PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (753444)

Clipboard (0)
None

Related Articles

1.  Crystallization and preliminary X-ray diffraction analysis of the complex of Kunitz-type tamarind trypsin inhibitor and porcine pancreatic trypsin 
A complex of tamarind trypsin inhibitor with porcine trypsin was crystallized and analyzed by X-ray diffraction.
The complex of Tamarindus indica Kunitz-type trypsin inhibitor and porcine trypsin has been crystallized by the sitting-drop vapour-diffusion method using ammonium acetate as precipitant and sodium acetate as buffer. The homogeneity of complex formation was checked by size-exclusion chromatography and further confirmed by reducing SDS–PAGE. The crystals diffracted to 2.0 Å resolution and belonged to the tetragonal space group P41, with unit-cell parameters a = b = 57.1, c = 120.1 Å. Preliminary X-ray diffraction analysis indicated the presence of one unit of inhibitor–trypsin complex per asymmetric unit, with a solvent content of 45%.
doi:10.1107/S1744309109041694
PMCID: PMC2777053  PMID: 19923745
tamarind trypsin inhibitor; porcine pancreatic trypsin; Kunitz-type inhibitors
2.  Isolation, purification, crystallization and preliminary crystallographic studies of chitinase from tamarind (Tamarindus indica) seeds 
A 34 kDa chitinase from tamarind (T. indica) seeds was purified, crystallized and characterized using X-ray diffraction.
A protein with chitinase activity has been isolated and purified from tamarind (Tamarindus indica) seeds. N-terminal amino-acid sequence analysis of this protein confirmed it to be an ∼34 kDa endochitinase which belongs to the acidic class III chitinase family. The protein was crystallized by the vapour-diffusion method using PEG 4000. The crystals belonged to the tetragonal space group P41, with two molecules per asymmetric unit. Diffraction data were collected to a resolution of 2.6 Å.
doi:10.1107/S1744309109006472
PMCID: PMC2664755  PMID: 19342775
chitinases; Tamarindus indicus
3.  Purification, crystallization and X-ray characterization of a Kunitz-type trypsin inhibitor protein from the seeds of chickpea (Cicer arietinum) 
The purification, characterization and crystallization of a trypsin inhibitor protein isolated from chickpea seeds are reported.
A Kunitz-type trypsin inhibitor protein (CPTI) purified from chickpea seeds was estimated to have a molecular mass of 18 kDa on SDS–PAGE. The IC50 value of CPTI was determined to be 2.5 µg against trypsin. The inhibitory activity of CPTI is 114 TIU (trypsin inhibitory units) per milligram of protein, which is high compared with those of other known Kunitz-type trypsin inhibitors from legumes. CPTI crystallized in three different orthorhombic crystal forms: P21212 form A, P21212 form B and P212121. The crystals of P21212 form A, with unit-cell parameters a = 37.2, b = 41.2, c = 104.6 Å, diffracted to 2.0 Å resolution at the home source and to 1.4 Å on beamline BM14 at the ESRF. Data were also collected from crystals grown in the presence of iodine. The Matthews coefficient for these crystals was calculated to be 2.37 Å3 Da−1, corresponding to a solvent content of 42%. The other two crystal forms (P21212 form B and P212121) diffracted comparatively poorly.
doi:10.1107/S1744309111015338
PMCID: PMC3107151  PMID: 21636920
chickpea; Kunitz-type trypsin inhibitors
4.  Crystallization and preliminary crystallographic characterization of the N-terminal Kunitz domain of boophilin 
The N-terminal Kunitz domain of boophilin, a specific thrombin inhibitor, was crystallized. The orthorhombic crystals had an unusually low solvent content and diffracted to beyond 0.87 Å resolution at a synchrotron source.
Boophilin is a tight-binding thrombin inhibitor composed of two canonical Kunitz-type domains in a tandem arrangement. Thrombin-bound boophilin can inhibit a second trypsin-like serine proteinase, most likely through the reactive loop of its N-terminal Kunitz domain. Here, the crystallization and preliminary crystallographic analysis of the isolated N-terminal domain of boophilin is reported. The crystals belonged to the orthorhombic space group P212121 and diffracted to beyond 1.8 Å resolution using a sealed-tube home source and to 0.87 Å resolution at a synchrotron source.
doi:10.1107/S1744309112005532
PMCID: PMC3325814  PMID: 22505414
thrombin inhibitors; noncovalent; blood coagulation; atomic resolution
5.  Crystallization and preliminary X-ray analysis of a novel Kunitz-type kallikrein inhibitor from Bauhinia bauhinioides  
Crystallization and preliminary X-ray diffraction studies are reported for a novel Kunitz-type protease inhibitor from B. bauhinioides which contains no disulfide bridges.
