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Related Experiment Videos

A rapid and effective procedure for screening protease mutants

I Venekei1, L Hedstrom, W J Rutter

  • 1Hormone Research Institute, University of California, San Francisco 94143-0534, USA.

Protein Engineering
|January 1, 1996
PubMed
Summary

This study presents a new method for screening active proteases in bacterial mutants. The technique efficiently identifies novel proteases with potential applications in biotechnology and enzyme evolution.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Screening for active proteases among numerous mutants is challenging.
  • Periplasmic protease activity is crucial for bacterial nitrogen metabolism.
  • Identifying novel proteases requires efficient screening methods.

Purpose of the Study:

  • To develop a simple and effective procedure for screening active proteases in large mutant libraries.
  • To enable the discovery of proteases with novel specificities and catalytic mechanisms.
  • To facilitate in vitro evolution studies of protease enzymes.

Main Methods:

  • Genetically screening bacterial transformants for periplasmic protease activity using a protein-based nitrogen source.
  • Employing activity staining and X-ray film digestion assays for verification and activity estimation.

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  • Utilizing polymerase chain reaction (PCR) for random mutagenesis of protease genes.

    • Main Results:

    • Successfully identified two active revertants from an inactive Asn102 trypsin mutant after screening ~4.4 x 10^4 random mutants.
    • The method demonstrated the ability to detect both activity and stability of expressed periplasmic proteases.
    • Calibration with wild-type and mutant trypsin and chymotrypsin confirmed method accuracy.

    Conclusions:

    • The developed screening procedure is efficient for identifying active proteases from mutant libraries.
    • This method is valuable for discovering proteases with engineered specificity or novel catalytic functions.
    • The technique supports advancements in enzyme engineering and in vitro evolution research.