title: Purification of FLAG-tagged eukaryotic initiation factor 2B complexes, subcomplexes, and fragments from Saccharomyces cerevisiae.
abstract: The eukaryotic initiation factor 2B (eIF2B) is a five-subunit guanine nucleotide exchange factor, that functions during translation initiation to catalyze the otherwise slow exchange of GDP for GTP on its substrate eIF2. Assays to measure substrate interaction and guanine nucleotide release ability of eIF2B require the complex to be purified free of interacting proteins. We have also found that a subcomplex of two subunits, gamma and epsilon or the largest one, epsilon alone, promotes this activity. Within eIF2Bepsilon, the catalytic center requires the C-terminal 200 residues only. Here, we describe our protocols for purifying the Saccharomyces cerevisiae eIF2B complexes and the catalytic subunit using FLAG-tagged proteins overexpressed in yeast cells. Using commercially available FLAG-affinity resin and high salt buffer, we are able to purify active eIF2B virtually free of contaminants.
authors.affiliation: Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom.
authors.forename: Sarah S
authors.initials: SS
authors.lastname: Mohammad-Qureshi
authors.forename: Raphaël
authors.initials: R
authors.lastname: Haddad
authors.forename: Karren S
authors.initials: KS
authors.lastname: Palmer
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Richardson
authors.forename: Edith
authors.initials: E
authors.lastname: Gomez
authors.forename: Graham D
authors.initials: GD
authors.lastname: Pavitt
mesh_terms: D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002846:Chromatography, Affinity; D003956:Dialysis; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D015870:Gene Expression; D005822:Genetic Vectors / Q000302:isolation & purification; D008954:Models, Biological; D046912:Multiprotein Complexes / Q000302:isolation & purification*; D009842:Oligopeptides; D010446:Peptide Fragments / Q000302:isolation & purification*; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification*; D010957:Plasmids / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics
chemical_list: D015852:Eukaryotic Initiation Factor-2; D046912:Multiprotein Complexes; D009842:Oligopeptides; D010446:Peptide Fragments; D010455:Peptides; D011993:Recombinant Fusion Proteins; C085706:FLAG peptide