A Kunitz-type protease inhibitor (BbKI) found in Bauhinia bauhinioides seeds has been overexpressed in Escherichia coli and crystallized at 293 K using PEG 4000 as the precipitant. X-ray diffraction data have been collected to 1.87 Å resolution using an in-house X-ray generator. The crystals of the recombinant protein (rBbKI) belong to the orthorhombic space group P212121, with unit-cell parameters a = 46.70, b = 64.14, c = 59.24 Å. Calculation of the Matthews coefficient suggests the presence of one monomer of rBbKI in the asymmetric unit, with a corresponding solvent content of 51% (V M = 2.5 Å3 Da−1). Iodinated crystals were prepared and a derivative data set was also collected at 2.1 Å resolution. Crystals soaked for a few seconds in a cryogenic solution containing 0.5 M NaI were found to be reasonably isomorphous to the native crystals. Furthermore, the presence of iodide anions could be confirmed in the NaI-derivatized crystal. Data sets from native and derivative crystals are being evaluated for use in crystal structure determination by means of the SIRAS (single isomorphous replacement with anomalous scattering) method.
doi:10.1107/S1744309105028496
PMCID: PMC1991323  PMID: 16511193
Kunitz-type kallikrein inhibitors; disulfide bridges; Bauhinia bauhinioides
6.  Crystallization and preliminary X-ray diffraction studies of Murraya koenigii trypsin inhibitor 
A Kunitz-type trypsin inhibitor purified from the seeds of Murraya koenigii has been crystallized by the sitting-drop vapour-diffusion method using PEG 8000 as the precipitating agent.
A Kunitz-type trypsin inhibitor purified from the seeds of Murraya koenigii has been crystallized by the sitting-drop vapour-diffusion method using PEG 8000 as the precipitating agent. The crystals belong to the tetragonal space group P43212, with unit-cell parameters a = b = 75.8, c = 150.9 Å. The crystals contain two molecules in the asymmetric unit with a V M value of 2.5 Å3 Da−1. Diffraction was observed to 2.65 Å resolution and a complete data set was collected to 2.9 Å resolution.
doi:10.1107/S1744309107011414
PMCID: PMC2330219  PMID: 17401205
Kunitz-type trypsin inhibitor; Murraya koenigii
7.  Crystallization and preliminary crystallographic studies of Schizolobium parahyba chymotrypsin inhibitor (SPCI) at 1.8 Å resolution 
Crystallization and preliminary crystallographic studies of Schizolobium parahyba chymotrypsin inhibitor (SPCI) at 1.8 Å resolution.
SPCI, a Kunitz-type chymotrypsin inhibitor, is a 180-amino-acid polypeptide isolated from Schizolobium parahyba seeds. This inhibitor has been characterized as a highly stable protein over a broad pH and temperature range. SPCI was crystallized using a solution containing 0.1 M sodium acetate trihydrate buffer pH 4.6, 33%(v/v) PEG 2000 and 0.2 M ammonium sulfate. Data were collected to 1.80 Å resolution from a single crystal of SPCI under cryogenic conditions. The protein crystallized in space group P21212, with unit-cell parameters a = 40.01, b = 71.58, c = 108.68 Å and an R merge of 0.052. The structure of SPCI has been solved by molecular replacement using the known structure of the Kunitz-type trypsin inhibitor from Delonix regia (PDB code 1r8n) as the search model.
