Note: Descriptions are shown in the official language in which they were submitted.
CA 02620935 2008-02-22
WO 2007/027947 PCT/US2006/034109
METHOD OF USING PORE-FORMING PEPTIDES FOR GENETIC
TRANSFORMATION, PROTEIN EXTRACTION, AND TRANSMEMBRANE
TRANSPORT
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Provisional Application
No.
60/713,941, filed September 1, 2005, the disclosure of which is incorporated
by
reference herein in its entirety.
BACKGROUND
[0002] Antimicrobial peptides (AMP's) comprise a diverse group of small
peptides
that participate in innate immunity in a wide variety of organisms (Reddy et
al. 2004).
Among the best characterized o~these are- the class of amphipathic cationic
peptides
thought to act by destabilizing bacterial membranes in order to disrupt
transmembrane
ion gradients leading to the energy starvation of the cell (Fernandez-Lopez et
al. 2001;
Sal-Man et al. 2002; Shai 2002). These peptides (and others) have attracted
much
interest due to their potential usefulness in treating infections,
particularly since they
are often effective against bacterial strains that have become resistant to
conventional
antibiotics (Reddy et al. 2004).
[0003] Much of the effort in identifying and characterizing antimicrobial
peptides has
been focused on increasing (for example, see (Tossi et al. 2000)) or narrowing
(Qiu et
al. 2003; Balaban et al. 2004) their target specificity, or reducing their
hemolytic
activity (Kondejewski et al. 1996; Giangaspero et al. 2001; Kondejewski et al.
2002)
CA 02620935 2008-02-22
WO 2007/027947 PCT/US2006/034109
in order to enhance their suitability for use as therapeutic agents. It has
been
suggested that the destabilization of bacterial membranes by AMP's leads to
formation of stable pores (Huang et al. 2004). If this is the case, then it
may be
possible to harness this phenomenon to transport particles across the
membrane.
[0004] Very few and limited approaches exist for the general, non-specific
transport
of substances or particles across membranes. Disclosed herein are methods of
using
antimicrobial peptides to mediate the transfer of particles into (in the case
of
transformation of plasmid DNA) or out of (in the case of extraction of
heterologously
expressed proteins) bacterial cells. Botli of these applications are subsets
of a general
mechanism for nonspecific transport of substances or particles across cell
membranes.
Further, the mechanism that underlies pore formation by these compounds allows
normally prepared competent cells to maintain their transformation efficiency
long
after standard (peptide-free) preparations have experienced severe reductions
in their
capacity for DNA uptake, vastly extending the shelf life of competent
bacterial cells, a
critical component of modern biological research.
SUMMARY
[0005] In certain embodiments, methods are provided for introducing a molecule
into
a prokaryotic cell by incubating the cell with the molecule in the presence of
one or
more pore-forming peptides. The types of molecules that may be introduced by
this
method include DNA, proteins, peptides, inorganic substances, or other small
molecules. In certain embodiments, the pore-forming peptide or peptides may
have a
[59157-8001/LA062350.036] -2-
CA 02620935 2008-02-22
WO 2007/027947 PCT/US2006/034109
length of 3 to 50 amino acids. Preferably, the concentration of the peptide or
peptides
present in the incubation mixture is not high enough to result in cell lysis
or death. In
certain embodiments, the peptide may be administered to the incubation mixture
in a
buffered solution, such as for example a saline solution. In certain
embodiments, the
buffered solution may include a divalent cation, a protease inhibitor, and/or
a
compound active in maintaining or altering the redox potential of the
solution. In
certain embodiments, the divalent cation may be selected from the group
consisting of
calcium, magnesium, or manganese.
