Biopanning of Phage Displayed Peptide Libraries for the Isolation of Cell-Specific Ligands

Division of Translational Research, Department of Internal Medicine and Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Methods in Molecular Biology (Impact Factor: 1.29). 02/2009; 504:291-321. DOI: 10.1007/978-1-60327-569-9_18
Source: PubMed
ABSTRACT
One limitation in the development of biosensors for the early detection of disease is the availability of high specificity and affinity ligands for biomarkers that are indicative of a pathogenic process. Within the past 10 years, biopanning of phage displayed peptide libraries on intact cells has proven to be a successful route to the identification of cell-specific ligands. The peptides selected from these combinatorial libraries are often able to distinguish between diseased cells and their normal counterparts as well as cells in different activation states. These ligands are small and chemical methodologies are available for regiospecific derivatization. As such, they can be incorporated into a variety of different diagnostic and therapeutic platforms. Here we describe the methods utilized in the selection of peptides from phage displayed libraries by biopanning. In addition, we provide methods for the synthesis of the selected peptides as both monomers and tetramers. Downstream uses for the peptides are illustrated.

Full-text

Available from: Michael Joseph Mcguire
Chapter 18
Biopanning of Phage Displayed Peptide Libraries
for the Isolation of Cell-Specific Ligands
Michael J. McGuire, Shunzi Li, and Kathlynn C. Brown
Summary
One limitation in the development of biosensors for the early detection of disease is the availability of
high specificity and affinity ligands for biomarkers that are indicative of a pathogenic process. Within
the past 10 years, biopanning of phage displayed peptide libraries on intact cells has proven to be a suc-
cessful route to the identification of cell-specific ligands. The peptides selected from these combinatorial
libraries are often able to distinguish between diseased cells and their normal counterparts as well as
cells in different activation states. These ligands are small and chemical methodologies are available for
regiospecific derivatization. As such, they can be incorporated into a variety of different diagnostic and
therapeutic platforms. Here we describe the methods utilized in the selection of peptides from phage dis-
played libraries by biopanning. In addition, we provide methods for the synthesis of the selected peptides
as both monomers and tetramers. Downstream uses for the peptides are illustrated.
Key words: Phage display, Peptides, Cell-targeting, Biopanning, Combinatorial library, Diagnostics,
Therapeutics, Quantum dots.
The development of biosensors for the detection of different dis-
ease states is dependent on the availability of high affinity and
specificity ligands for the desired cell type and/or biomarker. In
many applications, the accessibility of such ligands has been the
limiting factor in the development of the technology. To date,
antibodies have been the most common class of ligands utilized.
However, antibodies are expensive and can be difficult to modify.
Additionally, if the down-stream application is to detect particu-
lar cell types (i.e., a cancerous cell vs. its normal counterpart), the
antibody must bind to its target in the context of an intact cell.
1. Introduction
Avraham Rasooly and Keith E. Herold (eds.), Methods in Molecular Biology: Biosensors and Biodetection, Vol. 504
© Humana Press, a part of Springer Science + Business Media, LLC 2009
DOI:10.1007/978-1-60327-569-9_18
291
Page 1
292 McGuire, Li, and Brown
As such, our lab and others have turned toward peptide librar-
ies as a source of cell-specific ligands (1–16). In the same fashion
that phage displayed peptide libraries can be panned on purified
biomolecules, whole cells can be used as the bait for the peptide
library. This approach, often referred to as biopanning, results in
the isolation of peptides that display high cell-specificity; ligands
can be isolated that discriminate between cell types and disease
states. Furthermore, cell-specific peptides can be obtained with-
out the knowledge of a suitable cell surface biomarker. The pro-
tocol is amenable to a variety of different cell types, including
primary cells. To date, we have identified cell-specific peptides
for many different cell types including cells of the immune sys-
tem (2, 4), pancreatic β-cells (7), cardiac cells (3), tumor cells (5,
6), and pathogen-infected cells (8). Importantly, most peptides
selected in this manner are active outside of context of the phage,
retaining their cell-specificity and affinity. Furthermore, we have
shown that tetramerizing the peptides on a branched scaffold
can greatly enhance the peptides affinity for its target cell type
(5, 6, 8, 17, 18). These peptides can be employed for the deliv-
ery of fluorescent nanoparticles, as cell capture reagents for cell
enrichment, and as antibody replacements for flow cytometry. As
peptides are amenable to derivatization, we anticipate that these
cell-specific ligands will find utility in a variety of different biosen-
sor platforms.
1. Tissue culture cell line or primary cell of interest.
2. Tissue culture plates (12-well) for adherent cells.
3. Polypropylene centrifuge tubes (15 mL and 50 mL) for non-
adherent cells.
4. Microcentrifuge tubes (1.5 mL).
5. Cell scrapers.
6. Phage library (see Note 1) or amplification stock for each
round of panning.
7. RPMI media (or any cell-specific media) without serum.
8. Chloroquine stock (100×): Dissolve 55 mg chloroquine in
10 mL PBS for a final concentration of 10 mM. Filter steri-
lize the solution.
9. Protease inhibitor (25×) without EDTA (Roche).
10. Phosphate buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl,
10 mM Na
2
HPO
4
, 1.8 mM KH
2
PO
4
, pH 7.4.
2. Materials
2.1. Cell Culture
and Phage Panning
(Methods Outlined in
Subheadings 3.1
and 3.2)
Page 2
Biopanning of Phage Displayed Peptide Libraries 293
11. PBS
+
: Add 0.5 mM CaCl
2
and 10 mM MgCl
2
to PBS in this
order with stirring.
12. PBS
+
with 0.1% BSA: Add 0.1 g bovine serum albumin per
100 mL PBS
+
.
13. 0.1 M HCl–glycine, 0.9% NaCl pH adjusted to 2.2 with
glycine.
14. 1.5 M Tris–HCl, pH 8.8.
15. 30 mM Tris–HCl, pH 8.0.
1. Selective media for K91 bacterial stocks. We use M9-Pro
minimal medium prepared as follows: Mix 7.5 g agar +
430 mL water and autoclave solution. Cool agar solution to
55°C. Add 25 mL 20× M9 Salts, 5 mL 20% glucose, 50 μL
1 M CaCl
2
, 500 μL 1 M MgSO
4
, 100 μL 0.1% Thiamine,
2.5 mL 0.2 mg/mL biotin, 2.5 mL 1% uridine, 8 mL 1% leu-
cine, 8 mL 1% phenylalanine, 8 mL 1% threonine, 8 mL 1%
methionine, 8 mL 1% histidine, 8 mL 1% Tryptophan, and
8 mL 1% lysine. 20× M9 salts consist of 60 g Na
2
HPO
4
, 30 g
KH
2
PO
4
, 5 g NaCl, and 10 g NH
4
Cl.
2. LB media.
3. 100 mm and 150 mm LB-tet plates (12 μg/mL tetracycline).
4. Culture flasks for expansion and isolation of phage clones.
5. 20% PEG-8000 (Fisher Chemical) in 0.9% NaCl (see Note 2).
6. 65°C heating block.
7. Bacterial incubator with shaker.
8. Various centrifuge tubes and bottles.
9. Low (3,000 × g) and high speed (11,000 × g) centrifuges for
concentrating bacterial stocks and phage isolation.
10. Spectrophotometer to monitor bacterial cultures.
11. PBS prepared as described in Subheading 2.1.
1. BioRad iCycler or similar apparatus.
2. Sybr
®
green mastermix (see Note 3).
3. Optical PCR plates for real-time PCR.
4. Optical sealing tape for real-time PCR.
5. 8-channel pipette (5–50 μL).
6. Serial dilutions of previously characterized phage preparation
to generate a standard curve.
7. Specific primers to tetracycline resistance gene (see Note 4)
(a) Forward primer (tetR-F1): 5-CGAATAAGAAGGCTGG
CTCTGC-3.
(b) Reverse primer (tetR-R1): 5 -GCTGTGGGGCATTTTAC
TTTAGG-3.
2.2. Bacterial Culture
and Phage Amplifi-
cation and Titering
(Methods Outlined in
Subheadings 3.3
and 3.4)
18.2.3. Quantitative
Real-Time PCR for
Titering (Method Out-
lined in Subheading
18.3.5)
Page 3
294 McGuire, Li, and Brown
1. General materials for PCR mastermix preparation: 10×
polymerase buffer, 25 mM MgCl
2
, 10 mM dNTP mix, Taq
polymerase (GoTaq
®
DNA Polymerase 5 units/μL, Promega
Corp, or Choice
Taq, Denville Scientific).
2. Thermocycler.
3. Specific primers that flank library site
(a) For ward primer (fd-tet F1): 5 -GGGCGATGGTTGTTGT
CATTG-3.