doi:10.1107/S1744309107045393
PMCID: PMC2339747  PMID: 18007042
chymotrypsin inhibitor; Schizolobium parahyba; Kunitz-type inhibitors
8.  Digestive Proteolytic Activity in the Sunn Pest, Eurygaster integriceps  
The Sunn pest, Eurygaster integriceps Puton (Heteroptera: Scutelleridae), is one of the most important pests of wheat and causes considerable damage to this valuable crop annually. Digestive proteinase activity of adult insects was investigated using general and specific substrates and inhibitors. Proteolytic activity was low when the common conventional substrates, azoalbumin, azocasein and hemoglobin were used to assay salivary glands and midguts. Using the fluorescent casein substrate (BODIPY FL casein), total proteolytic activity was measured at different pH. Maximum proteolytic activity was detected at pH 7 (100%) and 8(65%) which suggested the presence of serine proteinases in the salivary glands. There was no detectable proteolytic activity in midgut extracts. The inhibitors; PMSF (inhibitor of serine proteinases) and TPCK (a specific chymotrypsin inhibitor) showed greater than 50% inhibitory effect on total proteolytic activity, however, TLCK (specific trypsin inhibitor) and E-64(specific cysteine proteinase inhibitor) did not inhibit total proteolytic activity. Using fluorescent specific substrates for serine and cysteine proteinases (Z-Arg-AMC, Z-Arg-Arg-AMC, Z-Arg-Phe-AMC and Suc-Ala-Ala-Pro-Phe-AMZ) revealed the presence of tryptic and chymotryptic activity in the salivary gland extract. Zymogram analysis under non-reducing SDS-PAGE conditions and using the substrate APNE showed at least 8 tryptic and chymotryptic activity bands in salivary gland extracts. A single high molecular weight band with tryptic activity (165 kDa) was detected using the substrate BApNA in a zymogram analysis uisng native-PAGE. Kinetic studies showed a km value of 0.6 mM for this enzyme against the substrate BApNA .The inhibitor TLCK decreased activity of the trypsin-like enzyme up to 73% and almost completely eliminated the only band related to this proteinase in the zymogram. Soybean Kunitz type trypsin inhibitor showed no effect on proteolytic activity of the trypsin-like serine proteinase. In general, the results revealed the presence of chymotrypsin- and trypsin-like serine proteinases in the salivary gland of E. integriceps, and it seems that the major total proteolytic activity is due to chymotrypsin proteinases.
doi:10.1673/031.009.7001
PMCID: PMC3011966  PMID: 20053125
wheat; digestive enzymes; salivary glands; proteinase
9.  Crystallization and preliminary X-ray analysis of a protease inhibitor from the latex of Carica papaya  
The Kunitz-type trypsin/chymotrypsin inhibitor isolated from C. papaya latex has been crystallized using the hanging-drop vapour-diffusion method. Two different crystal forms are observed, diffracting to 2.6 and 1.7 Å.
A Kunitz-type protease inhibitor purified from the latex of green papaya (Carica papaya) fruits was crystallized in the presence and absence of divalent metal ions. Crystal form I, which is devoid of divalent cations, diffracts to a resolution of 2.6 Å and belongs to space group P31 or P32. This crystal form is a merohedral twin with two molecules in the asymmetric unit and unit-cell parameters a = b = 74.70, c = 78.97 Å. Crystal form II, which was grown in the presence of Co2+, diffracts to a resolution of 1.7 Å and belongs to space group P212121, with unit-cell parameters a = 44.26, b = 81.99, c = 140.89 Å.
doi:10.1107/S1744309106046367
PMCID: PMC2225369  PMID: 17142906
protease inhibitors; detwinning
10.  Protective Effects of Recombinant Kunitz-Domain 1 of Human Tissue Factor Pathway Inhibitor-2 Against 2-Chloroethyl Ethyl Sulfide Toxicity In Vitro 
Eplasty  2007;8:e3.
