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 - 15 . (canceled)  
     
     
         16 . An isolated polypeptide consisting of a fragment of a native HCV NS2/3 protease, wherein the N-terminal residue of the fragment is at a position corresponding to a position between positions 815 to 906 in the HCV NS2/3 protease amino acid sequence set forth in SEQ ID No:2.  
     
     
         17 . An isolated polypeptide consisting of a fragment of a native HCV NS2/3 protease, wherein the N-terminal residue of the fragment is at a position corresponding to a position between positions 815 to 906 in the HCV NS2/3 protease amino acid sequence set forth in SEQ ID No:2 and the C-terminal residue of the fragment is at a position corresponding to position 1206 in the HCV NS2/3 protease amino acid sequence set forth in SEQ ID No:2.  
     
     
         18 . An isolated truncated HCV NS2/3 protease consisting of an amino acid sequence from residues 815 to 1206 in the HCV NS2/3 protease amino acid sequence set forth in SEQ ID No:2.  
     
     
         19 . An isolated truncated HCV NS2/3 protease consisting of an amino acid sequence from residues 827 to 1206 in the HCV NS2/3 protease amino acid sequence set forth in SEQ ID No:2.  
     
     
         20 . An isolated truncated HCV NS2/3 protease consisting of an amino acid sequence from residues 855 to 1206 in the HCV NS2/3 protease amino acid sequence set forth in SEQ ID No:2.  
     
     
         21 . An isolated truncated HCV NS2/3 protease consisting of an amino acid sequence from residues 866 to 1206 in the HCV NS2/3 protease amino acid sequence set forth in SEQ ID No:2.