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Recombinant Form (recombinant + form)
Selected AbstractsMolecular interactions of the neuronal GPI-anchored lipocalin LazarilloJOURNAL OF MOLECULAR RECOGNITION, Issue 5 2008Diego Sanchez Abstract Lazarillo, a glycoprotein involved in axon growth and guidance in the grasshopper embryo, is the only member of the lipocalin family that is attached to the cell surface by a GPI anchor. Recently, the study of Lazarillo homologous genes in Drosophila and mouse has revealed new functions in the regulation of lifespan, stress resistance and neurodegeneration. Here we report an analysis of biochemical properties of Lazarillo to gain insight into the molecular basis of its physiological function. Recombinant forms of the grasshopper protein were expressed in two different systems to test: (1) potential binding of several hydrophobic ligands; (2) protein,protein homophilic interactions; and (3) whether interaction with the function-blocking mAb 10E6 interferes with ligand binding. We tested 10 candidate ligands (retinoic acid, heme, bilirubin, biliverdin, ecdysterone, juvenile hormone, farnesol, arachidonic acid, linoleic acid and palmitic acid), and monitored binding using electrophoretic mobility shift, absorbance spectrum, and fluorimetry assays. Our work indicates binding to heme and retinoic acid, resulting in increased electrophoretic mobility, as well as to fatty acids, resulting in multimerization. Retinoic acid and fatty acids binding were confirmed by fluorescence titration, and heme binding was confirmed with absorbance spectrum assays. We demonstrate that Lazarillo oligomerizes in solution and can form clusters in the plasma membrane when expressed and GPI-anchored to the cell surface, however it is unable to mediate cell,cell adhesion. Finally, by ligand-mAb competition experiments we show that ligand-binding alone cannot be the key factor for Lazarillo to perform its function during axonal growth in the grasshopper embryo. Copyright © 2008 John Wiley & Sons, Ltd. [source] Comparison of the specificity, stability and individual rate constants with respective activation parameters for the peptidase activity of cruzipain and its recombinant form, cruzain, from Trypanosoma cruziFEBS JOURNAL, Issue 24 2001Wagner A. S. Judice The Trypanosoma cruzi cysteine protease cruzipain contains a 130-amino-acid C-terminal extension, in addition to the catalytic domain. Natural cruzipain is a complex of isoforms, because of the simultaneous expression of several genes, and the presence of either high mannose-type, hybrid monoantennary-type or complex biantenary-type oligosacharide chains at Asn255 of the C-terminal extension. Cruzipain and its recombinant form without this extension (cruzain) were studied comparatively in this work. S2 to S2, subsite specificities of these enzymes were examined using four series of substrates derived from the internally quenched fluorescent peptide Abz-KLRFSKQ-EDDnp (Abz, ortho -aminobenzoic acid; EDDnp, N -(2,4-dinitrophenyl)-ethylenediamine). Large differences in the kinetic parameters were not observed between the enzymes; however, Km values were consistently lower for the hydrolysis of most of the substrates by cruzain. No difference in the pH,activity profile between the two enzymes was found, but in 1 m NaCl cruzipain presented a kcat value significantly higher than that of cruzain. The activation energy of denaturation for the enzymes did not differ significantly; however, a negative entropy value was observed for cruzipain denaturation whereas the value for cruzain was positive. We determined the individual rate constants (k1, substrate diffusion; k,1, substrate dissociation; k2, acylation; k3, deacylation) and the respective activation energies and entropies for hydrolysis of Abz-KLRFSKQ-EDDnp determining the temperature dependence of the Michaelis,Menten parameters kcat/Km and kcat as previously described [Ayala, Y.M. & Di Cera, E. (2000) Protein Sci.9, 1589,1593]. Differences between the two enzymes were clearly detected in the activation energies E1 and E,1, which are significantly higher for cruzipain. The corresponding ,S1 and ,S,1 were positive and significantly higher for cruzipain than for cruzain. These results indicate the presence of a larger energy barrier for cruzipain relating to substrate diffusion and dissociation, which could be related to the C-terminal extension and/or glycosylation state of cruzipain. [source] Recombinant glycodelin carrying the same type of glycan structures as contraceptive glycodelin-A can be produced in human kidney 293 cellsbut not in Chinese hamster ovary cellsFEBS JOURNAL, Issue 15 2000Ingrid M. Van den Nieuwenhof We have produced human recombinant glycodelin in human kidney 293 cells and in Chinese hamster ovary (CHO) cells. Structural analyses by lectin immunoassays and fast atom bombardment mass spectrometry showed that recombinant human glycodelin produced in CHO cells contains only typical CHO-type glycans and is devoid of any of the N,N,- diacetyllactosediamine (lacdiNAc)-based chains previously identified in glycodelin-A (GdA). By contrast, human kidney 293 cells produced recombinant glycodelin with the same type of carbohydrate structures as GdA. The presence of a ,1,4- N- acetylgalactosaminyltransferase functioning in the synthesis of lacdiNAc-based glycans in human kidney 293 cells is concluded to be the cause of the occurrence of