Objective: Sulfur mustard is a well-known blistering chemical warfare agent that has been investigated for its toxicological mechanisms and an efficacious antidote. Since sulfur mustard injury involves dermal:epidermal separation, proteolytic enzymes were suspected to be involved for this separation and eventual blister development. Therefore, protease inhibitors could be of therapeutic utility against sulfur mustard injury. In this study, the effects of Kunitz-domain 1 of human tissue factor pathway inhibitor-2 were evaluated against the toxic effects of 2-chloroethyl ethyl sulfide, a surrogate agent of sulfur mustard. Tissue factor pathway inhibitor-2 is a 32-kDa serine protease inhibitor produced by a variety of cell types including human epidermal keratinocytes, fibroblasts, and endothelial cells. It consists of 3 Kunitz-domains and the first Kunitz-domain contains the putative P1 residue (arginine at position 24) responsible for protease inhibitory activity. Methods: Recombinant wild-type and R24Q mutant Kunitz-domain 1s were expressed in Escherichia coli and purified. The purified proteins were refolded, and their effects were tested in an in vitro human epidermal keratinocyte cell wounding assay. Results: Wild-type but not R24Q Kunitz-domain 1 inhibited the amidolytic activity of trypsin and plasmin. Wild-type Kunitz-domain1 was stable for 4 weeks at 42°C and for more than 8 weeks at room temperature. Wild-type Kunitz-domain 1 significantly improved wound healing of unexposed and 2-chloroethyl ethyl sulfide–exposed cells without influencing cell proliferation. Although R24Q Kunitz-domain 1 lacked trypsin and plasmin inhibitory activity, it promoted wound closure of untreated and 2-chloroethyl ethyl sulfide–treated cells but to a much lesser degree. Conclusion: These data suggest that wild-type Kunitz-domain 1 of human tissue factor pathway inhibitor-2 can be developed as a medical countermeasure against sulfur mustard cutaneous injury.
PMCID: PMC2206002  PMID: 18213399
11.  Protective Effects of Recombinant Kunitz-Domain 1 of Human Tissue Factor Pathway Inhibitor-2 Against 2-Chloroethyl Ethyl Sulfide Toxicity In Vitro 
Objective: Sulfur mustard is a well-known blistering chemical warfare agent that has been investigated for its toxicological mechanisms and an efficacious antidote. Since sulfur mustard injury involves dermal:epidermal separation, proteolytic enzymes were suspected to be involved for this separation and eventual blister development. Therefore, protease inhibitors could be of therapeutic utility against sulfur mustard injury. In this study, the effects of Kunitz-domain 1 of human tissue factor pathway inhibitor-2 were evaluated against the toxic effects of 2-chloroethyl ethyl sulfide, a surrogate agent of sulfur mustard. Tissue factor pathway inhibitor-2 is a 32-kDa serine protease inhibitor produced by a variety of cell types including human epidermal keratinocytes, fibroblasts, and endothelial cells. It consists of 3 Kunitz-domains and the first Kunitz-domain contains the putative P1 residue (arginine at position 24) responsible for protease inhibitory activity. Methods: Recombinant wild-type and R24Q mutant Kunitz-domain 1s were expressed in Escherichia coli and purified. The purified proteins were refolded, and their effects were tested in an in vitro human epidermal keratinocyte cell wounding assay. Results: Wild-type but not R24Q Kunitz-domain 1 inhibited the amidolytic activity of trypsin and plasmin. Wild-type Kunitz-domain1 was stable for 4 weeks at 42°C and for more than 8 weeks at room temperature. Wild-type Kunitz-domain 1 significantly improved wound healing of unexposed and 2-chloroethyl ethyl sulfide–exposed cells without influencing cell proliferation. Although R24Q Kunitz-domain 1 lacked trypsin and plasmin inhibitory activity, it promoted wound closure of untreated and 2-chloroethyl ethyl sulfide–treated cells but to a much lesser degree. Conclusion: These data suggest that wild-type Kunitz-domain 1 of human tissue factor pathway inhibitor-2 can be developed as a medical countermeasure against sulfur mustard cutaneous injury.
PMCID: PMC1937028  PMID: 17846661
12.  Isolation, Cloning and Structural Characterisation of Boophilin, a Multifunctional Kunitz-Type Proteinase Inhibitor from the Cattle Tick 
PLoS ONE  2008;3(2):e1624.
