An engineered cell line with a hRpn1-attached handle to isolate proteasomes

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Regulated protein degradation in eukaryotes is crucial and mediated by the 26S proteasome.
• The 26S proteasome comprises a regulatory particle (RP) and a core particle (CP), with numerous transiently interacting proteins.
• Previous methods for proteasome isolation involved overexpression or laborious purification, potentially altering native stoichiometry.

Purpose of the Study:

• To develop a straightforward method for purifying native 26S proteasomes from human cells.
• To create an engineered cell line for efficient isolation of proteasomes with a specific tag.

Main Methods:

• Engineered HCT116 colon cancer cells with a biotinylation handle on the RP subunit hRpn1/PSMD2.
• Purification of biotinylated 26S proteasomes using neutravidin agarose resin.
• Release of purified proteasomes via tobacco etch virus protease cleavage.

Main Results:

• Successfully isolated 26S proteasomes from the engineered HCT116 cell line using a simple protocol.
• Purified proteasomes demonstrated equivalent activity in degrading ubiquitinated p53 compared to commercial bovine proteasomes.
• The method preserves endogenous stoichiometry of cellular proteins, including the tagged hRpn1 subunit.

Conclusions:

• The engineered HCT116 cell line provides a simple and effective means to purify functional 26S proteasomes.
• This approach overcomes limitations of previous methods by maintaining native protein stoichiometry.
• The purified proteasomes are suitable for studying protein degradation mechanisms and proteasome interactions.