scholarly journals Expression, purification and crystallization of a fungal type III polyketide synthase that produces the csypyrones

2014 ◽  
Vol 70 (6) ◽  
pp. 730-733 ◽  
Author(s):  
Dengfeng Yang ◽  
Takahiro Mori ◽  
Takashi Matsui ◽  
Makoto Hashimoto ◽  
Hiroyuki Morita ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Polyketide Synthase ◽  
Fatty Acyl ◽  
Diffusion Method ◽  
Unit Cell Parameters ◽  
Type Iii Polyketide Synthase ◽  
Cell Parameters ◽  
Type Iii ◽  
Malonyl Coa ◽  
Vapour Diffusion

CsyB fromAspergillus oryzaeis a novel type III polyketide synthase that catalyzes the formation of csypyrone B1 [4-(3-acetyl-4-hydroxy-2-oxo-2H-pyran-6-yl)butyric acid] from fatty acyl-CoA, malonyl-CoA and acetoacetyl-CoA. Recombinant CsyB expressed inEscherichia coliwas crystallized by the sitting-drop vapour-diffusion method. The crystals belonged to spaceP21, with unit-cell parametersa= 70.0,b= 104.8,c= 73.5 Å, β = 114.4°.

scholarly journals Cloning, purification and preliminary X-ray crystallographic analysis of the OmpA-like domain of peptidoglycan-associated lipoprotein fromAcinetobacter baumannii

2012 ◽  
Vol 68 (11) ◽  
pp. 1351-1353 ◽  
Author(s):  
Jung Hyun Song ◽  
Woo Cheol Lee ◽  
Jeong Soon Park ◽  
Seung Il Kim ◽  
Je Chul Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Acinetobacter Baumannii ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Asymmetric Unit ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Vapour Diffusion ◽  
Strain Bl21

Peptidoglycan-associated lipoprotein (Pal) is one component of the Tol–Pal system that is involved in maintaining the integrity and stability of the outer membrane. The C-terminal OmpA-like domain of Pal interacts noncovalently with peptidoglycan. In this study, the OmpA-like domain of Pal fromAcinetobacter baumanniiwas overexpressed inEscherichia colistrain BL21 (DE3), purified and crystallized using the vapour-diffusion method. A native crystal diffracted to 1.4 Å resolution and belonged to space groupP61orP65, with unit-cell parametersa=b= 72.58,c= 44.65 Å, a calculated Matthews coefficient of 2.64 Å3 Da−1and one molecule per asymmetric unit.

scholarly journals Crystallization and preliminary X-ray analysis of the hypothetical deaminase RPB_0146 fromRhodopseudomonas palustrisHaA2

2014 ◽  
Vol 70 (11) ◽  
pp. 1560-1562
Author(s):  
Guofang Zhang ◽  
Dan Yu ◽  
Guodong Yang ◽  
Hui Dong ◽  
Tongcun Zhang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rhodopseudomonas Palustris ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters ◽  
Vapour Diffusion

RPB_0146, a putative deaminase fromRhodopseudomonas palustrisHaA2, was expressed inEscherichia coliBL21 (DE3) cells and purified using a His6tag by Ni2+-chelating affinity chromatography for X-ray crystallographic analysis. Diffraction-quality crystals were grown by the hanging-drop vapour-diffusion method at 289 K and diffracted to a resolution of 2.44 Å using a wavelength of 1.000 Å at the Photon Factory (KEK), Japan. The crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 66.26,b= 123.94,c= 155.95 Å.

Initiating a structural study of 2-keto-3-deoxy-6-phosphogluconate aldolase from Escherichia coli

1999 ◽  
Vol 55 (11) ◽  
pp. 1946-1948 ◽  
Author(s):  
Louise V. Buchanan ◽  
Nupur Mehta ◽  
Luka Pocivavsek ◽  
S. Niranjanakumari ◽  
Eric J. Toone ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Light Scattering ◽  
Dynamic Light Scattering ◽  
Recombinant Protein ◽  
Diffusion Method ◽  
Unit Cell Parameters ◽  
Cell Parameters ◽  
Vapour Diffusion ◽  
Kdpg Aldolase ◽  
Aldol Addition

2-Keto-3-deoxy-6-phosphogluconate aldolase (KDPG aldolase, E.C. 4.1.2.14) is a member of the pyruvate/phosphoenolpyruvate aldolase family. It is also a synthetically useful enzyme, capable of catalyzing the stereoselective aldol addition of pyruvate to a range of unnatural electrophilic substrates. The recombinant protein was purified by a two-step HPLC protocol involving anion-exchange and hydrophobic chromatography. Dynamic light-scattering experiments indicated the protein to be monodisperse. Crystals were obtained using the sitting-drop vapour-diffusion method, with PEG 6K as precipitant. Diffraction data were collected on a frozen crystal to a resolution of 2.26 Å on station PX9.6 at the Daresbury synchrotron. The crystal belongs to space group P212121, with unit-cell parameters a = 53.2, b = 77.9, c = 146.8 Å.

