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Rice Bowman–Birk inhibitor was expressed and crystallized.

Bowman–Birk inhibitors (BBIs) are cysteine-rich proteins with inhibitory activity against proteases that are widely distributed in monocot and dicot species. The expression of rice BBI from Oryza sativa is up-regulated and induced by pathogens or insects during germination of rice seeds. The rice BBI (RBTI) of molecular weight 15 kDa has been crystallized using the hanging-drop vapour-diffusion method. According to the diffraction of rice BBI crystals at a resolution of 2.07 Å, the unit cell belongs to space group P212121, with unit-cell parameters a = 74.37, b = 96.69, c = 100.36 Å. Preliminary analysis indicates four BBI molecules in an asymmetric unit, with a solvent content of 58.29%.

D. rostrata lectin was crystallized by hanging-drop vapor diffusion. The crystal belongs to the orthorhombic space group I222 and diffracted to 1.87 Å resolution.

Lectins from the Diocleinae subtribe (Leguminosae) are highly similar proteins that promote various biological activities with distinctly differing potencies. The structural basis for this experimental data is not yet fully understood. Dioclea rostrata lectin was purified and crystallized by hanging-drop vapour diffusion at 293 K. The crystal belongs to the orthorhombic space group I222, with unit-cell parameters a = 61.51, b = 88.22, c = 87.76 Å.  Assuming the presence of one monomer per asymmetric unit, the solvent content was estimated to be about 47.9%. A complete data set was collected at 1.87 Å resolution.

The crystallization and preliminary X-ray diffraction analysis of a red marine alga lectin isolated from H. musciformis is reported.

HML, a lectin from the red marine alga Hypnea musciformis, defines a novel lectin family. Orthorhombic crystals of HML belonging to space group P212121 grew within three weeks at 293 K using the hanging-drop vapour-diffusion method. A complete data set was collected at 2.4 Å resolution. HML is the first marine alga lectin to be crystallized.

XometC, a cystathionine γ-lyase-like protein from X. oryzae pv. oryzae and an antibacterial drug-target protein against bacterial blight, was cloned, purified and crystallized. Preliminary X-ray crystallographic analysis of XometC crystals was carried out.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight of rice (Oryza sativa L.), one of the most devastating diseases of rice in most rice-growing countries. XometC, a cystathionine γ-lyase (CGL) like protein that is an antibacterial drug-target protein against Xoo, was cloned, expressed, purified and crystallized. CGL catalyzes the second step in the reverse-transsulfuration pathway, which is essential for the metabolic interconversion of the sulfur-containing amino acids cysteine and methionine. Crystals of two different shapes, plate-shaped and pyramid-shaped, diffracted to 2.9 and 3.2 Å resolution and belonged to the primitive orthogonal space group P212121 and the tetragonal space group P41 (or P43), with unit-cell parameters a = 73.0, b = 144.9, c = 152.3 Å and a = b = 78.2, c = 300.7 Å, respectively. For the P212121 crystals, three or four monomers exist in the asymmetric unit with a corresponding V M of 3.02 or 2.26 Å3 Da−1 and a solvent content of 59.3 or 45.7%. For the P41 (or P43) crystals, four or five monomers exist in the asymmetric unit with a corresponding V M of 2.59 or 2.09 Å3 Da−1 and a solvent content of 52.5 or 40.6%.

The galactose-specific lectin from the seeds of a leguminous plant, D. lablab, has been crystallized. Molecular-replacement solution using 3.0 Å X-ray diffraction data showed the lectin to be a tetramer.

The galactose-specific lectin from the seeds of Dolichos lablab has been crystallized using the hanging-drop vapour-diffusion technique. The crystals belong to space group P1, with unit-cell parameters a = 73.99, b = 84.13, c = 93.15 Å, α = 89.92, β = 76.01, γ = 76.99°. X-ray diffraction data to a resolution of 3.0 Å have been collected under cryoconditions (100 K) using a MAR imaging-plate detector system mounted on a rotating-anode X-ray generator. Molecular-replacement calculations carried out using the available structures of legume lectins as search models revealed that the galactose-specific lectin from D. lablab forms a tetramer similar to soybean agglutinin; two such tetramers are present in the asymmetric unit.

The crystallization of rice α-amylase/subtilisin bifunctional inhibitor is reported.

