Hydrophobically-modified protein compositions and methods

Abstract

Hydrophobically-modified proteins and methods of making them are described. A hydrophobic moiety is attached to a surface amino acid residue of the protein. The hydrophobic moiety can be a lipid or a peptide. Alternatively, the protein can be derivatized by a wide variety of chemical reactions that append a hydrophobic structure to the protein. The preferred protein is of mammalian origin and is selected from the group consisting of Sonic, Indian, and Desert hedgehog. The hydrophobic moiety is used as a convenient tether to which may be attached a vesicle such as a cell membrane, liposome, or micelle.

1 . An isolated protein comprising an N-terminal amino acid and a C-terminal amino acid, wherein the protein is selected from:
 (a) a protein with an N-terminal cysteine that is appended with at least one hydrophobic moiety;   (b) a protein with an N-terminal amino acid that is not a cysteine appended with at least one hydrophobic moiety; or   (c) a protein with at least one hydrophobic moiety substituted for the N-terminal amino acid,   
       wherein the protein is not modified with a sterol. 
     
     
         2 . The protein of  claim 1 , wherein the hydrophobic moiety is a peptide comprising at least one hydrophobic amino acid. 
     
     
         3 . The protein of  claim 1 , wherein the hydrophobic moiety is a lipid. 
     
     
         4 . The protein of  claim 1 , wherein the protein further comprises a hydrophobic moiety substituted for, or appended to, the C-terminal amino acid. 
     
     
         5 . The protein of  claim 1 , wherein the protein is an extracellular signaling protein. 
     
     
         6 . The protein of  claim 1 , wherein the N-terminal amino acid is a functional derivative of a cysteine. 
     
     
         7 . The protein of  claim 1 , wherein the protein is modified at both the N-terminal amino acid and the C-terminal amino acid. 
     
     
         8 . The protein of  claims 4  or  7 , wherein the protein has a hydrophobic moiety substituted for, or appended to, at least one amino acid intermediate to the N-terminal and C-terminal amino acids. 
     
     
         9 . The protein of  claim 1 , wherein the protein has a hydrophobic moiety substituted for, or appended to, at least one amino acid intermediate to the N-terminal and C-terminal amino acids. 
     
     
         10 . The protein of  claim 3 , wherein the lipid moiety is a fatty acid selected from saturated and unsaturated fatty acids having between 2 and 24 carbon atoms. 
     
     
         11 . The protein of  claim 1 , wherein the protein is a hedgehog protein obtainable from a vertebrate source. 
     
     
         12 . The protein of  claim 1 , wherein the protein is a hedgehog protein. 
     
     
         13 . The protein of  claim 11 , wherein the vertebrate hedgehog protein is selected from any of a Sonic hedgehog protein, an Indian hedgehog protein, and a Desert hedgehog protein. 
     
     
         14 . The protein of  claim 1 , further comprising a vesicle in contact with the hydrophobic moiety. 
     
     
         15 . The protein of  claim 14 , wherein the vesicle is selected from the group consisting of a cell membrane, a micelle, and a liposome. 
     
     
         16 . The protein of  claim 12 , wherein the hedgehog protein comprises an amino acid sequence at least 90% identical to the amino acid sequence represented in any of SEQ ID NOS: 1-4. 
     
     
         17 . The protein of  claim 13 , wherein the hedgehog protein is missing between 1 and about 10 amino acids from the C-terminus thereof, when compared to a wild-type hedgehog protein. 
     
     
         18 . The protein of  claim 16 , wherein the hedgehog protein comprises an amino acid sequence at least 95% identical to the amino acid sequence represented in any of SEQ ID NOs: 1-4. 
     
     
         19 - 39 . (canceled) 
     
     
         40 . A method for modifying a physico-chemical property of a protein, comprising introducing at least one hydrophobic moiety to an N-terminal cysteine of the protein or to a functional equivalent of the N-terminal cysteine. 
     
     
         41 . The method of  claim 40 , further comprising contacting the hydrophobic moiety with a vesicle. 
     
     
         42 . The method of  claim 40 , wherein the hydrophobic moiety is either a lipid moiety selected from saturated and an unsaturated fatty acids having between 2 and 24 carbon atoms or is a hydrophobic protein. 
     
     
         43 . The method of  claim 40 , wherein the protein is a hedgehog protein. 
     
