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Related Experiment Videos

Engineering subtilisin BPN' for site-specific proteolysis.

P Carter1, B Nilsson, J P Burnier

  • 1Department of Biomolecular Chemistry, Genentech, Inc., South San Francisco, California 94080.

Proteins
|January 1, 1989
PubMed
Summary
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Engineered subtilisin variants with restricted specificity efficiently cleave fusion proteins at a designed site. These modified proteases offer precise control for biotechnological applications, enabling targeted protein release.

Area of Science:

  • Biochemistry
  • Protein Engineering
  • Enzymology

Background:

  • Wild-type subtilisin BPN' exhibits broad substrate specificity.
  • A histidine-to-alanine substitution at position 64 (H64A) restricts substrate recognition.
  • Further engineering is needed to enhance catalytic efficiency and specificity.

Purpose of the Study:

  • To engineer subtilisin variants with improved catalytic efficiency and specificity for site-specific proteolysis.
  • To create a fusion protein system for testing the engineered subtilisin variants.
  • To evaluate the utility of these variants for cleaving fusion proteins.

Main Methods:

  • Protein engineering of subtilisin BPN' by introducing mutations, including H64A.
  • Substrate optimization to identify favorable cleavage sequences.

Related Experiment Videos

  • Construction of a fusion protein linking Staphylococcus aureus protein A and Escherichia coli alkaline phosphatase.
  • Affinity purification of the fusion protein and subsequent cleavage by engineered subtilisin variants.

    • Main Results:

    • An H64A subtilisin variant with a 20-fold increase in catalytic efficiency (kcat/Km) was developed.
    • The engineered variants demonstrated efficient and exclusive cleavage at the designed target site in the fusion protein.
    • The cleavage liberated an alkaline phosphatase product of the correct size and N-terminal sequence.

    Conclusions:

    • Engineered subtilisin H64A variants offer exquisite substrate specificity, particularly on the N-terminal side of the cleavage site.
    • These variants are robust and remain active under various conditions (detergents, inhibitors, high temperatures, immobilization).
    • Subtilisin variants are highly attractive tools for site-specific proteolysis of fusion proteins in biotechnological applications.