C H A P T E R
A Peptide Carrier for the Delivery of
Biologically Active Proteins into
Mammalian Cells: Application to
the Delivery of Antibodies and
Therapeutic Proteins
May C. Morris, Julien Depollier, Frederic Heitz, and Gilles Divita
I. I N T R O D U C T I O N
In order to circumvent the technological problems
of gene delivery, an increasing interest is being taken
in designing novel strategies to deliver full-length
proteins into a large number of cells (Ford et al., 2001;
Wadia and Dowdy, 2002). However, the development
of peptide drugs and therapeutic proteins remains
limited by the poor permeability and the selectivity of
the cell membrane. Substantial progress has been
made in the development of cell-penetrating peptide-
based drug delivery systems that are able overcome
both extracellular and intracellular limitations (Morris
et al., 2000; Gariepy et al., 2001; Langel et al., 2002). A
series of small protein domains, termed protein trans-
duction domains (PTDs), have been shown to cross
biological membranes efficiently, independently of
transporters or specific receptors, and to promote the
delivery of peptides and proteins into cells (for review
see, Langel et al., 2002). The use of PTD-mediated
transfection has proven that "protein therapy" can
have a major impact on the future of therapies in a
variety of viral diseases and cancers. TAT protein
from immunodeficiency virus (HIV-1) (Schwarze et al.,
1999; Schwarze and Dowdy, 2000), the third ~ helix of
Antennapedia homeodomain (Derossi et al., 1994),
VP22 protein from herpes simplex virus (Elliott and
Cell Biology
13
O'Hare, 1997), and transportan (Pooga et al., 2001), as
well as polyarginine sequences (Futaki et al., 2000),
have been used successfully to improve the delivery of
covalently linked peptides or proteins into cells.
However, PTDs display a certain number of limita-
tions in that they all require cross-linking to the target
peptide or protein. Moreover, protein transduction
using the PTD-TAT-fusion protein system may require
denaturation of the protein prior to delivery, introduc-
ing an additional delay between time of delivery and
intracellular activation of the protein.
We have described a new strategy for the delivery
of full-length proteins and peptides into mammalian
cells based on a short amphipathic peptide carrier,
Pep-1. This peptide carrier allows the delivery of
several distinct proteins and peptides into different
cell lines in a fully biologically active form, without
the need for prior chemical covalent coupling or
denaturation steps (Morris et al., 2001). Pep-1 is a 21
residue peptide (KETWWETWWTEWSQPKKKRKV)
consisting of three domains with specific func-
tions: a hydrophobic tryptophan-rich motif
(KETWWETWWTEW), involved in the main interac-
tions with macromolecules and required for efficient
targeting to the cell membrane; a hydrophilic lysine-
rich domain (KKKRKV) derived from the nuclear
localization sequence (NLS) of simian virus 40 (SV-40)
large T antigen, required to improve intracellular
Copyright 2006, Elsevier Science (USA).
All rights reserved.
14 TRANSFER OF MACROMOLECULES
delivery and solubility of the peptide vector; and a
spacer domain (SQP), which improves the flexibility
and the integrity of both hydrophobic and hydrophilic
domains (Morris et al., 2001). Given that the mecha-
nism through which Pep-1 delivers macromolecules
does not involve the endosomal pathway, the degra-
dation of macromolecules delivered is significantly
limited and rapid dissociation of the Pep-I/macro-
molecule particle is favoured as soon as it has crossed
the cell membrane. This peptide-based protein
delivery strategy presents several advantages, includ-
ing rapid delivery of proteins into cells with very high
efficiency, stability in physiological buffers, lack of
toxicity, and lack of sensitivity to serum. Pep-1 tech-
nology constitutes a powerful tool for basic research,
and several studies have demonstrated that Pep-1
technology is extremely powerful for studying the role
of proteins and for targeting specific protein/protein
interactions in vitro as well as in vivo (Morris et al., 2001;
Gallo et al., 2002; Wu et al., 2002, Pratt and Kurch, 2002,
Aoshiba et al., 2003).
This article describes several protocols for the use of
noncovalent Pep-1 technology for the delivery of bio-
logically active proteins into mammalian cells in vitro.
II. MATERIALS A N D
IN S TRUMEN TATI ON S
A. Products
~-Galactosidase is from the Chariot kit (Cat. No.
30025) or the ~-galactosidase staining kit (Cat. No.
35001) from Active Motif (Carsbad-USA). Phosphate-
buffered saline (PBS) (Cat. No. 14190-169), cell culture
reagents Dulbecco's modified Eagle's medium
(DMEM) (Cat. No. 41965-062), glutamine, and strepto-
mycin/penicillin (Cat. No. 15140-130) are from Invitro-
gen Life Technologies (Carsbad, CA). Foetal bovine
serum (FBS) is from PERBIO (Lot 3264EHJ, Cat. No.
