Purification and Analysis of HCV NS3h Helicase
Mentor 1
David N. Frick
Location
Union Wisconsin Room
Start Date
29-4-2016 1:30 PM
End Date
29-4-2016 3:30 PM
Description
The Hepatitis C Virus (HCV) is a small enveloped positive-sense single-stranded RNA virus that infects millions of individuals worldwide. It is a prominent cause of liver cirrhosis, hepatocellular carcinoma, and chronic hepatitis. The blood bourn pathogen encodes a single polyprotein that is cleaved by both host and viral proteases into 10 nonstructural and structural proteins. The third HCV nonstructural protein (NS3) is a complex molecule that has been presumed as essential for viral replication, and has therefore been the target of antiviral therapy for HCV. NS3 contains a helicase whose activity is associated with 450 residues of the NS3 COOH-terminal. To understand the binding and association of NS3h with DNA when the DNA is unwound, the NS3h protein was purified as cyan fluorescent fusion protein (CFP-NS3h). After the purification of the fusion protein, a ATPase assay was used to assess the protein function. The results of the assay showed that the fluorescently marked CFP-NS3h had the same ATPase activity in the presence of PolyU RNA as a unmarked NS3h protein. Finally, FRET assays were performed to determine the binding capacity of the protein to DNA.
Purification and Analysis of HCV NS3h Helicase
Union Wisconsin Room
The Hepatitis C Virus (HCV) is a small enveloped positive-sense single-stranded RNA virus that infects millions of individuals worldwide. It is a prominent cause of liver cirrhosis, hepatocellular carcinoma, and chronic hepatitis. The blood bourn pathogen encodes a single polyprotein that is cleaved by both host and viral proteases into 10 nonstructural and structural proteins. The third HCV nonstructural protein (NS3) is a complex molecule that has been presumed as essential for viral replication, and has therefore been the target of antiviral therapy for HCV. NS3 contains a helicase whose activity is associated with 450 residues of the NS3 COOH-terminal. To understand the binding and association of NS3h with DNA when the DNA is unwound, the NS3h protein was purified as cyan fluorescent fusion protein (CFP-NS3h). After the purification of the fusion protein, a ATPase assay was used to assess the protein function. The results of the assay showed that the fluorescently marked CFP-NS3h had the same ATPase activity in the presence of PolyU RNA as a unmarked NS3h protein. Finally, FRET assays were performed to determine the binding capacity of the protein to DNA.