lacdiNAc-based glycans on glycodelin produced in these cells. Furthermore, human kidney 293 cells were found to be particularly suited for the production of recombinant glycodelin when they were cultured in high glucose media. Lowering the glucose concentration and the addition of glucosamine resulted in higher relative amounts of oligomannosidic-type glycans and complex glycans with truncated antennae. Human glycodelin is an attractive candidate for the development of a contraceptive agent, and this study gives valuable information for selecting the proper expression system and cell culture conditions for the production of a correctly glycosylated recombinant form. [source] Immunisation with non-integral OMPs promotes pulmonary clearance of Pseudomonas aeruginosaFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 2-3 2003Linda D. Thomas Abstract Pseudomonas aeruginosa is an opportunistic bacterial pathogen that can cause fatal acute lung infections in critically ill individuals. Lung damage due to chronic infections in cystic fibrosis sufferers is the major cause of morbidity and mortality in this group. The bacterium produces various immunomodulatory products that enable it to survive in the lung. Innate and increasing resistance to antibiotic therapy shown by this organism heightens the need for development of a vaccine. This study reports the identification of six non-integral protein antigens; Pa 13, azurin, acyl carrier protein (ACP), amidase, aminopeptidase and KatE, purified from a mucoid strain of P. aeruginosa. N-terminal amino acid sequencing was used to identify these proteins and, based on their ascribed functions, determined that their normal cellular location was cytosolic. A rat model of acute pulmonary infection was used to investigate the ability of these protein antigens to enhance pulmonary clearance of a live P. aeruginosa challenge. Mucosal immunisation with four of the six antigens significantly enhanced bacterial clearance from both the lavage fluid and lung tissue. The greatest level of clearance was demonstrated for the antigens; KatE, aminopeptidase and amidase. Enhanced bacterial clearance was maintained when the antigens amidase and aminopeptidase were produced in recombinant form. When delivered parenterally, aminopeptidase demonstrated its continued efficacy as a vaccine candidate. This study has demonstrated that non-integral outer membrane proteins are antigenic and protective and warrant further investigation as potential components of a vaccine. [source] Suppression of allergic airway inflammation in a mouse model by Der p2 recombined BCGIMMUNOLOGY, Issue 1pt2 2009Hai-Feng Ou-Yang Summary Allergic asthma is a chronic inflammatory disease mediated by T helper (Th)2 cell immune responses. Currently, immunotherapies based on both immune deviation and immune suppression, including the development of recombinant mycobacteria as immunoregulatory vaccines, are attractive treatment strategies for asthma. In our previous studies, we created a genetically recombinant form of bacille Calmette,Guerin (rBCG) that expressed Der p2 of house dust mites and established that it induced a shift from a Th2 response to a Th1 response in naive mice. However, it is unclear whether rBCG could suppress allergic airway inflammation in a mouse model. In this article we report that rBCG dramatically inhibited airway inflammation, eosinophilia, mucus production and mast cell degranulation in allergic mice. Analysis of interferon-, (IFN-,) and interleukin-4 (IL-4) levels in bronchoalveolar lavage fluid (BALF) and lung tissue revealed that the suppression was associated with a shift from a Th2 response to a Th1 response. At the same time, rBCG induced a CD4+ CD25+ Foxp3+ T-cell subtype that could suppress the proliferation of Th2 effector cells in vitro in an antigen-specific manner. Moreover, suppression of CD4+ CD25+ T cells could be adoptively transferred. Thus, our results demonstrate that rBCG induces both generic and specific immune responses. The generic immune response is associated with a shift from a Th2 to a Th1 cytokine response, whereas the specific immune response against Der p2 appears to be related to the expansion of transforming growth factor-, (TGF-,)-producing CD4+ CD25+ Foxp3+ regulatory T cells. rBCG can suppress asthmatic airway inflammation through both immune deviation and immune suppression and may be a feasible, efficient immunotherapy for asthma. [source] Enzyme Replacement Therapy for Murine Hypophosphatasia,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2008José Luis Millán PhD Abstract Introduction: Hypophosphatasia (HPP) is the inborn error of metabolism that features rickets or osteomalacia caused by loss-of-function mutation(s) within the gene that encodes the tissue-nonspecific isozyme of alkaline phosphatase (TNALP). Consequently, natural substrates for this ectoenzyme accumulate extracellulary including inorganic pyrophosphate (PPi), an inhibitor of mineralization, and pyridoxal 5,-phosphate (PLP), a co-factor form of vitamin B6. Babies with the infantile form of HPP often die with severe rickets and sometimes hypercalcemia and vitamin B6 -dependent seizures. There is no established medical treatment. Materials and Methods: Human TNALP was bioengineered with the C terminus extended by the Fc region of human IgG for one-step purification and a deca-aspartate sequence (D10) for targeting to mineralizing tissue (sALP-FcD10). TNALP-null