Rice bifunctional α-amylase/subtilisin inhibitor (RASI) can inhibit both α-­amylase from larvae of the red flour beetle (Tribolium castaneum) and subtilisin from Bacillus subtilis. The synthesis of RASI is up-regulated during the late milky stage in developing seeds. The 8.9 kDa molecular-weight RASI from rice has been crystallized using the hanging-drop vapour-diffusion method. According to 1.81 Å resolution X-ray diffraction data from rice RASI crystals, the crystal belongs to space group P21212, with unit-cell parameters a = 79.99, b = 62.95, c = 66.70 Å. Preliminary analysis indicates two RASI molecules in an asymmetric unit with a solvent content of 44%.

The 24 kDa egg lectin of Chinook salmon (Oncorhynchus tshawytscha) was purified by affinity chromatography from salmon eggs and crystallized by the hanging-drop vapor-diffusion method using 15/4 EO/OH (pentaerythritol ethoxylate) as a precipitant.

The 24 kDa egg lectin of Chinook salmon (Oncorhynchus tshawytscha) is released from the egg during the cortical reaction. The lectin functions in blocking polyspermy during the fertilization process. The egg lectin was purified by affinity chromatography from salmon eggs and crystallized by the hanging-drop vapor-diffusion method using 15/4 EO/OH (pentaerythritol ethoxylate) as a precipitant. The crystal diffracted synchrotron-radiation X-rays to 1.63 Å resolution. The crystal belongs to the monoclinic space group P21, with unit-cell parameters a = 93.0, b = 73.6, c = 113.6 Å, α = 90, β = 92.82, γ = 90°. The crystal is likely to contain eight molecules in the asymmetric unit (V M = 2.3 Å3 Da−1), corresponding to a solvent content of 45.5%. A self-rotation function suggests an arrangement with 222 point symmetry within the asymmetric unit.

Rice BGlu1 β-glucosidase was purified from recombinant E. coli and crystallized with and without the inhibitor 2-deoxy-2-fluoro-β-d-glucose. The crystals diffracted to 2.15 and 2.75 Å, respectively.

Rice (Oryza sativa) BGlu1 β-glucosidase was expressed in Escherichia coli with N-terminal thioredoxin and hexahistidine tags and purified by immobilized metal-affinity chromatography (IMAC). After removal of the N-terminal tags, cation-exchange and S-200 gel-filtration chromatography yielded a 50 kDa BGlu1 with >95% purity. The free enzyme and a complex with 2,4-dinitrophenyl-2-­deoxy-2-fluoro-β-d-glucopyranoside inhibitor were crystallized by microbatch and hanging-drop vapour diffusion. Small tetragonal crystals of BGlu1 with and without inhibitor grew in 18%(w/v) PEG 8000 with 0.1 M sodium cacodylate pH 6.5 and 0.2 M zinc acetate. Crystals of BGlu1 with inhibitor were streak-seeded into 23%(w/v) PEG MME 5000, 0.2 M ammonium sulfate, 0.1 M MES pH 6.7 to yield larger crystals. Crystals with and without inhibitor diffracted to 2.15 and 2.75 Å resolution, respectively, and had isomorphous orthorhombic unit cells belonging to space group P212121.

The cloning, overexpression, purification, crystallization and preliminary X-ray crystallographic analysis of triosephosphate isomerase from MRSA252 is reported.

Triosephosphate isomerase from methicillin-resistant Staphylococcus aureus (MRSA252) was cloned in pQE30 vector, overexpressed in Escherichia coli M15 (pREP4) cells and purified to homogeneity. The protein was crystallized from 1.6 M trisodium citrate dihydrate pH 6.5 using the hanging-drop vapour-diffusion method. The crystals belonged to space group P43212, with unit-cell parameters a = b = 79.15, c = 174.27 Å. X-ray diffraction data were collected and processed to a maximum resolution of 1.9 Å. The presence of two molecules in the asymmetric unit gave a Matthews coefficient (V M) of 2.64 Å3 Da−1, with a solvent content of 53.63%.

The cloning, overexpression, purification, crystallization and preliminary X-ray crystallographic analysis of glyceraldehyde-3-phosphate dehydrogenase from A. mylitta are reported.

Glyceraldehyde-3-phosphate dehydrogenase from Antheraea mylitta (AmGAPDH) was cloned in pQE30 vector, overexpressed in Escherichia coli M15 (pREP4) cells and purified to homogeneity. The protein was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to the orthorhombic space group I222, with unit-cell parameters a = 85.81, b = 133.72, c = 220.37 Å. X-ray diffraction data were collected and processed to a maximum resolution of 2.2 Å. The presence of three molecules in the asymmetric unit gave a Matthews coefficient (V M) of 2.80 Å3 Da−1, with a solvent content of 56.08%.

