Expression, purification and auto-activation of cathepsin E from insect cells

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Cathepsin E is an aspartic protease in the pepsin family.
• It shares similarities with Cathepsin D but has distinct tissue and cellular localization.
• Elevated Cathepsin E levels are associated with various tumors, notably pancreatic ductal adenocarcinoma.

Purpose of the Study:

• To establish a novel, high-yield protocol for purifying recombinant human Cathepsin E.
• To facilitate further research into Cathepsin E's structure-function relationship.
• To support the development of targeted therapeutic strategies.

Main Methods:

• Utilized the baculovirus expression system for recombinant protein production in Sf9 insect cells.
• Purified the inactive zymogen (procathepsin E) via ion-exchange, size exclusion, pepstatin-affinity, and heparin-affinity chromatography.
• Activated the purified zymogen at acidic pH to obtain active Cathepsin E.

Main Results:

• Achieved high-yield purification of recombinant human procathepsin E.
• Successfully activated the zymogen to yield active Cathepsin E.
• Confirmed that the purified enzyme's activity, stability, and molecular weight match native Cathepsin E.

Conclusions:

• A robust and efficient protocol for recombinant human Cathepsin E purification has been developed.
• This method enables the production of sufficient quantities of active enzyme for detailed investigation.
• The enhanced availability of Cathepsin E will advance understanding and therapeutic development for associated diseases.