Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

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
PubMed
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.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Addressing barriers to the prevention, diagnosis and treatment of hepatitis B and C in the face of persisting fiscal constraints in Europe: report from a high level conference.

Journal of viral hepatitis·2016
Same author

Depressive symptoms in chronic hepatitis C are associated with plasma apolipoprotein E deficiency.

Metabolic brain disease·2014
Same author

Successful control of nosocomial transmission of the USA300 clone of community-acquired meticillin-resistant Staphylococcus aureus in a UK paediatric burns centre.

The Journal of hospital infection·2013
Same author

Cerebral microglial activation in patients with hepatitis C: in vivo evidence of neuroinflammation.

Journal of viral hepatitis·2012
Same author

A comparison of 31P magnetic resonance spectroscopy and microbubble-enhanced ultrasound for characterizing hepatitis c-related liver disease.

Journal of viral hepatitis·2011
Same author

The state of hepatitis B and C in Europe: report from the hepatitis B and C summit conference*.

Journal of viral hepatitis·2011

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.

Related Experiment Videos

  • 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.