EP1929022A2

EP1929022A2 - Method of using pore-forming peptides for genetic transformation, protein extraction, and transmembrane transport

Info

Publication number: EP1929022A2
Application number: EP06790125A
Authority: EP
Inventors: Daniel Yarbrough; Wenyuan Shi; Fengxia Qi
Original assignee: University of California
Current assignee: University of California
Priority date: 2005-09-01
Filing date: 2006-08-30
Publication date: 2008-06-11
Also published as: CA2620935A1; WO2007027947A3; EP1929022A4; WO2007027947A2

Abstract

There are currently few effective approaches for the non-specific transport of molecules across cell membranes. The present application discloses methods of introducing molecules into and extracting molecules out of a cell in a non-specific manner using antimicrobial peptides. Further, the application discloses methods of utilizing antimicrobial peptides to enhance the competence of bacterial cells that have been rendered competent for genetic transformation.

Description

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 of 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) 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 maybe

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. Both 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- 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- [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 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.

[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 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 inhibitor, and/or a compound active in maintaining or altering the redox

[₅9157-8001/LA062₃50.036] -4- 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

In certain embodiments, the buffered solution may include a divalent cation, a

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

selected from the group consisting of calcium, magnesium, or manganese.

[591₅7-8001/LA062₃₅₀.036] -5- BRIEF DESCRIPTION OF THE DRAWINGS [0011] The Y-axis in all plots represents transformation efficiency.

[0012] Figure 1. (A) Transformation of E. coli using GlOKHC peptide. Maximal

increase in transformation efficiency is seen with 0.001 mg/mL GlOKHC 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.

nucleatum. (D) Comparison of various divalent cations in affecting the peptide-

mediated transformation of E. coli. CaCl₂ (shaded bars), MgCl₂ (striped bars), and

MnCl₂(unfilled bars). O.OOlmg/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 PiIA protein by lysis with hen egg white lysozyme (HEWL),

peptide-mediated extraction (GlO 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

GlOKHC or by HEWL lysis and M. xanthus PiIA protein was purified by Ni²⁺-affϊnity

chromatography prior to SDS-PAGE analysis. Despite the presence of protease

inhibitors, the HEWL lysate shows several strong low molecular weight bands

[S9157-8001/LA062350.03<5] -6- corresponding to PiIA 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 competent 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 GlOKHC

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

eukaryotic 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 transformation.

[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- 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 "enhance 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 competent 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.

[59157-8001/LA062350.036] -8- [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 experiment 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

antimicrobial 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.

mutans strain UA159 and F. nucleatum strain ATCC 23726. E. coli cells were

transformed with plasmid pGFPmut3 (Andersen et al. 1998), constitutively expressing

[₅91₅7-8001/LA0623₅0.036] -9- an enhanced variant of the Green Fluorescent Protein (GFP) and conferring

Ampicillin resistance. S. mutans cells were transformed with pVA838 (Macrina et al.

1982) encoding erythromycin resistance, and F. nucleatum cells were transformed

with pHS30 (a gift of Dr. J. Kinder-Haake) encoding thiamphenicol resistance. The

peptides used in this study were: GlOKHC (formerly GlOCatC, Eckert et al., 2006)

(KKHRKHRKHRKHGGSGGSKNLRRIIRKGIHIIKKYG, SEQ ID NO: 1), novispirin

GlO (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 wide variety of bacteria and specifically enhanced activity against

Pseudomonas species (Eckert et al., 2006). Bacterial cells were transformed in the

presence of 0.2-0.3 μg of plasmid DNA, 60 mM CaCl₂, and varying amounts of

peptide. As shown in Figure IA, the presence of 234 nM GlOKHC 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 GlOKHC against E. coli (Eckert et al.,

2006). A similar profile was observed when Streptococcus mutans cells were

transformed in the presence of GlOKHC (not shown), with a more modest 3-fold

increase in transformation efficiency at a peptide concentration of 2.3 μM (Figure

[₅91₅7-8001/LA0623₅0.036] -10- IB). GlOKHC did not greatly enhance the transformation of Fusobacterium

nucleatum (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 other 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 IB and C).

