| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 5516144 | Protein Expression and Purification | 2017 | 6 Pages |
•A procedure is presented for the purification of the HEAT-repeat domain of TOR.•Protein obtained from the thermophilic eukaryote Chaetomium thermophilum.•HEAT-repeat domain of TOR characterised by electrophoresis, SEC, and MS.•Protein suitable for high-resolution structural study.
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.
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