(b) Reverse primer (fd-tet B1): 5 -CTCATTTTCAGGGATAG
CAAGCC-3.
4. Agarose gel apparatus.
5. 100 bp ladder standards (Promega Corp, catalog # G2101 or
similar).
6. Exonuclease I (10 units/μL, New England Biolabs, or other
suitable vendor).
7. Shrimp alkaline phosphatase (1 unit/μL, New England
Biolabs or other suitable vendor).
8. BigDye
®
Terminator v3.1 (Applied Biosystems Inc).
9. Ethanol (70%).
10. Hi-Di
Formamide (Applied Biosystems Inc).
11. Sequencing stop/precipitation reagent: Prepare by mixing
125 mL 95% ethanol, 29 mL water, and 6 mL 3 M sodium
acetate, pH 5.2.
1. Materials outlined in Subheading 2.1 for the panning and
Subheading 2.2 or Subheading 2.3 for titering.
2. Isolated phage clones and a control phage clone displaying
an irrelevant peptide sequence. Alternatively, a phage clone
that displays no peptide (referred to as an “empty” phage)
can be employed.
3. Cell lines or primary cells of interest.
1. Symphony Synthesizer (Rainin Instruments, Protein Tech-
nologies, Inc. Woburn, MA) or other standard solid phase
peptide synthesizer.
2. Resins for solid phase synthesis: Rink Amide AM resin (substi-
tution level 0.71 mmol/g, Novabiochem, EMD Biosciences,
San Diego, CA); Fmoc
4
-Lys
2
-Lys-β-Ala-CLEAR
Acid
Resin, Fmoc
4
-Lys
2
-Lys-Lys(Biotin-PEG)-β-Ala-CLEAR
Acid Resin and Fmoc
4
-Lys
2
-Lys-Cys(Acm)-β-Ala-CLEAR
Acid Resin (substitution level 0.21 mmol/g, Peptides Inter-
national, Louisville, KY).
3. Fmoc amino acids required to synthesize desired peptide. Pre-
pare 200 mM amino acid solutions by dissolving 20 mmol Fmoc-
protected amino acids in DMF to final volume of 100 mL.
2.4. Colony PCR for
Sequence Determi-
nation (Outlined in
Subheading 3.6)
2.5. Selectivity and
Specificity
Determinations (Out-
lined in
Subheading 3.7)
2.6. Peptide
Synthesis (Outlined in
Subheadings
3.8–3.11)
Page 4
Biopanning of Phage Displayed Peptide Libraries 295
4. Coupling reagents: 2-(1H-Benzotriazole-1-yl)-1,1,3,3-te-
tramethyluronium hexafluorophosphate (HBTU), 1-Hy-
droxybenzotrizole (HOBt) all available from Novabiochem.
Prepare 200 mM solutions as follows: Weigh out 18.965 g
HBTU,6.755 g HOBt, and 11 mL NMM, Add DMF to final
volume of 250 mL.
5. If desired nonnatural amino acids can be incorporated into the
peptide. We routinely incorporate Fmoc-NH–(PEG)
11
–COOH
(C
42
H
65
NO
16
) (Polypure, Oslo, Norway), Fmoc-Glu(biotinyl-
PEG)–OH (C
40
H
55
N
5
O
10
S), and Fmoc-Lys(biotin)–OH
(C
31
H
38
N
4
O
6
S) (Novabiochem, EMD Biosciences, San Diego,
CA) (see Notes 5 and 6).
6. Piperidine in DMF (20%): 200 mL piperidine, 800 mL
DMF.
7. Cleavage cocktails (see Note 7):
(a) TFA: H
2
O:TIS (95%:2.5%:2.5%) prepared by mix-
ing 9.5 mL trifluoroacetic acid (TFA), 0.25 mL H
2
O,
0.25 mL triisopropylsilane (TIS). This cleavage cocktail
is used for the cleavage of linear synthesized tetrameric
peptide and maleimido activated cores.
(b) TFA: EDT:H
2
O:TIS (94%:2.5%:2.5%:1%) prepared by
mixing 9.4 mL TFA, 0.25 mL ethanedithiol (EDT),
0.25 mL H
2
O, 0.1 mL TIS. This cocktail is employed for
peptides containing a cysteine residue.
8. Diethyl ether.
9. Dichloromethane (DCM).
10. Dimethylformamide (see Note 8).
11. 3-Maleimidopropionic acid (Sigma-Aldrich Inc, St. Louis, MO).
1. PBS containing 0.01 M EDTA.
2. Argon for flushing solutions.
3. TFA/Anisole mixture (99:1).
4. Silver acetate (Sigma-Aldrich or other vendor).
5. Diethyl ether.
6. Dithiothreitol (0.2 M) prepared in 1 M acetic acid.
7. Guanidine hydrochloride (8 M).
1. HPLC solvent delivery system with binary gradient capabil-
ity and a UV detector.
2. Reversed-phase octadecylsilica (C18) column. In our
laboratory, we use the following columns: Preparative
column: Vydac RP-C18 column (250 mm length×22 mm
diameter, 10 μm particle size). Analytical column: Varian
RP-C18 column (250 mm length×4.6 mm diameter, 5 μm
particle size).
2.7. Removal of Group
from Selectively
Protected Cysteine
Residues (Outlined in
Subheading 3.12)
2.8. Peptide
Purification and Char-
acterization (Outlined
in Subheading 3.13)
Page 5
296 McGuire, Li, and Brown
3. Solvent filtration apparatus equipped with a 0.45 μm Teflon
filter (Such as Ultra-ware filter apparatus 300/1,000 mL
from Kontes glass company and 0.45 μm Teflon filters from
Millipore Corp.).
4. Syringe driven filter units, 0.22 μm porosity, 13 mm (Mil-
lipore Corp.).
5. Eluent A: H
2
O/0.1% TFA and eluent B: acetonitrile/0.1%TFA.
6. Access to mass spectrometer and/or peptide sequencing facility.
1. Selected phage clone and corresponding synthetic peptide.
2. Reagents indicated in Subheading 2.5.
1. Synthetic peptide prepared with incorporation of biotin.
2. Streptavidin-conjugated Qdots (emission wavelengths cho-
sen to match microscope emission filter set).
3. Microscope slides and coverslips.
4. Chamber slides (8-well) (for example, VWR, Inc., catalog #
62407–296) can be used to culture adherent cells prior to
staining.
5. Prolong
®
Gold antifade reagent with DAPI (Invitrogen).
6. Fluorescence microscope.
7. Streptavidin-coated magnetic beads (Dynabeads M280-SA,
Invitrogen, 6.7 × 10
8
beads/mL, 1 mg beads binds 700 pmoles
free biotin).
8. Cell isolation magnet (or strong magnet).
9. Streptavidin-phycoerythrin or streptavidin-FITC.
10. Flow cytometer (Cell Lab Quanta, Beckman Coulter or
other suitable instrument).
11. PBS+.
12. 0.4% Formalin: 37% formaldehyde, Sigma Chemical, diluted
1:10 in PBS, immediately prior to use.
13. Ethanol (70%).
14. Fingernail polish (any color).
The following information is based on our protocols for selec-
tion of peptide ligands for cell recognition and delivery. Selection
2.9. Inhibition of
Selected Phage Clone
by Cognate Peptide
(Outlined in
Subheading 3.14)
2.10. Applications of
Synthetic Peptides
(Outlined in
Subheadings 3.15–
3.18)
3. Methods
Page 6
Biopanning of Phage Displayed Peptide Libraries 297
of a peptide ligand, using our protocol, should be expected to
take 5–6 rounds of biopanning. During each round, cells are
incubated with a mixture of phage displaying different peptides.
Phage that do not bind or bind only to the surface of the cell
are washed away. Phage that bind to the cell and are internalized
by the cell are retained. These cell-internalized phage are ampli-
fied in bacteria, isolated, and used as the input in the next round
of biopanning. In each round of selection, the diversity of the
phage sample is reduced while the proportion of phage displaying
a peptide that mediates cell-specific binding is increased.
Once a phage displayed peptide has been selected using the
biopanning protocol, we characterize the binding selectivity and
cell specificity of that phage clone. Our determination of selectiv-
ity compares the binding and uptake of a cell-selected phage clone
with binding and uptake of a control phage clone that was ran-
domly selected from the library. This provides a rough estimate of
the affinity of the peptide. Additionally, it assures that the cellular
binding is due to the selected peptide and is not the result of non-
specific phage binding. The measurement of cell specificity involves
comparison of the selectivity index of a specific phage over a vari-
ety of different cell types. During the characterization process, we
also prepare chemically synthesized versions of the specific peptide,
monomeric and tetrameric, and test the utility of these constructs
as cell-binding reagents out of the context of phage presentation.
