Method of cross-linking peptides

Abstract

A method of cross-linking a peptide to form a homopolymer of the peptide, to immobilize the peptide on a solid phase and to enhance antigenicity of the peptide is disclosed. The method comprises the steps of preparing a fusion peptide by incorporating a cross-linking segment including a tetrapeptide sequence QXK(S/T) into the peptide and cross-linking the peptide by a glutaminase.

We claim:  
     
         1 . A method of cross-linking a peptide to form polymers of said peptide, comprising the steps of 
 (a) incorporating a cross-linking segment containing a Q-X-K-(S/T) sequence into said peptide to form a fusion polypeptide, wherein Q is glutamine, X is an amino acid having an aliphatic side chain, K is lysine and (S/T) is either serine or threonine; and    (b) cross-linking said fusion polypeptide by a transglutaminase in a cross-linking reaction buffer to form said polymers.    
     
     
         2 . The method of  claim 1 , wherein said cross-linking segment comprises at least two tandem repeats of said Q-X-K-(S/T) sequence.  
     
     
         3 . The method of  claim 2 , wherein said cross-linking segment comprises SEQ ID No: 3.  
     
     
         4 . The method of  claim 1 , wherein said Q-X-K-(S/T) sequence is SEQ ID No: 2.  
     
     
         5 . The method of  claim 1 , wherein said transglutaminase is from mouse coagulating gland fluid.  
     
     
         6 . The method of  claim 1 , wherein said transglutaminase is from human blood factor XIII.  
     
     
         7 . The method of  claim 1 , wherein said transglutaminase is guinea pig liver transglutaminase.  
     
     
         8 . A method of cross-linking a peptide to form polymers of said peptide, comprising the steps of incorporating a cross-linking segment containing SEQ ID No: 4 into said peptide to form a fusion polypeptide and cross-linking said fusion polypeptide by a transglutaminase in a cross-linking reaction buffer to form said polymers.  
     
     
         9 . A method of immobilize a peptide on a solid phase having a surface containing a primary amine moiety, comprising the steps of 
 (a) preparing a fusion polypeptide by incorporating a cross-linking segment containing a Q-X-K-(S/T) sequence into said peptide, wherein Q is glutamine, X is an amino acid having an aliphatic side chain, K is lysine and (S/T) is either serine or threonine; and    (b) cross-linking said fusion polypeptide with the surface of said solid phase in the presence of a cross-linking buffer by a transglutaminase to immobilize said fusion polypeptide.    
     
     
         10 . The method of  claim 9 , wherein said cross-linking segment comprises at least two tandem repeats of said Q-X-K-(S/T) sequence.  
     
     
         11 . The method of  claim 10 , wherein said cross-linking segment comprises SEQ ID No: 3.  
     
     
         12 . The method of  claim 9 , wherein said Q-X-K-(S/T) sequence is SEQ ID No: 2.  
     
     
         13 . The method of  claim 9 , wherein said transglutaminase is from mouse coagulating gland fluid.  
     
     
         14 . The method of  claim 9 , wherein said transglutaminase is from human blood factor XIII.  
     
     
         15 . The method of  claim 9 , wherein said transglutaminase is guinea pig liver transglutaminase.  
     
     
         16 . A method of enhancing antigenicity of a peptide, comprising the steps of 
 (a) preparing a fusion polypeptide by incorporating a cross-linking segment containing a Q-X-K-(S/T) sequence into said peptide, wherein Q is glutamine, X is an amino acid having an aliphatic side chain, K is lysine and (S/T) is either serine or threonine;    (b) cross-linking said fusion polypeptide by a transglutaminase in a cross-linking buffer to form polymers of said peptide; and    (c) immunizing an animal by injecting said animal with said polymers of said peptide to produce an antibody against said peptide.    
     
     
         17 . The method of  claim 16 , wherein said cross-linking segment comprises at least two tandem repeats of said Q-X-K-(S/T) sequence.  
     
     
         18 . The method of  claim 17 , wherein said cross-linking segment comprises SEQ ID No: 3.  
     
     
         19 . The method of  claim 16 , wherein said Q-X-K-(S/T) sequence is SEQ ID No: 2.  
     
     
         20 . The method of  claim 16 , wherein said transglutaminase is from mouse coagulating gland fluid.  
     
     
         21 . The method of  claim 16 , wherein said transglutaminase is from human blood factor XIII.  
     
     
         22 . The method of  claim 16 , wherein said transglutaminase is guinea pig liver transglutaminase.  
     
     
         23 . A peptide for cross-linking, comprising a -QX- sequence and a -XK(S/T) sequence, wherein Q is glutamine, X is an amino acid having an aliphatic side chain, K is lysine and (S/T) is either serine or threonine, said -QK- sequence and said -XK(S/T) sequence being spaced apart by at most 25 amino acid residues.  
     
     
         24 . The peptide of  claim 23  having a sequence identical to SEQ ID No: 4.  
     
     
         25 . A fusion polypeptide, comprising a cross-linking segment containing a Q-X-K-(S/T) sequence, wherein Q is glutamine, X is an amino acid having an aliphatic side chain, K is lysine and (S/T) is either serine or threonine.  
     
     
         26 . The fusion polypeptide of  claim 25 , wherein said cross-linking segment comprises at least two tandem repeats of said Q-X-K-(S/T) sequence.  
     
     
         27 . The fusion polypeptide of  claim 26 , wherein said cross-linking segment comprises SEQ ID No: 3.  
     
     
         28 . The fusion polypeptide of  claim 25 , wherein said Q-X-K-(S/T) sequence is SEQ ID No: 2.  
     
     
         29 . A fusion polypeptide, comprising a cross-linking segment containing a -QX- sequence and a -XK(S/T)- sequence, wherein Q is glutamine, X is an amino acid having an aliphatic side chain, K is lysine and (S/T) is either serine or threonine, said -QK- sequence and said -XK(S/T) sequence being spaced apart by at most 25 amino acid residues  
     
     
         30 . The fusion peptide of  claim 23 , wherein said cross-linking segment has a sequence identical to SEQ ID No: 4.