in LB. Dispense 1 ml diluted culture into culture tubes, one for each clone to be characterized. 10 clones from the third round are often sufcient to detect a consensus binding sequence. 2. Using a sterile wooden stick or pipet tip, stab a blue plaque and transfer to a tube containing diluted culture. Important: pick plaques from plates having no more than ~100 plaques. This will ensure that each plaque contains a single DNA sequence. Incubate tubes at 37C with shaking for 4.55 hours (no longer). Optional. In addition to sequencing individual clones, the entire pool of selected phage can be sequenced. This can yield a consensus binding sequence in a single step, but only if the common sequence elements appear in the same positions within the 12-residue "window in each clone. Add 10 l of the unamplied eluate to 1 ml diluted overnight culture and incubate at 37C with shaking for 4.55 hours. Transfer cultures to microcentrifuge tubes, centrifuge 30 seconds. Transfer the supernatant to a fresh tube and re-spin. Using a pipet, transfer the upper 80% of the supernatant to a fresh tube. This is the amplied phage stock and can be stored at 4C for several weeks with little loss of titer. For long-term storage, dilute 1:1 with sterile glycerol and store at 20C.

3. 4.

5.

Rapid purication of sequencing templates (22) This extremely rapid procedure produces template of sufcient purity for manual or automated dideoxy sequencing, without the use of phenol or chromatography. 1. Carry out the plaque amplication procedure described above. After the rst centrifugation step, transfer 500 l of the phage-containing supernatant to a fresh microfuge tube. Add 200 l PEG/NaCl. Invert to mix, and let stand at room temperature 10 minutes. Centrifuge 10 minutes, discard supernatant. Re-spin briey. Carefully pipet away any remaining supernatant. Suspend pellet thoroughly in 100 l Iodide Buffer and add 250 l ethanol. Incubate 10 minutes at room temperature. Short incubation at room temperature will preferentially precipitate single-stranded phage DNA, leaving most phage protein in solution. Spin 10 minutes, discard supernatant. Wash pellet in 70% ethanol, dry briey under vacuum. Suspend pellet in 30 l TE buffer [10 mM Tris-HCl (pH 8.0), 1 mM EDTA].

2. 3. 4. 5.

6. 7. 12

8.

5 l of the resuspended template should be sufcient for manual dideoxy sequencing with 35S or 33P, or automated cycle sequencing with dye-labeled dideoxynucleotides. More or less template may be required depending on the sequencing method used.

Sequencing guidelines 1. The 28 primer is recommended for manual dideoxy sequencing. The 96 primer should be used for automated sequencing. 2. The sequence being read corresponds to the anticodon strand of the template. Write out the complementary strand and check against the top strand sequence shown in Figure 3. Check that the 3rd position of each codon in the randomized region is G or T. Determine the amino acid sequence from this strand using the genetic code shown in Figure 4. TAG stop codons are suppressed by glutamine in ER2738 (supE), the strain originally used to produce the library. TAG should thus be considered a glutamine codon when translating (Figure 4).

3.

Assaying selected peptides for target binding by ELISA 1. When carrying out the plaque amplication for DNA sequencing (page 12), save the remaining phage-containing supernatants at 4C. 2. For each clone to be characterized, inoculate 20 ml of LB medium with ER2738 and incubate at 37C until slightly turbid. Alternatively, dilute an overnight culture of ER2738 1:100 in 20 ml LB. Add 5 l of phage supernatant to each culture and incubate at 37C with vigorous aeration for 4 1/2 hours. Transfer the culture to a centrifuge tube and spin 10 minutes at 10,000 rpm (Sorvall SS-34, Beckman JA-17 or equivalent). Transfer supernatant to a fresh tube and re-spin. Pipet the upper 80% of the supernatant to a fresh tube and add 1/6 volume of PEG/NaCl. Allow phage to precipitate at 4C for at least 1 hour or overnight. Spin PEG precipitation 15 minutes at 10,000 rpm at 4C. Decant supernatant, re-spin briey, and remove residual supernatant with a pipette. Suspend the pellet in 1 ml TBS. Transfer the suspension to a microcentrifuge tube and spin for 5 minutes at 4C to pellet residual cells. Transfer the supernatant to a fresh microcentrifuge tube and re-precipitate with 1/6 volume of PEG/NaCl. Incubate on ice 1560 minutes. Microcentrifuge for 10 minutes at 4C. Discard supernatant, re-spin briey, and remove residual supernatant with a micropipet. Suspend the pellet in 50 l TBS. Titer as described in General M13 Methods, pages 67. Store at 4C. 13