Depending on the down-stream applications of the ligand, we will
incorporate a unique cysteine for chemical modification or a biotin
moiety for use with streptavidin-based reagents.
1. Cells are seeded onto tissue culture wells 24–48 h before
panning. Only a single well is required for each panning
round. Once started, each round of the panning procedure
requires approximately 4–5 h to complete. Additional time
is required for bacterial plating for titer determination and
phage amplification.
2. 24–48 h before the phage biopanning will be conducted,
trypsinize cells from a propagation flask and seed cells in
12-well plate. On the day of panning, one well should be
90% confluent. The proper level of confluence is generally
obtained by seeding 100,000–150,000 cells in a well.
3. Begin the biopanning procedure by gently removing media
from the well. Wash cells with 1 mL RPMI media (or other
cell-appropriate media) without serum (tip plate to accumu-
late liquid on one side of the well so that media can be aspi-
rated without disturbing attached cells. Pipette wash media
gently to avoid dislodging cells. Remove wash media.
4. Gently add 1 mL/well media without serum and incubate
cells for 2 h at 37°C to clear cell surface receptors (referred
to as “clearing the receptors”).
3.1. Phage
Panning for Adherent
Cell Lines
Page 7
298 McGuire, Li, and Brown
5. Approximately 15 min before the end of the clearing step,
prepare the phage panning solution as follows:
(a) Chloroquine (10 μL) (100× stock).
(b) 40 μL protease inhibitor without EDTA (25× stock).
(c) 10–100 library equivalents of the phage library. The
phage library used for much of our work has a diversity
of 1×10
8
members (5, 19). Therefore, we add 1×10
9
1×10
10
phage to the input sample for round one. Thus,
each library member should be present in 10–100 cop-
ies in the input mixture. For each successive round of
biopanning we input 1.5×10
9
phage.
(d) Bring mixture to 1 mL final volume by addition of PBS
+
with 0.1% BSA.
6. Remove RPMI from the cells. Wash cells once with 1 mL
PBS
+
with 0.1% BSA that was prewarmed to 37°C. Remove
the wash solution from the cells.
7. Save 50 μL of the input phage solution for titer determina-
tion. Add the remainder of the phage solution to the cells
and incubate for 1 h at 37°C in a standard tissue culture CO
2
incubator.
8. After the 1 h incubation, aspirate the supernatant. We do not
save this solution containing unbound phage. Wash the cells
four times at room temperature:
(a) Add 1 mL PBS
+
with 0.1% BSA (room temperature)
(b) Incubate for 5 min.
(c) Aspirate buffer and repeat.
9. Acid elute/wash 1–2 times at room temperature:
(a) Add 1 mL 0.1 M HCl–glycine, pH 2.2 + 0.9% NaCl.
(b) Incubate for 5 min. Time could be reduced if the cell
line is fragile and lysis is problematic (see Note 9).
(c) If you are interested in phage that bind to the cell sur-
face but are not internalized, you can keep this acid wash
fraction when it is removed from the cells and amplify
the recovered phage as detailed below. Adjust the pH of
the acid wash material by addition of 1.5 M Tris–HCl,
pH 8.8 after it is removed from cells.
(d) Repeat acid wash once.
10. Remove the second acid wash and add 1 mL of 30 mM Tris–
HCl, pH 8.0 to the cells and incubate on ice for 30 min. This
hypotonic media is used to swell the cells and enhance lysis
and recovery of phage.
11. Freeze cells in plate. This is a suitable place to stop the pro-
tocol if needed.
Page 8
Biopanning of Phage Displayed Peptide Libraries 299
12. Thaw cells and scrape off plate. The freeze-thaw cycle dis-
rupts the cells and releases any internalized phage. This sam-
ple is referred to as the output fraction. Examine the well
under a microscope to ensure that the cells have been dis-
rupted. If freeze-thaw does not disrupt the cells, 0.1% Triton
X-100 or other detergent can be added to the hypotonic
buffer.
13. Set up amplification of output phage as well as titration of
input and output phage. Titer input, acid wash (if desired),
and output. Amplify output or acid wash (if desired).
1. The panning procedure requires approximately 4 h to com-
plete. The cells can be removed directly from a feeder flask
for the panning procedure. They do not have to be seeded
into a separate flask prior to the day of the panning proce-
dure.
2. Transfer cells from feeder flask to a 50-mL centrifuge tube
and pellet cells (Speed and time required for forming a good
pellet will vary with cell type).
3. Resuspend cells in 10 mL media without serum and pellet
again.
4. Resuspend cells in 10 mL media without serum and incubate
cells for 2 h at 37°C incubator to clear the receptors.
5. Count cells during clearing and determine volume needed
for 2 million cells.
6. Approximately 15 min before the end of the clearing step,
prepare the phage solution as detailed in step 5 of Subhead-
ing 3.1.
7. Pellet 2 million cells and wash one time with 10 mL PBS
+
with 0.1% BSA pre-warmed to 37°C.
8. Save 50 μL input solution for titer determination. Pellet
cells and resuspend the cell pellet gently in the remainder of
phage solution and incubate for 1 h at 37°C in tissue culture
incubator with 5% CO
2
.
9. At the end of the incubation, dilute the sample to 10 mL
with PBS
+
with 0.1% BSA (room temperature).
10. Wash the cells four times by centrifugation and resuspension.
Resuspend cells in 10 mL PBS
+
with 0.1% BSA and incubate
for 5 min at room temperature. Pellet cells.
11. Acid wash cells at room temperature:
(a) Resuspend cell pellet in 1 mL 0.1 M HCl–glycine, pH
2.2, 0.9% NaCl.
(b) Incubate 5 min (see Note 9).
(c) Pellet cells and remove supernatant.
3.2. Phage Panning for
Nonadherent Cells
Page 9
300 McGuire, Li, and Brown
(d) Repeat acid wash once. Remove supernatant solution.
12. Remove the second acid wash and add 1 mL of 30 mM Tris–
HCl, pH 8.0 to the cells and incubate on ice for 30 min.
13. Freeze cells and thaw. The freeze-thaw cycle disrupts the cell and
releases any phage. This is referred to as the output fraction.
14. Set up amplification and titration of phage. Titer input, acid
wash (if desired), and output. Amplify output and/or acid
wash (if desired).
1. Between successive rounds of phage biopanning, the output
phage sample (and/or the acid wash sample, if desired) must
be amplified. This procedure may be performed in parallel
with the phage titering as detailed in Subheading 3.3 or
may be performed independently.
2. On day one, pick a single K91 bacterial colony and inoculate
10–15 mL LB media without antibiotics for each sample that
will be amplified.
3. Culture bacteria at 37°C with shaking until an OD
600 nm
of
0.2–0.4 is obtained.
4. Spin down bacterial cells at 3,000 × g for 10 min at 4°C.
5. Resuspend the pellet in 1/10 the original volume using LB
media by pipetting up and down.
6. Add your phage sample to be amplified (the entire phage
sample – 50 μL aliquot removed for titration) to resuspended
K91 cells and incubate for 15 min at 37°C.
7. Dispense the complete mixture of phage-infected K91 cells
onto four, 150 mm LB-tet plates. Plate 1/4 of the bacterial
mix/plate.
8. Allow liquid to be absorbed into plate.
9. Invert plates and incubate at 37°C overnight.
10. On day two, harvest phage. Add 10 mL LB media to each of the
four LB-tet plates. Incubate for 10 min at room tempe-rature.
11. Scrape bacteria off the plate with a glass spreader. Collect all
the material from the four inoculated plates in a single 50 mL
centrifuge tube. Some of the media will not be recovered
from the plate.
12. Add 10 mL fresh LB media to one of the four plates. Use the
glass spreader to clean the plate further and transfer the wash
material to the second plate. Continue until all four plates
have been washed in this manner. Combine this wash mate-
rial with the original harvest in the 50 mL tube.
13. Centrifuge the harvested material to obtain a firm pellet of
bacterial cells (Example: 3,000 × g for 10 min at 4°C in Beckman
Coulter Allegra
®
25R centrifuge.).
3.2. Phage Panning for
Nonadherent Cells
Page 10
Biopanning of Phage Displayed Peptide Libraries 301
14. The infectious phage particles will be in the supernatant from
this centrifugation step. Transfer the supernatant to a fresh
centrifuge tube and measure the volume.
15. Add ¼ of the supernatant volume of 2.5 M NaCl + 20% PEG
8000 to the supernatant. Example: if volume of supernatant
is 40 mL, add 10 mL of the NaCl/PEG solution. Incubate
this mixture on ice for 1 h to precipitate the phage particles.
16. Collect the phage precipitate by centrifugation at 11,000 ×
g for 30 min at 4°C. The phage should produce a firm pellet
under these conditions.
