title: Cloning, expression, purification, crystallization and X-ray crystallographic analysis of CofB, the minor pilin subunit of CFA/III from human enterotoxigenic Escherichia coli.
abstract: Colonization factor antigen III (CFA/III) is one of the virulence factors of human enterotoxigenic Escherichia coli (ETEC) that forms the long, thin, proteinaceous fibres of type IV pili through assembly of its major and minor subunits CofA and CofB, respectively. The crystal structure of CofA has recently been reported; however, the lack of structural information for CofB, the largest among the known type IV pilin subunits, hampers a comprehensive understanding of CFA/III pili. In this study, constructs of wild-type CofB with an N-terminal truncation and the corresponding SeMet derivative were cloned, expressed, purified and crystallized. The crystals belonged to the rhombohedral space group R32, with unit-cell parameters a = b = 103.97, c = 364.57 Å for the wild-type construct and a = b = 103.47, c = 362.08 Å for the SeMet-derivatized form. Although the diffraction quality of these crystals was initially very poor, dehydration of the crystals substantially improved the resolution limit from ∼ 4.0 to ∼ 2.0 Å. The initial phase was solved by the single-wavelength anomalous dispersion (SAD) method using a dehydrated SeMet CofB crystal, which resulted in an interpretable electron-density map.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Kawahara
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Hiroya
authors.initials: H
authors.lastname: Oki
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Fukakusa
authors.affiliation: Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Maruno
authors.affiliation: Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kobayashi
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Motooka
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Tooru
authors.initials: T
authors.lastname: Taniguchi
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Honda
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Iida
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Shota
authors.initials: S
authors.lastname: Nakamura
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Tadayasu
authors.initials: T
authors.lastname: Ohkubo
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D054307:Enterotoxigenic Escherichia coli / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D036781:Fimbriae Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D010861:Fimbriae, Bacterial / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012645:Selenomethionine / Q000737:chemistry; D016415:Sequence Alignment; D055672:Static Electricity
chemical_list: C577578:CofA protein, E coli; D029968:Escherichia coli Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; C022924:colonization factor antigens; D036781:Fimbriae Proteins; D012645:Selenomethionine
keywords: CFA/III; CofB; ETEC; minor pilin; type IVb pili