CH30160-03). Potassium ferricyanide (Cat. No. P3667),
potassium ferrocyanide (Cat. No. P9387), magnesium
chloride (Cat. No. M2670), 5-bromo-4-chloro-3-indolyl-
~-D-galactopyranoside (X-gal; Cat. No. B4252), N, N-
dimethylformamide (DMF) (Cat. No. D4551), formal-
dehyde (Cat. No. F8775), and glutaraldehyde (Cat. No.
G5882) are from Sigma (St. Louis, MO).
B. Peptide Carrier Pep-l/Chariot
Pep-1 is 21 residue peptide KETWWETW
WTEWSQPKKKRKV (molecular mass 2907 Da). The
peptide is acetylated at it N terminus and carries a cys-
teamide group at its C terminus, both of which are
essential for the stability of the peptide and the trans-
duction mechanism (Morris et al., 2001). Pep-1 can be
synthesized in house or obtained from commercial
sources. Protocol for the synthesis and purification of
Pep-1 and derivatives are described in Mery et al.
(1993) and Morris et al. (1999, 2001). Also, Pep-1 is man-
ufactured under the name of Chariot by Active Motif
Inc. (Carlsbad, CA; http://www.activemotif.com, Cat.
No. 30025). The protein transduction kit contains
lyophilized Chariot and additional reagents required
for the transduction protocol, including a positive
transduction control (~-galactosidase Cat. No. 35001).
All the protocols described for Pep-1 are directly trans-
posable to Chariot.
III. PROCEDURES
A. Preparation of Pep-I/Protein or
Pep- 1/Peptide Complexes
This procedure is modified according to Morris et al.
(2001).
Solutions
1. Storage of Pep-1 powder: Pep-1 is stable at least one
year when stored at-20~ in a lyophilized form
2. Stock solution of Pep-l: Take the vial containing the
peptide powder out of the freezer and equilibrate for
30 min at room temperature without opening the vial.
This step is essential to limit hydration of the peptide
powder. Resuspend the peptide at a concentration of
2 m g / m l (concentration: 0.68 mM) in water or in PBS
buffer. Mix gently by tapping the tube or by vortexing
at low speed for 20 s.
3. Storage of the Pep-1 solution: Because repeated
freeze/thaw cycles can induce peptide aggregation, is
recommended to aliquot the Pep-1 stock solution into
tubes containing the amount you expect to use in a
typical experiment prior to freezing. The Pep-1 stock
solution is stable for about 2 months when stored at
-20~
Steps
1. Dilute the amount of protein or peptide in 100 gl
of PBS for each reaction. Use 0.5-1 gg of protein or
0.1-0.5 gg of peptide or low molecular weight protein
per transduction reaction. The protein or peptide
can be used at concentrations varying from 50 nM up
 PEPTIDE CARRIERFOR PROTEIN TRANSDUCTION
to 1 btM or higher depending on the biological
response expected.
2. For peptide or low molecular weight protein
(<10 kDa) transduction, the Pep-1 solution must be
diluted 1 : 10 in sterile H 2 0 (concentration: 68 btM). At
this stage, sonication of the peptide solution is recom-
mended, to limit aggregation, for 5 min in a water bath
sonicator. A probe sonicator can also be used: place the
tube in water and sonicate for I min at an amplitude
of 30%. In a tube dilute the appropriate volume of Pep-
1 into 100 btl of sterile water or PBS for each reaction.
For optimal transduction the molar ratio between Pep-
1 and protein or peptide is generally 15 : 1 to 20: 1.
3. Add 100 btl of diluted protein to 100 btl diluted
Pep-1. Mix gently by tapping the tube. It is necessary
to make the Pep-I/protein complex in a concentrated
solution, which will be added to the cells and then be
diluted to the final transduction volume (600 btl for 35-
mm culture plate).
4. Incubate at 37~ for 30 min to allow the Pep-
1/protein complex to form and then proceed immedi-
ately to the transduction experiments. At this stage,
Pep-I/protein complexes should not be stored.
B. Protein Transduction Protocol for
Adherent Cell Lines
The protocol is modified for 35-mm culture plates
according to Morris et al. (2001). The amount of
protein, Pep-l, transduction volume, and number of
cells should be adjusted accordingly to the size of the
culture plate used.
Steps
1. In a 6-well or a 35-mm tissue culture plate, seed
0.3 X 106 cells per well in 2 ml of complete growth
medium. Incubate the cells at 37~ in a humidified
atmosphere containing 5% CO2 until the cells are
50-70% confluent. It is recommended to pass the cells
the day before treatment for a better response follow-
ing transduction.
2. Aspirate the medium from the cells to be
transfected.
3. Wash the cells with PBS.
4. Overlay the cells with the 200 btl Pep-I/protein
complex. Add 400 btl of serum-free medium to the
overlay to achieve the final transduction volume of
600 btl for a 35-mm plate.
