title: Expression, purification and crystallization of the (3R)-hydroxyacyl-ACP dehydratase HadAB complex from Mycobacterium tuberculosis.
abstract: The (3R)-hydroxyacyl-ACP dehydratase HadAB, involved in the biosynthetic pathway for mycolic acid (MA) of Mycobacterium tuberculosis, catalyzes the third step in the fatty acid (FA) elongation cycle, which is an ideal and actual target for anti-tubercular agent. Though HadAB is predicted to be a member of the hotdog superfamily, it shares no sequence identity with typical hotdog fold isoenzyme FabZ. To characterize the significance of HadAB from the perspective of structural biology, large amount of pure HadAB complex is required for biochemical characterization and crystallization. Here, we used a unique expression and purification method. HadA and HadB were cloned separately and co-expressed in Escherichia coli. After GST affinity chromatography, two steps of anion exchange chromatography and gel filtration, the purity of the protein as estimated by SDS-PAGE was >95%. Using hanging-drop vapor-diffusion method, crystals were obtained and diffracted X-rays to 1.75Å resolution. The crystal belongs to space group P41212, with unit-cell parameters a=b=82.0Å, c=139.8Å, α=β=γ=90.0°.
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, People's Republic of China; University of Chinese Academy of Sciences, Beijing 100049, People's Republic of China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Dong
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, People's Republic of China.
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, People's Republic of China; University of Chinese Academy of Sciences, Beijing 100049, People's Republic of China.
authors.forename: Xiaodi
authors.initials: X
authors.lastname: Qiu
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, People's Republic of China; University of Chinese Academy of Sciences, Beijing 100049, People's Republic of China.
authors.forename: Chuanqiang
authors.initials: C
authors.lastname: Yan
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, People's Republic of China. Electronic address: lixm@sun5.ibp.ac.cn.
authors.forename: Xuemei
authors.initials: X
authors.lastname: Li
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D003460:Crystallization; D004746:Enoyl-CoA Hydratase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D004746:Enoyl-CoA Hydratase
keywords: Co-expression; Crystallization; Dehydratase; Drug target; HadAB; Mycobacterium tuberculosis; Mycolic acid