3. 4.

5.

6. 7. 8.

9.

10. Coat one row of ELISA plate wells for each clone to be characterized with 100200 l of 100 g/ml of target in 0.1 M NaHCO3 (pH 8.6). Incubate at 4C overnight in an air-tight humidied box (e.g., a sealable plastic box lined with wet paper towels). 11. Shake out excess target solution and slap plate face-down onto a paper towel. Fill each well completely with Blocking Buffer. Additionally, one row of uncoated wells per clone to be characterized should also be blocked in order to test for binding of each selected sequence to BSA-coated plastic. A second microtiter plate should also be blocked, which will be used for serial dilutions of phage before addition to the target-coated plate. The idea of doing dilutions in a separate blocked plate is to ensure that phage are not absorbed onto the target during the course of performing dilutions. Incubate the blocked plates at 4C, 1-2 hours. 12. Shake out the blocking buffer and wash each plate 6 times with 1X TBS/ Tween, slapping the plate face-down onto a clean section of paper towel each time. The percentage of Tween should be the same as the concentration used in the panning wash steps. 13. In the separate blocked plate, carry out four-fold serial dilutions of the phage in 200 l of TBS/Tween per well, starting with 1012 virions in the rst well of a row and ending with 2 x 105 virions in the 12th well. 14. Using a multichannel pipettor, transfer each row of diluted phage to the plate with the target. Incubate at room temperature for 12 hours with agitation. 15. Wash plate 6 times with 1X TBS/Tween as in step 12. 16. Dilute HRP-conjugated anti-M13 antibody (Pharmacia # 27-9411-01) 1:5,000 in blocking buffer. Add 200 l of diluted conjugate to each well and incubate at room temperature for 1 hour with agitation. 17. Wash 6 times with 1X TBS/Tween as in step 12. 18. Prepare the HRP substrate solution as follows: a stock solution of ABTS can be prepared in advance by dissolving 22 mg ABTS (Sigma #A1888) in 100ml of 50 mM sodium citrate, pH 4.0. Filter sterilize and store at 4C. Immediately prior to the detection step, add 36 l 30% H2O2 to 21 ml of ABTS stock solution per plate to be analyzed. 19. Add 200 l substrate solution to each well, incubate at room temperature for 1060 minutes. 20. Read plates using a microplate reader set at 405415 nm.

14

Alternate Panning Procedure (solution binding)

As an alternative to directly coating the plate with the target molecule, the target can be reacted with the phage in solution, followed by afnity capture of the phage-target complexes. Depending on the target, binding in solution can result in improved kinetics compared to surface binding and can bypass problems associated with partial denaturation of the target on the plastic surface. Afnity capture requires some sort of afnity tag on the target; this is typically accomplished by biotinylating the target and capturing the complexes with immobilized streptavidin. Biotinylation of Target 1. Dissolve 2 mg target protein in 1 ml of 50 mM NaHCO3 (pH 8.5). Other buffers may be used if necessary to maintain stability of target, but do NOT use buffers with free amine groups (i.e., Tris). 2. Immediately prior to use, dissolve 1 mg Sulfo-NHS-LC-Biotin (available from Pierce, cat. #21335) in 1 ml of water. Vortex vigorously. Add 74 l of this solution to the target solution. This reagent is a water-soluble activated ester of biotin that specically targets the -amine of solvent-accessible (surface) lysine residues. The remaining solution should be discarded, as the ester hydrolyzes rapidly upon storage. Place the tube on ice for 2 hours. Remove unreacted biotin by dialysis against a minimum of 2 1-liter changes of TBS, gel-ltration, or ultraltration through a Centricon apparatus (Amicon) or equivalent. Quantitate biotinylated protein by Bradford or Lowry assay. Biotinylation can be conrmed by Western detection or ELISA using commercially available anti-biotin antibody. The degree of biotinylation can be quantitated by a colorimetric assay based on displacement of the dye HABA [2-(4-hydroxy azobenzene)benzoic acid], also available from Pierce (cat.# 28010). Based on results obtained at NEB, the described reaction conditions result in an average of 2 biotinylated lysines per protein molecule.

