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An affinity tool for the isolation of endogenous active mTORC1 from various cellular sources.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2023 May; Vol. 299 (5), pp. 104644. Date of Electronic Publication: 2023 Mar 23. - Publication Year :
- 2023
-
Abstract
- The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of mammalian cell growth that is dysregulated in a number of human diseases, including metabolic syndromes, aging, and cancer. Structural, biochemical, and pharmacological studies that have increased our understanding of how mTORC1 executes growth control often relied upon purified mTORC1 protein. However, current immunoaffinity-based purification methods are expensive, inefficient, and do not necessarily isolate endogenous mTORC1, hampering their overall utility in research. Here we present a simple tool to isolate endogenous mTORC1 from various cellular sources. By recombinantly expressing and isolating mTORC1-binding Rag GTPases from Escherichia coli and using them as affinity probes, we demonstrate that mTORC1 can be isolated from mouse, bovine, and human sources. Our results indicate that mTORC1 isolated by this relatively inexpensive method is catalytically active and amenable to scaling. Collectively, this tool may be utilized to isolate mTORC1 from various cellular sources, organs, and disease contexts, aiding mTORC1-related research.<br />Competing Interests: Conflict of interest Y. H. I. is the founder of Araucaria Laboratories Inc.<br /> (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
- Subjects :
- Animals
Cattle
Humans
Mice
Mammals metabolism
Escherichia coli genetics
Escherichia coli Proteins genetics
Escherichia coli Proteins metabolism
Models, Molecular
Mechanistic Target of Rapamycin Complex 1 chemistry
Mechanistic Target of Rapamycin Complex 1 isolation & purification
Mechanistic Target of Rapamycin Complex 1 metabolism
Monomeric GTP-Binding Proteins chemistry
Monomeric GTP-Binding Proteins genetics
Monomeric GTP-Binding Proteins metabolism
Recombinant Proteins genetics
Recombinant Proteins metabolism
Biotechnology methods
Subjects
Details
- Language :
- English
- ISSN :
- 1083-351X
- Volume :
- 299
- Issue :
- 5
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 36965617
- Full Text :
- https://doi.org/10.1016/j.jbc.2023.104644