Purified active HCV NS2/3 protease

Abstract

A method for producing a refolded, inactive form of recombinantly produced NS2/3 protease which comprises the steps of: a) purifying the protease from inclusion bodies in the presence of a chaotropic agent; and b) refolding the purified protease by contacting it with a reducing agent and lauryldiethylamine oxide (LDAO) in the presence of reduced concentration of chaotropic agent or polar additive. The invention further comprises a method for activating this refolded inactive NS2/3 protease by adding an activation detergent. This method produces large amounts of the active NS2/3 protease to allow small molecules and ligands to be screened as potential inhibitors of NS2/3 protease, which may be useful as therapeutic agents against HCV.

1 . An isolated polypeptide consisting of a fragment of a native HCV NS2/3 protease precursor, wherein the amino terminus of the fragment is at a position corresponding to a position between positions 904 to 906 in the HCV NS2/3 protease precursor amino acid sequence set forth in SEQ ID NO:10 and the carboxyl terminus of the fragment is at a position corresponding to position 1206 in the HCV NS2/3 protease precursor amino acid sequence set forth in SEQ ID NO:10.  
   
   
       2 . The polypeptide according to  claim 1  where in the N-terminal residue of the fragment is the amino acid that is at position 904 in the HCV NS2/3 protease precursor.  
   
   
       3 . The polypeptide according to  claim 1  wherein the N-terminal residue of the fragment is the amino acid that is at position 906 in the HCV NS2/3 protease precursor.  
   
   
       4 . An isolated polypeptide consisting of an amino acid sequence that has 90% sequence identity over its length compared with a fragment of a native HCV NS2/3 protease precursor, wherein the amino terminus of the fragment is at a position between positions 904 to 906 in the HCV NS2/3 protease precursor amino acid sequence and the carboxyl terminus of the fragment is at position 1206 in the HCV NS2/3 protease precursor amino acid sequence, wherein the HCV NS2/3 protease precursor is encoded by a nucleic acid sequence encoding the HCV polyprotein of the HCV1b 40 strain set forth in SEQ ID NO:10.  
   
   
       5 . The polypeptide comprising the fragment of a native HCV NS2/3 protease precursor according to  claim 1 , and further comprising a fusion of one or more heterologous amino acid residues at either or both of its amino or carboxyl terminal.