title: Expression and crystallization of a bacterial glycoside hydrolase family 116 β-glucosidase from Thermoanaerobacterium xylanolyticum.
abstract: The Thermoanaerobacterium xylanolyticum gene product TxGH116, a glycoside hydrolase family 116 protein of 806 amino-acid residues sharing 37% amino-acid sequence identity over 783 residues with human glucosylceramidase 2 (GBA2), was expressed in Escherichia coli. Purification by heating, immobilized metal-affinity and size-exclusion chromatography produced >90% pure TxGH116 protein with an apparent molecular mass of 90 kDa on SDS-PAGE. The purified TxGH116 enzyme hydrolyzed the p-nitrophenyl (pNP) glycosides pNP-β-D-glucoside, pNP-β-D-galactoside and pNP-N-acetyl-β-D-glucopyranoside, as well as cellobiose and cellotriose. The TxGH116 protein was crystallized using a precipitant consisting of 0.6 M sodium citrate tribasic, 0.1 M Tris-HCl pH 7.0 by vapour diffusion with micro-seeding to form crystals with maximum dimensions of 120×25×5 µm. The TxGH116 crystals diffracted X-rays to 3.15 Å resolution and belonged to the monoclinic space group P2(1). Structure solution will allow a structural explanation of the effects of human GBA2 mutations.
authors.affiliation: Institute of Science, School of Biochemistry and Center for Biomolecular Structure, Function and Application, Suranaree University of Technology, 111 University Avenue, Nakhon Ratchasima Muang District, Nakhon Ratchasima 30000, Thailand.
authors.forename: Sompong
authors.initials: S
authors.lastname: Sansenya
authors.affiliation: Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Risa
authors.initials: R
authors.lastname: Mutoh
authors.affiliation: Institute of Science, School of Biochemistry and Center for Biomolecular Structure, Function and Application, Suranaree University of Technology, 111 University Avenue, Nakhon Ratchasima Muang District, Nakhon Ratchasima 30000, Thailand.
authors.forename: Ratana
authors.initials: R
authors.lastname: Charoenwattanasatien
authors.affiliation: Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Genji
authors.initials: G
authors.lastname: Kurisu
authors.affiliation: Institute of Science, School of Biochemistry and Center for Biomolecular Structure, Function and Application, Suranaree University of Technology, 111 University Avenue, Nakhon Ratchasima Muang District, Nakhon Ratchasima 30000, Thailand.
authors.forename: James R
authors.initials: JR
authors.lastname: Ketudat Cairns
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry*; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D046950:Thermoanaerobacterium / Q000201:enzymology*; D001617:beta-Glucosidase / Q000096:biosynthesis / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D001617:beta-Glucosidase
keywords: glycoside hydrolase family 116; thermophilic; β-glucosidase