Refolding of the full-length non-structural protein 3 of hepatitis C virus.
An easy and reproducible procedure for purification and refolding of the full-length non-structural protein 3 (NS3) from hepatitis C virus has been developed. Refolding was achieved by simply diluting the protein into a suitable buffer. Low protein concentration, high pH, highly reducing conditions, the presence of detergent, and low viscosity were important parameters for high refolding efficiency. Refolding was insignificantly affected by the presence of Zn(2+) in the refolding buffer, while the addition of NS4A cofactor inhibited refolding. A comparison of the kinetic parameters showed that the refolded enzyme is not as catalytically competent as the native enzyme. Nevertheless, the activity of the refolded NS3 protease was dependent on the specific NS4A-peptide cofactor and was inhibited by the specific substrate-based NS3 protease inhibitor, which indicates that the refolded NS3 can be appropriate for inhibitor screening. The yield of pure protein from the insoluble fraction of cell lysate was 6 mg/L of bacterial culture, which is 18 times higher than obtained from the soluble fraction. Improvement of the refolding conditions has resulted in a 50-fold higher activity of the protease as compared to refolding in buffer with neutral pH and no additives.
Poliakov
Anton
A
Department of Biochemistry, Uppsala University, Box 576, SE-751 23 Uppsala, Sweden.
Danielson
U Helena
UH
D002352:Carrier Proteins / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D015967:Gene Expression Regulation, Viral; D005990:Glycerol / Q000494:pharmacology; D016174:Hepacivirus / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D047908:Intracellular Signaling Peptides and Proteins; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013329:Structure-Activity Relationship; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D014764:Viral Proteins / Q000494:pharmacology; D017967:Zinc Compounds / Q000494:pharmacology; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D002352:Carrier Proteins; D002712:Chlorides; D003902:Detergents; D005990:Glycerol; D047908:Intracellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; D017967:Zinc Compounds; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C455041:NS4A cofactor peptide, Hepatitis C virus; C016837:zinc chloride