3. 4.

5.

Panning Follow the procedure on pages 810, but coat the plates with streptavidin [100 g/ml of streptavidin in 0.1 M NaHCO3 (pH 8.6)] instead of target. The blocking buffer should contain 0.1 g/ml streptavidin in order to complex any biotin in the BSA. Replace Step 7 in the panning procedure with the following: 7a. While plates are blocking (step 5), pre-complex phage with biotinylated target. Combine in microfuge tube 0.1 g biotinylated target (~10 nM nal for 25 kDa protein) and 1.5 x 1011 pfu of the input phage (=10 l of original library) in 400 l TBST. Alternate buffers (containing metal ions etc.) of similar ionic strength can be used if necessary for stabilizing target molecule. Incubate at room temperature for 1060 minutes. 15

7b. Add phage-target solution to the washed blocked plate. Incubate at room temperature for 10 minutes. 7c. Add biotin to a nal concentration of 0.1 mM and incubate an additional 5 minutes. This will displace any streptavidin-binding phage (displaying the HPQ sequence) from the plate. The off rate for the biotinylated target is sufciently slow that the target will not be displaced by the biotin. Continue with Step 8, page 8.

Alternate Epitope Mapping Procedure (solution binding with Protein A/G capture)
As an alternative to panning against an antibody that has been immobilized on a surface, the library can be reacted with the antibody in solution, followed by afnity capture of the antibody-phage complexes onto protein A and/or protein G agarose beads. In addition to requiring substantially less antibody per experiment than surface panning, solution panning can result in improved accessibility of the antigen binding site to phage-displayed peptides, as well as avoiding partial denaturation of the antibody on the plastic surface. Fortuitous selection of peptide sequences that specically bind protein A or protein G is avoided by alternating rounds of panning between protein A-agarose and protein G-agarose. For antibodies that do not bind well to protein A (sheep, goat, chicken and rat polyclonals, as well as human IgG3 and mouse IgG1 monoclonals), protein Gagarose can be used in all rounds. This procedure is easily adaptable for panning against any protein that is fused to an afnity tag (MBP, GST, polyhistidine, etc.), using the appropriate bead for afnity capture of the fusion. 1. Inoculate 10 ml LB medium with ER2738 (plating culture for titering). If amplifying the eluted phage on the same day, also inoculate 20 ml LB medium in a 250 ml Erlenmeyer ask (do not use a 50 ml conical tube) with ER2738. Incubate both cultures at 37C with vigorous shaking. Transfer 50 l of protein A-agarose (50% aqueous suspension) to a microfuge tube. Add 1 ml TBS + 0.1% Tween (TBST). Suspend resin by tapping tube or GENTLY vortexing. For polyclonal antibodies from goat, chicken, sheep or rat, or monoclonal antibodies of the human IgG3 subclass, use protein G-agarose for this step in all rounds. Protein A can be used for the mouse IgG1 subclass if the pH of the binding and wash buffer (TBS) is raised to 8.6. Pellet resin by centrifugation in a low-speed benchtop microcentrifuge (Capsulefuge or equivalent), 30 seconds. Carefully pipet away the supernatant, taking care not to disturb the resin pellet. Suspend resin in 1 ml Blocking Buffer. Incubate at 4C for 60 minutes, mixing occasionally. In the meantime, dilute 1.5 x 1011 phage virions (10 l of supplied library) and 300 ng of antibody to a nal volume of 200 l with TBST. The nal

2.

