| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | September 21, 1999 |
| PATENT TITLE |
Stable macroscopic membranes formed by self-assembly of amphiphilic peptides and uses therefor |
| PATENT ABSTRACT | Described herein is the self-assembly of amphiphilic peptides, i.e., peptides with alternating hydrophobic and hydrophilic residues, into macroscopic membranes. The membrane-forming peptides are greater than 12 amino acids in length, and preferably at least 16 amino acids, are complementary and are structurally compatible. Specifically, two peptides, (AEAEAKAK).sub.2 (ARARADAD).sub.2, were shown to self-assemble into macroscopic membranes. Conditions under which the peptides self-assemble into macroscopic membranes and methods for producing the membranes are also described. The macroscopic membranes have several interesting properties: they are stable in aqueous solution, serum, and ethanol, are highly resistant to heat, alkaline and acidic pH, chemical denaturants, and proteolytic digestion, and are non-cytotoxic. The membranes are potentially useful in biomaterial applications such as slow-diffusion drug delivery systems, artificial skin, and separation matrices, and as experimental models for Alzheimer's disease and scrapie infection. The sequence of the peptide, EAK16, was derived from a putative Z-DNA binding protein from yeast, called zuotin. The cloning and characterization of the ZUO1 gene are also described |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | August 22, 1994 |
| PATENT REFERENCES CITED |
Zhang et al., "Zuotin, a putative Z-DNA binding protein in Saccharomyces cerevisiae", The EMBO J. 11(10):3787-3796 (1992). Osterman, D.G. and Kaiser, E.T., "Design and Characterization of Peptides with Amphiphilic .beta.-Strand Structures", J. Cell. Biochem. 29:57-72 (1985). Brack, A. and Orgel, L.E., ".beta. structures of alternating polypeptides and their possible prebiotic significance", Nature 256:383-387 (1975). Brack, A. and Caille, A., Synthesis and .beta.-Conformation of Copolypeptides with Alternating Hydrophilic and Hydrophobic Residues:, Int. J. Peptide Protein Res. 11:128-139 (1978). Brack, A. and Barbier, B., "Early Peptidic Enzymes", Adv. Spac. Res. 9(6) : (6)83-(6)87 (1989). Marqusee S. and Baldwin, R.L., "Helix stabilization by Glu.sup.- . . . Lys.sup.+ salt bridges in short peptides of de novo design", Proc. Natl. Acad. Sci. USA 84:8898-8902 (1987). Marqusee, S., et al., "Unusually stable helix formation in short alanine-based peptides", Proc. Natl. Acad. Sci. USA 86:5286-5290 (1989). Padmanabhan, S., et al., "Relative helix-forming tendencies of nonpolar amino acids", Nature 344: 268-270 (1991). Seipke, G., et al., "Synthesis and Properties of Alternating Poly(Lys-Phe) and Comparison with the Random Copolymer Poly(Lys.sup.51, Phe.sup.49)", Biopolymers 13:1621-1633 (1974). St. Pierre, S., et al., "Conformational Studies of Sequential Polypeptides Containing Lysine and Tyrosine", Biopolymers 17:1837-1848 (1978). Peggion, E., et al., "Conformational Studies on Polypeptides. The Effect of Sodium Perchlorate on the Conformation of Poly-L-lysine and of Random Copolymers of L-Lysine and L-Phenylalanine in Aqueous Solution", Biopolymers 11:633-643 (1972). Trudelle, Y., "Conformational study of the sequential (Tyr-Glu) .sub..eta. copolymer in aqueous solution", Polymer 16:9-15 (1975). Rippon, W.B., et al., "Spectroscopic Characterization of Poly(Glu-Ala)", J. Mol. Biol. 75:369-375 (1973). Gay, N.J., et al., "A leucine-rich repeat peptide derived from the Drosophila Toll receptor forms extended filaments with a .beta.-sheet structure", FEBS 291:87-91 (1991). Hilbich, C., et al., "Aggregation and Secondary Structure of Synthetic Amyloid .beta.A4 Peptides of Alzheimer's Disease", J. Mol. Biol. 218:149-163 (1991). Halverson, K., "Molecular Determinants of Amyloid Deposition in Alzheimer's Disease: Conformational Studies of Synthetic .beta.