The purification, crystallization and preliminary X-ray crystallographic analysis of glyceraldehyde-3-phosphate dehydrogenase 1 (GAP1) from MRSA252 in the apo form is reported.

Glyceraldehyde-3-phosphate dehydrogenase 1 (GAP1) from methicillin-resistant Staphylococcus aureus (MRSA252) has been purified to homogeneity in the apo form. The protein was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space group P21, with unit-cell parameters a = 69.95, b = 93.68, c = 89.05 Å, β = 106.84°. X-ray diffraction data have been collected and processed to a maximum resolution of 2.2 Å. The presence of one tetramer in the asymmetric unit gives a Matthews coefficient (V M) of 1.81 Å3 Da−1 with a solvent content of 32%. The structure has been solved by molecular replacement and structure refinement is now in progress.

A thermostable multicopper oxidase from Thermus thermophilus HB27 (Tth-MCO) has been successfully crystallized using the sitting-drop and hanging-drop vapour-diffusion methods.

A thermostable multicopper oxidase from Thermus thermophilus HB27 (Tth-MCO) was successfully crystallized using the sitting-drop and hanging-drop vapour-diffusion methods. Crystallization conditions and preliminary X-ray diffraction data to 1.5 Å resolution obtained using synchrotron radiation at 100 K are reported. The crystals belonged to space group C2221, with unit-cell parameters a = 93.6, b = 110.3, c = 96.3 Å. A monomer in the asymmetric unit yielded a Matthews coefficient (V M) of 2.60 Å3 Da−1 and a solvent content of 53%. An inactive enzyme form, apo-Tth-MCO, was also crystallized and diffraction data were collected to 1.7 Å resolution. In addition, a second inactive form of the enzyme, Hg-Tth-MCO, was obtained by soaking apo-Tth-MCO crystals with mercury(II) chloride and data were collected to a resolution of 1.7 Å.

Canavalia boliviana lectin (Cbol) was purified using a Sephadex G-50 column and crystallized in the presence of X-Man by hanging-drop vapour diffusion at 293 K. After optimization, crystals suitable for diffraction were obtained using 0.1 M HEPES pH 7.5 and 3.0 M sodium formate.

Plant lectins are the most studied group of carbohydrate-binding proteins. Despite the high similarity between the members of the Diocleinae subtribe (Leguminosae) group, they present differing biological activities. Canavalia boliviana lectin (Cbol) was purified using a Sephadex G-50 column and crystallized in the presence of X-Man by hanging-drop vapour diffusion at 293 K. After optimization, crystals suitable for diffraction were obtained under the condition 0.1 M HEPES pH 7.5 and 3.0 M sodium formate. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 126.70, b = 66.64, c = 64.99 Å, α = 90.0, β = 120.8, γ = 90.0°. Assuming the presence of a dimer in the asymmetric unit, the solvent content was estimated to be about 46%. A complete data set was collected at 1.5 Å resolution.

The crystallization and characterization of a lectin isolated and purified from C. arietinum and possessing complex sugar specificity is reported.

The lectin isolated from mature seeds of Cicer arietinum (CAL) agglutinates pronase-treated rabbit and human erythrocytes and its haemagglutination activity is inhibited by fetuin and desialated fetuin but not by simple monosaccharides or oligosaccharides. The purified lectin is a dimer of molecular weight 43 000 Da composed of two identical subunits (MW 21 500), as confirmed by SDS–PAGE. The lectin has been crystallized using the hanging-drop vapour-diffusion method at 295 K over a well solution containing 0.2 M sodium acetate, 0.1 M sodium phosphate buffer pH 6.5 and 14%(w/v) polyethylene glycol 8000. The triangular prism-shaped crystals belong to space group R3 and have unit-cell parameters a = b = 81.2, c = 69.4 Å. The diffraction data are 93.8% complete to 2.3 Å Bragg spacing with an R merge of 0.103.

