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Microbatch Method (microbatch + method)
Selected AbstractsCharacterization, crystallization and preliminary X-ray analysis of bifunctional dihydrofolate reductase,thymidylate synthase from Plasmodium falciparumACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2004Penchit Chitnumsub The full-length pfdhfr-ts genes of the wild-type TM4/8.2 and the double mutant K1CB1 (C59R+S108N) from the genomic DNA of the corresponding Plasmodium falciparum parasite have been cloned into a modified pET(17b) plasmid and expressed in Escherichia coli BL21 (DE3) pLysS. Conditions for the expression and purification of the P. falciparum dihydrofolate reductase,thymidylate synthase (PfDHFR-TS) have been established that yield ,1,mg of the soluble active enzyme per litre of culture. The purified enzymes have been crystallized using a modified microbatch method with PEG 4000 as the primary precipitating agent. X-ray diffraction data were collected to 2.50 and 2.64,Å resolution under cryogenic conditions from single crystals of the two PfDHFR-TS proteins in complex with NADPH, dUMP and either Pyr30 or Pyr39. Preliminary X-ray analysis indicated that the crystals belong to the orthorhombic space group P212121, with two molecules per asymmetric unit and ,52% solvent content (VM, 2.6,Å3,Da,1). The use of a particular type of baby oil in the microbatch setup appeared to be beneficial to PfDHFR-TS crystallization and a preliminary comparison with another commonly used oil is described. [source] Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of the putative SAICAR synthetase (PH0239) from Pyrococcus horikoshii OT3ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2010Kavyashree Manjunath 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 P31, with unit-cell parameters a = b = 95.62, c = 149.13,Å. Assuming the presence of a hexamer in the asymmetric unit resulted in a Matthews coefficient (VM) of 2.3,Å3,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. [source] Crystallization and preliminary X-ray diffraction of the DEAD-box protein Mss116p complexed with an RNA oligonucleotide and AMP-PNPACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2009Mark Del Campo The Saccharomyces cerevisiae DEAD-box protein Mss116p is a general RNA chaperone which functions in mitochondrial group I and group II intron splicing, translation and RNA-end processing. For crystallization trials, full-length Mss116p and a C-terminally truncated protein (Mss116p/,598,664) were overproduced in Escherichia coli and purified to homogeneity. Mss116p exhibited low solubility in standard solutions (,1,mg,ml,1), but its solubility could be increased by adding 50,mMl -arginine plus 50,mMl -glutamate and 50% glycerol to achieve concentrations of ,10,mg,ml,1. Initial crystals were obtained by the microbatch method in the presence of a U10 RNA oligonucleotide and the ATP analog AMP-PNP and were then improved by using seeding and sitting-drop vapor diffusion. A cryocooled crystal of Mss116p/,598,664 in complex with AMP-PNP and U10 belonged to space group P21212, with unit-cell parameters a = 88.54, b = 126.52, c = 55.52,Å, and diffracted X-rays to beyond 1.9,Å resolution using synchrotron radiation from sector 21 at the Advanced Photon Source. [source] Purification, crystallization and data collection of the apoptotic nuclease endonuclease GACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2009Sei Mee Yoon Endonuclease G (EndoG) is a mitochondrial enzyme that responds to apoptotic stimuli by translocating to the nucleus and cleaving chromosomal DNA. EndoG is the main apoptotic endonuclease in the caspase-independent pathway. Mouse EndoG without the mitochondrial localization signal (amino-acid residues 1,43) was successfully overexpressed, purified and crystallized using a microbatch method under oil. The initial crystal (type I) was grown in the presence of the detergent CTAB and diffracted to 2.8,Å resolution, with unit-cell parameters a = 72.20, b = 81.88, c = 88.66,Å, , = 97.59° in a monoclinic space group. The crystal contained two monomers in the asymmetric unit, with a predicted solvent content of 46.6%. Subsequent mutation of Cys110 improved the stability of the protein significantly and produced further crystals of types II, III and IV with space groups C2, P41212 (or P43212) and P212121, respectively, in various conditions. This suggests the critical involvement of this conserved cysteine residue in the crystallization process. [source] Purification, crystallization and preliminary X-ray analysis of NtdA, a putative pyridoxal phosphate-dependent aminotransferase from Bacillus subtilisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2009K. E. Van Straaten NtdA is a putative sugar aminotransferase that is required for the synthesis of 3,3,-neotrehalosadiamine. The enzyme was purified to homogeneity by means of Ni2+ -affinity chromatography and was crystallized using the microbatch method. X-ray diffraction data were collected from a single crystal to 2.3,Å resolution at the Canadian Light Source (CLS). The crystals belonged to space group P21, with unit-cell parameters a = 50.3, b = 106.7, c = 96.7,Å, , = 96.2°, and contained two molecules per asymmetric unit. [source] Crystallization and preliminary X-ray crystallographic studies of the Z-DNA-binding domain of a PKR-like kinase (PKZ) in complex with Z-DNAACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2009Doyoun Kim PKZ, a PKR-like eIF2, kinase, consists of a Z-DNA-specific binding domain (Z,) and an eIF2, kinase domain. The kinase activity of PKZ is modulated by the binding of Z, to Z-DNA. The mechanisms underlying Z-DNA binding and the subsequent stimulation of PKZ raise intriguing questions. Interestingly, the Z-DNA-binding domain of PKZ from goldfish (Carassius auratus; caZ,PKZ) shows limited sequence homology to other canonical Z, domains, suggesting that it may have a distinct Z-DNA-recognition mode. In this study, the Z-DNA-binding activity and stoichiometry of caZ,PKZ were confirmed using circular dichroism (CD). In addition, preliminary X-ray studies of the caZ,PKZ,Z-DNA complex are reported as the first step in the determination of its three-dimensional structure. Bacterially expressed recombinant caZ,PKZ was purified and crystallized with Z-DNA at 295,K using the microbatch method. X-ray diffraction data were collected to 1.7,Å resolution with an Rmerge of 7.4%. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 55.54, b = 49.93, c = 29.44,Å, , = 96.5°. Structural analysis of caZ,PKZ,Z-DNA will reveal the binding mode of caZ,PKZ to Z-DNA and its relevance to other Z-DNA-binding proteins. [source] Crystallization and preliminary X-ray diffraction studies on the catalytic domain of the chick retinal neurite-inhibitory factor CRYP-2ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2005T. S. Girish The receptor protein tyrosine phosphatase CRYP-2 has been shown to be an inhibitory factor for the growth of retinal axons in the chick. The extracellular receptor domain of CRYP-2 contains eight fibronectin repeats and studies using the extracellular domain alone demonstrated the chemorepulsive effect on retinal neurons. The precise role of the intracellular catalytic domain and the mechanism by which its activity is regulated is not known. Determination of the structure of the catalytic domain of CRYP-2 was proposed in an effort to understand the downstream signal transduction mechanism in this system. The cloning, expression, purification and crystallization of the catalytic domain of CRYP-2 are now reported. Preliminary crystallographic studies were performed on the diamond-shaped crystals, which grew under oil using the microbatch method at 298,K. Native X-ray diffraction data were collected to 2.9,Å resolution on a home source. The crystals belong to the trigonal space group P3121, with unit-cell parameters a = b = 68.26, c = 244.95,Å. Assuming the presence of two molecules per asymmetric unit, the VM value was 2.7,Å3,Da,1 and the solvent content was 54.8%. [source] | ||||||||||