[0006] In certain embodiments, methods are provided for extracting a molecule
from
a prokaryotic or eukaryotic cell by incubating the cell with one or more pore-
forming
peptides. The types of molecules that may-be extracted by this method include
DNA,
proteins, peptides, inorganic substances, or other small molecules. In certain
embodiments, the pore-forming peptide or peptides may have a length of 3 to 50
amino acids. Preferably, the concentration of the peptide or peptides present
in the
incubation mixture is not high enough to result in cell lysis or death. In
certain
embodiments, the peptide may be administered to the incubation mixture in a
buffered
solution, such as for example a saline solution. In certain embodiments, the
buffered
solution may include a divalent cation, a protease inhibitor, and/or a
compound active
in maintaining or altering the redox potential of the solution. In certain
embodiments,
the divalent cation may be selected from the group consisting of calcium,
magnesium,
or manganese.
[59157-8001/LA062350.036] -3-
CA 02620935 2008-02-22
WO 2007/027947 PCT/US2006/034109
[0007] In certain embodiments, methods are provided for enhancing the
competence
of a bacterial cell that has been rendered competent for genetic
transformation by
incubating the cell with one or more pore-forming peptides. The resultant
enhancement in competence of the cell may be an increase in the transformation
efficiency of the cell, an extension of the time period over which the cell
maintains
competence, or some combination thereof. In certain einbodiments, the pore-
forming
peptide or peptides may have a length of 3 to 50 amino acids. Preferably, the
concentration of the peptide or peptides present in the incubation mixture is
not high
enough to result in cell lysis or death. In certain embodiments, the peptide
may be
administered to the incubation mixture in a buffered solution, such as for
example a
saline solution. In certain embodiments, the buffered solution may include a
divalent
cation, a protease inhibitor, and/or a compound active in maintaining or
altering the
redox potential of the solution. In certain embodiments, the divalent cation
may be
selected from the group consisting of calcium, magnesium, or manganese.
[0008] In certain embodiments, a kit is provided for introducing a molecule
into a
cell. The kit includes one or more pore-forming peptides and instructions for
use. In
certain embodiments, the pore-forming peptide or peptides may be 3 to 50 amino
acids
in lengtli. In certain embodiments, the pore-forming peptide or peptides may
be in a
buffered solution, or a buffered solution may be provided for solubilizing the
peptide.
In certain embodiments, the buffered solution may include a divalent cation, a
protease inhibitor, and/or a conipound active in maintaining or altering the
redox
[59157-80011LA062350.036] -4-
CA 02620935 2008-02-22
WO 2007/027947 PCT/US2006/034109
potential of the solution. In certain embodiments, the divalent cation may be
selected
from the group consisting of calcium, magnesium, or manganese.
[0009] In certain embodiments, a kit is provided for extracting a molecule
from a
cell. The kit includes one or inore pore-forming peptides and instructions for
use. In
certain embodiments, the pore-forming peptide or peptides may be 3 to 50 amino
acids
in length. In certain embodiments, the pore-forming peptide or peptides may be
in a
buffered solution, or a buffered solution may be provided for solubilizing the
peptide.
In certain embodiments, the buffered solution may include a divalent cation, a
protease inllibitor, and/or a compound active in maintaining or altering the
redox
potential of the solution. In certain embodiments, the divalent cation may be
selected
from the group consisting of calcium, magnesium, or manganese.
[0010] In certain embodiments, a kit is provided for enhancing the
transformation
competence of a bacterial cell that has been rendered competent for genetic
transformation. The kit includes one or more pore-forming peptides and
instructions
for use. In certain embodiments, the pore-forming peptide or peptides may be 3
to 50
amino acids in length. In certain embodiments, the pore-forming peptide or
peptides
may be in a buffered solution, or a buffered solution may be provided for
dissolving
the peptide. In certain embodiments, the buffered solution may include a
divalent
cation, a protease inhibitor, and/or a compound active in maintaining or
altering the
redox potential of the solution. In certain embodiments, the divalent cation
may be
selected from the group consisting of calcium, magnesium, or manganese.
[59157-8001/LA062350.036] -5-
CA 02620935 2008-02-22
WO 2007/027947 PCT/US2006/034109
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] The Y-axis in all plots represents transformation efficiency.
[0012] Figure 1. (A) Transformation of E. coli using G10KHC peptide. Maximal
increase in transformation efficiency is seen with 0.001 mg/mL G10KHC peptide.