3.

4. 5. 16

concentration of antibody is 10 nM. Incubate at room temperature for 20 minutes. 6. 7. Following the blocking reaction, pellet the resin as in Step 3 and wash 4 times with 1 ml TBST, pelleting resin each time. Transfer the phage-antibody mixture to the tube containing the washed resin. Mix gently and incubate at room temperature for 15 minutes, mixing occasionally. Pellet resin as in Step 3, discard supernatant, and wash 10 times with 1 ml TBST. Elute bound phage by suspending pelleted resin in 1 ml 0.2 M Glycine-HCl, pH 2.2, 1 mg/ml BSA. Incubate 10 minutes at room temperature.

8. 9.

10. Centrifuge elution mixture 1 minute in benchtop microcentrifuge. Carefully transfer the supernatant to a new microfuge tube, taking care not to disturb the pelleted resin. 11. Immediately neutralize the eluate with 150 l 1 M Tris-HCl, pH 9.1. 12. Titer a small aliquot of the eluate on LB/IPTG/Xgal plates as described in General M13 Methods, pages 67. 13. The remaining eluate should be amplied: Add the eluate to the 20 ml ER2738 culture (should be early-log at this point) and incubate at 37C with vigorous shaking for 4.5 hours. (Alternatively, the eluate can be stored at 4C overnight and amplied the next day. In this case, set up an overnight culture of ER2738 in LB-Tet. The next day dilute the overnight culture 1:100 in 20 ml LB in a 250 ml Erlenmeyer ask and add the unamplied eluate. Incubate at 37C with vigorous shaking for 4.5 hours.) 14. Transfer the culture to a centrifuge tube and spin 10 minutes at 10,000 rpm (Sorvall SS-34, Beckman JA-17 or equivalent) at 4C. Transfer the supernatant to a fresh tube and re-spin. 15. Pipet the upper 80% of the supernatant to a fresh tube and add 1/6 volume of 20% PEG, 2.5 M NaCl. Allow phage to precipitate at 4C for 2 hours or overnight. 16. Spin PEG precipitation 15 minutes at 10,000 rpm at 4C. Decant supernatant, re-spin briey, and remove residual supernatant with a pipette. 17. Suspend the pellet in 1 ml TBS. Transfer the suspension to a microcentrifuge tube and spin for 5 minutes at 4C to pellet residual cells. 18. Transfer the supernatant to a fresh microcentrifuge tube and re-precipitate with 1/6 volume of PEG/NaCl. Incubate on ice 1560 minutes. Microcentrifuge for 10 minutes at 4C. Discard supernatant, re-spin briey, and remove residual supernatant with a micropipet. 19. Suspend the pellet in 200 l TBS, 0.02% NaN3. Microcentrifuge 1 minute to pellet any remaining insoluble matter. Transfer the supernatant to a fresh tube. This is the amplied eluate. 17

20. Titer the amplied eluate on LB/IPTG/Xgal plates as described in General M13 Methods, pages 67. Store at 4C. 21. The next day, count blue plaques and determine phage titer. Use this value to calculate an input volume corresponding to 12 x 1011 pfu. If the titer is too low, succeeding rounds of panning can be carried out with as little as 109 pfu of input phage. 22. Carry out a second round of panning: repeat Steps 121 using 12 x 1011 pfu of the rst round amplied eluate as input phage. Use protein G-agarose beads instead of protein A beads, and raise the Tween concentration in the binding and wash steps to 0.5% (v/v). 23. Carry out a third round of panning: repeat Steps 112 using 12 x 1011 pfu of the second round amplied eluate as input phage. Use protein A-agarose if you used protein A for the rst round, and keep the Tween concentration at 0.5% (v/v) in the binding and wash steps. Time the titering step so the plates are incubated no longer than 18 hours, as deletions may occur if the plates are incubated longer. 24. Amplify the phage from 10 or more plaques and prepare templates for DNA sequencing as described on pages 1213. 25. If no consensus sequence is evident from the third round sequenced clones, amplify the remaining third round eluate (Steps 1321) and carry out a fourth round of panning, using protein G-agarose.