     
         44 . The method of  claim 43 , wherein the hedgehog protein is selected from the group consisting of Sonic, Indian and Desert hedgehog. 
     
     
         45 . The method of  claim 43 , wherein the hedgehog has an amino acid sequence according to any one of SEQ ID NOS: 1-4. 
     
     
         46 - 49 . (canceled) 
     
     
         50 . A method for modifying a protein having a biological activity and containing an N-terminal cysteine, comprising reacting the N-terminal cysteine with a fatty acid thioester to form an amide, wherein such modification enhances the protein's biological activity. 
     
     
         51 . The method of  claim 50 , wherein the protein is a hedgehog protein. 
     
     
         52 . The method of  claim 51 , wherein the hedgehog protein is selected from the group consisting of Sonic, Indian, Desert hedgehog, and functional variants thereof. 
     
     
         53 . A method for modifying a protein having a biological activity and containing an N-terminal cysteine, comprising reacting the N-terminal cysteine with a maleimide group, wherein such modification enhances the protein's biological activity. 
     
     
         54 . The method of  claim 53 , wherein the protein is a hedgehog protein. 
     
     
         55 . The method of  claim 54 , wherein the hedgehog protein is selected from the group consisting of Sonic, Indian, Desert hedgehog, and functional variants thereof. 
     
     
         56 . A method for modifying a protein having a biological activity comprising appending an hydrophobic peptide to the protein. 
     
     
         57 . The method of  claim 56 , wherein the hydrophobic peptide is appended to an amino acid of the protein selected from the group consisting of the N-terminal amino acid, the C-terminal amino acid, an amino acid intermediate between the N-terminal amino acid and the C-terminal amino acid, and combinations of the foregoing. 
     
     
         58 . The method of  claim 69 , wherein the protein is a hedgehog protein. 
     
     
         59 . The method of  claim 71 , wherein the hedgehog protein is selected from the group consisting of Sonic, Indian and Desert hedgehog. 
     
     
         60 - 62 . (canceled) 
     
     
         63 . The method of  claim 57 , wherein the step of appending comprises replacing at least the N-terminal amino acid of the protein with at least one hydrophobic amino acid. 
     
     
         64 . The method of  claim 63 , wherein the at least one hydrophobic amino acid is a plurality of isoleucine residues. 
     
     
         65 . The method of  claim 63 , further comprising chemically modifying at least one of the isoleucine residues. 
     
     
         66 - 67 . (canceled) 
     
     
         68 . A method for modifying a protein having a biological activity and containing an N-terminal cysteine, comprising reacting the N-terminal cysteine with a substituted acetamide group, wherein such modification enhances the protein's biological activity. 
     
     
         69 . The method of  claim 68 , wherein the protein is a hedgehog protein. 
     
     
         70 . The method of  claim 69 , wherein the hedgehog protein is selected from the group consisting of Sonic, Indian, Desert hedgehog, and functional variants thereof. 
     
     
         71 . A method for modifying a protein having a biological activity and containing an N-terminal cysteine, comprising reacting the N-terminal cysteine with a haloketone group, wherein such modification enhances the protein's biological activity. 
     
     
         72 - 86 . (canceled) 
     
     
         87 . The protein of  claim 1 , wherein the protein is selected from:
 (a) a protein with an N-terminal amino acid that is not a cysteine appended with at least one hydrophobic moiety; or   (b) a protein with at least one hydrophobic moiety substituted for the N-terminal amino acid,   
       wherein the protein is not modified with a sterol. 
     
     
         88 . The protein of  claim 1 , wherein an amino acid intermediate to the N-terminal and C-terminal amino acids is substituted with a cysteine. 
     
     
         89 . The protein of  claim 88 , further comprising a hydrophobic moiety appended to said cysteine. 
     
     
         90 . The protein of  claim 8 , wherein the at least one amino acid intermediate to the N-terminal and C-terminal amino acids is a cysteine. 
     
     
         91 . The protein of  claim 9 , wherein the at least one amino acid intermediate to the N-terminal and C-terminal amino acids is a cysteine. 
     
     
         92 . The protein of  claim 88 , wherein the protein is a hedgehog protein selected from any of a Sonic hedgehog protein, an Indian hedgehog protein, and a Desert hedgehog protein. 
     
     
         93 . The protein of  claim 89 , wherein the protein is a hedgehog protein selected from any of a Sonic hedgehog protein, an Indian hedgehog protein, and a Desert hedgehog protein. 
     
