An affinity tool for the isolation of endogenous active mTORC1 from various cellular sources

Area of Science:

• Cellular Biology
• Molecular Biology
• Biochemistry

Background:

• The mechanistic target of rapamycin complex 1 (mTORC1) regulates mammalian cell growth.
• Dysregulation of mTORC1 is implicated in diseases like metabolic syndromes, aging, and cancer.
• Current methods for purifying mTORC1 are often expensive, inefficient, and may not isolate endogenous protein.

Purpose of the Study:

• To develop a simple and cost-effective tool for isolating endogenous mTORC1.
• To enable broader research into mTORC1's role in various cellular contexts and diseases.

Main Methods:

• Recombinant expression and isolation of mTORC1-binding Rag GTPases from Escherichia coli.
• Utilizing these Rag GTPases as affinity probes for mTORC1 purification.

Main Results:

• Demonstrated successful isolation of endogenous mTORC1 from mouse, bovine, and human cellular sources.
• The isolated mTORC1 is catalytically active and the method is scalable.
• The purification method is relatively inexpensive compared to existing techniques.

Conclusions:

• A novel, accessible tool for isolating endogenous mTORC1 has been developed.
• This tool facilitates research on mTORC1 in diverse biological samples and disease states.
• The method supports scalable and cost-effective mTORC1 purification for scientific investigation.