17. Pour off and discard the supernatant making sure no stand-
ing liquid is left in the centrifuge tube. Tilt the centrifuge
tube to drain off any residual PEG solution. Leave the tube
inverted for 1 h at 4°C. Residual PEG solution will make it
more difficult to completely resuspend the phage pellet.
18. After draining for 1 h, put the tube upright and add 1 mL
PBS to the pellet. Incubate on ice for 30 min. During this
incubation, tilt the tube so that the pellet is completely cov-
ered with PBS.
19. Gently resuspend the phage pellet using a 1 mL pipette.
Do not vortex. Vortexing concentrated phage solution may
result in shearing of the phage. Mix the samples so that there
are no visible chunks or cakes in the sample. Transfer the
resuspended phage to a clean 1.5 mL microcentrifuge tube.
20. Pellet insoluble debris by centrifugation at 16,000 × g for
2 min in a bench top microcentrifuge.
21. Transfer supernatant to a fresh microcentrifuge tube and
incubate at 65°C for 15 min in a water bath or heating block
to kill any bacteria remaining in the sample. Do not extend
time of this incubation or the phage will lose infectivity.
22. Pellet insoluble debris by centrifugation at 16,000 × g for 2 min.
23. Transfer supernatant to a clean tube. Discard pellet. Mix and
dispense aliquots of the purified phage to clean microcen-
trifuge tubes. Label tube with the cell type, panning round
number, date and operator’s initials.
24. Store aliquots of the phage preparation at −80°C until use.
25. Before using on cells, set up bacterial titration to determine
the yield of infective phage. The titration should be per-
formed as detailed in Subheading 3.3 for the input phage
sample except that more dilute samples are required to infect
with bacteria. We typically dilute amplified phage prepara-
tions 10
−2
, 10
−4
, 10
−6
, 10
−7
, and10
−8
. The samples diluted
10
−6
, 10
−7
, and10
−8
are used to infect K91 bacterial cells and
aliquots of these infections are plated.
26. For amplification of individual phage clones (see Note 10).
Page 11
302 McGuire, Li, and Brown
1. We maintain K91 cells on minimal media minus proline sup-
plemented with 0.5 μg/mL thiamine. The bacteria grow
slowly on these plates, generally requiring 2 days of culture
to produce suitable colonies. Each day that a titration will
be performed, start a liquid culture of the bacteria from a
single colony.
2. Pick a single colony and inoculate 5–10 mL LB media with-
out antibiotics.
3. Culture bacteria at 37°C with shaking until an OD
600 nm
of
0.4 is obtained. If culture goes past an OD
600 nm
of 0.6, the
culture should be diluted approximately tenfold with LB
media and continue culturing until the proper optical den-
sity is obtained.
4. If bacterial cells are ready before samples that will be titered,
place bacterial cells on ice until needed. If placed on ice,
rewarm the cells to 37°C prior to mixing with phage sam-
ples.
5. Prepare serial dilution of input phage sample (50 μL aliquot
was saved for titering):
(a) Add 10 μL input phage to 990 μL LB media (=10
−2
dilu-
tion).
(b) Add 10 μL of 10
−2
input phage dilution to 990 μL LB
media (=10
−4
dilution).
(c) Add 100 μL of 10
−4
input phage dilution to 900 μL LB
media (=10
−5
dilution).
(d) Add 100 μL of 10
−5
input phage dilution to 900 μL LB
media (=10
−6
dilution).
6. For titration of biopanning output samples:
(a) Mix the freeze-thaw cell lysate well by flicking.
(b) Add 50 μL of cell lysate to 450 μL LB media (=10
−1
dilu-
tion).
(c) Add 100 μL of 10
−1
dilution to 900 μL LB media (=10
−2
dilution).
(d) For the early rounds of panning, these first two dilutions
should be adequate. After round three, an additional
tenfold dilution is suggested.
7. Dispense 900 μL K91 bacterial cells to sterile tubes for
phage infection. For input phage samples, dispense aliq-
uots to be infected with the 10
−4
, 10
−5
, and 10
−6
dilutions,
respectively. For the output phage samples, dispense
aliquots to be infected with the 10
−1
and 10
−2
dilutions,
respectively. Dispense one K91 aliquot that will serve as a
noninfected control.
3.4. Bacterial cell
Culture and Phage
Titration (See Note 11)
Page 12
Biopanning of Phage Displayed Peptide Libraries 303
8. Add 100 μL of diluted phage sample to 900 μL K 91 cells.
Incubate at 37°C for 15 min (see Note 12). Label LB-tet
plates for each sample. We inoculate 2 LB-tet plates for each
infected K91 sample, one plate with 100 μL and second plate
with 50 μL.
9. Add 100 μL of each infected K91 sample onto a separate,
labeled LB-tet plate and spread evenly.
10. Add 50 μL of each infected K91 sample onto a separate,
labeled LB-tet plate and spread evenly.
11. Additionally, plate 100 μL of uninfected K91 cells as a con-
tamination control. After overnight incubation, these con-
trol plates should not have any colonies.
12. Allow plates to dry before inverting.
13. Incubate at 37°C overnight to allow colonies to grow.
14. After overnight incubation, count and record the number
of colonies present on each plate. There should not be any
colonies on the uninfected K91 cell plates. If colonies are
present on these plates, the source of contamination needs
to be eliminated and the samples need to be titered again.
15. Calculate titer of each sample. For each plate, use the for-
mula: (# of colonies × dilution factor× 10 for dilution into
K91)/mL of K91 mixture plated = colony forming units
(cfu)/mL in the original sample. Calculate the independent
determinations for each sample and average them to obtain
sample titer.
16. The output titer plates from biopanning round 3 and sub-
sequent rounds are saved for DNA sequence analysis and
determination of phage displayed peptide sequences (see
Subheading 3.6).
1. This protocol assumes the use of the BioRad iCycler. Adjust-
ments to the protocol may be required using other devices.
We use the generalized Sybr
®
green detection system that
does not require the generation of independent-labeled
probes.
2. Turn on the lamp, camera, and thermal cycler at least 30 min
before a reaction starts to stabilize per manufacturer’s sug-
gestion.
3. Prepare standards and samples. Our calibration standard for
titration consists of a series of tenfold dilutions of phage. We
routinely generate this calibration standard line of infectious
phage that range from 100 to 1 × 10
9
phage/mL. We have also
run ssDNA and dsDNA preparations of phage in q-PCR.
4. Prepare the master mix for the number of reactions needed.
3.5. Real-Time,
Quantitative-PCR
Phage Titration
(See Note 13)
Page 13
304 McGuire, Li, and Brown
Component Volume per sample (µL)
2× IQ SYBR
®
Green Supermix 50
10 µM forward primer 3
10 µM reverse primer 3
H
2
O34
Total 90
5. Dispense 90 μL aliquots of mastermix to clean PCR tubes (1
aliquot/sample or DNA standard). Add 10 μL of standard
DNA or phage sample to 90 μL of the master mix.
6. Mix the 100 μL complete reaction mixture and dispense the
reaction mixture into three wells of a 96-well plate that is
optically suitable for q-PCR at 25 μL/well using an 8-chan-
nel pipette.
7. Spin down the plate to exclude bubbles at the bottom of the
wells.
8. Enter the PCR Protocol and Plate Setup files from the iCy-
cler or appropriate software. We use a 3-step protocol for the
individual amplification cycles:
Procedure Temperature (°C) Time
Hot start 95 3 min
Amplification cycles
Denature 95 30 s
Anneal 55 30 s
Extension 72 30 s
40 cycles
Denature before melt curve analysis 95 1 min
Annealing before melt curve analysis 55 1 min
Melt curve analysis 0.5 up 10 s
80 cycles
End 4 Hold
(e) The software for the iCycler (and other real-time thermo-
cyclers) will automatically determine the parameters of the
standard line and calculate the values of phage titers for each
sample. Attention should be paid to the threshold parameter
established by the software, the slope of the standard line
(should be close to 3.2–3.3), and the melt curve analysis,
which indicates the specificity of the amplified product. The
PCR products can be evaluated by agarose gel electrophore-
sis if there is a question about amplification specificity.
Page 14
Biopanning of Phage Displayed Peptide Libraries 305
1. Dispense 50 μL water to 16 PCR tubes.
2. Label and pick 16-well-spaced colonies on a titer plate of
interest (see Note 14).
3. Using a plastic pipette tip or toothpick, stab each colony and
mix it into a different tube prepared in step 1.
4. Heat the samples at 95°C for 5 min to lyse bacteria and dena-
ture proteins.
5. Cool the samples while preparing PCR master mix contain-
ing 100 μL 10× PCR buffer without MgCl
2
, 60 μL 25 mM
MgCl
2
, 20 μL 10 mM dNTP mix, 20 μL 10 μM forward
primer (fd-tet F1), 20 μL 10 μM reverse primer (fd-tet B1),
730 μL water and 10 μL Taq polymerase.