18

Appendix
Optimizing peptide binding interactions There are several variables affecting the stringency of selection during panning. Depending on the interaction being studied, adjustment of the stringency of selection or elution may be necessary to obtain a consensus binding sequence. 1. Detergent: The presence of detergent (typically Tween-20) in the binding and wash buffers reduces nonspecic interactions between the phage and the target and/or blocking agent (BSA). Lower Tween concentrations in early rounds will result in higher eluate titers, and the stringency can be gradually increased with each round by raising the Tween concentration stepwise to a maximum of 0.5%. In side-by-side experiments, however, we have obtained identical consensus sequences when Tween concentrations were held constant (0.5%) or increased stepwise (0.1, 0.3, 0.5%) in 3 rounds of panning. The use of lower Tween concentrations in earlier rounds is recommended when the interaction under study is so specic that the eluate titer (i.e., the number of bound sequences) in early rounds is expected to be very low. Temperature: Depending on whether the binding interaction is enthalpically or entropically driven, stringency can be increased or decreased by raising the temperature of the binding step, respectively. Suggested binding temperatures to try: 4C, room temperature and 37C. Temperatures as high as 55C may be used without inactivating the phage. Binding and Elution time: With shorter binding times, selection of peptides with rapid on rates (kon) is favored. Conversely, with longer elution times, selection of peptides with slow off rates (koff) is favored. Since the equibrium association constant Ka for binding of the peptide to the target is equal to kon/koff, stringency can be increased by shortening the binding time and lengthening the elution time. In later rounds the elution can be carried out in 2 stages: phage eluted in a short period of time are discarded and phage eluting in a longer period are taken on to the next round or plated. Do not elute for longer than 10 minutes if eluting with pH 2.2 glycine buffer, as the infectivity of the phage will be reduced. Target concentration: If panning against the target in solution, the stringency can be increased by lowering the concentration of target (23). An initial target concentration of 10 nM is recommended; this can be lowered to 1 nM in later rounds for selection of ligands with nanomolar binding afnity. Number of rounds: With each round of panning and amplication, the pool of phage becomes enriched in favor of sequences that bind to the target. Maintaining a constant input phage concentration in each round results in a stepwise increase in the number of particles displaying a given sequence until a point is reached where most or all of the eluted particles display a consensus binding sequence. Depending on the interaction being studied and the applied stringency, this usually happens after 2 or 3 rounds. If no clear consensus sequence emerges after 3 rounds, the 3rd round eluate should be amplied and a fourth round of panning carried out. 19

2.

3.

4.

5.

6.

Choice of Library: If, following three or four rounds of panning, there is no clear consensus ligand sequence (or all of the plaques are white), it is possible that the library simply does not contain any clones that bind tightly enough to the target. One explanation is that the ideal ligand sequence is not statistically represented in the library (see pp. 2122). Alternatively, if the peptide does not bind with high enough afnity to be selected, the Ph.D.-C7C library might work better. In this library, all the displayed peptides are structurally constrained in a 7-residue disulde loop, allowing a productive binding conformation to bind more tightly than the same sequence expressed in a linear library (due to the improved entropy of binding). Lastly, if the ligand only requires a few binding contacts spread over a short region, then the Ph.D.-7 linear library might be a better choice. As the Ph.D.-12 library provides an increased window of randomness, it allows the target to select sequences with multiple weak contacts, instead of a few strong interactions. Therefore the Ph.D.-7 library might force the appearance of a consensus sequence. Unfortunately, in the absence of detailed structural information about the target-ligand interaction, it is impossible to predict which library will yield the most productive ligands. For a continually updated list of Frequently Asked Questions (FAQ), please check our Web Site at http://www.neb.com.