5. Incubate at 37~ in a humidified atmosphere
containing 5% CO2 for I h.
6. Add I ml of complete growth medium to the
cells. Do not remove the Pep-I/protein complex. Then
15
incubate at 37~ in a humidified atmosphere contain-
ing 5% CO2 for 30 min to 24 h, depending on the cel-
lular response expected. Peptides and proteins are
fully released in the cells 1 and 2 h later, respectively.
7. Process the cells for observation or detection
assays. Cells may be fixed or observed directly using
live imaging technology.
8. For a 6-well plate, dilute Pep-1 into 600 btl of
water or PBS and the protein into 600 btl of PBS. Mix
the two solutions and then proceed as described in
steps 1-7. For multiple transductions, do not use a mix
that exceeds the volume required for six transductions
per tube, as this may cause aggregation.
C. Protein Transduction Protocol for
Suspension Cells
These conditions were optimized using several
cell lines for the delivery of protein of size ranging
from 5 to 100 kDa. However, efficient transduction
may require optimization of Pep-1 concentration,
cell numbers, and exposure time of cells to the
Pep-i/protein complex.
Steps
1. The same number of cells recommended for
seeding adherent cells is recommended for suspension
cells (confluency between 50 and 70%). Culture cells in
standard medium in 35-mm dishes or 6-well plates.
2. Form the Pep-I/protein or Pep-1/peptide com-
plexes as described for adherent cells steps 1-4 in
section III,A.
3. Collect the suspension cells by centrifugation at
400 g for 5 min. Remove the supernatant.
4. Wash the cells twice with PBS.
5. Centrifuge at 400 g for 5 min to pellet the cells.
Remove the supernatant.
6. Solubilize the cell pellet in the Pep-I/protein or
peptide complex solution (200 btl). Add serum-free
medium to achieve a final transduction volume of
600 btl.
7. Incubate at 37~ in a humidified atmosphere
containing 5% CO2 for I h.
8. Add complete growth medium to the cells. Do
not remove the Pep-I/protein or peptide complex.
Continue to incubate at 37~ in a humidified atmos-
phere containing 5% CO2 for 1 to 24 h depending on
the expected cellular response. As described for adher-
ent cells, peptides and proteins are fully released in the
cells 1 and 2 h later, respectively.
9. Process the cells for observation or detection
assays. Cells may be fixed or observed directly using
live imaging spectroscopy.
16 TRANSFER OF MACROMOLECULES
D. Application: Transduction of ~-
Galactosidase as a Control Protein
Solutions
1. Preparation of ~3-galactosidase: [5-Galactosidase is
provided in a lyophilized form and stored at-20~
Make a stock solution at 0.25 m g / m l in water. Aliquot
into several tubes to avoid repeated freeze/thaw
cycles, taking into account that 0.5-1.0~tg of ~-
galactosidase is used per transduction reaction on
35-mm plates. Store the stock solution at-20~
2. Staining solution: Prepare stock solutions in water.
Mix 400 mM potassium ferricyanide (13.2 g for 100 ml),
400 mM potassium ferrocyanide (16.9 g for 100ml),
200 mM of magnesium chloride (4.6 g for 10 ml), and
20 m g / m l X-Gal resuspended in DMF. Store solutions
at-20~ in the dark. The staining solution should be
prepared freshly: 250 ~tl of 400 mM potassium ferri-
cyanide (4 mM final), 250 ~tl of 400 mM potassium
ferrocyanide (4 mM final), 250 ~tl ml of 200 mM mag-
nesium chloride (2 mM final), 1.25 ml X-Gal (20 m g / m l
in DMF) (1 m g / m l final), and 23 ml of PBS.
3. Fixation solution: Prepare a 10-fold concentrated
solution in PBS (10X) containing 20% formaldehyde
and 2% glutaraldehyde. Store at 20~
1. Transduction Protocol
Steps
1. Dilute 6 ~1 of Pep-1 stock solution (0.68 mM) in
100 ~tl of sterile water.
2. Dilute 0.5 ~tg of [5-Gal in 100 ~tl of PBS.
3. Add the Pep-1 solution to the [5-Gal solution and
mix gently
4. Incubate at 37~ for 30 min to allow the Pep-l/[5-
Gal complex to form.
5. Use the transduction protocol described in
Section III,B (steps 1-7) for adherent cell lines and in
Section III,C (steps 1-8) for suspension cell lines.
2. Staining Protocol
Steps
1. After 2 to 3 h, remove the growth medium from
the transfected cells.
2. Rinse the cells three times with PBS.
3. Add fixing Solution. (Dilute the 10X stock in
sterile water to make a 1X solution.) Incubate at room
temperature for 5 rain and rinse extensively with PBS.