-Protein Fragments", Biochemistry 29:2639-2644 (1990). Gasset, M., et al., "Pertubation of the secondary structure of the scrapie prion protein under conditions that alter infectivity", Proc. Natl. Acad. Sci. USA 90:1-5 (1993). Lizardi, P.M., "Genetic Polymorphism of Silk Fibroin Studied by Two-Dimensional Translation Pause Fingerprints", Cell 18:581-589 (1979). Thomas, E.L., "Gigamolecules in Flatland", Science 259:43-45 (1993). Stupp, S.I., "Synthesis of Two-Dimensional Polymers", Science 259:59-63 (1993). Gulik-Krzywicki, T., et al., "Electron microscopic study of supramolecular liquid crystalline polymers formed by molecular recognition-directed self-assembly from complementary chiral components", Proc. Natl. Acad. Sci. USA 90: 163-167 (1993). Smith, Garnet G. and Peck, Garnet E., "Continuous-Flow System for Determination of Diffusion Coefficients: Use of a Natural Membrane," J. of Pharmaceutical Sciences 65(5):727-732 (1976). Physicians' Desk Reference, 47th Edition, Medical Economics Data, Montvale, NJ, p. 2598 (1993). Peppas, Nicholas A. and Langer, Robert, "New Challenges in Biomaterials," Science 263:1715-1720 (1994). Lehn, Jean-Marie, "Self-assembly of double helical, triple helical and deoxyribonucleo-helicate architectures," Chemistry & Biology, Introductory issue, xviii-xix (1994). Frechet, Jean M., "Functional Polymers and Dendrimers: Reactivity, Molecular Architecture, and Interfacial Energy," Science 263:1710-1719 (1994). Clery, Daniel, "After Years in the Dark, Electric Plastic Finally Shines," Science 263:1700-1703 (1994). Hynes, Richard O., "Integrins: Versatility, Modulation, and Signaling in Cell Adhesion," Cell 69:11-25 (1992). Yamada, Kenneth M., "Adhesive Recognition Sequences," J. of Biological Chemistry 266 (20):12809-12812 (1991). Prieto, Anne L. et al., "Multiple integrins mediate cell attachment to cytotactin/tenascin", Proc. Natl. Acad. Sci. USA 90:10154-10158 (1993). Cima, Linda G. and Langer, Robert, "Engineering Human Tissue", Chemical Engineering Progress 89(6): 46-54 (1993). Barrera, Denise et al. "Synthesis and RGD Peptide Modification of a New Biodegradable Copolyner: Poly(lactic acid-co-lysine)," J. Am. Chem. Soc. 115:11010-11011 (1993). Den Dunnen, W. F. A. et al., "A new PLLA/PCL copolymer for nerve regeneration", J. of Materials Science: Materials in Medicine 4:521-525 (1993). Lin, Horng-Ban et al. "Synthesis, surface, and cell-adhesion properties of polyurethanes containing covalently grafted RGD-peptides," J. of Biomedical Materials Research 28:329-342 (1994). Dagani, Ron, "Biodegradable copolymer eyed as tissue matrix," C&EN p. 5 (1993). WPI Accession No. 92-313679/38 (JP 4-221395) (1992). WPI Accession No. 92-313678/38 (JP-4-221394) (1992). |
| PATENT GOVERNMENT INTERESTS |
GOVERNMENT FUNDING This work was supported by Grant No. NIH-5R37-CA04186 from the National Institutes of Health and by Grant No. N00014-90-J-4075 from the Office of Naval Research. The U.S. Government has certain rights in this invention |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
We claim: 1. A method for in vitro cell culture comprising: a) adding a macroscopic membrane which in formed by self-assembly of amphiphilic peptides in an aqueous solution containing monovalent metal cations, wherein the peptides are 12 or more amino acids in length, have alternating hydrophobic and hydrophilic amino acids, and are complementary and structurally compatible, to a call culture medium comprising cells, thereby forming a membrane/culture mixture; b) maintaining the mixture under conditions sufficient for cell growth. 2. The method of claim 1 wherein the peptides are homogeneous. 3. The method of claim 1 wherein the macroscopic membrane was formed by self-assembly of a peptide (residues 310 to 317 of SEQ ID NO:2) having the sequence (Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys).sub.n, where n is greater than or equal to 2. 4. The method of claim 1 wherein the macroscopic membrane was formed by a peptide (residues 1 to 8 of SEQ ID NO:40) having the sequence (Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala).sub.n, where n is greater than or equal to 2. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
BACKGROUND Macroscopic membranes play an important role in many biological processes at both the cellular and organismic level. In addition, membranes are used in a number of medical, research, and industrial applications. Physiologically compatible membranes would be especially valuable for biomedical products. At present, the self-assembly of peptides into macroscopic membranes has not been reported. SUMMARY OF THE INVENTION The invention relates to the discovery that a new class of biomaterials derived from peptides related to the yeast DNA binding protein zuotin. The oligopeptides are generally stable in aqueous solutions and self-assemble into large, extremely stable macroscopic structures or matrices when exposed to physiological levels of salt. The biomaterials are visible to the naked eye when stained with a dye, Congo Red, and can form sheet-like or fibril structures which have high tensile strength. These materials are substantially resistant to change in pH, heat, and enzymatic proteolysis. The biomaterials can have a fibrous microstructure with small pores as revealed