scholarly journals Recombinant ACHT1 fromArabidopsis thaliana: crystallization and X-ray crystallographic analysis

2017 ◽  
Vol 73 (7) ◽  
pp. 382-385 ◽  
Author(s):  
Weimin Pan ◽  
Junchao Wang ◽  
Ye Yang ◽  
Lin Liu ◽  
Min Zhang
Keyword(s):  
Escherichia Coli ◽  
Arabidopsis Thaliana ◽  
Disulfide Bonds ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Unit Cell Parameters ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Vapour Diffusion

Thioredoxins (Trxs) play important roles in chloroplasts by linking photosynthetic light reactions to a series of plastid functions. They execute their function by regulating the oxidation and reduction of disulfide bonds. ACHT1 (atypical cysteine/histidine-rich Trx1) is a thylakoid-associated thioredoxin-type protein found in theArabidopsis thalianachloroplast. Recombinant ACHT1 protein was overexpressed inEscherichia coli, purified and crystallized by the vapour-diffusion method. The crystal diffracted to 1.7 Å resolution and a complete X-ray data set was collected. Preliminary crystallographic analysis suggested that the crystals belonged to space groupC2221, with unit-cell parametersa = 102.7,b= 100.6,c= 92.8 Å.

scholarly journals Expression, purification, crystallization and preliminary X-ray analysis of the human NORE1 SARAH domain

2012 ◽  
Vol 68 (7) ◽  
pp. 813-815 ◽  
Author(s):  
Hye Jin Kim ◽  
Eunha Hwang ◽  
Young-Hyun Han ◽  
Saehae Choi ◽  
Woo Cheol Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Kinase ◽  
Unit Cell ◽  
Diffusion Method ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Apoptotic Protein ◽  
Vapour Diffusion

NORE1 is an important tumour suppressor in human cancers that interacts with the pro-apoptotic protein kinase MST1/2 through SARAH domains. The SARAH domain (residues 366–413) of human NORE1 was expressed inEscherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystal diffracted to 2.7 Å resolution and belonged to space groupP6122, with unit-cell parametersa=b= 73.041,c = 66.092 Å, α = β = 90, γ = 120°.

scholarly journals Expression, purification and crystallization of a dye-decolourizing peroxidase fromDictyostelium discoideum

2014 ◽  
Vol 70 (2) ◽  
pp. 252-255 ◽  
Author(s):  
Amrita Rai ◽  
Roman Fedorov ◽  
Dietmar J. Manstein
Keyword(s):  
Escherichia Coli ◽  
Substrate Specificity ◽  
Dictyostelium Discoideum ◽  
Electron Acceptor ◽  
Diffusion Method ◽  
Asymmetric Unit ◽  
Unit Cell Parameters ◽  
Cell Parameters ◽  
Vapour Diffusion ◽  
Environmental Relevance

Dye-decolourizing peroxidases are haem-containing peroxidases with broad substrate specificity. Using H2O2as an electron acceptor, they efficiently decolourize various dyes that are of industrial and environmental relevance, such as anthraquninone- and azo-based dyes. In this study, the dye-decolourizing peroxidase DdDyP fromDictyostelium discoideumwas overexpressed inEscherichia colistrain Rosetta(DE3)pLysS, purified and crystallized using the vapour-diffusion method. A native crystal diffracted to 1.65 Å resolution and belonged to space groupP41212, with unit-cell parametersa=b= 141.03,c = 95.56 Å, α = β = γ = 90°. The asymmetric unit contains two molecules.

scholarly journals Purification, crystallization and preliminary X-ray crystallographic studies of Rv3899c fromMycobacterium tuberculosis

2015 ◽  
Vol 71 (1) ◽  
pp. 107-109 ◽  
Author(s):  
Yingjia Song ◽  
Jianghui Liu ◽  
De-Feng Li ◽  
Honglin Li ◽  
Shihua Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Tuberculosis ◽  
Hypothetical Protein ◽  
Diffusion Method ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Chromatographic Techniques ◽  
Vapour Diffusion

Rv3899c, a hypothetical protein fromMycobacterium tuberculosisthat is conserved within the mycobacteria, is predicted to be secreted and has been found in culture filtrates. Here, Rv3899c has been cloned, expressed inEscherichia coliand purified using standard chromatographic techniques. The hanging-drop vapour-diffusion method with PEG 3350 as a precipitant was used to crystallize the protein. N-terminal sequencing results showed that the amino-acid sequence of the crystallized protein began with GATAG, indicating that it is a fragment containing residues 184–410 of Rv3899c. Rv3899c184–410crystals exhibited the symmetry of space groupP212121, with unit-cell parametersa= 49.88,b= 54.72,c= 75.52 Å, α = β = γ = 90°, and diffracted to a resolution of 1.90 Å.

