title: Purification, crystallization and preliminary X-ray crystallographic analysis of the CIDE-N domain of Fsp27.
abstract: Fsp27, a member of the CIDE protein family which is selectively expressed in adipocytes, has emerged as a novel regulator for unilocular lipid droplet (LD) formation, lipid metabolism, differentiation of adipocytes and insulin sensitivity. An LD is a subcellular compartment that is used by adipocytes for the efficient storage of fats. The CIDE-N domain of Fsp27 functions as a recruitment platform that induces the correct configuration of the Fsp27 CIDE-C domain to facilitate LD fusion. This study reports the high-yield expression of the mouse Fsp27 CIDE-N domain in Escherichia coli; a two-step purification protocol with high efficiency was established and crystallographic analysis was performed. The purity of the recombinant Fsp27 was >95% as assessed by SDS-PAGE. Crystals were obtained at 291 K using 28% polyethylene glycol 4000 as a precipitant. Diffraction data were collected to 1.92 Å resolution and the crystal belonged to space group P6(5), with unit-cell parameters a=b=63.3, c=37.4 Å, α=β=90, γ=120°. The components of the crystal were identified by ion-trap LC/MS/MS spectrometric analysis. The structure has been solved by molecular replacement and refinement is in progress.
authors.affiliation: National Engineering Laboratory for AIDS Vaccine, Jilin University, Changchun 130012, People's Republic of China.
authors.forename: Xiaodan
authors.initials: X
authors.lastname: Wang
authors.forename: Bo
authors.initials: B
authors.lastname: Zhang
authors.forename: Duo
authors.initials: D
authors.lastname: Xu
authors.forename: Jinlan
authors.initials: J
authors.lastname: Gao
authors.forename: Linfang
authors.initials: L
authors.lastname: Wang
authors.forename: Zhi
authors.initials: Z
authors.lastname: Wang
authors.forename: Yaming
authors.initials: Y
authors.lastname: Shan
authors.forename: Xianghui
authors.initials: X
authors.lastname: Yu
mesh_terms: D017667:Adipocytes / Q000166:cytology / Q000378:metabolism; D000818:Animals; D003460:Crystallization; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D050356:Lipid Metabolism; D051379:Mice; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D011506:Proteins; D011994:Recombinant Proteins; C525805:fat-specific protein 27, mouse