Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Cloning, expression, purification and preliminary X-ray analysis of a putative metagenome-derived lipase.

ABSTRACT: LipS is a novel thermostable putative lipase that was isolated from a metagenomic library using functional screening methods. The corresponding gene shows high similarity to that encoding a putative but uncharacterized esterase from Symbiobacterium thermophilum IAM14863 (99% nucleotide-sequence similarity). Two different constructs of the recombinant lipase were crystallized. Crystals belonging to space group P4(2)2(1)2 diffracted X-ray radiation to 2.8?Å resolution and crystals belonging to space group P4 diffracted to 2.0?Å resolution. The most probable content of their asymmetric units were two molecules (P4(2)2(1)2) and four or five molecules (P4), respectively.

SUBMITTER: Fersini F

PROVIDER: S-EPMC3412774 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Cloning, expression, purification and preliminary X-ray analysis of a putative metagenome-derived lipase.

Fersini Francesco F Dall'Antonia Yuliya Y Chow Jennifer J Streit Wolfgang R WR Mueller-Dieckmann Jochen J

Acta crystallographica. Section F, Structural biology and crystallization communications 20120731 Pt 8

LipS is a novel thermostable putative lipase that was isolated from a metagenomic library using functional screening methods. The corresponding gene shows high similarity to that encoding a putative but uncharacterized esterase from Symbiobacterium thermophilum IAM14863 (99% nucleotide-sequence similarity). Two different constructs of the recombinant lipase were crystallized. Crystals belonging to space group P4(2)2(1)2 diffracted X-ray radiation to 2.8 Å resolution and crystals belonging to spa ...[more]

PMID: 22869123

Similar Datasets

Cloning, purification, crystallization and preliminary X-ray studies of a carbohydrate-binding module (CBM_E1) derived from sugarcane soil metagenome.

Project description:In recent years, owing to the growing global demand for energy, dependence on fossil fuels, limited natural resources and environmental pollution, biofuels have attracted great interest as a source of renewable energy. However, the production of biofuels from plant biomass is still considered to be an expensive technology. In this context, the study of carbohydrate-binding modules (CBMs), which are involved in guiding the catalytic domains of glycoside hydrolases for polysaccharide degradation, is attracting growing attention. Aiming at the identification of new CBMs, a sugarcane soil metagenomic library was analyzed and an uncharacterized CBM (CBM_E1) was identified. In this study, CBM_E1 was expressed, purified and crystallized. X-ray diffraction data were collected to 1.95 Å resolution. The crystals, which were obtained by the sitting-drop vapour-diffusion method, belonged to space group I23, with unit-cell parameters a = b = c = 88.07 Å.

| S-EPMC4157425 | biostudies-literature

GPX3 from Arabidopsis thaliana: cloning, expression, purification, crystallization and preliminary X-ray analysis.

Project description:The Arabidopsis thaliana glutathione peroxidase 3 (GPX3) gene encodes a glutathione peroxidase with roles in H2O2 homeostasis and signalling. The GPX3 gene sequence was cloned into pGEX-6P1 and overexpressed in Escherichia coli. The GPX3 protein was purified to homogeneity in two chromatographic steps. Various lengths of the GPX3 sequence were used to obtain proteins that yielded crystals using vapour-diffusion techniques, but only GPX3ΔN36 (lacking 36 amino acids from the N-terminus) showed a good diffraction pattern. Its crystals diffracted to 2.8 Å resolution and belonged to space group P65, with unit-cell parameters a = b = 98.241, c = 42.057 Å.

| S-EPMC3818038 | biostudies-literature

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of recombinant human fumarase.

Project description:Human fumarase (HsFH) is a well-known citric acid cycle enzyme and is therefore a key component in energy metabolism. Genetic studies on human patients have shown that polymorphisms in the fumarase gene are responsible for diseases such as hereditary leiomyomatosis and renal cell cancer. As a first step in unravelling the molecular basis of the mechanism of fumarase deficiency in genetic disorders, the HsFH gene was cloned in pET-28a, heterologously expressed in Escherichia coli, purified by nickel-affinity chromatography and crystallized using the vapour-diffusion technique. X-ray diffraction experiments were performed at a synchrotron source and the structure was solved at 2.1?Å resolution by molecular replacement.

| S-EPMC3943092 | biostudies-literature

Cloning, expression, purification, crystallization and preliminary X-ray analysis of Mycoplasma genitalium protein MG289.

Project description:Mycoplasma genitalium is a human pathogen that is associated with nongonococcal urethritis in men and cervicitis in women. The cloning, expression, purification and crystallization of the protein MG289 from M. genitalium strain G37 are reported here. Crystals of MG289 diffracted X-rays to 2.8 A resolution. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 49.7, b = 90.9, c = 176.1 A. The diffraction data after processing had an overall R(merge) of 8.7%. The crystal structure of Cypl, the ortholog of MG289 from M. hyorhinis, has recently been determined, providing a reasonable phasing model; molecular replacement is currently under way.

| S-EPMC2795598 | biostudies-literature

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of AerF from Microcystis aeruginosa, a putative reductase participating in aeruginosin biosynthesis.

