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Related Experiment Videos

Proteinase trapping: screening for viral proteinase mutants by alpha complementation.

H D Liebig1, T Skern, M Luderer

  • 1Institute of Biochemistry, University of Vienna, Austria.

Proceedings of the National Academy of Sciences of the United States of America
|July 15, 1991
PubMed
Summary

Researchers developed a novel in vivo screening system to identify active viral proteinase mutants. This system efficiently detects both intramolecular and intermolecular cleavage activities, applicable to various specific proteinases.

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Area of Science:

  • Molecular Biology
  • Virology
  • Biochemistry

Background:

  • Viral polyproteins are often processed by self-cleaving proteinases.
  • Understanding proteinase activity is crucial for viral replication studies.

Purpose of the Study:

  • To develop a versatile in vivo screening system for viral proteinase activity.
  • To enable high-density screening of both active and inactive proteinase mutants.

Main Methods:

  • Utilized a lacZ gene fragment from M13mp18 for an in vivo assay.
  • Employed alpha complementation of beta-galactosidase for screening.
  • Introduced random mutations into the 2A proteinase gene via PCR amplification.

Main Results:

  • Achieved a 5% efficiency in obtaining M13 phage defective in alpha complementation.

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  • Identified mutated 2A genes in screened mutants.
  • Demonstrated successful intermolecular cleavage screening using the developed system.

    • Conclusions:

    • The developed system allows for high-density screening of viral proteinase mutants.
    • The system is versatile, accommodating both intramolecular and intermolecular cleavage.
    • This screening approach is potentially applicable to other highly specific proteinases.