1. Cloning, expression, purification, and characterisation of the HEAT-repeat domain of TOR from the thermophilic eukaryote Chaetomium thermophilum.
- Author
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Robinson GC, Vegunta Y, Gabus C, Gaubitz C, and Thore S
- Subjects
- Protein Domains, Recombinant Proteins, Repetitive Sequences, Amino Acid, TOR Serine-Threonine Kinases biosynthesis, TOR Serine-Threonine Kinases chemistry, TOR Serine-Threonine Kinases genetics, TOR Serine-Threonine Kinases isolation & purification, Chaetomium enzymology, Chaetomium genetics, Cloning, Molecular, Fungal Proteins biosynthesis, Fungal Proteins chemistry, Fungal Proteins genetics, Fungal Proteins isolation & purification, Gene Expression
- Abstract
The Target of Rapamycin Complex is a central controller of cell growth and differentiation in eukaryotes. Its global architecture has been described by cryoelectron microscopy, and regions of its central TOR protein have been described by X-ray crystallography. However, the N-terminal region of this protein, which consists of a series of HEAT repeats, remains uncharacterised at high resolution, most likely due to the absence of a suitable purification procedure. Here, we present a robust method for the preparation of the HEAT-repeat domain, utilizing the thermophilic fungus Chaetomium thermophilum as a source organism. We describe construct design and stable expression in insect cells. An efficient two-step purification procedure is presented, and the purified product is characterised by SEC and MALDI-TOF MS. The methods described pave the way for a complete high-resolution characterisation of this elusive region of the TOR protein., (Copyright © 2017 Elsevier Inc. All rights reserved.)
- Published
- 2017
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