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Expression, purification, and partial characterization of HCV RNA polymerase
Z H Yuan1, U Kumar, H C Thomas
1Department of Medicine, St. Mary's Medical School, Imperial College of Science, Technology and Medicine, London, United Kingdom.
Biochemical and Biophysical Research Communications
|March 6, 1997
Summary
Researchers successfully purified Hepatitis C Virus (HCV) NS5B RNA polymerase from Escherichia coli inclusion bodies. The purified enzyme exhibits RNA polymerase activity, crucial for studying viral replication and developing new antiviral drugs.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Hepatitis C Virus (HCV) NS5B protein is essential for viral RNA replication.
- Expression of recombinant HCV NS5B in Escherichia coli often results in insoluble inclusion bodies.
- Efficient purification of active HCV NS5B is critical for biochemical studies and drug discovery.
Purpose of the Study:
- To develop a method for high-level expression and purification of active Hepatitis C Virus NS5B RNA polymerase.
- To characterize the enzymatic activity of the purified HCV NS5B protein.
Main Methods:
- Expressed Hepatitis C Virus NS5B gene in Escherichia coli, yielding both fusion and single proteins.
- Isolated inclusion bodies, followed by solubilization, purification, and refolding of the expressed protein.
- Assayed RNA polymerase activity using polyA/oligoU template, Mg++, and tested for rifampicin resistance.
Main Results:
- Achieved high-level expression of HCV NS5B protein, sequestered in inclusion bodies.
- Developed a successful protocol for purification and refolding, yielding 80-90% pure protein.
- Demonstrated that the purified HCV NS5B possesses RNA polymerase activity, dependent on poly A and Mg++, and resistant to rifampicin.
Conclusions:
- The developed method enables the production of purified, active Hepatitis C Virus NS5B RNA polymerase.
- The availability of this enzyme facilitates further research into HCV replication mechanisms.
- This purified enzyme serves as a basis for screening and developing novel antiviral therapeutics against HCV.