(B)
Peptide-mediated transformation of multiple bacterial species. All three
species tested
showed at least a 2.5-fold increase in the number of transformants over the
baseline
level. (C) Effectiveness of various peptides in enhancing the transformation
of F.
nucleatuna. (D) Comparison of various divalent cations in affecting the
peptide-
mediated transformation of E. coli. CaC12 (shaded bars), MgC12 (striped bars),
and
MnC12(unfilled bars). 0.001mg/mL peptide causes increased transformation
efficiency with all three cations.
[0013] Figure 2. (A) SDS-PAGE results showing the extraction of the
heterologously
expressed M. xanthus PilA protein by lysis with hen egg white lysozyme (HEWL),
peptide-mediated extraction (G10 and GlOKHC), and Freeze/Thaw extraction
(F/T).
Lane marked "Pellet" contains whole cell extracts. Note that HEWL lysis
results in
release of large amounts of additional proteins while the purity of the
peptide-
extracted samples is much higher. (B) SDS-PAGE results showing residual
proteolytic activity in HEWL lysates. Extracts were prepared either by
extraction with
G10KHC or by HEWL lysis and M. xanthus PilA protein was purified by Ni2+-
affmity
chromatography prior to SDS-PAGE analysis. Despite the presence of protease
inhibitors, the HEWL lysate shows several strong low molecular weight bands
[59157-8001/LA062350.036] -6-
CA 02620935 2008-02-22
WO 2007/027947 PCT/US2006/034109
corresponding to Pi1A proteolysis products, while the protein from the GlOKHC
extract remains mostly intact.
[0014] Figure 3. Comparison of the loss of transformation efficiency in
normally
prepared coinpetent cells over time. Over the course of 12 months, peptide-
free
preparations of chemically competent E. coli JM- 109 cells experience a 2-3
fold loss
of transformation efficiency (unfilled bars). Identically prepared cells with
G10KHC
peptide included experience no loss of transformation efficiency after 12
months
(shaded bars). '
DETAILED DESCRIPTION
[0015] Provided herein are methods of using pore-forming antimicrobial
peptides to
deliver molecules to a prokaryotic cell or extract molecules from a
prokaryotic or
eulcaryotic cell. Molecules that may be delivered or extracted using these
methods
include proteins, peptides, nucleic acids, small molecules and/or inorganic
substances.
Also provided herein are methods of using one or more pore-forming peptides to
enhance the competence of a bacterial cell that has been rendered competent
for
genetic transformation. In addition, kits are provided that include one or
more pore-
forming peptides for use in delivering a molecule to a prokaryotic cell,
extracting a
molecule from a prokaryotic or eukaryotic cell, or enhancing the competence of
a
bacterial cell that has been rendered competent for genetic transforma.tion.
[0016] A"pore-forming peptide" as used herein refers to any peptide capable of
causing leakage of cellular membranes. The ability of a peptide to cause
leakage of a
[59157-8001/LA062350.036] -7-
CA 02620935 2008-02-22
WO 2007/027947 PCT/US2006/034109
cellular membrane may be determined by, for example, by observing and/or
measuring the leakage of fluorescent dyes or colorimetric dyes from
artificially
formed lipid or phospholipid vesicles (Haas et al. 2004).
[0017] To "eiihance the competence of a bacterial cell that has been rendered
competent for genetic transformation" as used herein refers to increasing the
transformation capability of the cell, the timeframe over which the cell is
competent
for transformation, or both. The enhancement in competence may be carried out
after
the cell has been made coinpetent for genetic transformation, or may be
carried out
simultaneously with the process of rendering the cell competent.
