Method for stabilization of proteins using non-natural amino acids

Abstract

The present invention provides a method for producing modified stable polypeptides introducing at least one non-natural amino acid into the hydrophobic region of the polypeptide. The thermal and chemical stability of such polypeptides is improved compared to those properties of its corresponding wild type proteins. The invention further provides purified leucine zipper and coiled-coil proteins in which the leucine residues have been replaced with 5,5,5-trifluoroleucines, and the modified proteins so produced demonstrate increased thermal and chemical stability compared to their corresponding wild-type natural proteins.

1 .- 14 . (canceled) 
     
     
         15 . An artificial polypeptide, comprising a coiled coil structure wherein at least one hydrophobic amino acid residue in a hydrophobic core region of an α-helix of the coiled coil structure is substituted with a hydrophobic non-natural amino acid selected from 5,5,5-trifluoroleucine, 5,5,5,5′,5′,5′-hexafluoroleucine, 2-amino-4-methyl-4-pentenoic acid, 2-amino-3,3,3-trifluoro-methylpentanoic acid, 2-amino-3-methyl-5,5,5-tri-fluoropentanoic acid, 2-amino-3-methyl-4-pentenoic acid, trifluorovaline, or hexafluorovaline, and wherein the artificial polypeptide retains activity and exhibits a higher ratio of folded to unfolded polypeptide as compared to its corresponding wild type polypeptide under denaturing conditions. 
     
     
         16 . The artificial polypeptide according to  claim 15 , wherein the at least one amino acid residue is substituted with a non-natural amino acid selected from 5,5,5-trifluoroleucine or 5,5,5,5′,5′,5′-hexafluoroleucine. 
     
     
         17 . The artificial polypeptide according to  claim 15 , wherein the at least one amino acid residue is substituted with a non-natural amino acid selected from 2-amino-4-methyl-4-pentenoic acid, 2-amino-3,3,3-trifluoro-methylpentanoic acid, 2-amino-3-methyl-5,5,5-tri-fluoropentanoic acid, or 2-amino-3-methyl-4-pentenoic acid. 
     
     
         18 . The artificial polypeptide according to  claim 15 , wherein the at least one amino acid residue is replaced with a non-natural amino acid selected from trifluorovaline or hexafluorovaline. 
     
     
         19 . The artificial polypeptide according to  claim 15 , wherein the non-natural amino acid is more hydrophobic than the amino acid residue that is substituted. 
     
     
         20 . The artificial polypeptide according to  claim 15 , wherein the substituted amino acid residue is leucine, isoleucine, or valine. 
     
     
         21 . The artificial polypeptide according to  claim 16 , wherein the substituted amino acid residue is leucine. 
     
     
         22 . The artificial polypeptide according to  claim 17 , wherein the substituted amino acid residue is isoleucine. 
     
     
         23 . The artificial polypeptide according to  claim 15 , wherein the denaturing condition is elevated temperature, a denaturing chemical, extreme solution pH, a non-physiological environment, or any combination thereof. 
     
     
         24 . The artificial polypeptide according to  claim 15 , wherein the substituted amino acid residue is at a d-position of the α-helix structure of the artificial polypeptide. 
     
     
         25 . The artificial polypeptide according to  claim 15 , wherein the substituted wild type protein is a leucine zipper protein, a membrane protein, a cytokine, or an enzyme. 
     
     
         26 . A method for increasing polypeptide stability, the method comprising substituting at least one hydrophobic amino acid residue in a hydrophobic core region of an α-helix of a coiled coil structure of the polypeptide with a hydrophobic non-natural amino acid selected from 5,5,5-trifluoroleucine, 5,5,5,5′,5′,5′-hexafluoroleucine, 2-amino-4-methyl-4-pentenoic acid, 2-amino-3,3,3-trifluoro-methylpentanoic acid, 2-amino-3-methyl-5,5,5-tri-fluoropentanoic acid, 2-amino-3-methyl-4-pentenoic acid, trifluorovaline, or hexafluorovaline, thereby producing an artificial polypeptide that retains activity and has increased stability relative to its corresponding wild type polypeptide under denaturing conditions. 
     
     
         27 . The method according to  claim 26 , wherein the non-natural amino acid is 5,5,5-trifluoroleucine or 5,5,5,5′,5′,5′-hexafluoroleucine. 
     
     
         28 . The method according to  claim 26 , wherein the non-natural amino acid is 2-amino-4-methyl-4-pentenoic acid, 2-amino-3,3,3-trifluoro-methylpentanoic acid, 2-amino-3-methyl-5,5,5-tri-fluoropentanoic acid, or 2-amino-3-methyl-4-pentenoic acid. 
     
     
         29 . The method according to  claim 26 , wherein the non-natural amino acid is trifluorovaline or hexafluorovaline. 
     
     
         30 . The method according to  claim 26 , wherein the denaturing condition is elevated temperature, a denaturing chemical, extreme solution pH, a non-physiological environment, or any combination thereof. 
     
     
         31 . The method according to  claim 26 , wherein the substituted amino acid residue is at a d-position of the α-helix structure of the artificial polypeptide. 
     
     
         32 . The method according to  claim 26 , wherein the substituted wild type protein is a leucine zipper protein, a membrane protein, a cytokine, or an enzyme.