20

Amino Acid Distribution of the Ph.D.-12 Library

A total of 104 clones from the naive library were sequenced. Six clones (5.8%) did not contain a displayed peptide insert. The overall amino acid distribution from the remaining 1176 sequenced codons (98 clones x 12 random codons) is as follows: Amino Acid Codons Arg Leu Ser Ala Gly Pro Thr Gln Val Asn Asp Cys Glu His Ile Lys Met Phe Trp Tyr CGK, AGG CTK, TTG TCK, AGT GCK GGK CCK ACK CAG, TAG

Expected frequency* 9.4% 9.4% 9.4% 6.2% 6.2% 6.2% 6.2% 6.2% 6.2% 3.1% 3.1% 3.1% 3.1% 3.1% 3.1% 3.1% 3.1% 3.1% 3.1% 3.1%

Observed frequency 4.7% (55/1176) 9.3% (109/1176) 10.0% (118/1176) 6.0% (71/1176) 2.6% (30/1176) 12.2% (143/1176) 11.1% (131/1176) 5.1% (60/1176) 3.9% (46/1176) 4.6% (54/1176) 2.8% (33/1176) 0.5% (6/1176) 3.1% (36/1176) 6.3% (74/1176) 3.4% (40/1176) 2.8% (33/1176) 2.6% (30/1176) 3.3% (39/1176) 2.2% (26/1176) 3.6% (42/1176)

GTK AAT GAT TGT GAG CAT ATT AAG ATG TTT TGG TAT

*Expected frequency = # codons for that amino acid 32 codons x 100%. Note use of reduced genetic code NNK (32 codons) in library construction. Arginines and single cysteinesin the random peptide sequence interfere with secretion of pIII and phage infectivity, respectively; consequently, clones with peptides containing Arg or Cys are selected against (24).

The stop codon TAG is suppressed by Gln in the strain used to propagate the library.

21

Calculation of Representation of a Given Peptide Motif in Library

The likelihood that the Ph.D.-12 peptide library contains a given peptide motif canbe calculated using the preceding empirical data as follows: 1. Multiply the observed frequencies of each residue in the motif (expressed as decimal values) from the Table to obtain the absolute probability p of obtaining that sequence. If the motif is less than 12 residues in length, p shouldbe multiplied by the number of ways the motif can appear within the 12-residue window (i.e., 7 for a 6-mer, 8 for a 5-mer etc.). Multiply p by the complexity of the library n (2.7 x 109) to obtain the expectednumber of independent clones within the library that display the desired motif. The probability P(k) that the library contains exactly k clones displaying the desired motif can be calculated using the Poisson distribution, P(k) = ek/k!. For k = 0 (i.e. the library does not contain the desired motif), this reduces to P(0) = e = enp. The probability P(k >0) that the library contains at least one independent clonedisplaying the desired motif is therefore P(k >0) = 1 P(0) = 1 enp.

2.

3.

4.

Example: What is the likelihood that the Ph.D. library contains the hexapeptide motif Gly-Ala-Arg-Cys-Ile-Ala? 1. From the Table, p(Gly-Ala-Arg-Cys-Ile-Ala) = (.026)(.060)(.047)(.005) (.034)(.060) x 7 = 5.2 x 109. It is necessary to multiply by 7 because the desired hexapeptide motif can begin at positions 1 through 7 of the library.

2.

= np = (2.7 x 109)(5.2 x 109) = 14. We expect that approximately 14independent clones in the library display the sequence Gly-Ala-Arg-Cys-Ile-Ala.
P(k>0) = 1 enp = 1 e14 = 0.9999. The probability that the library contains at least one copy of the desired peptide approaches 100%.