6. Dispense 48 μL PCR mastermix to 16 new PCR tubes.
7. Add 2 μL of lysed bacterial colony sample to each aliquot of
mastermix.
8. Perform PCR. We use the following protocol:
Procedure Temperature(°C) Time
Hot start 95 2 min
Amplification cycles
Denature 95 30 s
Anneal 55 30 s
Extension 72 30 s
35 cycles
Final extension 72 5 min
End 4 Hold
9. Evaluate PCR products on 1% agarose gel. Single products
of approximately 450 bp are expected.
10. Remove dNTPs and oligonucleotide primers from the
PCR product by combining 20 μL of PCR product with
2 μL exonuclease I and 2 μL shrimp alkaline phosphatase
(SAP). Incubate at 37°C for 30 min followed by 15 min
at 85°C.
11. After exonuclease I and SAP treatment, the PCR product
can serve directly as template in a dideoxy terminator DNA
sequencing reaction. We use BigDye
®
Terminator v3.1 in the
following mixture: 5 μL treated product, 1 μL 10 μM primer
(fd-tet F1), 3 μL 5× reaction buffer (supplied with BigDye
®
Terminator), 2 μL BigDye
®
Terminator Mix, and 9 μL water.
BigDye
®
reactions are prepared in a 96-well plate.
12. Perform sequencing PCR using the following conditions:
3.6. Colony PCR for
Determination of
Displayed Peptide
Sequence
Page 15
306 McGuire, Li, and Brown
Procedure Temperature(°C) Time
Hot start 95 2 min
Amplification cycles
Denature 95°C 30 s
Anneal 55°C 30 s
Extension 72°C 30 s
30 cycles
Final extension 72°C 5 min
End 4°C Hold
13. Purify BigDye reaction by adding 80 μL sequencing stop/
precipitation reagent.
14. Collect precipitates by centrifugation at 3,000 × g for 30 min.
15. Invert plate onto a paper towel to collect liquid.
16. Centrifuge inverted plate at 100 × g for 1 min to remove
liquid from plate.
17. Wash precipitates with 150 μL of 70% ethanol. Centrifuge at
3,000 × g for 10 min.
18. Invert plate onto a paper towel and centrifuge at 100 × g for
1 min to remove liquid from plate.
19. Add 26 μL Hi-Di formamide
to each well.
20. Heat to 95°C for 5 min.
21. Cool plate and load onto ABI 3100 automated sequencer or
other suitable sequencer.
22. Translate DNA sequence to peptide sequence for the segment
encoding pIII protein at the point of the library insertion.
1. The selectivity determination is performed in a manner simi-
lar to the phage biopanning protocols for adherent and non-
adherent cells detailed in Subheadings 3.1 and 3.2, with
some important modifications.
2. Selectivity determinations require two matched wells of adher-
ent cells or two aliquots of nonadherent cells. The adherent
cells should be seeded onto wells 24–48 h before the experi-
ment. For nonadherent cells, use 2 million cells/phage.
3. On the day of the experiment, the receptors are cleared by
incubating cells in serum-free media for 2 h.
4. Approximately 15 min before the end of the clearing step,
prepare the specific phage and control phage as separate
solutions, each as follows:
3.7. Selectivity and
Specificity
Determinations
(See Note 15)
Page 16
Biopanning of Phage Displayed Peptide Libraries 307
(a) 10 μL Chloroquine (100× stock)
(b) 40 μL protease inhibitor without EDTA (25× stock)
(c) 1 × 10
8
phage as input (see Note 16).
(d) Bring each mixture to 1 mL final volume by addition of
PBS
+
with 0.1% BSA.
5. Wash cells once with 1 mL PBS
+
with 0.1% BSA that was pre-
warmed to 37°C. Remove the wash solution from the cells.
6. Save 50 μL of the input phage solution for titer determination.
Add the remainder of the phage solution to the cells and incubate
for 10 min at 37°C in a standard tissue culture CO
2
incubator.
7. After the 10 min incubation, aspirate the supernatant directly from
adherent cells. Pellet nonadherent cells for each wash cycle.
8. Add 1 mL PBS
+
with 0.1% BSA (room temperature)
9. Incubate for 5 min.
10. Aspirate buffer and repeat step 8–10 4 times to remove all
weakly bound phage.
11. Acid elute/wash 1–2 times at room temperature: add 1 mL
0.1 M HCl–glycine, pH 2.2 + 0.9% NaCl.
12. Incubate for 5 min. Time could be reduced if the cell line is
fragile and lysis is problematic (see Note 9).
13. Repeat acid wash once.
14. Remove the second acid wash and add 1 mL of 30 mM Tris–
HCl, pH 8.0 to the cells and incubate on ice for 30 min.
15. Freeze cells in plate or centrifuge tube. This is a suitable
place to stop the protocol if needed.
16. Thaw cells and scrape off plate, if needed. This sample is
referred to as the output fraction.
17. Set up titration of input and output phage for the control
and specific phage as detailed in Subheading 3.3.
18. The following calculations are used to compare phage binding
to cells:
(a) Output/input ratio (O/I) = output titer of phage/input
titer of phage applied to cells.
(b) Selectivity index = (O/I for specific phage clone)/(O/I
for control phage).
(c) Specificity is determined as the selectivity index for a
specific phage clone tested on a panel of cell lines and
primary cells.
1. Monomeric peptide syntheses are performed on a Sym-
phony
®
peptide synthesizer by Fmoc solid-phase peptide
synthesis but all methods reported here can be adapted to
other solid phase synthesizers.
3.8. Monomeric Pep-
tide Synthesis
Page 17
308 McGuire, Li, and Brown
2. For a 0.1 mmol synthesis, place 141 mg Rink Amide AM
resin (substitution level 0.71 mmol/g) in the peptide syn-
thesis reaction vessel. Place the reaction vessel in one of the
positions of the synthesizer. Add 2.5 mL DMF to swell the
resin. Flush with nitrogen to form a suspension of resin for
30 min (see Note 17). Drain off DMF.
3. Repeat step 2 three times.
4. Add 2.5 mL Piperidine in DMF (20%) to deprotect the resin
by removing the Fmoc moieties. Flush with nitrogen for
10 min. Drain off reagent. Repeat twice.
5. Add 2.5 mL DMF to the resin. Flush with nitrogen for 30 s.
Drain off DMF. Repeat six times.
6. Add 2.5 mL Fmoc-protected amino acids in DMF (200 mM)
to the deblocked peptidyl resin. Add 2.5 mL HBTU, HOBt,
and NMM in DMF (200 mM) to the resin. Flush with nitro-
gen for 45 min. Drain off reagent.
7. Add 2.5 mL DMF to the resin. Flush with nitrogen for 30 s.
Drain off DMF. Repeat six times.
8. Repeat the cycle from step 4 to step 7 for the next amino
acid coupling until the completion of the peptide synthesis
(see Notes 18 and 19).
9. Add 2.5 mL of 20% piperidine in DMF to the resin. Flush
with nitrogen for 10 min. Drain off reagent. Repeat twice.
10. Add 2.5 mL DMF to the resin. Flush with nitrogen for 30 s.
Drain off DMF. Repeat six times.
11. Add 2.5 mL DCM to the resin. Flush with nitrogen for 30 s.
Drain off DCM. Repeat nine times.
12. Dry the resin for 10 min.
13. Place the dry resin in a 50 mL round bottom flask. Add 5 mL
cleavage cocktail TFA: EDT:H
2
O:TIS (94%:2.5%:2.5%:1%)
to the resin. For cysteine containing peptides use TFA:
EDT:H
2
O:TIS (94%:2.5%:2.5%:1%) (see Notes 7 and 20).
Flush flask with nitrogen, stopper, and leave to stand at room
temperature with occasional shaking for 3 h.
14. Remove the resin by filtration under reduced pressure
through a sintered glass funnel. Wash the resin twice with
3 mL cleavage cocktail (see Note 21).
15. Combine filtrates and transfer to an appropriate sized round-
bottomed flask. Concentrate the TFA and scavenger mixture
quickly to a volume of approximately 3 mL on a rotatory
evaporator (see Note 22).
16. Fill a 50 mL conical tube about two-thirds full with cold die-
thyl ether. Add the concentrated peptide/TFA solution into
cold ether using a Pasteur pipette. Place the cold ether with
the peptide precipitate at −80°C freezer overnight.
Page 18
Biopanning of Phage Displayed Peptide Libraries 309
17. Centrifuge the cold ether with the peptide precipitate solu-
tion at 4°C for 10 min at 2,800–3,000 × g. Carefully decant
the ether from the tube (see Note 23).