Xoo0352, which encodes d-alanine-d-alanine ligase A (DdlA), from X. oryzae pv. oryzae was cloned, purified and crystallized. Preliminary X-ray crystallographic analysis of DdlA crystals was performed.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight (BB), which is one of the most devastating diseases of rice in most rice-growing countries. d-­Alanine-d-alanine ligase A (DdlA), coded by the Xoo0352 gene, was expressed, purified and crystallized. DdlA is an enzyme that is involved in d-­alanine metabolism and the biosynthesis of an essential bacterial peptidoglycan precursor, in which it catalyzes the formation of d-alanyl-d-alanine from two d-­alanines, and is thus an attractive antibacterial drug target against Xoo. The DdlA crystals diffracted to 2.3 Å resolution and belonged to the primitive tetragonal space group P43212, with unit-cell parameters a = b = 83.0, c = 97.6 Å. There is one molecule in the asymmetric unit, with a corresponding V M of 1.88 Å3 Da−1 and a solvent content of 34.6%. The initial structure was determined by molecular replacement using d-alanine-d-alanine ligase from Staphylococcus aureus (PDB code 2i87) as a template model.

Glycerol kinase from the hyperthermophilic archaeon Thermococcus kodakaraensis was crystallized and preliminary crystallographic studies of the crystals were performed.

Glycerol kinase from the hyperthermophilic archaeon Thermococcus kodakaraensis was crystallized and preliminary crystallographic studies of the crystals were performed. Crystals were grown at 293 K by the sitting-drop vapour-diffusion method. Native X-ray diffraction data were collected to 2.4 Å resolution using synchrotron radiation at station BL44XU of SPring-8. The crystal belongs to the rhombohedral space group R3, with unit-cell parameters a = b = 217.48, c = 66.48 Å. Assuming the presence of two molecules in the asymmetric unit, the V M value was 2.7 Å3 Da−1 and the solvent content was 54.1%. The protein was also cocrystallized with substrates and diffraction data were collected to 2.7 Å resolution.

Crystals of the carbohydrate-recognition domain of human langerin were obtained that diffracted synchrotron radiation to 1.5 Å resolution.

Langerin, a lectin that is specific to Langerhans cells, interacts with glyco­conjugates through its carbohydrate-recognition domain (CRD). This carbo­hydrate binding occurs by an avidity-based mechanism that is enabled by the neck domain responsible for trimerization. Langerin binds HIV through its CRD and thus plays a protective role against its propagation by the internalization of virions in Birbeck granules. Here, the overproduction, purification and crystallization of the langerin CRD is reported. Crystals obtained by the hanging-drop vapour-diffusion method allowed the collection of a complete data set to 1.5 Å resolution and belonged to the tetragonal space group P42, with unit-cell parameters a = b = 79.55, c = 90.14 Å.

Recombinant human CLEC-2 was crystallized in the orthorhombic space group P212121 and X-ray diffraction data were collected to 2.0 Å.

The human C-type lectin-like protein CLEC-2 has recently been shown to be expressed on the surface of platelets and to function as a receptor for the snake-venom protein rhodocytin. The C-type lectin-like domain (CTLD) of CLEC-2 was expressed in Escherichia coli, refolded and purified. Crystals of this recombinant CLEC-2 were grown by sitting-drop vapour diffusion using polyethylene glycol (PEG) 6000 as a precipitant. After optimization, crystals were grown which diffracted to 2.0 Å using in-house radiation (λ = 1.5418 Å). These crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 35.407, b = 55.143, c = 56.078 Å. The presence of one molecule per asymmetric unit is consistent with a crystal volume per unit weight (V M) of 1.82 Å3 Da−1 and a solvent content of 32.6%. These results suggest that crystals producing diffraction of this quality will be suitable for the structural determination of human CLEC-2.

The sulfide:quinone oxidoreductase from A. ferrooxidans ATCC 23270 was overexpressed in E. coli and purified. Crystallization and preliminarily X-ray crystallographic analysis were performed for the recombinant enzyme.

The gene product of open reading frame AFE_1293 from Acidithiobacillus ferrooxidans ATCC 23270 is annotated as encoding a sulfide:quinone oxido­reductase, an enzyme that catalyses electron transfer from sulfide to quinone. Following overexpression in Escherichia coli, the enzyme was purified and crystallized using the hanging-drop vapour-diffusion method. The native crystals belonged to the tetragonal space group P42212, with unit-cell parameters a = b = 131.7, c = 208.8 Å, and diffracted to 2.3 Å resolution. Preliminary crystallographic analysis indicated the presence of a dimer in the asymmetric unit, with an extreme value of the Matthews coefficient (V M) of 4.53 Å3 Da−1 and a solvent content of 72.9%.

