Cloning, expression and characterisation of murine procathepsin E

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Cathepsin E is an aspartic proteinase involved in various physiological and pathological processes.
• Understanding its structure and function is crucial for developing targeted therapies.

Purpose of the Study:

• To isolate and sequence the cDNA encoding murine procathepsin E.
• To produce and characterize recombinant mouse cathepsin E.
• To investigate the enzyme's quaternary structure and potential for further studies.

Main Methods:

• cDNA isolation and sequencing of murine procathepsin E.
• Recombinant protein expression in Escherichia coli.
• Enzyme activity characterization using synthetic substrates and inhibitors.
• Analysis of enzyme quaternary structure (homodimer formation).

Main Results:

• Successful isolation, sequencing, and recombinant expression of mouse cathepsin E.
• Characterization of enzyme activity and inhibitor interactions.
• Identification of the recombinant enzyme predominantly existing as a disulfide-linked homodimer.
• The homodimer formation is attributed to a unique N-terminal cysteine residue.

Conclusions:

• Recombinant murine cathepsin E can be produced in E. coli.
• The enzyme forms a disulfide-linked homodimer, similar to human cathepsin E.
• The availability of recombinant enzyme and cDNA facilitates future structural studies and knockout mouse generation.