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Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD
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Engineering a novel endopeptidase based on SARS 3CL(pro).

Chih-Jung Kuo1, Yan-Ping Shih, Daphne Kan

  • 1Institute of Biological Chemistry, Academia Sinica and Core Facility of Recombinant Protein Production, Taipei, Taiwan.

Biotechniques
|January 1, 2010
PubMed
Summary
This summary is machine-generated.

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A novel mutant 3C-like protease (3CLpro) enhances recombinant protein purification by efficiently cleaving fusion tags. This engineered protease offers improved performance over existing methods for applications in E. coli and yeast.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Virology

Background:

  • Severe acute respiratory syndrome-coronavirus (SARS-CoV) 3C-like protease (3CLpro) is crucial for viral replication.
  • 3CLpro cleaves viral polyproteins at specific Gln- [downward arrow] (Ser, Ala, Gly) sites.

Purpose of the Study:

  • To engineer a mutant 3CLpro (T25G) with an expanded S1' binding pocket.
  • To evaluate its efficiency in cleaving substrates with larger P1' residues, such as methionine.
  • To develop vectors for its application in recombinant protein purification.

Main Methods:

  • Site-directed mutagenesis was used to create the T25G mutant of SARS-CoV 3CLpro.
  • Enzyme kinetics (k(cat)/K(m)) were measured for wild-type and mutant proteases with various substrates.

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  • Expression vectors for fusion proteins incorporating the T25G recognition site were constructed for E. coli and yeast.
  • Tag cleavage efficiency was compared between 3CLpro (T25G) and TEV protease.

    • Main Results:

    • The T25G mutant exhibited a 43.5-fold increase in catalytic efficiency (k(cat)/K(m)) for substrates with a methionine at P1'.
    • Vectors enabling expression of fusion proteins with the Ala-Val-Leu-Gln [downward arrow]Met recognition site were successfully constructed.
    • The PstI restriction site was utilized for cloning to avoid extraneous N-terminal amino acids.
    • 3CLpro (T25G) demonstrated a 3-fold improvement in tag cleavage efficiency compared to TEV protease.

    Conclusions:

    • The engineered 3CLpro (T25G) is a highly efficient protease for cleaving larger P1' residues.
    • It is suitable for tag removal in recombinant protein purification systems using E. coli and yeast.
    • This mutant protease offers an improved alternative to existing tag cleavage enzymes like TEV protease.