Inhibitors of coagulation factors from blood-feeding animals display a wide variety of structural motifs and inhibition mechanisms. We have isolated a novel inhibitor from the cattle tick Boophilus microplus, one of the most widespread parasites of farm animals. The inhibitor, which we have termed boophilin, has been cloned and overexpressed in Escherichia coli. Mature boophilin is composed of two canonical Kunitz-type domains, and inhibits not only the major procoagulant enzyme, thrombin, but in addition, and by contrast to all other previously characterised natural thrombin inhibitors, significantly interferes with the proteolytic activity of other serine proteinases such as trypsin and plasmin. The crystal structure of the bovine α-thrombin·boophilin complex, refined at 2.35 Å resolution reveals a non-canonical binding mode to the proteinase. The N-terminal region of the mature inhibitor, Q16-R17-N18, binds in a parallel manner across the active site of the proteinase, with the guanidinium group of R17 anchored in the S1 pocket, while the C-terminal Kunitz domain is negatively charged and docks into the basic exosite I of thrombin. This binding mode resembles the previously characterised thrombin inhibitor, ornithodorin which, unlike boophilin, is composed of two distorted Kunitz modules. Unexpectedly, both boophilin domains adopt markedly different orientations when compared to those of ornithodorin, in its complex with thrombin. The N-terminal boophilin domain rotates 9° and is displaced by 6 Å, while the C-terminal domain rotates almost 6° accompanied by a 3 Å displacement. The reactive-site loop of the N-terminal Kunitz domain of boophilin with its P1 residue, K31, is fully solvent exposed and could thus bind a second trypsin-like proteinase without sterical restraints. This finding explains the formation of a ternary thrombin·boophilin·trypsin complex, and suggests a mechanism for prothrombinase inhibition in vivo.
doi:10.1371/journal.pone.0001624
PMCID: PMC2230226  PMID: 18286181
13.  Cloning, purification, crystallization and preliminary X-ray crystallographic analysis of a cyclophilin A-like protein from Piriformospora indica  
Cyclophin A like protein from Piriformospora indica involved in salt-stress tolerance was cloned, overexpressed, purified and crystallized. The crystals obtained diffracted X-rays to 2 Å resolution and belonged to space group C2221.
Cyclophilins are widely distributed both in eukaryotes and prokaryotes and have a primary role as peptidyl-prolyl cis–trans isomerases (PPIases). This study focuses on the cloning, expression, purification and crystallization of a salinity-stress-induced cyclophilin A (CypA) homologue from the symbiotic fungus Piriformospora indica. Crystallization experiments in the presence of 56 mM sodium phosphate monobasic monohydrate, 1.34 M potassium phosphate dibasic pH 8.2 yielded crystals that were suitable for X-ray diffraction analysis. The crystals belonged to the orthorhombic space group C2221, with unit-cell parameters a = 121.15, b = 144.12, c = 110.63 Å. The crystals diffracted to a resolution limit of 2.0 Å. Analysis of the diffraction data indicated the presence of three molecules of the protein per asymmetric unit (V M = 4.48 Å3 Da−1, 72.6% solvent content).
doi:10.1107/S1744309112018131
PMCID: PMC3370917  PMID: 22684077
cyclophilin A; Piriformospora indica; salinity stress
14.  Purification, crystallization and preliminary X-ray analysis of rice BGlu1 β-glucosidase with and without 2-deoxy-2-fluoro-β-d-glucoside 
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.
doi:10.1107/S1744309106027084
PMCID: PMC2242908  PMID: 16880561
BGlu1 β-glucosidase; 2,4-dinitrophenyl-2-­deoxy-2-fluoro-β-d-glucopyranoside; rice
15.  Purification, crystallization and preliminary crystallographic studies of SPCI–chymotrypsin complex at 2.8 Å resolution 
S. parahyba chymotrypsin inhibitor in complex with chymotrypsin has been purified and crystallized using the vapour-diffusion method and preliminarily analysis has been performed using X-ray diffraction.
A binary complex of the Schizolobium parahyba chymotrypsin inhibitor (SPCI) with chymotrypsin was purified by size-exclusion chromatography and crystallized by the sitting-drop vapour-diffusion method with 100 mM MES–NaOH pH 5.5, 20%(w/v) PEG 6000, 200 mM LiCl as precipitant and 200 mM nondetergent sulfobetaine molecular weight 201 Da (NDSB-201) as an additive. SPCI is a small protein with 180 amino-acid residues isolated from S. parahyba seeds and is able to inhibit chymotrypsin at a 1:1 molar ratio by forming a stable complex. X-ray data were collected to 2.8 Å resolution from a single crystal of the SPCI–chymotrypsin binary complex under cryogenic conditions. The crystal belongs to space group P212121, with unit-cell parameters a = 45.28, b = 64.57, c = 169.23 Å, and the R merge is 0.122 for 11 254 unique reflections. A molecular-replacement solution was found using the preliminary crystal structure of SPCI and the structure of chymotrypsin (PDB code 4cha) independently as search models.
doi:10.1107/S1744309108026870
PMCID: PMC2564883  PMID: 18931434
proteinase inhibitors; Kunitz inhibitors; Schizolobium parahyba
16.  Purification, crystallization and preliminary X-ray characterization of a haemagglutinin from the seeds of Jatropha curcas  
A novel haemagglutinin from Jatropha curcas seeds is purified and crystallized. X-ray diffraction data collected from the rod-shaped crystals were processed in the orthorhombic space group P212121 and the crystals diffracted to 2.8 Å resolution at 103 K.