mice (Akp2,/,), an excellent model for infantile HPP, were treated from birth using sALP-FcD10. Short-term and long-term efficacy studies consisted of once daily subcutaneous injections of 1, 2, or 8.2 mg/kg sALP-FcD10 for 15, 19, and 15 or 52 days, respectively. We assessed survival and growth rates, circulating levels of sALP-FcD10 activity, calcium, PPi, and pyridoxal, as well as skeletal and dental manifestations using radiography, ,CT, and histomorphometry. Results:Akp2,/, mice receiving high-dose sALP-FcD10 grew normally and appeared well without skeletal or dental disease or epilepsy. Plasma calcium, PPi, and pyridoxal concentrations remained in their normal ranges. We found no evidence of significant skeletal or dental disease. Conclusions: Enzyme replacement using a bone-targeted, recombinant form of human TNALP prevents infantile HPP in Akp2,/, mice. [source] Quasispecies analysis of novel HIV-1 recombinants of subtypes A and G reveals no similarity to the mosaic structure of CRF02_AG,JOURNAL OF MEDICAL VIROLOGY, Issue 9 2007Rebecca L.R. Powell Abstract HIV-1 circulating recombinant form (CRF) 02_AG is responsible for greater than 65% of HIV-1 infections in Cameroon and is widespread across West and West-Central Africa. The parental subtypes A1 and G cocirculate in this part of Africa, and high rates of infection predispose to the generation of AG unique recombinant forms (URFs). Little is known as to whether A1 and G can recombine and thrive in vivo with breakpoints other than those characteristic of CRF02_AG. In this study, six unique recombinant viruses of subtypes A1 and G were identified in two individuals in Cameroon. A 1.5 kb fragment of the reverse transcriptase (RT) region of pol (HXB2 location 2,612,4,159) and the entire env gene (HXB2 location 6,202,9,096) were evaluated by phylogenetic and breakpoint analyses. Each URF was found to have breakpoints different than CRF02_AG, indicating that A and G gene segments are functionally compatible with more than one pattern of recombination. Furthermore, contemporaneous, cultured viruses from these individuals were analyzed, revealing different proportions of URFs compared to those found in plasma, possibly indicating compart mentalization and/or phenotypic variation among the URFs. CRF02_AG emerged from West-Central Africa to become a highly successful viral strain. As such, monitoring the spread of newly emerging AG recombinants is critical not only for understanding the epidemiology of HIV-1, but also in the design of future therapeutics and vaccines appropriate to this part of Africa, and globally. J. Med. Virol. 79:1270,1285, 2007. © Wiley-Liss, Inc. [source] Nerve Tissue-Specific (GLUD2) and Housekeeping (GLUD1) Human Glutamate Dehydrogenases Are Regulated by Distinct Allosteric MechanismsJOURNAL OF NEUROCHEMISTRY, Issue 5 2000Implications for Biologic Function Abstract: Human glutamate dehydrogenase (GDH), an enzyme central to the metabolism of glutamate, is known to exist in housekeeping and nerve tissue-specific isoforms encoded by the GLUD1 and GLUD2 genes, respectively. As there is evidence that GDH function in vivo is regulated, and that regulatory mutations of human GDH are associated with metabolic abnormalities, we sought here to characterize further the functional properties of the two human isoenzymes. Each was obtained in recombinant form by expressing the corresponding cDNAs in Sf9 cells and studied with respect to its regulation by endogenous allosteric effectors, such as purine nucleotides and branched chain amino acids. Results showed that L-leucine, at 1.0 mM, enhanced the activity of the nerve tissue-specific (GLUD2-derived) enzyme by ,1,600% and that of the GLUD1-derived GDH by ,75%. Concentrations of L-leucine similar to those present in human tissues (,0.1 mM) had little effect on either isoenzyme. However, the presence of ADP (10-50 ,M) sensitized the two isoenzymes to L-leucine, permitting substantial enzyme activation at physiologically relevant concentrations of this amino acid. Nonactivated GLUD1 GDH was markedly inhibited by GTP (IC50 = 0.20 ,M), whereas nonactivated GLUD2 GDH was totally insensitive to this compound (IC50 > 5,000 ,M). In contrast, GLUD2 GDH activated by ADP and/or L-leucine was amenable to this inhibition, although at substantially higher GTP concentrations than the GLUD1 enzyme. ADP and L-leucine, acting synergistically, modified the cooperativity curves of the two isoenzymes. Kinetic studies revealed significant differences in the Km values obtained for ,-ketoglutarate and glutamate for the GLUD1- and the GLUD2-derived GDH, with the allosteric activators differentially altering these values. Hence, the activity of the two human GDH is regulated by distinct allosteric mechanisms, and these findings may have implications for the biologic functions of these isoenzymes. [source] Factor Xa is highly protected from antithrombin,fondaparinux and antithrombin,enoxaparin when incorporated into the prothrombinase complexJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 6 2003N. Brufatto Summary., Antithrombin and its cofactor, heparin, target both the product of prothrombin activation by prothrombinase, thrombin, as well as the enzyme responsible for the reaction, factor (F)Xa. These studies were carried out to quantify the effects of each of the prothrombinase components on the half-life of FXa in the presence of antithrombin and the low-molecular-weight