3.

22

References
1. 2. 3. 4. 5. Parmley, S. F. and Smith, G. P. (1988) Gene 73, 305318. Smith, G. P. and Scott, J. K. (1993) Methods Enzymol. 217, 228257. Reviewed in Cortese et al. (1995) Curr. Opin. Biotechnol. 6, 7380. Scott, J. K. and Smith, G. P. (1990) Science 249, 386390. Cwirla, S. E., Peteres, E. A., Barrett, R. W. and Dower, W. J. (1990) Proc. Natl. Acad. Sci. USA 87, 63786382. 6. Felici, F., Castagnoli, L., Musacchio, A., Jappelli, R. and Cesarini, G. (1991) J. Mol. Biol. 222, 301310. 7. Hong, S. S. and Boulanger, P. (1995) EMBO J. 14, 47144727. 8. Devlin, J. J., Panganiban, L. C. and Devlin, P. E. (1990) Science 249, 404406. 9. Oldenburg, K. R., Loganathan, D., Goldstein, I. J., Schultz, P. G. and Gallop, M. A. (1992) Proc. Natl. Acad. Sci. USA 89, 53935397. 10. Scott, J. K., Loganathan, D., Easley, R. B., Gong, X. and Goldstein, I. J. (1992) Proc. Natl. Acad. Sci USA 89, 53985402. 11. Hoess, R., Brinkmann, U., Handel, T. and Pastan, I. (1993) Gene 128, 4349. 12. ONeil, K. T., Hoess, R. H., Jackson, S. A., Ramachandran, N. S., Mousa, S. A. and DeGrado, W. F. (1992) Proteins 14, 509515. 13. Doorbar, J. and Winter, G. (1994) J. Mol. Biol. 244, 361369. 14. Goodson, R. J., Doyle, M. V., Kaufman, S. E. and Rosenberg, S. (1994) Proc. Natl. Acad. Sci. USA 91, 712971331. 15. Barry, M. A., Dower, W. J. and Johnston, S. A. (1996) Nature Medicine 2, 299305. 16. Smith, M. M., Shi, L. and Navre, M. (1995) J. Biol. Chem. 270, 64406449. 17. Barbas, S. M. and Barbas, C. F. (1994) Fibrinolysis 8, Suppl. 1, 245252 18. Lowman, H. B., Bass, S. H. Simpson, N. and Wells, J. A. (1991) Biochemistry 30, 1083210838. 19. Roberts, B. L. et al. (1992) Proc. Natl. Acad. Sci. USA 89, 24292433. 20. Soumillion, P., Jespers, L., Bouchet, M., Marchand-Brynaert, J., Winter, G. and Fastrez, J. (1994) J. Mol. Biol. 237, 415422. 21. Choo, Y. and Klug, A. (1995) Curr. Opin. Biotechnol. 6, 431436 22. Wilson, R. K. (1993) Biotechniques 15, 414422. 23. Barrett, R. W., et al. (1992) Anal. Biochem. 204, 357364 24. Peters, E. A., Schatz, P. J., Johnson, S. S. and Dower, W. J. (1994) J.Bacteriol 176, 42964305.

23

Also Available from NEB:

Ph.D.-7 Phage Display Peptide Library Kit #E8100S 10 Panning Experiments Ph.D.-C7C Phage Display Peptide Library Kit #E8120S 10 Panning Experiments Ph.D.-7 Phage Display Peptide Library #E8102L 50 Panning Experiments Ph.D.-12 Phage Display Peptide Library #E8111L 50 Panning Experiments Ph.D.-C7C Phage Display Peptide Library #E8121L 50 Panning Experiments Ph.D. Peptide Display Cloning System #E8101S vector and extension primer 28 gIII sequencing primer #S1258S 0.5 A260 unit 96 gIII sequencing primer #S1259S 0.5 A260 unit ER2738 Host Strain #E4104S Glycerol culture, not competent

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