The plant Jatropha curcas (Euphorbiaceae) is an important source of biofuel from the inedible oil present in its toxic seeds. The toxicity arises from the presence of curcin, a ribosome-inactivating protein showing haemagglutination activity. In this communication, the purification, crystallization and preliminary X-ray characterization are reported of a small protein isolated from J. curcas seeds with a molecular mass of ∼10 kDa that agglutinates rabbit erythrocytes. The protein was crystallized using the hanging-drop vapour-diffusion method and also by the microbatch method in 72-well HLA plates, using PEG 8000 as the precipitant in both conditions. X-ray diffraction data collected from the rod-shaped crystals were processed in the orthorhombic space group P212121. The crystals diffracted to 2.8 Å resolution at 103 K.
doi:10.1107/S1744309111038218
PMCID: PMC3232132  PMID: 22139159
Jatropha curcas; haemagglutinins
17.  Structure of the recombinant BPTI/Kunitz-type inhibitor rShPI-1A from the marine invertebrate Stichodactyla helianthus  
The recombinant BPTI/Kunitz-type inhibitor rShPI-1A from the Caribbean sea anemone S. helianthus has been crystallized and its structure has been refined to 2.5 Å resolution and compared with known X-ray structures of BPTI-like Kunitz-type inhibitors.
The BPTI/Kunitz-type inhibitor family includes several extremely potent serine protease inhibitors. To date, the inhibitory mechanisms have only been studied for mammalian inhibitors. Here, the first crystal structure of a BPTI/Kunitz-type inhibitor from a marine invertebrate (rShPI-1A) is reported to 2.5 Å resolution. Crystallization of recombinant rShPI-1A required the salt-induced dissociation of a trypsin complex that was previously formed to avoid intrinsic inhibitor aggregates in solution. The rShPI-1A structure is similar to the NMR structure of the molecule purified from the natural source, but allowed the assignment of disulfide-bridge chiralities and the detection of an internal stabilizing water network. A structural comparison with other BPTI/Kunitz-type canonical inhibitors revealed unusual ϕ angles at positions 17 and 30 to be a particular characteristic of the family. A significant clustering of ϕ and ψ angle values in the glycine-rich remote fragment near the secondary binding loop was additionally identified, but its impact on the specificity of rShPI-1A and similar molecules requires further study.
doi:10.1107/S1744309112039085
PMCID: PMC3515366  PMID: 23143234
protease inhibitors; BPTI/Kunitz-type domains
18.  Screening of Various Botanical Extracts for Antioxidant Activity Using DPPH Free Radical Method 
Aiming at the exploration of herbal use by society, crude extracts of the seeds of some commonly used medicinal plants (Vitis vinifera, Tamarindus indica and Glycin max) were screened for their free radical scavenging properties using ascorbic acid as standard antioxidant. Free radical scavenging activity was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical. The overall antioxidant activity of grape seeds (Vitis vinifera) was the strongest, followed in descending order by soybean (Glycin max) and tamarind (Tamarindus indica). The seeds extract of Vitis vinifera, Glycin max and Tamarindus indica showed 85.61%, 83.45% and 79.26%, DPPH scavenging activity respectively.
PMCID: PMC3847382  PMID: 24311867
Antioxidant activity; DPPH; free-radical; Vitis vinifera; Glycin max; Tamarindus indica
19.  Cloning, expression, purification, crystallization and preliminary X-ray analysis of the human RuvBL1–RuvBL2 complex 
A truncated variant of the human RuvBL1–RuvBL2 complex was cloned, expressed, purified and crystallised. Synchrotron diffraction data to 4 Å resolution were used to carry out a preliminary crystallographic analysis of the complex.