     
         94 . The protein of  claim 90 , wherein the protein is a hedgehog protein selected from any of a Sonic hedgehog protein, an Indian hedgehog protein, and a Desert hedgehog protein. 
     
     
         95 . The protein of  claim 91 , wherein the protein is a hedgehog protein selected from any of a Sonic hedgehog protein, an Indian hedgehog protein, and a Desert hedgehog protein. 
     
     
         96 . The protein of  claim 92 , wherein the hedgehog protein comprises an amino acid sequence at least 90% identical to the amino acid sequence represented in any of SEQ ID NOs: 1-4. 
     
     
         97 . The protein of  claim 93 , wherein the hedgehog protein comprises an amino acid sequence at least 90% identical to the amino acid sequence represented in any of SEQ ID NOs: 1-4. 
     
     
         98 . The protein of  claim 94 , wherein the hedgehog protein comprises an amino acid sequence at least 90% identical to the amino acid sequence represented in any of SEQ ID NOs: 1-4. 
     
     
         99 . The protein of  claim 95 , wherein the hedgehog protein comprises an amino acid sequence at least 90% identical to the amino acid sequence represented in any of SEQ ID NOs: 1-4. 
     
     
         100 . The protein of  claim 2 , wherein the hydrophobic moiety is a peptide comprising at least two hydrophobic amino acids. 
     
     
         101 . The protein of  claim 2 , wherein the hydrophobic moiety is a peptide consisting of one hydrophobic amino acid. 
     
     
         102 . The protein of  claim 2 , wherein the hydrophobic moiety is a peptide consisting of two hydrophobic amino acids. 
     
     
         103 . The protein of  claim 2 , wherein the peptide comprises an isoleucine. 
     
     
         104 . The protein of  claim 100 , wherein the peptide comprises at least two isoleucines. 
     
     
         105 . The protein of  claim 101 , wherein the hydrophobic amino acid is isoleucine. 
     
     
         106 . The protein of  claim 102 , wherein the hydrophobic amino acids are isoleucine. 
     
     
         107 . The protein of  claim 100 , wherein the protein is a hedgehog protein selected from any of a Sonic hedgehog protein, an Indian hedgehog protein, and a Desert hedgehog protein. 
     
     
         108 . The protein of  claim 107 , wherein the hedgehog protein comprises an amino acid sequence at least 90% identical to the amino acid sequence represented in any of SEQ ID NOs: 1-4. 
     
     
         109 . The protein of  claim 101 , wherein the protein is a hedgehog protein selected from any of a Sonic hedgehog protein, an Indian hedgehog protein, and a Desert hedgehog protein. 
     
     
         110 . The protein of  claim 109 , wherein the hedgehog protein comprises an amino acid sequence at least 90% identical to the amino acid sequence represented in any of SEQ ID NOs: 1-4. 
     
     
         111 . The protein of  claim 102 , wherein the protein is a hedgehog protein selected from any of a Sonic hedgehog protein, an Indian hedgehog protein, and a Desert hedgehog protein. 
     
     
         112 . The protein of  claim 111 , wherein the hedgehog protein comprises an amino acid sequence at least 90% identical to the amino acid sequence represented in any of SEQ ID NOs: 1-4. 
     
     
         113 . The protein of  claim 103 , wherein the protein is a hedgehog protein selected from any of a Sonic hedgehog protein, an Indian hedgehog protein, and a Desert hedgehog protein. 
     
     
         114 . The protein of  claim 113 , wherein the hedgehog protein comprises an amino acid sequence at least 90% identical to the amino acid sequence represented in any of SEQ ID NOs: 1-4. 
     
     
         115 . The protein of  claim 104 , wherein the protein is a hedgehog protein selected from any of a Sonic hedgehog protein, an Indian hedgehog protein, and a Desert hedgehog protein. 
     
     
         116 . The protein of  claim 115 , wherein the hedgehog protein comprises an amino acid sequence at least 90% identical to the amino acid sequence represented in any of SEQ ID NOs: 1-4. 
     
     
         117 . The protein of  claim 105 , wherein the protein is a hedgehog protein selected from any of a Sonic hedgehog protein, an Indian hedgehog protein, and a Desert hedgehog protein. 
     
     
         118 . The protein of  claim 117 , wherein the hedgehog protein comprises an amino acid sequence at least 90% identical to the amino acid sequence represented in any of SEQ ID NOs: 1-4.