Site specific protein modification

Abstract

Processes for conjugating proteins with polyethylene glycol are disclosed. The disclosed processes provide modified proteins having little or no decrease in their activity and include the steps of deleting at least one amino acid residue on the protein, replacing the at least one amino acid residue with an amino acid residue that does not react with polyethylene glycol, and contacting the protein with polyethylene glycol under conditions sufficient to conjugate the polyethylene glycol to the protein. This advantageous retention of a desired protein activity is attributed to the availability of one or more protein binding sites which is unaltered in the conjugation process and thus remains free to interact with a binding partner ligand or cognate subsequent to the conjugation process.

1 - 18 . (canceled)  
     
     
         19 . A pegylated p75 TNFR mutant polypeptide comprising a replacement of the lysine that corresponds to amino acid 108 of SEQ ID NO:8 with an arginine, wherein said pegylated p75 TNFR mutant polypeptide neutralizes TNF-alpha.  
     
     
         20 . The pegylated p75 TNFR mutant polypeptide of claim  1 , further comprising a replacement of the lysine that corresponds to amino acid 120 of SEQ ID NO:8 with an arginine, wherein said pegylated p75 TNFR mutant polypeptide neutralizes TNF-alpha.  
     
     
         21 . A method of neutralizing TNF-alpha comprising contacting TNF-alpha with a pegylated p75 TNFR mutant polypeptide of claim  1  or claim  2 .  
     
     
         22 . A p75 TNFR, the improvement comprising replacement of the lysine that corresponds to amino acid 108 of SEQ ID NO:8 with an arginine.  
     
     
         23 . The p75 TNFR of claim  4 , further comprising replacement of the lysine that corresponds to amino acid 120 of SEQ ID NO:8 with an arginine.  
     
     
         24 . The p75 TNFR of claim  4  or claim  5 , further comprising pegylation of one or more lysine residues.