18. Add 50 mL fresh diethyl ether, seal and shake the tube to
resuspend the peptide. Centrifuge for 10 min under the same
conditions. Repeat three times.
19. Decant the ether from the tube. Put the tube in the hood for
aproximately15 min to evaporate trace amounts of residual
ether. Put the tube in the vacuum desiccator and dry the
peptide under vacuum for 3 h. Weigh the peptide.
1. Tetrameric peptides syntheses are performed on a Symphony
®
synthesizer (Rainin Instruments, Protein Technologies, Inc.
Woburn, MA) by Fmoc solid-phase stepwise peptide synthe-
sis on trilysine core. These instructions are easily adaptable to
other automated peptide synthesis instruments.
2. For a 0.1 mmol synthesis, place 476 mg Fmoc
4
-Lys
2
-Lys-β-
Ala-CLEAR
Acid Resin (substitution level 0.21 mmol/g)
in the peptide synthesis reaction vessel (see Note 25). Place
the reaction vessel in one of the positions of the synthesizer.
Add 5 mL DMF to swell the resin. Flush with nitrogen to
form a suspension of resin for 30 min (see Note 17). Drain
off DMF.
3. Repeat step 2 three times.
4. Add 5 mL of 20% piperidine in DMF to remove the Fmoc
protecting groups on the resin. Flush with nitrogen for
10 min. Drain off reagent. Repeat twice.
5. Add 5 mL DMF to the resin. Flush with nitrogen for 30 s.
Drain off DMF. Repeat six times.
6. Add 5 mL Fmoc-protected amino acids in DMF (200 mM)
to the deblocked peptidyl resin. Add 5 mL HBTU, HOBt,
and NMM in DMF (200 mM) to the resin. Flush with nitrogen
for 45 min. Drain off reagent.
7. Repeat steps 5 and 6 (see Note 26).
8. Add 5 mL DMF to the resin. Flush with nitrogen for 30 s.
Drain off DMF. Repeat six times.
9. Repeat the cycle from step 4 to step 8 for the next amino
acid coupling until the completion of the peptide synthesis.
10. Add 5 mL Piperidine in DMF (20%) to the resin. Flush with
nitrogen for 10 min. Drain off reagent. Repeat twice.
11. Add 5 mL DMF to the resin. Flush with nitrogen for 30 s.
Drain off DMF. Repeat six times.
12. Add 5 mL DCM to the resin. Flush with nitrogen for 30 s.
Drain off DCM. Repeat nine times.
13. Continue from step 12, Subheading 3.8.
3.9. Linear
Tetrameric Peptide
Synthesis (See Note 24)
Page 19
310 McGuire, Li, and Brown
1. Maleimido activated core syntheses are performed on a Sym-
phony
®
synthesizer by Fmoc solid-phase peptide synthesis.
These methods can easily be adapted for other solid phase
peptide synthesizers.
2. For a 0.1 mmol synthesis, place 476 mg Fmoc
4
-Lys
2
-Lys-β-
Ala-CLEAR
Acid Resin, Fmoc
4
-Lys
2
-Lys-Lys(Biotin-PEG)
-β-Ala-CLEAR
Acid Resin and Fmoc
4
-Lys
2
-Lys-Cys(Acm)-
β-Ala-CLEAR
Acid Resin, respectively (substitution level
0.21 mmol/g) in the peptide synthesis reaction vessel (see
Notes 27 and 28). Place the reaction vessel in one of the
positions of the synthesizer. Add 5 mL DMF to swell the
resin. Flush with nitrogen to form a suspension of resin for
30 min. Drain off DMF.
3. Repeat step 2 three times.
4. Add 5 mL of 20% piperidine in DMF as a deprotection rea-
gent to remove Fmoc protecting groups from the resin. Flush
with nitrogen for 10 min. Drain off reagent. Repeat twice.
5. Add 5 mL DMF to the resin. Flush with nitrogen for 30 s.
Drain off DMF. Repeat six times.
6. Add 2.5 mL 3-maleimidopropionic acid in DMF (200 mM)
to the deblocked peptidyl resin. Add 2.5 mL HBTU, HOBt,
and NMM in DMF (200 mM) to the resin. Flush with nitrogen
for 24 h. Drain off reagent.
7. Continue from step 10, Subheading 3.98.
1. Pipette 1.5 mL 1× PBS/0.01 M EDTA in a microcentrifuge
tube. Purge with Argon for 3 min.
2. Dissolve 8 μmol of monomeric peptide containing a unique
cysteine and 1 μmol of maleimido activated core in Ar-purged
1× PBS/0.01 M EDTA. Vortex it at room temperature for
2 h (see Notes 5 and 29).
1. Dissolve 1 μmol tetrameric peptide possessing an acetamido-
methyl (Acm) protecting group in 1 mL of TFA/anisole (99:1).
2. Add 28 mg of silver acetate. Stir the solution at 4°C for 2 h.
3. Concentrate under argon to 0.5 mL.
4. Add 5 mL cold diethyl ether. Centrifuge the cold ether with
the peptide precipitate solution at 4°C for 10 min.
5. Decant the ether from the tube.
6. Add 1 mL of 0.2 M dithiothreitol prepared in 1 M acetic acid.
Vortex solution at room temperature for 3 h.
7. Add 0.5 mL of 8 M guanidine hydrochloride. Filter the solu-
tion by syringe driven filter unit (0.22 μm porosity). Purify
the peptide by HPLC.
3.10. Core Synthesis
for Convergent
Tetrameric Synthesis
3.11. Tetrameric
Peptide Synthesis by
Convergent Strategy
(Scheme 18.I)
3.12. Removal of an
Acetamidomethyl
Group from a Uniquely
Placed Cysteine
Residue
Page 20
Biopanning of Phage Displayed Peptide Libraries 311
1. Filter eluents through a 0.45 μm Teflon
®
filter before use.
2. Dissolve 15 mg of crude peptide in 2 mL of Buffer A. Filter
the sample through a 0.22 μm filter.
3.13. Peptide
Purification and
Characterization
Scheme 18.1. Convergent synthesis of tetrameric cell-binding peptides. A biotin or a unique cysteine can be incorpo-
rated into the peptide for use in other applications. A PEG moiety is placed between the trilysine branch and the selected
peptide to increase aqueous solubility.
Page 21
312 McGuire, Li, and Brown
3. Equilibrate the HPLC preparative column under the follow-
ing initial conditions. Solvent: buffer A. Flow rate: 10 mL/
min. Detection wavelength: 220 nm.
4. Once a stable baseline is obtained, inject 2 mL of the sample
and use the elution profile in a linear gradient (referred to as
Method A): 0–1 min, 90%A, 10%B; 1–61 min, eluent B was
increased from 10 to 40% at a flow rate of 10 mL/min.
5. Collect the target peptide peak which is generally the major
peak (see Note 30).
6. Lyophilize fractions containing the peptide product. If avail-
able, the fractions can be analyzed by MALDI MS to deter-
mine which fractions to collect.
7. For analytical HPLC, dissolve 1 mg of purified peptide in
1 mL of Buffer A. Filter the sample through a 0.22 μm filter.
8. Equilibrate the HPLC analytical column under the following
initial conditions. Solvent: buffer A. Flow rate: 1 mL/min.
Detection wavelength: 220 nm.
9. Once a stable baseline is obtained, inject 100 μL of the sam-
ple and use the elution profile in a linear gradient (referred
to as Method B): 0–1 min, 90%A, 10%B; 1–51 min, eluent B
was increased from 10 to 60% at a flow rate of 1 mL/min.
10. Confirm the peptide mass by mass spectrometry. For ease we
typically perform matrix assisted laser desorption ionization
time of flight mass spectrometry (MALDI MS) (see Note 31).
11. Edman N-terminal sequence analysis can be performed to
further confirm the sequence of the peptide.
1. Perform phage uptake experiments in the same manner as
outlined in Subheading 3.7 except that the free peptide
is added to the phage solution before addition to the cells.
No prior addition of the peptide to the cells is required. We
typically cover a peptide concentration range from 1 nM to
10 μM (see Note 33).
2. Titer the common input sample and the individual output
samples as detailed in Subheading 3.3.
3. Calculate the output phage to input phage ratio in the pres-
ence of the peptide compared with the same ratio without
added peptide.
1. In a final volume of 100 μL PBS, streptavidin-Qdots (200 nM)
are mixed with 600 nM biotinylated peptide and incubated
for 2 h at room temperature. This incubation is performed
on the day the Qdots are to be used. Control SA-Qdots
can be prepared using no peptide, an irrelevant sequence
peptide or a scrambled version of the specific peptide.