[0018] The formulations disclosed herein may include appropriate buffers and
__ additional factors that may_be necessary for the stability of the particles
or substances
to be transferred across the membrane, such as a stabilization buffer in the
case of
protein extraction, or a divalent cation in the case of genetic transformation
with
plasmid DNA. Destabilization of the membrane by the pore-forming peptide
allows
the formation of pores (Huang et al. 2004), through which molecules or
substances
may be transferred. For example, pore formation facilitates the leakage of
bacterially
expressed soluble protein into the surrounding medium without requiring cell
lysis,
while insoluble protein and most cellular proteins remain in the cell and are
removed
by centrifugation. In another example, pore formation facilitates the transfer
of DNA
from the surrounding medium into the interior of bacterial cells, causing
genetic
transformation.
[59 1 57-800 1/LA062350.036] -8-
CA 02620935 2008-02-22
WO 2007/027947 PCT/US2006/034109
[0019] The methods disclosed herein eliminate several tedious and time-
consuming
steps in the protein purification process. By selectively allowing extraction
of soluble
proteins, it simultaneously allows for extraction and partial purification of
desired
proteins. Since many native proteolytic enzymes are retained within the cell,
degradation of protein samples by these enzymes is reduced. Destabilization of
bacterial cell membranes by repeated cycles of freezing and thawing has been
shown
to cause a similar extraction (Johnson et al. 1994), but involves multiple
steps, is more
time consuming, and shows less consistent results from experinient to
experiment than
the proposed method. The methods disclosed herein also allow the elimination
of
several time-consuming steps in the genetic transformation process, and make
it
possible to_ transform species of bacteria other than common laboratory
strains, in
which transformation protocols have not yet been developed. Further, addition
of
antiinicrobial peptides to conventionally prepared chemically competent
bacterial cells
prevents the natural loss of transformation efficiency that occurs over time
during
storage of these cells. This method also allows the transport across membranes
of any
substance or soluble particle of appropriate size without causing cell death,
an
application for which there are few acceptable methods or reagents currently
in use.
Examples
[0020] Transformation experiments were carried out using E. coli strain JM-
109, S.
nautans strain UA159 and F. nucleatuna strain ATCC 23726. E. coli cells were
transformed with plasmid pGFPmut3 (Andersen et al. 1998), constitutively
expressing
[59 1 57-8 00 1/LA062350.036] -9-
CA 02620935 2008-02-22
WO 2007/027947 PCT/US2006/034109
an enhanced variant of the Green Fluorescent Protein (GFP) and conferring
Ampicillin resistance. S. inutans cells were transformed with pVA838 (Macrina
et al.
1982) encoding erythromycin resistance, and F. nucleatunz cells were
transformed
witli pHS30 (a gift of Dr. J. Kinder-Haake) encoding thiamphenicol resistance.
The
peptides used in this study were: G10KHC (formerly G10CatC, Eckert et al.,
2006)
(KKHRKHRKHRKHGGSGGSKNLRRIIRKGIHIIKKYG, SEQ ID NO:1), novispirin
Gl0 (Sawai et al. 2002) (KNLRRIIRKGIHIIKKYG, SEQ ID NO:2), #48 (J. He,
unpublished), and PL-135 (R. Lehrer, unpublished).
Example 1: Enhancement of Bacterial Transformation Efficiency by AMP's:
[0021] GlOKHC is an engineered antimicrobial peptide with moderate activity
against-a widevariety of bacteria and specifical-ly enhanced activity against
Pseudomonas species (Eckert et al., 2006). Bacterial cells were transformed in
the
presence of 0.2-0.3 g of plasmid DNA, 60 mM CaC12, and varying amounts of
peptide. As shown in Figure 1A, the presence of 234 nM GIOKHC peptide induced
a
25-fold increase in the transformation efficiency of E. coli, while lower
concentrations
were less effective at promoting transformation. Higher concentrations were
also less
effective, probably due to reduced cell survival as the concentration neared 5
mg/mL,
previously determined to be above the MIC for G10KHC against E. coli (Eckert
et al.,
2006). A similar profile was observed when Streptococcus mutans cells were
transformed in the presence of G10KHC (not shown), with a more modest 3-fold
increase in transformation efficiency at a peptide concentration of 2.3 .M
(Figure
[59157-8001/LA062350.036] -10-
CA 02620935 2008-02-22
WO 2007/027947 PCT/US2006/034109
1B). G10KHC did not greatly enhance the transformation of Fusobacterium
nucleatuin (a bacterium that is extraordinarily difficult to transform using
conventional methods), leading only to a 2-fold increase in transformation
efficiency
(data not shown), so the effectiveness of otlzer peptides in transforming this
bacterium
was investigated. The peptide PL-135 was found to be ineffective in enhancing
transformation of F. nucleatum due to its pronounced toxicity against this
organism,
while peptide #48 was found to cause a 5-fold enhancement in transformation
efficiency (Figures 1B and C).