scholarly journals Crystallization and preliminary X-ray diffraction analysis of orotate phosphoribosyltransferase from the human malaria parasitePlasmodium falciparum

2012 ◽  
Vol 68 (2) ◽  
pp. 244-246 ◽  
Author(s):  
Yasuhide Takashima ◽  
Eiichi Mizohata ◽  
Keiji Tokuoka ◽  
Sudaratana R. Krungkrai ◽  
Yukiko Kusakari ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Orotic Acid ◽  
Diffusion Method ◽  
Tetragonal Symmetry ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Orotate Phosphoribosyltransferase ◽  
X Ray ◽  
Cell Parameters ◽  
Vapour Diffusion

Orotate phosphoribosyltransferase (OPRT) catalyzes the Mg2+-dependent condensation of orotic acid (OA) with 5-α-D-phosphorylribose 1-diphosphate (PRPP) to yield diphosphate (PPi) and the nucleotide orotidine 5′-monophosphate. OPRT fromPlasmodium falciparumproduced inEscherichia coliwas crystallized by the sitting-drop vapour-diffusion method in complex with OA and PRPP in the presence of Mg2+. The crystal exhibited tetragonal symmetry, belonging to space groupP41orP43, with unit-cell parametersa=b= 49.15,c = 226.94 Å. X-ray diffraction data were collected to 2.5 Å resolution at 100 K using a synchrotron-radiation source.

scholarly journals Crystallization and preliminary X-ray diffraction analysis of the Csu pili CsuC–CsuA/B chaperone–major subunit pre-assembly complex fromAcinetobacter baumannii

2015 ◽  
Vol 71 (6) ◽  
pp. 770-774 ◽  
Author(s):  
Natalia Pakharukova ◽  
Minna Tuittila ◽  
Sari Paavilainen ◽  
Anton Zavialov
Keyword(s):  
Diffusion Method ◽  
X Ray Diffraction ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
Gram Negative ◽  
X Ray ◽  
Cell Parameters ◽  
Abiotic Surfaces ◽  
Vapour Diffusion ◽  
Fimbrial Adhesins

The attachment of many Gram-negative pathogens to biotic and abiotic surfaces is mediated by fimbrial adhesins, which are assembledviathe classical, alternative and archaic chaperone–usher (CU) pathways. The archaic CU fimbrial adhesins have the widest phylogenetic distribution, yet very little is known about their structure and mechanism of assembly. To elucidate the biogenesis of archaic CU systems, structural analysis of the Csu fimbriae, which are used byAcinetobacter baumanniito form stable biofilms and cause nosocomial infection, was focused on. The major fimbriae subunit CsuA/B complexed with the CsuC chaperone was purified from the periplasm ofEscherichia colicells co-expressing CsuA/B and CsuC, and the complex was crystallized in PEG 3350 solution using the hanging-drop vapour-diffusion method. Selenomethionine-labelled CsuC–CsuA/B complex was purified and crystallized under the same conditions. The crystals diffracted to 2.40 Å resolution and belonged to the hexagonal space groupP6422, with unit-cell parametersa=b= 94.71,c = 187.05 Å, α = β = 90, γ = 120°. Initial phases were derived from a single anomalous diffraction (SAD) experiment using the selenomethionine derivative.

scholarly journals Crystallographic studies of aspartate racemase fromLactobacillus sakeiNBRC 15893

2015 ◽  
Vol 71 (8) ◽  
pp. 1012-1016 ◽  
Author(s):  
Tomomi Fujii ◽  
Takae Yamauchi ◽  
Makoto Ishiyama ◽  
Yoshitaka Gogami ◽  
Tadao Oikawa ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Low Temperatures ◽  
Diffusion Method ◽  
Lactobacillus Sakei ◽  
Unit Cell Parameters ◽  
Cell Parameters ◽  
Aspartate Racemase ◽  
Vapour Diffusion ◽  
Brewing Process ◽  
Resolution Structure

Aspartate racemase catalyzes the interconversion between L-aspartate and D-aspartate and belongs to the PLP-independent racemases. The enzyme from the lactic acid bacteriumLactobacillus sakeiNBRC 15893, isolated fromkimoto, is considered to be involved in D-aspartate synthesis during the brewing process of Japanese sake at low temperatures. The enzyme was crystallized at 293 K by the sitting-drop vapour-diffusion method using 25%(v/v) PEG MME 550, 5%(v/v) 2-propanol. The crystal belonged to space groupP3121, with unit-cell parametersa=b= 104.68,c= 97.29 Å, and diffracted to 2.6 Å resolution. Structure determination is under way.

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