The complex of RuvBL1 and its homologue RuvBL2, two evolutionarily highly conserved eukaryotic proteins belonging to the AAA+ (ATPase associated with diverse cellular activities) family of ATPases, was co-expressed in Escherichia coli. For crystallization purposes, the flexible domains II of RuvBL1 and RuvBL2 were truncated. The truncated RuvBL1–RuvBL2 complex was crystallized using the hanging-drop vapour-diffusion method at 293 K. The crystals were hexagonal-shaped plates and belonged to either the orthorhombic space group C2221, with unit-cell parameters a = 111.4, b = 188.0, c = 243.4 Å and six monomers in the asymmetric unit, or the monoclinic space group P21, with unit-cell parameters a = 109.2, b = 243.4, c = 109.3 Å, β = 118.7° and 12 monomers in the asymmetric unit. The crystal structure could be solved by molecular replacement in both possible space groups and the solutions obtained showed that the complex forms a dodecamer.
doi:10.1107/S174430910802558X
PMCID: PMC2531268  PMID: 18765919
RuvBL1; RuvBL2; ATPases
20.  Purification, crystallization and preliminary X-ray analysis of CMS1MS2: a cysteine proteinase from Carica candamarcensis latex 
CMS1MS2, a cysteine proteinase from C. candamarcensis, displays high amidase activity against the substrate BAPNA. The enzyme was purified and crystallized by the hanging-drop method and preliminary diffraction data were collected to 1.8 Å resolution.
Cysteine proteinases from the latex of plants of the family Caricaceae are widely used industrially as well as in pharmaceutical preparations. In the present work, a 23 kDa cysteine proteinase from Carica candamarcensis latex (designated CMS1MS2) was purified for crystallization using three chromatography steps. The enzyme shows about fourfold higher activity than papain with BAPNA as substrate. Crystals suitable for X-ray diffraction experiments were obtained by the hanging-drop method in the presence of PEG and ammonium sulfate as precipitants. The crystals are monoclinic (space group P21), with unit-cell parameters a = 53.26, b = 75.71, c = 53.23 Å, β = 96.81°, and diffract X-rays to 1.8 Å resolution.
doi:10.1107/S174430910801172X
PMCID: PMC2496862  PMID: 18540057
Carica candamarcensis; Caricaceae; cysteine proteinases
21.  Structure of the SARS coronavirus main proteinase as an active C2 crystallographic dimer 
An orthorhombic crystal form of the SARS CoV main proteinase diffracting to a resolution of 1.9 Å is reported. The conformation of residues in the catalytic site indicates an active enzyme.
The 34 kDa main proteinase (Mpro) from the severe acute respiratory syndrome coronavirus (SARS-CoV) plays an important role in the virus life cycle through the specific processing of viral polyproteins. As such, SARS-CoV Mpro is a key target for the identification of specific inhibitors directed against the SARS virus. With a view to facilitating the development of such compounds, crystals were obtained of the enzyme at pH 6.5 in the orthorhombic space group P21212 that diffract to a resolution of 1.9 Å. These crystals contain one monomer per asymmetric unit and the biologically active dimer is generated via the crystallographic twofold axis. The conformation of the catalytic site indicates that the enzyme is active in the crystalline form and thus suitable for structure-based inhibition studies.
doi:10.1107/S1744309105033257
PMCID: PMC1978130  PMID: 16511208
protease; crystallographic dimer; SARS coronavirus
22.  Preliminary crystallographic analysis of the N-­terminal domain of FILIA, a protein essential for embryogenesis 
The preliminary crystallographic analysis of the N-terminal domain of FILIA is described in this paper. FILIA is a component of subcortical maternal complex, which plays critical roles in embryogenesis.
FILIA is a component of the subcortical maternal complex that is essential for early stage embryogenesis. Its 6×His-tagged N-terminal domain was expressed in Escherichia coli and purified to homogeneity. Two types of crystals formed under different crystallization conditions during screening. Orthorhombic crystals appeared in a solution containing 1.4 M ammonium sulfate, 0.1 M Tris pH 8.2 and 12% glycerol, while tetragonal crystals were obtained using 15% PEG 4000 mixed with 0.1 M HEPES pH 7.5 and 15% 2-propanol. High-quality diffraction data were collected from the two crystal forms to resolutions of 1.8 and 2.2 Å, respectively, using synchrotron radiation. The Matthews coefficients indicated that the P212121 and P41212 crystals contained two molecules and one molecule per asymmetric unit, respectively. A selenomethionine-substituted sample failed to crystallize under the native conditions, but another ortho­rhombic crystal form was obtained under different conditions and anomalous diffraction data were collected.
