High yield expression of catalytically active USP18 (UBP43) using a Trigger Factor fusion system

Area of Science:

• Biochemistry
• Virology
• Molecular Biology

Background:

• The ubiquitin-like protein ISG15 modification inhibits viral infections.
• USP18 is the primary protease that removes ISG15 from target proteins.
• Inhibiting USP18 could be a novel antiviral strategy, but active protein production was previously limited.

Purpose of the Study:

• To develop a heterologous expression system for catalytically active USP18.
• To enable high-throughput screening for USP18 inhibitors.
• To facilitate biochemical and structural characterization of USP18.

Main Methods:

• A chaperone-based fusion system was employed for high-level heterologous expression of USP18 in E. coli.
• Purification of USP18 was achieved via a single-step IMAC using a His6-tag.
• Enzymatic activity and specificity assays were performed using ISG15-conjugated substrates and ISG15-AMC.

Main Results:

• High-level expression and purification of catalytically active USP18 were achieved.
• The expressed USP18 demonstrated efficient hydrolysis of ISG15-AMC and ISG15-conjugated substrates.
• USP18 showed specific activity towards ISG15, confirmed by covalent adduct formation with ISG15 vinyl sulfone, but not ubiquitin vinyl sulfone.

Conclusions:

• A chaperone fusion system successfully yields difficult-to-express proteins like USP18.
• The produced USP18 is suitable for establishing high-throughput small molecule inhibitor screens.
• This work provides a foundation for further biochemical and structural studies of USP18.