title: Recombinant expression, purification, crystallization and preliminary X-ray diffraction analysis of Haemophilus influenzae BamD and BamCD complex.
abstract: The Bam machinery, which is highly conserved from bacteria to humans, is well recognized as the apparatus responsible for the insertion and folding of most outer membrane proteins in Gram-negative bacteria. In Escherichia coli, the Bam machinery consists of five components (i.e. BamA, BamB, BamC, BamD and BamE). In comparison, there are only four partners in Haemophilus influenzae: a BamB homologue is not found in its genome. In this study, the recombinant expression, purification, crystallization and preliminary X-ray diffraction analysis of H. influenzae BamD and BamCD complex are reported. The genes encoding BamC and BamD were cloned into a pET vector and expressed in E. coli. Affinity, ion-exchange and gel-filtration chromatography were used to obtain high-purity protein for further crystallographic characterization. Using the hanging-drop vapour-diffusion technique, BamD and BamCD protein crystals of suitable size were obtained using protein concentrations of 70 and 50 mg ml(-1), respectively. Preliminary X-ray diffraction analysis showed that the BamD crystals diffracted to 4.0 Å resolution and belonged to space group P212121, with unit-cell parameters a = 54.5, b = 130.5, c = 154.7 Å. The BamCD crystals diffracted to 3.8 Å resolution and belonged to space group I212121, with unit-cell parameters a = 101.6, b = 114.1, c = 234.9 Å.
authors: State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui-Jin Hospital affiliated to Shanghai JiaoTong University School of Medicine, 197 Ruijin Er Road, Shanghai 200025, People's Republic of China.
authors: Jintang
authors: J
authors: Lei
authors: State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui-Jin Hospital affiliated to Shanghai JiaoTong University School of Medicine, 197 Ruijin Er Road, Shanghai 200025, People's Republic of China.
authors: Xun
authors: X
authors: Cai
authors: State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui-Jin Hospital affiliated to Shanghai JiaoTong University School of Medicine, 197 Ruijin Er Road, Shanghai 200025, People's Republic of China.
authors: Xiaodan
authors: X
authors: Ma
authors: State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui-Jin Hospital affiliated to Shanghai JiaoTong University School of Medicine, 197 Ruijin Er Road, Shanghai 200025, People's Republic of China.
authors: Li
authors: L
authors: Zhang
authors: State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui-Jin Hospital affiliated to Shanghai JiaoTong University School of Medicine, 197 Ruijin Er Road, Shanghai 200025, People's Republic of China.
authors: Yuwen
authors: Y
authors: Li
authors: State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui-Jin Hospital affiliated to Shanghai JiaoTong University School of Medicine, 197 Ruijin Er Road, Shanghai 200025, People's Republic of China.
authors: Xue
authors: X
authors: Dong
authors: Department of Pediatrics, Children's Hospital of Philadelphia and the Perelman School of Medicine at the University of Pennsylvania, Philadelphia, USA.
authors: Joseph
authors: J
authors: St Geme
authors: State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui-Jin Hospital affiliated to Shanghai JiaoTong University School of Medicine, 197 Ruijin Er Road, Shanghai 200025, People's Republic of China.
authors: Guoyu
authors: G
authors: Meng
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D003460:Crystallization; D006193:Haemophilus influenzae / Q000737:chemistry*; D008958:Models, Molecular; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins
keywords: BamCD complex; BamD; Haemophilus influenzae