Project description:The 2-carboxy-6-hydroxyoctahydroindole moiety is an essential residue for the antithrombotic activity of aeruginosins, which are a class of cyanobacteria-derived bioactive linear tetrapeptides. The biosynthetic pathway of the 2-carboxy-6-hydroxyoctahydroindole moiety has not yet been resolved. AerF was indicated to be involved in the biosynthesis of the 2-carboxy-6-hydroxyoctahydroindole moiety. This study reports the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of AerF from Microcystis aeruginosa with a C-terminal His6 tag. The crystal diffracted to a maximum resolution of 1.38 Å and belonged to the tetragonal space group P4322, with unit-cell parameters a = b = 101.581, c = 116.094 Å. The calculated Matthews coefficient and solvent content of the crystal were 2.47 Å(3) Da(-1) and 50.32%, respectively. The initial model of the structure was obtained by the molecular-replacement method and refinement of the structure is in progress.

| S-EPMC4388185 | biostudies-literature

Cloning, expression, purification and preliminary X-ray crystallographic analysis of mouse protein arginine methyltransferase 7.

Project description:Protein arginine methyltransferase 7 (PRMT7) is a unique but less characterized member of the family of protein arginine methyltransferases (PRMTs) that plays a role in male germline gene imprinting. PRMT7 is the only known PRMT member that catalyzes the monomethylation but not the dimethylation of the target arginine residues and harbours two catalytic domains in tandem. PRMT7 genes from five different species were cloned and expressed in Escherichia coli and Sf21 insect cells. Four gave soluble proteins from Sf21 cells, of which two were homogeneous and one gave crystals. The mouse PRMT7 structure was solved by the single anomalous dispersion method using a crystal soaked with thimerosal that diffracted to beyond 2.1?Å resolution. The crystal belonged to space group P4(3)2(1)2, with unit-cell parameters a = b = 97.4, c = 168.1?Å and one PRMT7 monomer in the asymmetric unit. The structure of another crystal form belonging to space group I222 was solved by molecular replacement.

| S-EPMC3943109 | biostudies-literature

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of YvoA from Bacillus subtilis.

Project description:The putative transcriptional regulator protein YvoA (BSU35030) from Bacillus subtilis was cloned and heterologously expressed in Escherichia coli. The protein was purified by immobilized metal-affinity chromatography and size-exclusion chromatography and subsequently crystallized. A complete native data set was collected to 2.50 A resolution. The crystals belonged to the monoclinic space group C2 and preliminary analysis of the diffraction data indicated the presence of approximately 12 molecules per asymmetric unit.

| S-EPMC2664774 | biostudies-literature

Cloning, expression, purification, crystallization and preliminary X-ray analysis of Thermus aquaticus succinyl-CoA synthetase.

Project description:Succinyl-CoA synthetase (SCS) is an enzyme of the citric acid cycle and is thus found in most species. To date, there are no structures available of SCS from a thermophilic organism. To investigate how the enzyme adapts to higher temperatures, SCS from Thermus aquaticus was cloned, overexpressed, purified and crystallized. Attempts to crystallize the enzyme were thwarted by proteolysis of the beta-subunit and preferential crystallization of the truncated form. Crystals of full-length SCS were grown after the purification protocol was modified to include frequent additions of protease inhibitors. The resulting crystals, which diffract to 2.35 A resolution, are of the protein in complex with Mn2+-GDP.

| S-EPMC2335007 | biostudies-literature

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of the putative SAICAR synthetase (PH0239) from Pyrococcus horikoshii OT3.

Project description:The study of proteins involved in de novo biosynthesis of purine nucleotides is central in the development of antibiotics and anticancer drugs. In view of this, a protein from the hyperthermophile Pyrococcus horikoshii OT3 was isolated, purified and crystallized using the microbatch method. Its primary structure was found to be similar to that of SAICAR synthetase, which catalyses the seventh step of de novo purine biosynthesis. A diffraction-quality crystal was obtained using Hampton Research Crystal Screen II condition No. 34, consisting of 0.05 M cadmium sulfate hydrate, 0.1 M HEPES buffer pH 7.5 and 1.0 M sodium acetate trihydrate, with 40%(v/v) 1,4-butanediol as an additive. The crystal belonged to space group P3(1), with unit-cell parameters a = b = 95.62, c = 149.13 A. Assuming the presence of a hexamer in the asymmetric unit resulted in a Matthews coefficient (V(M)) of 2.3 A(3) Da(-1), corresponding to a solvent content of about 46%. A detailed study of this protein will yield insights into structural stability at high temperatures and should be highly relevant to the development of antibiotics and anticancer drugs targeting the biosynthesis of purine nucleotides.

| S-EPMC2815687 | biostudies-literature

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of bacterioferritin A from Mycobacterium tuberculosis.

Project description:Bacterioferritins (Bfrs) comprise a subfamily of the ferritin superfamily of proteins that play an important role in bacterial iron storage and homeostasis. Bacterioferritins differ from ferritins in that they have additional noncovalently bound haem groups. To assess the physiological role of this subfamily of ferritins, a greater understanding of the structural details of bacterioferritins from various sources is required. The gene encoding bacterioferritin A (BfrA) from Mycobacterium tuberculosis was cloned and expressed in Escherichia coli. The recombinant protein product was purified by affinity chromatography on a Strep-Tactin column and crystallized with sodium chloride as a precipitant at pH 8.0 using the vapour-diffusion technique. The crystals diffracted to 2.1 A resolution and belonged to space group P4(2), with unit-cell parameters a = 123.0, b = 123.0, c = 174.6 A.

| S-EPMC2376407 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data