heparins (enoxaparin, Aventis, Laval, Quebec, Canada) or the heparin pentasaccharide (fondaparinux, Organon Sanofi-Synthelabo, Cypress, TX, USA). Experiments were carried out using a recombinant form of prothrombin in which the active site serine has been mutated to cysteine and subsequently labeled with fluorescein. This mutant allowed calculation of the second order rate constant for inhibition of FXa by antithrombin in such a way that competition for antithrombin by thrombin is eliminated and competition for FXa by prothrombin is accounted for. Intrinsic rate constants for the inhibition of FXa by antithrombin,enoxaparin and antithrombin,fondaparinux, in the presence of the various prothrombinase components, were calculated. Addition of phospholipid had no significant effect on the second order rate constant for inhibition of FXa by antithrombin, while addition of FVa appeared to be mildly protective. Further addition of prothrombin however, caused profound protection of FXa, increasing its half-life from 1.1 to 353 s in the case of fondaparinux, and from 0.4 to 42 s in the case of enoxaparin. Similar results were reported for unfractionated heparin previously [1]. Therefore, in the presence of unfractionated heparin, fondaparinux, or enoxaparin, prothrombinase is profoundly protected from antithrombin. [source] Comparative enzymology of native and recombinant house dust mite allergen Der p 1ALLERGY, Issue 3 2009J. Zhang Background:, The cysteine peptidase activity of group 1 house dust mite allergens is important for their allergenicity and may offer new therapeutic targets for allergy treatment. Hitherto, the design of specific inhibitors has been impeded because the availability of pure, fully active allergens has limited the implementation of drug screening campaigns. Similarly, investigation of the mechanisms by which peptidase allergens promote sensitization has also been restricted. Our aim was to compare the enzymology of recombinant and native forms of Der p 1 to establish if an easily expressed recombinant form of Der p 1 could be used as a drug discovery tool. Methods:, Enzymatic activity of natural and recombinant Der p 1 was compared fluorimetrically using a novel specific substrate (ADZ 50,059) and a novel specific active site titrant (ADZ 50,000). The effect of recombinant Der p 1 prodomain on the catalytic activity of both Der p 1 preparations was also examined. Results:, Although differing substantially in molecular weight, the enzymological properties of recombinant and native Der p 1 were indistinguishable. Our data show clearly by experiment that, in contrast to some suggestions, Der p 1 is not an enzyme of bifunctional mechanism. Conclusion:, The catalytic activity of Der p 1 is tolerant of glycosylation differences that occur at N150 when the protein is expressed in Pichia pastoris. This suggests that this recombinant protein may be suitable for drug design studies and in the elucidation of how peptidase activity promotes sensitization to peptidase and nonpeptidase bystander allergens. [source] Polyspecific malaria antibodies present at the time of infection inhibit the development of immunity to malaria but antibodies specific for the malaria merozoite surface protein, MSP1, facilitate immunityPARASITE IMMUNOLOGY, Issue 5 2002Wenbao Zhang Summary Serum taken from mice immune to malaria as a result of infection and drug cure, or from mice immunized with a recombinant form of the merozoite surface protein, MSP1, can provide passive protection of recipient mice against the lethal parasite, Plasmodium yoelii YM. However, recipients of MSP1-immune serum go on to develop long-term immunity, whereas recipients of serum from mice naturally immune to malaria rapidly lose their resistance to infection. We demonstrate that ,infection/cure' serum suppresses the development of both antibody and cell-mediated parasite-specific responses in recipients, whereas these develop in recipients of MSP1-specific antibodies. These data have profound implications for our understanding of the development of malaria immunity in babies who passively acquire antibodies from their mothers. [source] A novel missense mutation causing abnormal LMAN1 in a Japanese patient with combined deficiency of factor V and factor VIII,AMERICAN JOURNAL OF HEMATOLOGY, Issue 11 2009Takayuki Yamada Combined deficiency of coagulation factor V (FV) and factor VIII (FVIII) (F5F8D) is an inherited bleeding disorder characterized by a reduction in plasma concentrations of FV and FVIII. F5F8D is genetically linked to mutations in either LMAN1 or MCFD2. Here, we investigated the molecular basis of F5F8D in a Japanese patient, and identified a novel missense mutation (p.Trp67Ser, c.200G>C) in the LMAN1, but no mutation in the MCFD2. The amount of LMAN1 in Epstein-Barr virus-immortalized lymphoblasts from the patient was found to be almost the same as that in cells from a normal individual. Interestingly, an anti-MCFD2 antibody did not co-immunoprecipitate the mutant LMAN1 with MCFD2 in lymphoblasts from the patient, suggesting the affinity of MCFD2 for the mutant LMAN1 is weak or abolished by the binding of the anti-MCFD2 antibody. In addition, a Myc/6×His-tagged recombinant form of wild-type LMAN1 could bind to D-mannose, but that of the mutant could not. The p.Trp67Ser mutation was located in the carbohydrate recognition domain (CRD), which is thought to participate in the selective binding of LMAN1 to the D-mannose of glycoproteins as well as the EF-motif of MCFD2. Taken together, it was suggested that the p.Trp67Ser mutation might affect the molecular chaperone function of LMAN1, impairing affinity for D-mannose as well as for MCFD2, which may be responsible for F5F8D in the patient. This is the first report of F5F8D caused by a qualitative defect of LMAN1 due to a missense mutation in LMAN1. Am. J. Hematol. 