The cloning, overexpression, purification, crystallization and preliminary X-ray crystallographic analysis of glyceraldehyde-3-phosphate dehydrogenase 1 (GAP1) from MRSA252 are reported.

Glyceraldehyde-3-phosphate dehydrogenase 1 from methicillin-resistant Staphylo­coccus aureus (MRSA252) was cloned in pQE30 vector, overexpressed in Escherichia coli M15(pREP4) cells and purified to homogeneity. The protein was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space group P21, with unit-cell parameters a = 65.23, b = 95.58, c = 87.91 Å, β = 106.5°. X-ray diffraction data were collected and processed to a maximum resolution of 2.0 Å. The presence of one tetramer in the asymmetric unit gave a Matthews coefficient (V M) of 1.78 Å3 Da−1 and a solvent content of 31%. The structure was solved by molecular replacement and structure refinement is now in progress.

Phosphoribosylglycinamide formyltransferase (PurN) from Streptococcus mutans was expressed in E. coli, purified and studied crystallographically.

Phosphoribosylglycinamide formyltransferase (PurN) from Streptococcus mutans was recombinantly expressed in Escherichia coli. An effective purification protocol was established. The purified protein, which had a purity of >95%, was identified by SDS–PAGE and MALDI–TOF MS. The protein was crystallized using the vapour-diffusion method in hanging-drop mode with PEG 3350 as the primary precipitant. X-ray diffraction data were collected to 2.1 Å resolution. Preliminary X-ray analysis indicated that the crystal belonged to space group P212121, with unit-cell parameters a = 52.25, b = 63.29, c = 131.81 Å.

The crystallization of xylose reductase from C. tropicalis is reported.

Xylose reductase (XR), which requires NADPH as a co-substrate, catalyzes the reduction of d-xylose to xylitol, which is the first step in the metabolism of d-­xylose. The detailed three-dimensional structure of XR will provide a better understanding of the biological significance of XR in the efficient production of xylitol from biomass. XR of molecular mass 36.6 kDa from Candida tropicalis was crystallized using the hanging-drop vapour-diffusion method. According to X-ray diffraction data from C. tropicalis XR crystals at 2.91 Å resolution, the unit cell belongs to space group P31 or P32. Preliminary analysis indicated the presence of four XR molecules in the asymmetric unit, with 68.0% solvent content.

Champedak mannose-binding lectin was crystallized at 293 K. Crystallization conditions and preliminary X-ray diffraction analysis are reported.

Mannose-binding lectin from champedak (Artocarpus integer) is a homotetra­mer with a single-monomer molecular weight of 16 800 Da. Previous work has shown it to bind IgE and IgM, as well as being a mitogen of T cells in humans. Champedak mannose-binding lectin has successfully been used to detect altered glycosylation states of serum proteins. The protein was crystallized at 293 K in space group P212121 (unit-cell parameters a = 76.89, b = 86.22, c = 95.37 Å) and the crystals diffracted to 2.0 Å resolution.

Leucine aminopeptidase, an exopeptidase that hydrolyzes leucine from the N-terminus of polypeptides, from X. oryzae pv. oryzae was cloned, expressed and crystallized.

Xanthomonas oryzae pv. oryzae (Xoo) causes the serious disease bacterial blight in rice. The pepA (Xoo0834) gene from Xoo is one of around 100 genes that have been selected for the design of antibacterial drugs. The pepA gene encodes leucine aminopeptidase (LAP), an exopeptidase that catalyzes the hydrolysis of leucine residues from the N-terminus of a protein or peptide. This enzyme was expressed in Escherichia coli, purified and crystallized, and preliminary X-ray structural studies have been carried out. The LAP crystal diffracted to 2.6 Å resolution and belonged to the cubic space group P213. The unit-cell volume of the crystal was compatible with the presence of two monomers in the asymmetric unit.

The expression, purification and preliminary X-ray diffraction studies of a chitin-binding domain of the chitinase from P. furiosus are reported.

The crystallization and preliminary X-ray diffraction analysis of the chitin-binding domain of chitinase from a hyperthermophilic archaeon, Pyrococcus furiosus, are reported. The recombinant protein was prepared using an Escherichia coli overexpression system and was crystallized by the hanging-drop vapour-diffusion method. An X-ray diffraction data set was collected to 1.70 Å resolution. The crystal belonged to space group P43212 or P41212. The unit-cell parameters were determined to be a = b = 48.8, c = 85.0 Å.