[0022] The requirement for calcium in the transformation solution was also

investigated. Solutions were prepared containing calcium, magnesium, manganese, or

no divalent cation at all. As shown in Figure ID, manganese, calcium and magnesium

have equivalent effects hi 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

[₅91₅7-8Q01/LA062350.036] -1 1- 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 GlOKHC,

GlO, 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 PiIA protein is extremely vulnerable to proteolysis, showing

significant degradation even upon lysis of PilA-expressing cells. Purification of PiIA

extracts was carried out in order to more clearly illustrate this phenomenon: as shown

in figure 2B, PiIA purified from HEWL lysates contained significant contamination

likely corresponding to the N-terminal proteolytic fragment of this protein. PiIA

purified from GlOKHC 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- extraction of soluble heterologous proteins in minutes rather than the hours required

for freeze/thaw methods.

Example 3: Preservation of conventionally prepared 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).

GlOKHC was added to half of the sample at a final 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 μl aliquots and both preparations were

stored at -70⁰C. 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 GlOKHC significantly extends the useful life of conventionally prepared

chemically competent bacterial cells.

[59157-8001/LA062350.036] -13- REFERENCES

Andersen, J. B., C. Sternberg, et al. (1998). Appl Environ Microbiol 64(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). Phvs 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 Biotechnol 21(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 Eng 15(3): 225-32.

Shai, Y. (2002). Biopolymers 66(4): 236-48.

Tossi, A., L. Sandri, et al. (2000). Biopolvmers 55(1): 4-30.

[₅9157-8001/LA0623₅0.036] -14-

Claims

What is claimed is:

1. A method of introducing a molecule into a cell comprising incubating said cell with said molecule in the presence of one or more pore-forming peptides.

2. The method of claim 1 , wherein said cell is prokaryotic.

3. The method of claim 1, wherein said molecule is selected from the group consisting of DNA, protein, peptide, small molecule, or inorganic substance.

4. The method of claim 1, wherein said one or more pore-forming peptides range in length from 3 to 50 amino acids.

5. The method of claim 1 , wherein said one or more pore-forming peptides are added to the incubation mixture in a buffered solution.

6. The method of claim 5, wherein said buffered solution is a saline solution.

7. The method of claim 5, wherein said buffered solution includes one more ingredients selected from the group consisting of one or more divalent cations, one or more protease inhibitors, one or more compounds capable of altering the redox potential of the solution, and one or more compounds capable of maintaining the redox potential of the solution.

8. A method of extracting a molecule from a cell comprising incubating said cell in the presence of one or more pore-forming peptides.

9. The method of claim 8, wherein said cell is prokaryotic.

10. The method of claim 8, wherein said cell is eukaryotic.

[₅9157-8001/LA062350.036] -15-

11. The method of claim 8, wherein said molecule is selected from the group consisting of DNA, protein, peptide, small molecule, or inorganic substance.

12. The method of claim 8, wherein said one or more pore-forming peptides range in length from 3 to 50 amino acids.

13. The method of claim 8, wherein said one or more pore-forming peptides are added to the incubation mixture in a buffered solution.

14. The method of claim 13, wherein said buffered solution is a saline solution.

15. The method of claim 13, wherein said buffered solution includes one more ingredients selected from the group consisting of one or more divalent cations, one or more protease inhibitors, one or more compounds capable of altering the redox potential of the solution, and one or more compounds capable of maintaining the redox potential of the solution.

16. A method of enhancing the competence of a bacterial cell that has been rendered competent for genetic transformation comprising incubating said cell with one or more pore-forming peptides.

17. The method of claim 16, wherein said molecule is selected from the group consisting of DNA, protein, peptide, small molecule, or inorganic substance.

18. The method of claim 16, wherein said one or more pore-forming peptides range in length from 3 to 50 amino acids.

19. The method of claim 16, wherein said one or more pore-forming peptides are added to the incubation mixture in a buffered solution.

[59157-8001/LA062350.036] -16-

20. The method of claim 19, wherein said buffered solution is a saline solution.

21. The method of claim 19, wherein said buffered solution includes one more ingredients selected from the group consisting of one or more divalent cations, one or more protease inhibitors, one or more compounds capable of altering the redox potential of the solution, and one or more compounds capable of maintaining the redox potential of the solution.

22. A kit for introducing a molecule into a cell comprising one or more pore-forming peptides and instructions for use.

23. A kit for extracting a molecule from a cell comprising one or more pore- forming peptides and instructions for use.

24. A kit for enhancing the competence of a bacterial cell that has been rendered competent for genetic transformation comprising one or more pore-forming peptides and instructions for use.

[591₅7-8001/LA0₆₂3₅0.036] -17-