3.14. Inhibition of
Phage Uptake by Free
Synthetic Peptides
(See Note 32)
3.15. Microscopy/Qdot
Delivery (See Note 34;
Fig. 1)
Page 22
Biopanning of Phage Displayed Peptide Libraries 313
2. At the end of the SA-Qdot-peptide incubation, unoccupied
streptavidin sites on the Qdots are quenched by the addition
of excess biotin (25 μL of a 20 μM stock) and incubated for
15 min at room temperature.
3. The mixture is diluted to 1 mL with PBS
+
with 0.1% BSA to
obtain a 20 nM Qdot solution for cell uptake.
4. Cells are incubated with Qdots on chamber slides or in poly-
propylene tubes for 10 min to 2 h.
5. At the end of the incubation, the solution containing Qdots
is removed and cells are washed briefly four times in PBS
+
with 0.1% BSA. The wash solution is added and then is
removed without further incubation.
Fig. 18.1. A tetrameric lung cancer targeting peptide can selectively deliver a fluorescent nanoparticle to its target cells.
The peptide TP H1299.2 was isolated by biopanning on the large cell lung cancer cell line H1299. H1299 cells were
incubated with 20 nM of the tetrameric TP H1299.2 peptide conjugated to SAQDot605 (D)–(F) or 20 nM tetrameric
control peptide conjugated to SAQDot605 (A)–(C). Bright field images (A), (D) and nuclear staining images (B), (E) of
the corresponding fields are shown. The fluorescence of the Qdot was visualized at 200-fold magnification on a Nikon
TE2000 fluorescent scope. Higher magnification (×1,000) shows cell surface binding of the tetrameric TP H1299.2
peptide-SAQDot605 conjugate as well internalized particles (G)–(I). (Reproduced from ref. 5 with permission from
Elsevier Science.).
Page 23
314 McGuire, Li, and Brown
6. Cells are briefly washed with 0.1 M HCl–glycine, pH 2.2.
+ 0.9% NaCl. Acid wash is removed after addition without
further incubation.
7. Cells are washed with PBS and excess liquid is removed from
the slide.
8. Cells can be viewed without fixation or they can be fixed
using 0.4% formalin solution, 70% ethanol or cold acetone
solution if desired. In this case, a 5 min incubation in fixative
is followed by PBS washes to remove fixative solution.
9. Incubation chamber is removed from chamber slide and
Prolong
®
Gold antifade reagent with DAPI stain is added
to the samples. If suspension grown cells were used, samples
can be spotted by hand onto slide or by using a CytoSpin
centrifuge, if available.
10. Add cover slip to slide and seal with fingernail polish.
11. Observe samples under microscope.
1. Phage coated plates can be prepared by incubating phage
solution (10
6
phage/mL) in wells at 4°C overnight. For
12-well plates, use 1 mL phage solution per well. For 96-well
plates, use 0.1 mL per well.
2. Phage solution is flicked from plate and residual binding sites
are masked by incubation of the wells (filled to capacity) in
0.1% BSA in PBS (1 h to overnight).
3. Nonadherent cells are removed from their culture flask and
washed by centrifugation in PBS
+
with 0.1% BSA. Cells are
dispensed to wells containing specific phage or control phage
or no phage and incubated for 10 min to 1 h.
4. Wells are washed four times with PBS
+
with 0.1% BSA to
remove unbound cells.
5. The number of captured cells can be determined by direct
cell counting – in the well or after release using nonenzy-
matic cell release solution or trypsinization. Alternatively,
cell numbers can be determined by lysis and assay for a
specific cellular component such as ATP. ATP content can
be assayed using the commercially available reagent that
requires only a single reagent addition to the wells (CellTit-
erGlo
, Promega).
6. The captured cells can be further characterized by down-
stream processes for gene or protein expression.
1. Cells are cleared by incubation for 2 h in RPMI without
serum as detailed in Subheadings 3.1 and 3.2.
2. 50 μL Streptavidin-coated Dynabeads
®
are washed twice by
suspension in 1 mL PBS and capture with a magnet for 5 min
and removal of the wash media by aspiration.
3.16. Capture of Cells
with Phage-Coated
Tissue Culture Wells
3.17. Capture of Cells
with Peptide-Coated
Magnetic Beads
Page 24
Biopanning of Phage Displayed Peptide Libraries 315
3. The magnetic beads are suspended in 1 mL PBS and allowed
to react with biotin-modified synthetic peptide (50 nM) for
30 min at room temperature.
4. The ligand-coated beads are washed twice in RPMI without
serum to block any remaining streptavidin sites (RPMI con-
tains 200 mg biotin/L).
5. Magnetic beads are washed once in PBS
+
with 0.1% BSA,
then resuspended in 1 mL PBS
+
with 0.1% BSA, and mixed
with cells.
6. Cells are incubated with the ligand-coated magnetic beads
for 15 min at 37°C.
7. Nonadherent cells that take up magnetic beads are captured
on the magnet (5 min at room temperature).
8. Adherent cells are released from wells using enzyme-free cell
dissociation buffer (GIBCO) before capture on the magnet.
Time of dissociation will vary with the cell type. Some cells
are easily released after a 5 min incubation on ice in dissocia-
tion buffer while other are released more effectively at room
temperature or even 37°C.
9. Cells are washed by suspension in PBS
+
with 0.1% BSA by
release from the magnet and recapture.
10. Captured cells are then suitable for additional analysis.
1. Cell samples are cleared by incubation in RPMI without
serum for 2 h.
2. During last 15 min of clearing, peptide solution is prepared
containing:
(a) 10 μL Chloroquine (100× stock).
(b) 40 μL protease inhibitor without EDTA (25× stock).
(c) Synthetic peptide construct with biotin – concentration
required varies with cell and peptide combination. No
peptide addition and control peptide solutions are pre-
pared separately.
(d) PBS
+
with 0.1% BSA to volume of 1 mL/sample.
3. Incubate cells with peptide for 15 min at 37°C.
4. Wash the cells in PBS
+
with 0.1% BSA four times at room
temperature.
5. Cells are diluted in PBS
+
with 0.1% BSA containing strepta-
vidin–phycoerythrin (SA-PE) at 1:100 dilution in the
mix.
6. SA-PE conjugate is added and cells are incubated for 15 min
at room temperature.
7. Stained cells are washed once and resuspended in fresh PBS
+
with 0.1% BSA.
3.18. Flow
Cytometry
Page 25
316 McGuire, Li, and Brown
8. Adherent cells can be released from the wells by incubation
in enzyme-free cell dissociation buffer as detailed in Sub-
heading 3.17.
9. Cell staining is evaluated by flow cytometry. Cells can be
counterstained with viability marker such as Alexafluor
488-Annexin V added to resuspension buffer and incubating
for 5 min at room temperature prior to loading of sample.
1. The phage library used in most of our selections for cell
ligands displays a 20 mer peptide at the amino-terminus of
the minor coat protein pIII (19). We have selected peptides
from other phage display libraries (5), including the Ph.D.
12-mer library available from New England Biolabs(MJM
and KCB unpublished results).
2. 20% PEG-8000 in 0.9% NaCl solution should be prepared
in advance. We routinely prepare 500–1,000 mL of this solu-
tion. PEG-8000 dissolves slowly even with constant stirring.
Preparation of the solution can take several hours. After all
of the PEG-8000 is in solution, filter-sterilize the solution
using 0.22 μm membranes (Millipore Corp., catalog # SCG-
P05RE). This process is also relatively slow because of the
viscous nature of the solution.
3. Sybr
®
green mixes for q-PCR are available from a number
of companies. We have found the DyNAmo
-Sybr
®
Green
qPCR kit (Finnzymes Inc., Distributed in USA through
New England Biolabs) to produce the most consistent results
in amplification of phage DNA recovered from mammalian
cells and tissues. Some mammalian tissues appear to have a
factor that inhibits the quantitative amplification of phage
DNA from crude extracts. In this case we have purified
total DNA from the tissue, using genomic DNA isolation
kits from Qiagen, prior to qPCR. Even using purified DNA,
inhibition was observed in some tissues. The recommended
Sybr
®
green mix uses a polymerase that appears to be less
subject to inhibition by these tissues.
4. Although we amplify a region of the Tet resistance gene, other
constant regions of the phage genome can be amplified.
5. Fmoc-NH–(PEG)
11
–COOH is incorporated to increase the
water solubility of the peptide if necessary. We routinely place
this PEG linker between the trilysine core and the targeting
peptide in the tetrameric constructs.
4. Notes
Page 26
Biopanning of Phage Displayed Peptide Libraries 317
6. In contrast to Fmoc-Lys(biotin)–OH, Fmoc-Glu(biotinyl-
PEG)–OH has excellent solubility in DMF and other sol-
vents used in solid phase peptide synthesis. The PEG-spacer
restricts hindrance between the peptide and avidin, leading
to better biotin binding.
