title: Expression, purification, and crystallization of Schizosaccharomyces pombe eIF2B.
abstract: Tight control of protein synthesis is necessary for cells to respond and adapt to environmental changes rapidly. Eukaryotic translation initiation factor (eIF) 2B, the guanine nucleotide exchange factor for eIF2, is a key target of translation control at the initiation step. The nucleotide exchange activity of eIF2B is inhibited by the stress-induced phosphorylation of eIF2. As a result, the level of active GTP-bound eIF2 is lowered, and protein synthesis is attenuated. eIF2B is a large multi-subunit complex composed of five different subunits, and all five of the subunits are the gene products responsible for the neurodegenerative disease, leukoencephalopathy with vanishing white matter. However, the overall structure of eIF2B has remained unresolved, due to the difficulty in preparing a sufficient amount of the eIF2B complex. To overcome this problem, we established the recombinant expression and purification method for eIF2B from the fission yeast Schizosaccharomyces pombe. All five of the eIF2B subunits were co-expressed and reconstructed into the complex in Escherichia coli cells. The complex was successfully purified with a high yield. This recombinant eIF2B complex contains each subunit in an equimolar ratio, and the size exclusion chromatography analysis suggests it forms a heterodecamer, consistent with recent reports. This eIF2B increased protein synthesis in the reconstituted in vitro human translation system. In addition, disease-linked mutations led to subunit dissociation. Furthermore, we crystallized this functional recombinant eIF2B, and the crystals diffracted to 3.0 Å resolution.
authors.affiliation: Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.|RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045, Japan.|RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Kashiwagi
authors.affiliation: Graduate School of Engineering, University of Hyogo, Himeji, 671-2280, Japan.
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Shigeta
authors.affiliation: Graduate School of Engineering, University of Hyogo, Himeji, 671-2280, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Imataka
authors.affiliation: Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan. takuhiro.ito@riken.jp.|RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045, Japan. takuhiro.ito@riken.jp.|RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045, Japan. takuhiro.ito@riken.jp.
authors.forename: Takuhiro
authors.initials: T
authors.lastname: Ito
authors.affiliation: Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan. yokoyama@riken.jp.|RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045, Japan. yokoyama@riken.jp.|RIKEN Structural Biology Laboratory, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045, Japan. yokoyama@riken.jp.
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
mesh_terms: D002850:Chromatography, Gel; D003460:Crystallization; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D020717:Eukaryotic Initiation Factor-2B / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D020717:Eukaryotic Initiation Factor-2B; D021122:Protein Subunits; D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins
keywords: Eukaryotic translation initiation factor; Guanine-nucleotide exchange factor; eIF2B