[0022] The requirement for calcium in the transfomlation solution was also
investigated. Solutions were prepared containing calcium, magnesium,
manganese, or
no divalent-cation at all. As shown in Figure 1D; manganese, calcium and
magnesium
have equivalent effects in promoting transformation of E. coli, while samples
containing no divalent cations yielded no transformants. This may reflect the
need to
neutralize charge repulsion between the heavily negatively charged DNA
molecules
and negatively charged bacterial surfaces or may be a result of more complex
interactions between divalent cations and bacterial membrane components (for
example, see (Huang et al. 1995)).
Example 2: Extraction of Heterologously Expressed Proteins by AMP's:
[0023] In order to determine whether peptide-induced membrane destabilization
could be useful in the extraction and purification of heterologously expressed
proteins,
E. coli cells expressing the PiIA protein from Myxococcus xanthus were
subjected to
[59157-8001/LA062350.036] -11-
CA 02620935 2008-02-22
WO 2007/027947 PCT/US2006/034109
sub-lethal concentrations of antimicrobial peptides. Figure 2A shows a
comparison of
this technique with lysis by hen egg white lysozyme (HEWL) and freeze-thaw
extraction: HEWL lysis lead to a release of large amounts of protein as well
as
miscellaneous cell contents, while extracts from cells treated with 1 mg/mL
G10KHC,
G10, or a single freeze-thaw cycle showed significant amounts of protein at a
much
higher level of purity. Thus, the use of antimicrobial peptides not only
allows
extraction of heterologously expressed proteins, but can facilitate the
purification
process as well: given the level of purity seen in these samples, for some
applications
further purification may not be necessary. Further, the cells used to produce
the
protein were not killed by the peptide treatment and could be readily grown on
-selective medium following extraction (data not shown). This was not the case
with
cells subjected to HEWL lysis or freeze-thaw extraction.
[0024] The M. xanthus PilA protein is extremely vulnerable to proteolysis,
showing
significant degradation even upon lysis of PiIA-expressing cells. Purification
of PilA
extracts was carried out in order to more clearly illustrate this phenomenon:
as shown
in figure 2B, PilA purified from HEWL lysates contained significant
contamination
likely corresponding to the N-terminal proteolytic fragment of this protein.
PilA
purified from G10KHC extracts were nearly free of this problem.
[0025] Somewhat similar results can often be obtained by repeated cycles of
freezing
and thawing of the cells, though this method is time consuming and sometimes
gives
irregular results. The peptide-based extraction protocol used here provides
consistent
[59157-8001/LA062350.036] -12-
CA 02620935 2008-02-22
WO 2007/027947 PCT/US2006/034109
extraction of soluble heterologous proteins in minutes rather than the hours
required
for freeze/thaw methods.
Example 3: Preservation of conventionally ~repared chemically competent cells
by
AMP's:
[0026] To examine the effect of AMP's on the competence of conventionally
prepared high-efficiency chemically competent cells, a sample of E. coli
Strain JM-
109 cells was prepared according to standard techniques (Ausubel et al. 1997).
G l OKHC was added to half of the sample at a fmal concentration of 0.001
mg/mL,
while an equal volume of buffer (described in Ausubel et al. 1997) was added
to the
other half. Samples were divided into 100 gl aliquots and both preparations
were
stored at -70 C. Test transformations were carried out,- showing no initial
difference
in transformation efficiency between the peptide treated and untreated
samples,
consistent with similar experiments carried out both previously and
subsequently.
