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Related Experiment Videos

Prochymosin expression in Bacillus subtilis.

D Parente1, F de Ferra, G Galli

  • 1Genetic Engineering and Microbiology Laboratory, Eniricerche S.p.A., Milan, Italy.

FEMS Microbiology Letters
|January 15, 1991
PubMed
Summary
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Bacillus subtilis was engineered to express prochymosin (PC) intracellularly and secreted it. Intracellular PC was insoluble but refoldable, while secreted PC was properly folded but at low levels.

Area of Science:

  • Biotechnology
  • Molecular Biology
  • Enzyme Engineering

Background:

  • Prochymosin (PC) is a key enzyme precursor in the dairy industry.
  • Efficient expression systems are needed for recombinant enzyme production.
  • Bacillus subtilis offers a robust platform for industrial protein expression.

Purpose of the Study:

  • To develop and optimize expression systems for prochymosin (PC) in Bacillus subtilis.
  • To investigate both intracellular and secreted expression strategies.
  • To enhance enzyme yield and functionality.

Main Methods:

  • Cloning of the PC sequence into Bacillus subtilis using two distinct plasmid constructions.
  • Intracellular expression utilizing plasmid pSM316.
  • Modification of the 5' gene region to create a two-cistron expression system.

Related Experiment Videos

  • Fusion of PC cDNA to a subtilisin leader sequence for secretion.
  • Expression under the control of the B. subtilis neutral protease promoter.

    • Main Results:

    • Intracellular PC expression resulted in an insoluble form, requiring denaturing and refolding for activity.
    • A two-cistron system enhanced intracellular PC expression levels.
    • Secreted PC was properly folded but obtained at low yields.
    • The subtilisin leader sequence facilitated PC secretion.

    Conclusions:

    • Bacillus subtilis can be engineered for both intracellular and secreted prochymosin production.
    • Optimization of expression strategies is crucial for enzyme yield and form.
    • Further development is needed to increase secretion efficiency for industrial applications.