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Related Experiment Videos

An expression system for trypsin.

J R Vasquez1, L B Evnin, J N Higaki

  • 1Department of Pharmaceutical Chemistry, University of California, San Francisco 94143.

Journal of Cellular Biochemistry
|March 1, 1989
PubMed
Summary
This summary is machine-generated.

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Researchers expressed rat anionic trypsin in E. coli using a phoA promoter and signal peptide for periplasmic secretion. Active trypsin was produced, enabling easier screening and purification of this serine protease.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Engineering

Background:

  • Heterologous expression of eukaryotic enzymes in prokaryotic systems presents challenges in folding and secretion.
  • Rat anionic trypsin, a eukaryotic serine protease, requires specific folding and disulfide bond formation for activity.

Purpose of the Study:

  • To develop a system for the efficient heterologous expression and secretion of rat anionic trypsin and its mutants in Escherichia coli.
  • To facilitate the screening and purification of active trypsin for further functional studies and protein engineering.

Main Methods:

  • Utilized the bacterial alkaline phosphatase (phoA) promoter for inducible or constitutive expression of trypsin in E. coli.
  • Replaced the eukaryotic signal peptide with the phoA signal peptide to direct trypsinogen secretion to the periplasmic space.

Related Experiment Videos

  • Modified the trypsinogen gene by deleting the activation hexapeptide to achieve active trypsin in the periplasm.
  • Incorporated a transcription terminator to enhance expression levels.

    • Main Results:

    • Successfully secreted trypsinogen to the E. coli periplasm using the phoA signal peptide.
    • Achieved expression of active rat anionic trypsin in the periplasm, indicating correct folding and disulfide bond formation.
    • Observed a twofold increase in expression levels with a transcription terminator.
    • Found that increased plasmid copy number reduced expression levels.

    Conclusions:

    • The phoA signal peptide effectively directs mammalian trypsinogen secretion into the E. coli periplasm.
    • Periplasmic expression of active trypsin demonstrates successful zymogen activation, correct folding, and disulfide bond formation.
    • Periplasmic localization simplifies screening of modified trypsin variants and facilitates purification to homogeneity and crystallinity.