by electron microscopy. A small peptide termed EAK16 (AEAEAKAKAEAEAKAK, aa 310-325 of SEQ ID NO: 2) was discovered serendipitously to self-assemble into stable macroscopic membranes and filaments in the presence of millimolar concentrations of salt. This invention relates to the self-assembly of peptides into stable macroscopic membranes and filaments. Peptides which form membranes are characterized as being amphiphilic, e.g., having alternating hydrophobic and hydrophilic amino acid residues; greater than 12 amino acids, and preferably at least 16 amino acids; complementary and structurally compatible. Complementary refers to the ability of the peptides to interact through ionized pairs and/or hydrogen bonds which form between their hydrophilic side-chains, and structurally compatible refers to the ability of complementary peptides to maintain a constant distance between their peptide backbones. Peptides having these properties participate in intermolecular interactions which result in the formation and stabilization of .beta.-sheets at the secondary structure level and interwoven filaments at the tertiary structure level. Both homogeneous and heterogeneous mixtures of peptides characterized by the above-mentioned properties can form stable macroscopic membranes and filaments. Peptides which are self-complementary and self-compatible can form membranes in a homogeneous mixture. Heterogeneous peptides, including those which cannot form membranes in homogeneous solutions, which are complementary and/or structurally compatible with each other can also self-assemble into macroscopic membranes and filaments. Peptides which can self-assemble into macroscopic membranes and filaments, the conditions under which membrane and filament formation occurs, and methods for producing the membranes and filaments are described and included in this invention. Macroscopic membranes and filaments formed of the peptide EAK16 have been found to be stable in aqueous solution, in serum, and in ethanol and are highly resistant to degradation by heat, alkaline and acidic pH (i.e., stable at pH 1.5-11), chemical denaturants (e.g., guanidine-HCl, urea and sodium dodecyl sulfate), and proteases in vitro (e.g., trypsin, .alpha.-chymotrypsin, papain, protease K, and pronase). The membranes and filaments have also been found to be non-cytotoxic. The membranes are thin, transparent and resemble high density felt under high magnification. Being composed primarily of protein, the membranes and filaments can be digested and metabolized in animals and people. They have a simple composition, are permeable, and are easy and relatively inexpensive to produce in large quantities. The membranes and filaments can also be produced and stored in a sterile condition. Thus, the macroscopic membranes and filaments provided by this invention are potentially useful as biomaterial for medical products, as vehicles for slow-diffusion drug delivery, as separation matrices, for supporting in vitro cell attachment and growth, for supporting artificial tissue, e.g., for in vivo use, and for other uses requiring permeable and water-insoluble material. Furthermore, the salt-induced assembly of the peptides into insoluble and protease-resistant protein filaments with a .beta.-sheet secondary structure is similar in some respects to the formation of the neurofibrillary filaments and amyloid plaques associated with Alzheimer's disease and the formation of scrapie prion protein filaments liver cirrhosis, kidney amyloidosis, and other protein confirmational diseases. The formation of the macroscopic membranes and filaments can, therefore, be useful as a model system, e.g. to study these pathological processes. For example, such a model system can be used to identify drugs which inhibit filament formation and are thus useful for treating Alzheimer's disease and scrapie infection. Peptide EAK16 was derived from a region of a yeast protein, zuotin, which exhibits a high affinity for DNA in the left-handed Z conformation. Zuotin was identified by a gel shift assay for Z-DNA binding proteins developed by the Applicants. Applicants further cloned and sequenced the gene encoding zuotin. Characterization of zuotin revealed that the protein is a potential substrate for several protein kinases and identified a putative DNA-binding domain. This invention also includes all or biologically active portions of the zuotin protein and DNA encoding zuotin. |
| PATENT EXAMPLES | This data is not available for free |
| PATENT PHOTOCOPY | Available on request |
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