Stable macroscopic membranes formed by self-assembly of amphiphilic peptides and uses therefor

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) 2 (ARARADAD) 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.

1 . A method for in vitro cell culture comprising: 
 (a) adding a macroscopic membrane which is formed by self-assembly of amphiphilic peptides in an aqueous solution containing monovalent metal cations to a cell 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 greater than 12 amino acids in length, have alternating hydrophobic and hydrophilic amino acids, and are complementary and structurally compatible.  
     
     
         3 . The method of  claim 2  wherein the hydrophilic amino acids are selected from amino acids which can form ionized pairs and amino acids which can form hydrogen bonds.  
     
     
         4 . The method of  claim 1  wherein the peptides are homogeneous.  
     
     
         5 . The method of  claim 1  wherein the macroscopic membrane was formed by self-assembly of a peptide having the sequence (Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys) n , where n is greater than or equal to 2.  
     
     
         6 . The method of  claim 1  wherein the macroscopic membrane formed by a peptide having the sequence (Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala) n , where n is greater than or equal to 2.  
     
     
         7 . The method of  claim 1  wherein the macroscopic membrane is not substantially affected by a condition selected from the group consisting of: 
 a) aqueous solution;    b) serum;    c) ethanol;    d) dilution of the peptide;    e) concentration of the monovalent metal cations;    f) temperature up to 90° C.;    g) pH from 1 to about 11;    h) up to 10% sodium dodecyl sulfate;    i) up to 7 M guanidine hydrochloride;    j) up to 8 M urea; and    k) active protease.    
     
     
         8 . The method of  claim 7  wherein the aqueous solution is selected from water, a salt solution, and tissue culture medium.  
     
     
         9 . The method of  claim 7  wherein the protease is trypsin, α-chymotrypsin, papain, protease K or pronase.  
     
     
         10 . The method of  claim 1  wherein the cells are mammalian cells.  
     
     
         11 . The method of  claim 1  wherein the cells are human cells.  
     
     
         12 . A method for forming a macroscopic membrane comprising combining peptides, which are greater than 12 amino acids in length have alternating nonpolar and hydrophilic amino acids, and are complementary and structurally compatible, with monovalent metal cations in an aqueous solution under conditions suitable for self-assembly of the peptide into the macroscopic membrane.  
     
     
         13 . The method of  claim 12  wherein the peptides are homogeneous.  
     
     
         14 . The method of  claim 13  wherein the peptides have a sequence characterized by (Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys) n , where n is bigger than or equal to 2.  
     
     
         15 . The method of  claim 13  wherein the peptides have a sequence characterized by (Ala-Arg-Ala-Arg-Ala-Asp-Ala-Asp) n , where n is bigger than or equal to 2.  
     
     
         16 . The method of  claim 12  wherein the peptide is chemically synthesized.  
     
     
         17 . The method of  claim 12  wherein the monovalent metal cations are selected from Li + , Na + , and K + .  
     
     
         18 . The method of  claim 12  wherein the peptides are added to an aqueous solution containing the monovalent metal cations.  
     
     
         19 . The method of  claim 18  wherein the aqueous solution is phosphate-buffered saline.  
     
     
         20 . The method of  claim 12  wherein the suitable conditions comprise the absence of an inhibitor of the self-assembly of the peptides into the macroscopic membrane.  
     
     
         21 . The method of  claim 20  wherein the inhibitor is a divalent metal cation.  
     
     
         22 . The method of  claim 20  wherein the inhibitor is sodium dodecyl sulfate.  
     
     
         23 . The method of  claim 12  wherein the suitable conditions comprise a pH of less than 12.  
     
     
         24 . A method for slow-diffusion delivery of a drug comprising administering the drug in a vehicle comprising a macroscopic membrane formed by self-assembly of amphiphilic peptides.  
     
     
         25 . The method of  claim 24  wherein the peptides have, a sequence characterized by (Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys) n , where n is greater than or equal to 2.  
     
     
         26 . The method of  claim 24  wherein the peptides have a sequence characterized by (Ala-Arg-Ala-Arg-Ala-AspAla-Asp) n , where n is greater than or equal to 2.  
     
     
         27 . The method of  claim 24  wherein the drug is administered orally.  
     
     
         28 . A method for identifying a drug which inhibits the self-assembly of amphiphilic peptides into a macroscopic membrane comprising: 
 a) combining a drug with the amphiphilic peptides and monovalent metal cations in an aqueous solution under conditions which would be suitable for self-assembly of the amphiphilic peptides into a macroscopic membrane in the absence of the drug; and    b) detecting decreased membrane formation, wherein decreased membrane formation in the presence of the drug indicates that the drug inhibits the self-assembly of the amphiphilic peptides into a macroscopic membrane.    
     
     
         29 . A drug identified by the method of  claim 28 .  
     
     
         30 . Substantially pure DNA having all or a portion of the nucleotide sequence of  FIG. 12C  (SEQ ID NO: 1).  
     
     
         31 . Substantially pure DNA which encodes a protein having all or a biologically active portion of the amino acid sequence of  FIG. 1  (SEQ ID NO: 2).  
     
     
         32 . Isolated protein having all or a biologically active portion of the amino acid sequence of  FIG. 1  (SEQ ID NO: 2).  
     
     
         33 . A reagent comprising the isolated protein of  claim 32 , which is a substrate for a protein kinase selected from the group consisting of: 
 a) CDC28;    b) casein kinase II;    c) cAMP-dependent protein kinase;    d) tyrosine kinase; and    e) protein kinase C.    
     
     
         34 . A reagent comprising the isolated protein of  claim 32 , which binds left-handed Z-DNA.  
     
     
         35 . The reagent of  claim 34  wherein the protein has a portion of the amino acid sequence of  FIG. 1  (SEQ ID NO: 2) from amino acids 306 to 339.  
     
     
         36 . A reagent which converts B-DNA to Z-DNA comprising a peptide selected from the group consisting of: 
 a) KAKAK (SEQ ID NO: 29);    b) KAK; and    c) KAHAK (SEQ ID NO: 30).    
     
     
         37 . A reagent which converts B-DNA to Z-DNA comprising the peptide KAKAX (SEQ ID NO: 31) where X is any amino acid.