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HCl pH (hcl + ph)
Selected AbstractsCrystallization and preliminary X-ray crystallographic analysis of p24, a component of the potato nuclear factor PBF-2ACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2002Darrell Desveaux The Solanum tuberosum (potato) nuclear factor PBF-2 is implicated in pathogen-induced expression of the pathogenesis-related gene PR-10a. Crystals of the DNA-binding component of PBF-2, p24, have been obtained at 277,K in 20,mM Tris,HCl pH 8.0. Recombinant protein with a His tag at its C-terminus was overexpressed in Escherichia coli in the presence and absence of selenomethionine and was purified using a combination of HiTrap affinity columns and gel-filtration chromatography. Crystals suitable for structural analysis were obtained for both native and selenomethionine-labelled proteins and yielded diffraction data at 100,K that were processed to 2.3 and 2.8,Å resolution, respectively. The p24 protein crystals belong to space group P212121, with unit-cell parameters a = 69.4,(69.1), b = 89.4,(90.5), c = 144.1,(144.3),Å. The asymmetric unit contains four protomers, giving a crystal volume per protein mass (VM) of 2.23,Å3,Da,1 and a solvent content of 45% by volume. [source] A novel 40,kDa protein from goat mammary secretions: purification, crystallization and preliminary X-ray diffraction studiesACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2001P. Kumar A novel 40,kDa protein has been purified from dry secretions of the mammary gland of goats. The first 15 N-terminal residues were sequenced and showed a sequence identity of 30% to a novel 39,kDa whey protein from bovine mammary secretions. The protein was crystallized by the microdialysis method. Protein was dissolved to a concentration of 40,mg,ml,1 in 0.025,M Tris,HCl pH 8.0 and equilibrated with the same buffer containing 19%(v/v) ethanol. The crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 66.1, b = 107.8, c = 63.2,Å and one molecule per asymmetric unit. Intensity data were collected to 2.9,Å resolution, with a completeness of 95%. Since no similar model is available in the protein structure database, heavy-atom derivatives have been prepared and three-dimensional structure determination using the isomorphous replacement method is in progress. [source] Purification, crystallization and preliminary crystallographic analysis of very-long-chain acyl-CoA dehydrogenase from Caenorhabditis elegansACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2010Zhijie Li Acyl-CoA dehydrogenase [acyl-CoA:(acceptor) 2,3-oxidoreductase; EC 1.3.99.3] catalyzes the first reaction step in mitochondrial fatty-acid ,-oxidation. Here, the very-long-chain acyl-CoA dehydrogenase from Caenorhabditis elegans (cVLCAD) has been cloned and overexpressed in Escherichia coli strain BL21 (DE3). Interestingly, unlike other very-long-chain acyl-CoA dehydrogenases, cVLCAD was found to form a tetramer by size-exclusion chromatography coupled with in-line static light-scattering, refractive-index and ultraviolet measurements. Purified cVLCAD (12,mg,ml,1) was successfully crystallized by the hanging-drop vapour-diffusion method under conditions containing 100,mM Tris,HCl pH 8.0, 150,mM sodium chloride, 200,mM magnesium formate and 13% PEG 3350. The crystal has a tetragonal form and a complete diffraction data set was collected and processed to 1.8,Å resolution. The crystal belonged to space group C2, with unit-cell parameters a = 138.6, b = 116.7, c = 115.3,Å, , = , = 90.0, , = 124.0°. A self-rotation function indicated the existence of one noncrystallographic twofold axis. A preliminary molecular-replacement solution further confirmed the presence of two molecules in one asymmetric unit, which yields a Matthews coefficient VM of 2.76,Å3,Da,1 and a solvent content of 55%. [source] Expression, purification, crystallization and preliminary X-ray analysis of rice (Oryza sativa L.) Os4BGlu12 ,-glucosidaseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2010Sompong Sansenya Rice (Oryza sativa L.) Os4BGlu12, a glycoside hydrolase family 1 ,-glucosidase (EC 3.2.1.21), was expressed as a fusion protein with an N-terminal thioredoxin/His6 tag in Escherichia coli strain Origami B (DE3) and purified with subsequent removal of the N-terminal tag. Native Os4BGlu12 and its complex with 2,4-dinitrophenyl-2-deoxy-2-fluoro-,- d -glucopyranoside (DNP2FG) were crystallized using 19% polyethylene glycol (3350 or 2000, respectively) in 0.1,M Tris,HCl pH 8.5, 0.16,M NaCl at 288,K. Diffraction data sets for the apo and inhibitor-bound forms were collected to 2.50 and 2.45,Å resolution, respectively. The space group and the unit-cell parameters of the crystal indicated the presence of two molecules per asymmetric unit, with a solvent content of 50%. The structure of Os4BGlu12 was successfully solved in space group P43212 by molecular replacement using the white clover cyanogenic ,-glucosidase structure (PDB code 1cbg) as a search model. [source] Production, purification, crystallization and preliminary X-ray diffraction analysis of the HIV-2-neutralizing V3 loop-specific Fab fragment 7C8ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2009Hannes Uchtenhagen 7C8 is a mouse monoclonal antibody that is specific for the third hypervariable loop (V3 loop) of the human immunodeficiency virus type 2 (HIV-2) associated protein gp125. Fab fragments of 7C8 effectively neutralize HIV-2. 