2009. © 2009 Wiley-Liss, Inc. [source] Determination of the disulfide bond pattern of the endogenous and recombinant angiogenesis inhibitor endostatin by mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 14 2001Harald John Endostatin, a C-terminal fragment of collagen XVIII, is a promising protein drug which is in development for cancer therapy due to its anti-angiogenic activity. Although several endogenous molecular forms of human endostatin differing in their N-terminal length and their post-translational modifications (18.5,22,kDa) have been discovered, only one recombinant form of 20,kDa is used in clinical trials. This protein, recombinantly expressed in Pichia pastoris, contains four cysteines forming two disulfide bonds (Cys1-Cys4 and Cys2-Cys3). In contrast, there are conflicting data about the disulfide pattern of endogenous material. This report presents the disulfide analyses of both the endogenous circulating endostatins isolated from human hemofiltrate and the recombinant protein. The determination of the disulfide pattern was performed by Edman degradation, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and electrospray ionization ion trap mass spectrometry (ESI-ITMS) performed in the off-line nanospray mode. All native and recombinant endostatins exhibited a Cys1-Cys4 (Cys162 -Cys302) and Cys2-Cys3 (Cys264 -Cys294) linkage. For a clear discussion of fragmented disulfide-bridged peptide chains obtained from MSn experiments, a modified general nomenclature is proposed. Copyright © 2001 John Wiley & Sons, Ltd. [source] The I-antigens of Ichthyophthirius multifiliis are GPI-Anchored ProteinsTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 3 2001THEODORE G. CLARK ABSTRACT. The parasitic ciliate Ichthyophthirius multifiliis has abundant surface membrane proteins (i-antigens) that when clustered, trigger rapid, premature exit from the host. Similar antigens are present in free-living ciliates and are GPI-anchored in both Paramecium and Tetrahymena. Although transmembrane signalling through GPI-anchored proteins has been well-documented in metazoan cells, comparable phenomena have yet to be described in protists. Since premature exit of Ichthyophthirius is likely to involve a transmembrane signalling event, we sought to determine whether i-antigens are GPI-anchored in these cells as well. Based on their solubility properties in Triton X-114, the i-antigens of Ichthyophthirius are amphiphilic in nature and partition with the detergent phase. Nevertheless, following treatment of detergent lysates with phospholipase C, the same proteins become hydrophilic. Concomitantly, they are recognized by antibodies against a cross-reacting determinant exposed on virtually all GPI-anchored proteins following cleavage with phospholipase C. Finally, when expressed in recombinant form in Tetrahymena thermophila, full-length i-antigens are restricted to the membrane, while those lacking hydrophobic C-termini are secreted from the cell. Taken together, these observations argue strongly that the i-antigens of Ichthyophthirius multifiliis are, in fact, GPI-anchored proteins. [source] Expression, crystallization and preliminary X-ray crystallographic studies of Klebsiella pneumoniae maltohexaose-producing ,-amylaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 12-2 2004Mitsuru Momma A recombinant form of Klebsiella pneumoniae maltohexaose-producing ,-amylase has been overexpressed in Escherichia coli and purified to homogeneity. Crystals were obtained at 293,K by the microbatch technique using 80,mM sodium/potassium phosphate buffer pH 6.2 containing 8% polyethylene glycol 3000, 4% polyethylene glycol 3350 and 40,mM sodium thiocyanate. Crystals of the overexpressed recombinant enzyme diffracted to better than 2.5,Å resolution at 95,K using a synchrotron-radiation source. The crystals belong to the primitive monoclinic space group P21, with unit-cell parameters a = 74.8, b = 107.6, c = 82.2,Å, , = 96.2°. Assuming the presence of two molecules per asymmetric unit, the VM value for the crystal was 2.3,Å3,Da,1, indicating a solvent content of 47%. [source] Expression, crystallization and preliminary X-ray crystallographic studies of Arthrobacter globiformis inulin fructotransferaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2003Mitsuru Momma A recombinant form of Arthrobacter globiformis inulin fructotransferase (DFAIII-producing) has been overexpressed in Escherichia coli and purified to homogeneity. Crystals were obtained at 293,K by the hanging-drop vapour-diffusion technique using 0.1,M Na HEPES pH 7.5 buffer containing 1.5,M lithium sulfate as a precipitant. Crystals of the recombinant wild-type enzyme diffracted to better than 1.5,Å at 100,K using a synchrotron-radiation source at the Photon Factory. The crystal belonged to space group R32, with unit-cell parameters a = b = 92.02, c = 229.82,Å in the hexagonal axes. Assuming the presence of one molecule in the asymmetric unit, the VM value for the crystal was 2.15,Å3,Da,1, indicating