Preparation of Deallergenized Proteins and Permuteins

Abstract

Modified proteins are disclosed that maintain enzymatic and insecticidal activity while displaying reduced or eliminated allergenicity. Epitopes which bind to anti-patatin antibodies were identified, and removed via site directed mutagenesis. Tyrosines were observed to generally contribute to the allergenic properties of patatin proteins. Removal of glycosylation sites was observed to reduce or eliminate antibody binding. Permuteins are also disclosed which have a rearranged amino acid sequence while retaining enzymatic activity. Deallergenized proteins and permuteins can be used as insecticidal materials, as nutritional supplements, and as immunotherapeutic agents.

1 - 53 . (canceled) 
     
     
         54 . A method for decreasing allergen eliciting properties of a native protein comprising the steps of:
 a) identifying a patient exhibiting an allergic sensitivity to said native protein and obtaining serum from said patient;   b) exposing synthetic overlapping peptides representative of said native protein to said patient serum to identify peptides exhibiting epitopes which bind IgE present within said patient serum;   c) producing variant peptides exhibiting alanine scanning or rational scanning amino acid substitutions based on peptides from step (b), wherein said variant peptides exhibit decreased IgE binding compared to peptides from step (b), said amino acid substitutions comprising result effective substitutions;   d) modifying the amino acid sequence of said native protein to contain one or more of said result effective substitutions, thereby obtaining a modified protein; and   e) isolating and purifying the modified protein comprising said one or more result effective amino acid substitutions;   
       wherein the modified protein exhibits decreased allergen eliciting properties of said native protein as determined by reduced binding of IgE when exposed to said patient serum when compared with said native protein. 
     
     
         55 . The method according to  claim 54 , wherein said native protein is selected from the group consisting of SEQ ID NO:6, SEQ ID NO:278, SEQ ID NO:279, SEQ ID NO:280, SEQ ID NO:281, SEQ ID NO:282, SEQ ID NO:286, SEQ ID NO:287, SEQ ID NO:288, SEQ ID NO:289, SEQ ID NO:290, SEQ ID NO:291, SEQ ID NO:292, and SEQ ID NO:293. 
     
     
         56 . A method for producing a modified acyl lipid hydrolase protein comprising:
 a) identifying a patient exhibiting an allergic sensitivity to a native acyl lipid hydrolase protein and obtaining serum from said patient;   b) exposing synthetic overlapping peptides representative of said native acyl lipid hydrolase protein to said patient serum to identify peptides exhibiting epitopes which bind immunoglobulins present within said patient serum, said immunoglobulins exhibiting a binding specificity for said native acyl lipid hydrolase protein;   c) producing variant peptides exhibiting alanine scanning or rational scanning amino acid substitutions based on peptides from step (b), wherein said variant peptides exhibit decreased immunoglobulin binding compared to peptides from step (b), said amino acid substitutions comprising result effective substitutions;   d) modifying the amino acid sequence of said native acyl lipid hydrolase protein to contain one or more of said result effective substitutions, thereby obtaining a modified acyl lipid hydrolase protein; and   e) isolating and purifying the modified acyl lipid hydrolase protein comprising said one or more result effective amino acid substitutions;   
       wherein the modified acyl lipid hydrolase protein exhibits reduced binding of immunoglobulins when exposed to said patient serum when compared with said native acyl lipid hydrolase protein. 
     
     
         57 - 60 . (canceled) 
     
     
         61 . The method according to  claim 56 , wherein said native acyl lipid hydrolase protein is selected from the group consisting of SEQ ID NO:6, SEQ ID NO:278, SEQ ID NO:279, SEQ ID NO:280, SEQ ID NO:281, SEQ ID NO:282, SEQ ID NO:286, SEQ ID NO:287, SEQ ID NO:288, SEQ ID NO:289, SEQ ID NO:290, SEQ ID NO:291, SEQ ID NO:292, and SEQ ID NO:293. 
     
     
         62 . The method according to  claim 54 , wherein said synthetic overlapping peptides are selected from the group consisting of SEQ ID NOs:16-104. 
     
     
         63 . The method according to  claim 56 , wherein said synthetic overlapping peptides are selected from the group consisting of SEQ ID NOs:16-104. 
     
     
         64 . The method according to  claim 54 , wherein said epitopes contain tyrosine and said result effective substitutions include substitutions of alanine or phenylalanine for tyrosine. 
     
     
         65 . The method according to  claim 56 , wherein said epitopes contain tyrosine and said result effective substitutions include substitutions of alanine or phenylalanine for tyrosine. 
     
     
         66 . The method for decreasing allergen eliciting properties of a native protein according to  claim 54 , wherein said modified protein is used in immunotherapy. 
     
     
         67 . The method for producing a modified acyl lipid hydrolase protein according to  claim 56 , wherein said modified acyl lipid hydrolase protein is used in immunotherapy.