7C8 was expressed and purified from a hybridoma cell line in order to establish the molecular basis underlying the specificity of the 7C8 antibody for the V3 loop as well as the specific role of the elongated third complementarity-determining region of the heavy chain (CDRH3). The antibody was digested with papain and Fab fragments were purified using size-exclusion chromatography. Hanging-drop vapour-diffusion crystallization techniques were employed and the protein was crystallized in 50,mM ammonium sulfate, 100,mM Tris,HCl pH 8.5, 25%(w/v) PEG 8000 and 2.5%(w/v) PEG 400 at 275,K. The analysed crystals belonged to the rhombohedral space group P3221, with unit-cell parameters a = b = 100.1, c = 196.8,Å, and diffracted to 2.7,Å resolution. [source] Purification, crystallization and preliminary structural analysis of nucleoside diphosphate kinase from Bacillus anthracisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2007Gauri Misra Bacillus anthracis nucleoside diphosphate kinase (BaNdk) is an enzyme whose primary function is to maintain deoxynucleotide triphosphate (dNTP) pools by converting deoxynucleotide diphosphates to triphosphates using ATP as the major phosphate donor. Although the structures of Ndks from a variety of organisms have been elucidated, the enzyme from sporulating bacteria has not been structurally characterized to date. Crystals of the B. anthracis enzyme were grown using the vapour-diffusion method from a hanging drop consisting of 2,µl 10,mg,ml,1 protein in 50,mM Tris,HCl pH 8.0, 50,mM NaCl, 5,mM EDTA equilibrated against 500,µl reservoir solution consisting of 2.25,M ammonium formate and 0.1,M HEPES buffer pH 7.25. Diffraction data extending to 2.0,Å were collected at room temperature from a single crystal with unit-cell parameters a = b = 107.53, c = 52.3,Å. The crystals are hexagonal in shape and belong to space group P6322. The crystals contain a monomer in the asymmetric unit, which corresponds to a Matthews coefficient (VM) of 2.1,Å3,Da,1 and a solvent content of about 36.9%. [source] Overexpression, purification, crystallization and preliminary structural studies of p -coumaric acid decarboxylase from Lactobacillus plantarumACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2007Blanca De Las Rivas The substrate-inducible p -coumaric acid decarboxylase (PDC) from Lactobacillus plantarum has been overexpressed in Escherichia coli, purified and confirmed to possess decarboxylase activity. The recombinant His6 -tagged enzyme was crystallized using the hanging-drop vapour-diffusion method from a solution containing 20%(w/v) PEG 4000, 12%(w/v) 2-propanol, 0.2,M sodium acetate, 0.1,M Tris,HCl pH 8.0 with 0.1,M barium chloride as an additive. Diffraction data were collected in-house to 2.04,Å resolution. Crystals belonged to the tetragonal space group P43, with unit-cell parameters a = b = 43.15, c = 231.86,Å. The estimated Matthews coefficient was 2.36,Å3,Da,1, corresponding to 48% solvent content, which is consistent with the presence of two protein molecules in the asymmetric unit. The structure of PDC has been determined by the molecular-replacement method. Currently, the structure of PDC complexed with substrate analogues is in progress, with the aim of elucidating the structural basis of the catalytic mechanism. [source] Crystallization and preliminary structure analysis of the blue laccase from the ligninolytic fungus Panus tigrinusACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2005Marta Ferraroni The blue laccase from the white-rot basidiomycete fungus Panus tigrinus, an enzyme involved in lignin biodegradation, has been crystallized. P. tigrinus laccase crystals grew within one week at 296,K using the sitting-drop vapour-diffusion method in 22%(w/v) PEG 4000, 0.2,M CaCl2, 100,mM Tris,HCl pH 7.5. The crystals belong to the monoclinic space group P21, with unit-cell parameters a = 54.2, b = 111.6, c = 97.1, , = 97.7°, and contain 46% solvent. A complete native data set was collected to 1.4,Å resolution at the copper edge. Molecular replacement using the Coprinus cinereus laccase structure (PDB code 1hfu) as a starting model was performed and initial electron-density maps revealed the presence of a full complement of copper ions. Model refinement is in progress. The P. tigrinus laccase structural model exhibits the highest resolution available to date and will assist in further elucidation of the catalytic mechanism and electron-transfer processes for this class of enzymes. [source] | ||||||||||