Title |
A Yeast Purification System for Human Translation Initiation Factors eIF2 and eIF2Bε and Their Use in the Diagnosis of CACH/VWM Disease
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Published in |
PLOS ONE, January 2013
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DOI | 10.1371/journal.pone.0053958 |
Pubmed ID | |
Authors |
Rogerio A. de Almeida, Anne Fogli, Marina Gaillard, Gert C. Scheper, Odile Boesflug-Tanguy, Graham D. Pavitt |
Abstract |
Recessive inherited mutations in any of five subunits of the general protein synthesis factor eIF2B are responsible for a white mater neurodegenerative disease with a large clinical spectrum. The classical form is called Childhood Ataxia with CNS hypomyelination (CACH) or Vanishing White Matter Leukoencephalopathy (VWM). eIF2B-related disorders affect glial cells, despite the fact that eIF2B is a ubiquitous protein that functions as a guanine-nucleotide exchange factor (GEF) for its partner protein eIF2 in the translation initiation process in all eukaryotic cells. Decreased eIF2B activity measured by a GEF assay in patients' immortalised lymphocytic cells provides a biochemical diagnostic assay but is limited by the availability of eIF2 protein, which is classically purified from a mammalian cell source by column chromatography. Here we describe the generation of a recombinant expression system to produce purified human eIF2 from yeast cells. We demonstrate that human eIF2 can function in yeast cells in place of the equivalent yeast factor. We purify human eIF2 and the C-terminal domain of human eIF2Bε using affinity chromatography from engineered yeast cells and find that both function in a GEF assay: the first demonstration that this human eIF2Bε domain has GEF function. We show that CACH/VWM mutations within this domain reduce its activity. Finally we demonstrate that the recombinant eIF2 functions similarly to eIF2 purified from rat liver in GEF assays with CACH/VWM eIF2B-mutated patient derived lymphocytic cells. |
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Researcher | 8 | 24% |
Student > Bachelor | 5 | 15% |
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Lecturer > Senior Lecturer | 1 | 3% |
Other | 2 | 6% |
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Chemistry | 1 | 3% |
Other | 0 | 0% |
Unknown | 2 | 6% |