7. Cleavage cocktails should always be prepared fresh. The cleav-
age procedure generally takes 2–4 h to perform. However,
some protecting groups are quite stable to TFA depending
on the location and number in a sequence, requiring up to
12 h of treatment for complete removal.
8. Amine impurities that could possibly remove the Fmoc
group include dimethylamine found in DMF. It is recom-
mended that DMF is protected under nitrogen or freshly
purified before use.
9. Although most cells we have tested have been stable during
the acid wash step of the protocol, some cells are lysed by this
treatment (primary cardiac myocytes and A20 B cell lymphoma
cells as examples). In these cases, we have deleted this step
from the protocol, reduced the incubation time in the acid, or
only performed a single acid wash. If necessary to isolate only
internalized phage, others have treated the cells with subtilisin
or other proteases to inactivate surface bound phage (16).
10. Since the output phage sample is a mixture of individual
library members, we prefer to amplify the phage mixtures as
colonies on large LB-tet plates. This will allow the individ-
ual members to grow without interference from competing
phage. For single phage clones, we grow infected E. coli in
liquid media (LB + 12 μg/mL tetracycline). The preparation
of phage from liquid cultures is the same as from plates from
step 13 of Subheading 3.4.
11. Titration is used to determine the number of infectious phage
particle per milliliter of solution. It is used in determining
the volume of a phage stock that will be added to a pan-
ning solution as well as the actual phage number in the input
and output samples from a round of panning or comparative
binding. Phage infection of E. coli requires that the bacteria
express pili. With fd-tet phage, the phage confer tetracycline
resistance on the bacteria. Bacteria without phage will not
grow on the LB-tet plates. The fd-tet phage do not cause
cell lysis. In fact, only phage-infected bacteria will produce
isolated colonies on LB-tet plates.
12. During this step, phages are allowed to infect bacteria. Do
not incubate the samples for more than 15 min. Place sam-
ples on ice immediately after removal from the incubator.
Bacteria should be plated before cells have time produce
progeny phage.
Page 27
318 McGuire, Li, and Brown
13. We use real-time quantitative PCR for three distinct pur-
poses. The first is to determine phage copy number after
the phage have lost the ability to infect bacteria. For example,
phage infectivity decreases rapidly after injection into a mam-
malian host. Second, we have used q-PCR to determine the
presence of a specific phage clone in a mixture of phage. This
has allowed us to add mixtures of phage in an experiment and
have one phage serve as an internal control. Third, we have
used q-PCR to determine phage levels in a large number
of samples simultaneously. We can determine total phage
copy number using sets of primers directed at a nucleotide
sequence in the backbone of the phage DNA. Additionally,
we can determine the copy number of a specific phage clone
using one primer directed at the nucleotide sequence encod-
ing the displayed peptide and a second primer for a sequence
from the phage backbone. Not all nucleotide sequences for
the displayed peptides have been suitable for generating use-
ful clone-specific primer sequences.
14. Titration plates with well-spaced and defined colonies can be
used for determination of displayed peptide sequence. We
initiate sequencing with the output of the third round of
biopanning. DNA sequencing is also performed to verify the
identity of an amplified phage clone. Since we use an ABI
3100 DNA sequencer that has a 16 capillary array, we rou-
tinely sequence samples in groups of 16.
15. To determine the initial success of a phage display peptide selec-
tion, we compare the binding and uptake of a specific phage
with the binding and uptake of a randomly chosen, control
phage using the same cells employed for the selection. The
randomly chosen, control phage mimics the binding character-
istics of the whole phage display library in these assays. The
selectivity value is the ratio of specific phage output/input
and the control phage output/input. The evaluation of
specificity compares the selectivity values of an individual
phage across a battery of cell lines or primary cells.
16. Determinations of selectivity and specificity are comparative
binding assays based on the ratio of the number of phage
taken up by a cell divided by the number of phage to which
the cells were exposed. A specific phage clone is being
compared with a control phage that represents a nonspecific
component of phage uptake by a cell sampling its environment.
Therefore, it is important to match the amount of specific
and control phage used as input. For these assays, the input
phage should be added at 1 × 10
8
phage/sample. Deviations
from the input phage number will distort the ratios used for
comparison and produce artificially high or low selectivity
indices.
Page 28
Biopanning of Phage Displayed Peptide Libraries 319
17. We recommend adjusting the N
2
flow to 10 psi. Higher flow
rates flush the resin to the top of the reaction vessel resulting
in incomplete coupling.
18. Sometimes the coupling reaction of an activated carboxy
group and a deprotected amino group is difficult to accom-
plish. These difficult couplings are usually sequence-depend-
ent and not residue-specific. In these cases, a double coupling
is required (i.e., repeat step 5–6 before step 7).
19. Fmoc-NH–(PEG)
11
–COOH, Fmoc-Glu(biotinyl-PEG)–OH
and Fmoc-Lys(biotin)–OH are coupled in the same fashion
as the natural Fmoc-protected amino acids.
20. Always handle thiol-containing substances in proper ventila-
tion. These compounds have an offensive odor that can be
neutralized with bleach. After the final cleavage operation,
rinse all glassware, pipettes, and tubes that came into contact
with scavengers with bleach before taking them out of the
hood.
21. The expended resin should not be discarded but retained, in
case it should prove necessary to repeat the cleavage reaction.
Many times during a poor extraction step, peptide remains
adhered to the resin beads and must extracted with an alter-
native solvent.
22. Temperature of the water bath should be below 37°C.
23. The diethyl ether washes should be retained until the yield of
product has been established. If a poor yield is obtained, the
washings should be evaporated under vacuum to dryness.
24. Linear synthesis of the tetrameric peptides is only recom-
mended for shorter peptides, on the order of ten amino acids
or less in length. We strongly prefer the synthetic route out-
lined Subheading 3.11.
25. We strongly encourage reducing the substitution of the resin
for tetrameric peptide synthesized linearly, longer molecules
(>30 residues) or for peptides rich in β-structural elements
to a substitution value lower than 0.25 mmol/g of resin.
26. A double coupling is usually preformed to increase the step-
wise coupling yield and avoid deletion contaminants.
27. For the maleimido activated core synthesis, the substitution
of the resin does not have to be lower than 0.25 mmol/g. A
normal substitution between 0.5 and −0.8 mmol/g will suf-
fice the synthesis.
28. The resin of choice depends on the down-stream application
of the peptide. We frequently attach the peptides to a desired
support, bead, or molecule via a unique cysteine located
before the trilysine branch point. The Acm protecting group
can be removed as described in Subheading 3.12 without
Page 29
320 McGuire, Li, and Brown
loss of the peptide branches. If using streptavidin conjugates,
the biotin containing resin is used instead.
29. After 2 h reaction time, the reaction solution must be puri-
fied by HPLC immediately. Otherwise, self-oxidized peptide
dimer side products occur as a result of disulfide formation.
30. For the tetrameric peptide synthesized by linear strategy, the
RP-HPLC trace typically shows one broad main peak. For
monomeric peptides, the RP-HPLC trace usually shows one
clear main peak. The occurrence of peaks with longer reten-
tion times than the main peak is suggestive of incomplete
removal of protecting groups. For the tetrameric peptide syn-
thesized by convergent strategy, the RP-HPLC trace typically
shows two major peaks. The peak with the lower retention
time is excess amount of monomeric peptide. The other with
the higher retention time is the product tetrameric peptide.
31. For monomeric peptides, the mass is confirmed by MALDI
MS using a-cyano-4-hydroxycinnamic acid as the matrix
using a Voyager DE
Pro instrument in reflector mode.
MALDI Mass Spectra of tetrameric peptide is obtained in
linear mode using sinapinic acid as matrix.
32. To determine whether the peptide is functional outside of the
phage particle, we determine whether the peptide can block
cell binding of its cognate phage. This assay is particularly
useful when the cellular target of the peptide is unknown.
Half maximal phage blocking can be determined as a meas-
ure of peptide affinity.
33. Do not use DMSO or DMF for preparation of peptide
stock solutions. These compounds appear to produce a
higher level of cell uptake of phage when added to cell-phage
mixtures. Incorporation of a PEG moiety improves the
peptide’s water solubility and often alleviates the need for
organic co-solvents.
34. Cell-binding of the selected phage clones can be determined
by fluorescent microscopy utilizing anti-phage antibodies (2, 6).
However, since our goal is to rapidly translate the peptide
outside of the context of the phage, we most often move
directly to using the free peptide for these studies.
This work was supported by the National Cancer Institute of the
NIH (1RO1CA106646 and R211R21CA114157-01).
Acknowledgments
Page 30
Biopanning of Phage Displayed Peptide Libraries 321
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Page 31
  • Source
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