Open AccessPurification, crystallization and preliminary X‐ray crystallographic analysis of the CIDE‐N domain of Fsp27
Author(s) -
Wang Xiaodan,
Zhang Bo,
Xu Duo,
Gao Jinlan,
Wang Linfang,
Wang Zhi,
Shan Yaming,
Yu Xianghui
Publication year - 2012
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309112043989
Subject(s) - polyethylene glycol , crystallization , escherichia coli , fusion protein , crystallography , recombinant dna , chemistry , crystal (programming language) , yield (engineering) , resolution (logic) , materials science , biochemistry , gene , artificial intelligence , metallurgy , organic chemistry , computer science , programming language
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 P 6 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.