a solvent content of 42.8%. Selenomethionine-derivative crystals belonged to a different space group, C2, with unit-cell parameters a = 159.32, b = 91.92, c = 92.58,Å, , = 125.06. Matthews coefficient calculations suggested that the C2 selenomethionine-derivative crystal contained three molecules per asymmetric unit. [source] Purification, crystallization and molecular symmetry of CDP- d -glucose 4,6-dehydratase from Yersinia pseudotuberculosisACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2002Erik M. Vogan The enzyme CDP- d -glucose 4,6-dehydratase (EC 4.2.1.45) is an NAD+ -dependent oxidoreductase which catalyzes the irreversible conversion of CDP- d -glucose to CDP-4-keto-6-deoxy- d -glucose. The product of this reaction is an intermediate in the synthesis of all CDP-linked 3,6-dideoxyhexoses, an important class of antigenic determinants found in the lipopolysaccharide layer of Gram-negative bacteria. Crystals of a recombinant form of this enzyme from Yersinia pseudotuberculosis have been grown in two crystal forms, both possessing pseudo-translational non-crystallographic symmetry, with dramatically different diffraction characteristics. A complete 1.8,Å data set has been collected from the primitive orthorhombic crystal form, for which the non-crystallographic symmetry is described in detail. [source] Crystallization and preliminary X-ray diffraction analysis of a [2Fe,2S] ferredoxin (FdVI) from Rhodobacter capsulatusACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2001Jean Armengaud A [2Fe,2S] ferredoxin found in the photosynthetic bacterium Rhodobacter capsulatus has been purified in recombinant form from Escherichia coli. This protein, called FdVI, resembles ferredoxins involved in iron,sulfur cluster biosynthesis in various prokaryotic and eukaryotic cells. Purified recombinant FdVI was recovered in high yields and appeared to be indistinguishable from the genuine R. capsulatus ferredoxin based on UV,visible absorption and EPR spectroscopy and mass spectrometry. FdVI has been crystallized in the oxidized state by a sitting-drop vapour-diffusion technique using sodium formate as precipitant. Seeding larger drops from a previous hanging-drop-grown small crystal resulted in the formation of long red,brown prismatic needles. Preliminary X-ray diffraction analysis indicated that FdVI crystals are orthorhombic and belong to the space group P212121, with unit-cell parameters a = 45.87, b = 49.83, c = 54.29 Å. [source] Crystallization of agGST1-6, a recombinant glutathione S -transferase from a DDT-resistant strain of Anopheles gambiaeACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2001Pamela H. Roberts Glutathione S -transferases (GSTs) belong to a family of detoxification enzymes that conjugate glutathione to various xenobiotics, thus facilitating their expulsion from the cell. GST activity is elevated in many insecticide-resistant insects, including the DDT-resistant malaria vector Anopheles gambiae. Crystals of the recombinant form of a GST from A. gambiae, agGST1-6, have been grown in at least five different crystal forms, with a broad range of diffraction resolution limits. A complete 2.0,Å data set has been collected on a C -centered orthorhombic crystal form with unit-cell parameters a = 99.0, b = 199.4, c = 89.6,Å. A search for heavy-atom derivatives has been initiated, along with phase-determination efforts by molecular replacement. [source] Purification and crystallization of human Cu/Zn superoxide dismutase recombinantly produced in the protozoan Leishmania tarentolaeACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010Emerich Mihai Gazdag The rapid and inexpensive production of high-quality eukaryotic proteins in recombinant form still remains a challenge in structural biology. Here, a protein-expression system based on the protozoan Leishmania tarentolae was used to produce human Cu/Zn superoxide dismutase (SOD1) in recombinant form. Sequential integration of the SOD1 expression cassettes was demonstrated to lead to a linear increase in expression levels to up to 30,mg per litre. Chromatographic purification resulted in 90% pure recombinant protein, with a final yield of 6.5,mg per litre of culture. The protein was crystallized and the structures of two new crystal forms were determined. These results demonstrate the suitability of the L. tarentolae expression system for structural research. [source] Structure of photosynthetic glyceraldehyde-3-phosphate dehydrogenase (isoform A4) from Arabidopsis thaliana in complex with NADACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2010Simona Fermani The crystal structure of the A4 isoform of photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Arabidopsis thaliana, expressed in recombinant form and complexed with NAD, is reported. The crystals, which were grown in 2.4,M ammonium sulfate and 0.1,M sodium citrate, belonged to space group I222. The asymmetric unit includes ten subunits, i.e. two independent tetramers plus a dimer that generates a third tetramer by a crystallographic symmetry operation. The crystal structure was solved by molecular replacement and refined to an R factor of 23.7% and an Rfree factor of 28.9% at 2.6,Å resolution. In the final model, each subunit binds one NAD+ molecule and two sulfates, which occupy the Ps and the Pi anion-binding sites. Detailed knowledge of this structure is instrumental for structural investigation of supramolecular complexes