doi:10.1107/S1744309110031994
PMCID: PMC2935241  PMID: 20823540
FILIA; embryogenesis
23.  Cloning, purification, crystallization and preliminary structural studies of penicillin V acylase from Bacillus subtilis  
An unannotated protein reported from B. subtilis has been expressed in E. coli and identified as possessing penicillin V acylase activity. The crystallization and preliminary crystallographic analysis of this penicillin V acylase is presented.
Penicillin acylase proteins are amidohydrolase enzymes that cleave penicillins at the amide bond connecting the side chain to their β-lactam nucleus. An unannotated protein from Bacillus subtilis has been expressed in Escherichia coli, purified and confirmed to possess penicillin V acylase activity. The protein was crystallized using the hanging-drop vapour-diffusion method from a solution containing 4 M sodium formate in 100 mM Tris–HCl buffer pH 8.2. Diffraction data were collected under cryogenic conditions to a spacing of 2.5 Å. The crystals belonged to the orthorhombic space group C2221, with unit-cell parameters a = 111.0, b = 308.0, c = 56.0 Å. The estimated Matthews coefficient was 3.23 Å3 Da−1, corresponding to 62% solvent content. The structure has been solved using molecular-replacement methods with B. sphaericus penicillin V acylase (PDB code ) as the search model.
doi:10.1107/S1744309105017987
PMCID: PMC1952454  PMID: 16511127
Ntn hydrolase; penicillin V acylase; conjugated bile-salt hydrolase
24.  Cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of an inositol monophosphatase family protein (SAS2203) from Staphylococcus aureus MSSA476 
The cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of an inositol monophosphatase family protein (SAS2203) from S. aureus MSSA476 is reported.
The gene product of the sas2203 ORF of Staphylococcus aureus MSSA476 encodes a 30 kDa molecular-weight protein with a high sequence resemblance (29% identity) to tetrameric inositol monophosphatase from Thermotoga maritima. The protein was cloned, expressed, purified to homogeneity and crystallized. Crystals appeared in several conditions and good diffraction-quality crystals were obtained from 0.2 M Li2SO4, 20% PEG 3350, 0.1 M HEPES pH 7.0 using the sitting-drop vapour-diffusion method. A complete diffraction data set was collected to 2.6 Å resolution using a Rigaku MicroMax-007 HF Cu Kα X-­ray generator and a Rigaku R-AXIS IV++ detector. The diffraction data were consistent with the orthorhombic space group P212121, with unit-cell parameters a = 49.98, b = 68.35, c = 143.79 Å, α = β = γ = 90°, and the crystal contained two molecules in the asymmetric unit.
doi:10.1107/S1744309111003496
PMCID: PMC3080153  PMID: 21505244
SAS2203; Staphylococcus aureus MSSA476; inositol monophosphatase family
25.  Expression, purification, crystallization and preliminary X-ray diffraction analysis of Arabidopsis thaliana cyclophilin 38 (AtCyp38) 
Crystallization of Arabidopsis thaliana cyclophilin 38. The crystal diffracts X-rays to 2.5 Å resolution.
AtCyp38 is one of the highly divergent multidomain cyclophilins from Arabidopsis thaliana. A recombinant form of AtCyp38 (residues 83–437) was expressed in Escherichia coli and purified to homogeneity. The protein was crystallized using the vapour-batch technique with PEG 6000 and t-butanol as precipitants. Crystals of recombinant AtCyp38 diffracted X-rays to better than 2.5 Å resolution at 95 K using a synchrotron-radiation source. The crystal belongs to the C-centred orthorhombic space group C2221, with unit-cell parameters a = 58.2, b = 95.9, c = 167.5 Å, and contains one molecule in the asymmetric unit. The selenomethionine derivative of the AtCyp38 protein was overexpressed, purified and crystallized in the same space group and data were collected to 3.5 Å at the NSLS synchrotron. The structure is being solved by the MAD method.
doi:10.1107/S1744309105037681
PMCID: PMC1978155  PMID: 16511242
cyclophilins; PPIases; AtCyp38

Results 1-25 (753444)