After 12 months, transformation efficiency was compared again, showing a 2-3
fold
decrease in the transformation efficiency of the untreated cells relative to
the peptide-
treated cells (see figure 3). The peptide-treated cells showed essentially no
change in
transformation efficiency after 12 months of storage. Thus, the addition of
0.001
mg/mL G10KHC significantly extends the useful life of conventionally prepared
chemically competent bacterial cells.
[59157-8001[LA062350.036] -13-
CA 02620935 2008-02-22
WO 2007/027947 PCT/US2006/034109
REFERENCES
Andersen, J. B., C. Sternberg, et al. (1998). App1 Environ Microbio164(6):
2240-6.
Ausubel, F. M., R. Brent, et al., Eds. (1997). Current Protocols in Molecular
Biology,
John Wiley & Sons.
Balaban, N., Y. Gov, et al. (2004). Antimicrob Agents Chemother 48(7): 2544-
50.
Eckert, R., F. Qi, et al. (2006). Antimicrob Agents Chemother 50(4): 1480-8.
Fernandez-Lopez, S., H. S. Kim, et al. (2001). Nature 412(6845): 452-5.
Giangaspero, A., L. Sandri, et al. (2001). Eur J Biochem 268(21): 5589-600.
Haas, D. H. and R. M. Murphy (2004). J Pept Res 63(1): 9-16.
Huang, H. W., F. Y. Chen, et al. (2004). Phys Rev Lett 92(19): 198304.
Huang, R. and R. N. Reusch (1995). J Bacteriol 177(2): 486-90.
Johnson, B. H. and M. H. Hecht (1994). Biotechnology (N Y) 12(13): 1357-60.
Kondejewski, L. H., S. W. Farmer, et al. (1996). J Biol Chem 271(41): 25261-8.
Kondejewski, L. H., D. L. Lee, et al. (2002). J Biol Chem 277(1): 67-74.
Macrina, F. L., J. A. Tobian, et al. (1982). Gene 19(3): 345-53.
Qiu, X. Q., H. Wang, et al. (2003). Nat Biotechno121(12): 1480-5.
Reddy, K. V., R. D. Yedery, et al. (2004). Int J Antimicrob Agents 24(6): 536-
47.
Sal-Man, N., Z. Oren, et al. (2002). Biochemistry 41(39): 11921-30.
Sawai, M. V., A. J. Waring, et al. (2002). Protein En~ 15(3): 225-32.
Shai, Y. (2002). Biopolymers 66(4): 236-48.
Tossi, A., L. Sandri, et al. (2000). Biopolymers 55(1): 4-30.
[59157-8001/LA062350.036] -14-
CA 02620935 2008-02-22
WO 2007/027947 PCT/US2006/034109
wo00 sequence listing.txt
SEQUENCE LISTING
<110> The Regents of the university of California
Yarbrough, Daniel K.
shi, wenyuan
Qi, Fengxia
<120> Method of using pore-forming peptides for genetic transformation,
protein extraction, and transmembrane transport
<130> 59157.8001.W000
<150> US 60/713,941
<151> 2005-09-01
<160> 2
<170> Patentin version 3.3
<210> 1
<211> 36
<212> PRT
<213> Artificial
<220>
<223> chemically synthesized peptide
<400> 1
Lys Lys His Arg Lys His Arg Lys His Arg Lys His Gly Gly Ser Gly
1 5 10 15
Gly Ser--Lys Asn Leu Arg Arg 11e 11e Arg Lys Gly 11e-His-Ile_11e.
20 25 30
Lys Lys Tyr Gly
<210> 2
<211> 18
<212> PRT
<213> Artificial
<220>
<223> chemically synthesized peptide
<400> 2
Lys Asn Leu Arg Arg Ile Ile Arg Lys Gly Ile His Ile Ile Lys Lys
1 5 10 15
Tyr Gly
Page 1
