CA2620935A1 - Method of using pore-forming peptides for genetic transformation, protein extraction, and transmembrane transport - Google Patents

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 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) 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-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 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-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-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-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 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-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-[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-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] -10-1B). 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-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-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-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-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 Gly Ser--Lys Asn Leu Arg Arg 11e 11e Arg Lys Gly 11e-His-Ile_11e.

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 Tyr Gly

Claims (24)

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.

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, wlierein 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.

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.