4. Add a freshly made staining solution to the cells.
5. Incubate the cells at 37~ for 30 rain to 2 h. Cells
can be kept overnight at room temperature before
analysis.
6. Analyze the percentage of cells transfected with
~-galactosidase under a microscope.
E. Application: Delivery of Fluorescently
Labelled Antibodies
Pep-1 strategy has been used for the delivery of
antibodies into living cells. The protocol was opti-
mized according to Morris et al. (2001) and to the
Chariot guideline manual (www.activemotif.com).
Steps
1. Detection of fluorescently labelled antibodies in
cells requires a large amount of antibody to be deliv-
ered. Depending on the sensitivity required to detect
your antibody, it may be necessary to increase the
amount of Pep-1 and antibody used. Using fluores-
cently labelled antibodies, concentrations in the final
transduction volume of 0.1 ~tM for antibody and 5 ~tM
for Pep-1 are recommended.
2. Incubate Pep-1 and antibody solution for 30 min
at 37~
3. Overlay onto cultured cells for 1 to 3 h as
described in Section III,C or III,D.
4. Extensively wash and observe cells by fluores-
cence microscopy. There is no need to fix the cells for
observation.
IV. COMMENTS
A large variety of proteins, antibodies, and peptides
have been successfully introduced into different cell
lines using Pep-I/Chariot strategy and have been
shown to retain their biological activity (Morris et al.,
2001; Buster et al., 2002, Jurney et al., 2002; Gallo et al.,
2002; Wu et al., 2002; Pratt and Kinch, 2002; Aoshiba
et al., 2003). An update of published work using
Chariot strategy is available on the Web site of Active
Motif.
The advantages of the Pep-1 technology are directly
associated with the mechanism through which this
carrier promotes the delivery of proteins and peptides
into cells.
a. The fact that Pep-l-mediated protein transduc-
tion is independent of the endosomal pathway signifi-
cantly limits degradation and preserves the biological
activity of the internalized protein for prolonged time
periods.
b. An important property of Pep-l-based protein
delivery is that it allows for the relatively rapid intro-
duction of proteins into cells. That there is no need
of covalent coupling for the formation of Pep-l/
macromolecule particles favours rapid release of pep-
tides or proteins into the cytoplasm as soon as the cell
 PEPTIDE CARRIER FOR PROTEIN TRANSDUCTION
membrane has been crossed. Then the final localization
of the macromolecule is determined by its inherent
intracellular targeting properties. Entry of P e p - l /
macromolecule complexes into the cell occurs in a short
time (10 min), and release of macromolecules varies
from 1 to 3 h, depending on the nature of the protein
and on its affinity for the target (Morris et al., 2001).
c. It is important that the process of complex for-
mation between the Pep-I/protein was performed in
the absence of serum to limit interactions with serum
proteins. However, the transduction process itself is
not affected by the presence of serum, which is a con-
siderable advantage for most biological applications
(Morris et al., 2001).
V. PITFALLS
1. Although this protocol was tested on several
cells lines, conditions for efficient protein transduction
should also be optimized for each cell line, including
reagent concentration, cell number, and exposure time
of cells to the Pepl/macromolecule complexes. [3-
Galactosidase should always be used as a positive
control of transduction.
2. Low protein transduction efficiency may be asso-
ciated with several parameters.
a. For adherent cells, the optimal confluence is
about 50-60%; a higher confluence (90%) re-
duces the transduction efficiency dramatically.
b. Conditions for the formation of Pep-I/macro-
molecule complexes are critical and should be
respected. Special attention should be paid to
the recommended volumes, incubation times
for the formation of Pep-1/macromolecule
complexes, and time of exposure of these com-
plexes to cells.
c. Transduced macromolecules m a y be cyto-
toxic. Transfect the macromolecule at a lower
concentration. Compare untransfected cells,
cells incubated with Pep-1 alone, and cells
with macromolecule alone. Transfect the [3-
galactosidase control protein.
3. Pep-1 interacts via hydrophobic and hydrophilic
interactions. Each peptide or protein will have a dif-
ferent hydrophobicity pattern and may require a dif-
ferent amount of Pep-1. The association of Pep-1 with
proteins depends on the structure and biophysical
properties of both components of the complex, and
thus complex formation may vary between different
combinations of Pep-1 and macromolecules (Morris
et al., 2001). The size of the protein or peptide may
also affect the decaging procedure, and low efficiency
17
may be observed for peptides shorter than 15
residues.
4. Diluting Pep-1 in dimethyl sulfoxide (DMSO)
may improve the delivery of high molecular weight
proteins and antibodies. Pep-1 should first be diluted
in 60% DMSO and then combined with the antibody
or protein dilution. The final concentration of DMSO
should not exceed 20%. However, as this amount of
DMSO may be toxic, it may need to be lowered for
certain cell lines.
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