of A4 -GAPDH, phosphoribulokinase and CP12, which are involved in the regulation of photosynthesis in the model plant A. thaliana. [source] Crystallization and preliminary X-ray analysis of a d -alanyl- d -alanine ligase (EcDdlB) from Escherichia coliACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2010Sarah Batson A recombinant form of Escherichia coli DdlB (EcDdlB) has been prepared and cocrystallized with ADP and d -alanyl- d -alanine to represent the ternary complex of EcDdlB. Furthermore, EcDdlB has been cocrystallized under the same conditions with the ligands ATP and d -alanyl- d -alanine, representing the product-inhibited complex. The crystals belonged to space group P212121, with unit-cell parameters a = 53.0, b = 97.6, c = 109.5,Å and a = 51.2, b = 97.8, c = 110.1,Å, respectively, and both contained two molecules in the asymmetric unit. Complete data sets were collected to 1.5 and 1.4,Å resolution, respectively, from single crystals under cryogenic conditions using synchrotron radiation. [source] Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the collagen-binding region of RspB from Erysipelothrix rhusiopathiaeACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2010Aribam Swarmistha Devi RspB is a surface adhesin of Erysipelothrix rhusiopathiae. A recombinant form of the collagen-binding region of this protein, RspB(31,348), has been overexpressed in Escherichia coli in native and selenomethionine-derivative forms and purified using affinity and gel-permeation chromatography. Thin plate-like crystals were obtained by the hanging-drop vapour-diffusion method using the same condition for both forms. The native crystals diffracted to a resolution of 2.5,Å using an in-house X-ray source, while the selenomethionine-derivative crystals diffracted to a resolution of 2.2,Å using synchrotron radiation. The crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 46.19, b = 66.65, c = 101.72,Å, , = 94.11°. [source] Crystallization of human complement component C3b in the presence of a staphylococcal complement-inhibitor protein (SCIN)ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2009Brandon L. Garcia Staphylococcus aureus secretes a number of small proteins that effectively attenuate the human innate immune response. Among these, the staphylococcal complement-inhibitor protein (SCIN) disrupts the function of the complement component 3 (C3) convertase that is initiated through either the classical or the alternative pathway and thereby prevents amplification of the complement response on the bacterial surface. Recent studies have shown that SCIN may affect the activities of the C3 convertase by binding in an equimolar fashion to C3b, which is itself an integral although non-enzymatic component of the convertase. In order to better understand the nature of the C3b,SCIN interaction, the hanging-drop vapor-diffusion technique was used to crystallize human C3b in the presence of a recombinant form of SCIN. These crystals diffracted synchrotron X-rays to approximately 6,Å Bragg spacing and grew in a primitive tetragonal space group (P41212 or P43212; unit-cell parameters a = b = 128.03, c = 468.59,Å). Cell-content analysis of these crystals was consistent with the presence of either two 1:1 complexes or a single 2:2 assembly in the asymmetric unit, both of which correspond to a solvent content of 51.9%. By making use of these crystals, solution of the C3b,SCIN structure should further our understanding of complement inhibition and immune evasion by this pathogen. [source] Expression of a plant-derived peptide harboring water-cleaning and antimicrobial activitiesBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2003M. Suarez Abstract Drinking water is currently a scarce world resource, the preparation of which requires complex treatments that include clarification of suspended particles and disinfection. Seed extracts of Moringa oleifera Lam., a tropical tree, have been proposed as an environment-friendly alternative, due to their traditional use for the clarification of drinking water. However, the precise nature of the active components of the extract and whether they may be produced in recombinant form are unknown. Here we show that recombinant or synthetic forms of a cationic seed polypeptide mediate efficient sedimentation of suspended mineral particles and bacteria. Unexpectedly, the polypeptide was also found to possesses a bactericidal activity capable of disinfecting heavily contaminated water. Furthermore, the polypeptide has been shown to efficiently kill several pathogenic bacteria, including antibiotic-resistant isolates of Staphylococcus, Streptococcus, and Legionella species. Thus, this polypeptide displays the unprecedented feature of combining water purification and disinfectant properties. Identification of an active principle derived from the seed extracts points to a range of potential for drinking water treatment or skin and mucosal disinfection in clinical settings. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 13,20, 2003. [source] Structure of Staphylococcus aureus 1,4-dihydroxy-2-naphthoyl-CoA synthase (MenB) in complex with acetoacetyl-CoAACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2007Venkatasubramanian Ulaganathan Vitamin K2, or menaquinone, is an essential cofactor for many organisms and the enzymes involved in its biosynthesis are potential antimicrobial drug targets. One of these enzymes, 1,4-dihydroxy-2-naphthoyl-CoA synthase (MenB) from the pathogen Staphylococcus aureus, has been obtained in recombinant form and its quaternary structure has been analyzed in solution. Cubic crystals of the enzyme allowed a low-resolution structure (2.9,Å) to be determined. The asymmetric unit consists of two subunits and a crystallographic threefold axis of symmetry generates a hexamer consistent with size-exclusion chromatography. Analytical ultracentrifugation indicates the presence of six states in solution, monomeric through to hexameric, with the dimer noted as being particularly stable. MenB displays the crotonase-family fold with distinct N- and C-terminal domains and a flexible segment of structure around the active site. The smaller C-terminal domain plays an important role in oligomerization and also in substrate binding. The presence of acetoacetyl-CoA in one of the two active sites present in the asymmetric unit indicates how part of the substrate binds and facilitates comparisons with the structure of Mycobacterium tuberculosis MenB. [source] Expression, purification and preliminary X-ray characterization of dl -2-haloacid dehalogenase from Methylobacterium sp.ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2007dl -2-Haloacid dehalogenase from Methylobacterium sp. CPA1 (dl -DEX Mb) is a unique enzyme that catalyzes the dehalogenation reaction without the formation of an ester intermediate. A recombinant form of dl -DEX Mb has been expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystal belongs to the hexagonal space group P63, with unit-cell parameters a = b = 186.2, c = 114.4,Å. The crystals are likely to contain between four and eight monomers in the asymmetric unit, with a VM value of 4.20,2.10,Å3,Da,1. A self-rotation function revealed peaks on the , = 180° section. X-ray data have been collected to 1.75,Å resolution. [source] Expression, purification, crystallization and preliminary X-ray analysis of Pseudomonas fluorescens AlgKACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2007Carrie-Lynn Keiski AlgK is an outer-membrane lipoprotein involved in the biosynthesis of alginate in Pseudomonads and Azotobacter vinelandii. A recombinant form of Pseudomonas fluorescens AlgK with a C-terminal polyhistidine affinity tag has been expressed and purified from the periplasm of Escherichia coli cells and diffraction-quality crystals of AlgK have been grown using the hanging-drop vapour-diffusion method. The crystals grow as flat plates with unit-cell parameters a = 79.09, b = 107.85, c = 119.15,Å, , = 96.97°. The crystals exhibit the symmetry of space group P21 and diffract to a minimum d -spacing of 2.5,Å at Station X29 of the National Synchrotron Light Source, Brookhaven National Laboratory. On the basis of the Matthews coefficient (VM = 2.53,Å3,Da,1), four protein molecules are estimated to be present in the asymmetric unit. [source] Crystallization and preliminary X-ray characterization of aminopeptidase N from Escherichia coliACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2006Yuko Onohara A recombinant form of aminopeptidase N (molecular weight 99,kDa) from Escherichia coli was crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitating agent. The crystals belong to the hexagonal space group P3121, with unit-cell parameters a = b = 120.5, c = 171.0,Å. The crystals are most likely to contain one molecule in the asymmetric unit, with a VM value of 3.62,Å3,Da,1. Diffraction data were collected to 2.0,Å resolution using Cu,K, radiation from a rotating-anode X-ray generator. [source] Production of native and modified recombinant Der p 1 molecules in tobacco plantsCLINICAL & EXPERIMENTAL ALLERGY, Issue 5 2009D. Burtin Summary Background As a complex molecule requiring post-translational processing, it has been difficult to produce the Der p 1 major allergen from the Dermatophagoides pteronyssinus house dust mite in a recombinant form. Objective Here, we tested whether transgenic tobacco plants are suitable to express Der p 1, either as a wild-type molecule or as variants lacking N -glycosylation sites (Gly,) and/or cysteine protease activity (Enz,). Methods Using Agrobacterium tumefaciens -based transformation, pro Der p 1 molecules bearing mutations within either the N -glycosylation sites (N34Q, N150Q) and/or the cysteine protease-active site (C132V) were expressed in tobacco plants. After purification by ion exchange chromatography, allergens were characterized using immunoblotting, circular dichroism (CD), as well as basophil and T lymphocyte stimulation assays. Results Four forms of recombinant Der p 1 (i.e. wild-type Gly+/Enz+, as well as Gly,/Enz+, Gly+/Enz, or Gly,/Enz, variants) were successfully expressed in tobacco leaves as pro Der p 1 molecules. Spontaneous cleavage of the pro-peptide was observed in tobacco leaf extracts for all forms of recombinant Der p 1 (r Der p 1). CD confirmed that all r Der p 1 molecules, with the exception of the Gly,/Enz, variant, exhibited secondary structures comparable to the natural protein. A cysteine protease activity was associated only with the Gly+/Enz+ form. All these molecules exhibit a profile similar to natural Der p 1 with respect to IgE immunoreactivity, basophil activation and T cell recognition. Conclusion A tobacco plant expression system allows the production of various forms of mature Der p 1, which could be used for diagnostic or immunotherapeutic purposes. [source] | ||||||||||||||||