title: Cloning, expression, purification, and characterisation of the HEAT-repeat domain of TOR from the thermophilic eukaryote Chaetomium thermophilum.
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.
authors.affiliation: Department of Molecular Biology, University of Geneva, 30 quai Ernest Ansermet, 1211 Geneva, Switzerland.
authors.forename: Graham C
authors.initials: GC
authors.lastname: Robinson
authors.affiliation: Department of Molecular Biology, University of Geneva, 30 quai Ernest Ansermet, 1211 Geneva, Switzerland.
authors.forename: Yogesh
authors.initials: Y
authors.lastname: Vegunta
authors.affiliation: Department of Molecular Biology, University of Geneva, 30 quai Ernest Ansermet, 1211 Geneva, Switzerland.
authors.forename: Caroline
authors.initials: C
authors.lastname: Gabus
authors.affiliation: Department of Molecular Biology, University of Geneva, 30 quai Ernest Ansermet, 1211 Geneva, Switzerland.
authors.forename: Christl
authors.initials: C
authors.lastname: Gaubitz
authors.affiliation: Department of Molecular Biology, University of Geneva, 30 quai Ernest Ansermet, 1211 Geneva, Switzerland; INSERM U-1212, CNRS UMR-5320, Université de Bordeaux, 146 rue Léo Saignat, 33000 Bordeaux, France. Electronic address: stephane.thore@inserm.fr.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Thore
mesh_terms: D002597:Chaetomium / Q000201:enzymology / Q000235:genetics; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D000072417:Protein Domains; D011994:Recombinant Proteins; D020449:Repetitive Sequences, Amino Acid; D058570:TOR Serine-Threonine Kinases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D058570:TOR Serine-Threonine Kinases
keywords: Chaetomium thermophilum; Mass spectrometry; MultiBac expression system; Purification; TOR; Target of rapamycin signalling