scholarly journals Heterologous Expression of Functional Ptr ToxA

2000 ◽  
Vol 13 (4) ◽  
pp. 456-464 ◽  
Author(s):  
Robert P. Tuori ◽  
Thomas J. Wolpert ◽  
Lynda M. Ciuffetti
Keyword(s):  
Fusion Protein ◽  
Heterologous Expression ◽  
Specific Activity ◽  
Maximal Activity ◽  
Cysteine Residue ◽  
Binding Studies ◽  
Reading Frame ◽  
Pyrenophora Tritici Repentis ◽  
Ptr Toxa

Ptr ToxA, a proteinaceous host-selective toxin (HST) produced by the fungus Pyrenophora tritici-repentis, was expressed in Escherichia coli and purified as a polyhistidine-tagged, fusion protein (NC-FP). NC-FP, consisting of both the N and C domains of the ToxA open reading frame (ORF), is produced as an insoluble protein in E. coli at approximately 10 to 16 mg per liter of culture. Following in vitro refolding, NC-FP elicits cultivar-specific necrosis in wheat, with a specific activity similar to that of native Ptr ToxA. A fusion protein consisting of only the C domain has approximately 10 to 20% of the activity of native Ptr ToxA. These data suggest that (i) the N domain is important for maximal activity of Ptr ToxA, (ii) the N domain does not function to eliminate activity of the protoxin, and (iii) post-translational modifications of Ptr ToxA are not essential for activity. A C domain construct with a cysteine residue mutated to glycine is inactive. This, plus the observation that toxin activity is sensitive to reducing agents, provides evidence that the two cysteine residues in Ptr ToxA are involved in a disulfide bond that is essential for activity. The heterologous expression of Ptr ToxA provides a valuable tool for addressing a number of issues such as receptor binding studies, structure/function studies, and screening wheat cultivars for disease resistance.

scholarly journals Characterization of the ToxB Gene from Pyrenophora tritici-repentis

2001 ◽  
Vol 14 (5) ◽  
pp. 675-677 ◽  
Author(s):  
J. Patrick Martinez ◽  
Sean A. Ottum ◽  
Shaukat Ali ◽  
Leonard J. Francl ◽  
Lynda M. Ciuffetti
Keyword(s):  
Amino Acid ◽  
Pichia Pastoris ◽  
Heterologous Expression ◽  
North Dakota ◽  
Signal Peptide ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Pyrenophora Tritici Repentis ◽  
Putative Signal Peptide

The ToxB gene was cloned and characterized from a race 5 isolate of Pyrenophora tritici-repentis from North Dakota. ToxB contains a 261-bp open reading frame that encodes a 23 amino acid putative signal peptide and a 64 amino acid host-selective toxin, Ptr ToxB. Analysis of Ptr ToxB from heterologous expression in Pichia pastoris confirms that ToxB encodes a host-selective toxin.

scholarly journals Three cytosolic NAD-malate dehydrogenase isoforms of Arabidopsis thaliana: on the crossroad between energy fluxes and redox signaling

2020 ◽  
Vol 477 (19) ◽  
pp. 3673-3693
Author(s):  
Aleksandra Liszka ◽  
Regina Schimpf ◽  
Krupskaya Ivannova Cartuche Zaruma ◽  
Annika Buhr ◽  
Thorsten Seidel ◽  
...  
Keyword(s):  
Malate Dehydrogenase ◽  
Nuclear Translocation ◽  
Specific Activity ◽  
Redox Homeostasis ◽  
Disulfide Bridge ◽  
Glycolytic Enzyme ◽  
Cysteine Residue ◽  
Oxidative Modification ◽  
Animal Cells

In yeast and animal cells, mitochondrial disturbances resulting from imbalances in the respiratory chain require malate dehydrogenase (MDH) activities for re-directing fluxes of reducing equivalents. In plants, in addition to mitochondria, plastids use malate valves to counterbalance and maintain redox-homeostasis. Arabidopsis expresses three cytosolic MDH isoforms, namely cyMDH1, cyMDH2, and cyMDH3, the latter possessing an N-terminal extension carrying a unique cysteine residue C2. In this study, redox-effects on activity and structure of all three cyMDH isoforms were analyzed in vitro. cyMDH1 and cyMDH2 were reversibly inactivated by diamide treatment, accompanied by dimerization via disulfide-bridge formation. In contrast, cyMDH3 forms dimers and higher oligomers upon oxidation, but its low specific activity is redox-independent. In the presence of glutathione, cyMDH1 and cyMDH2 are protected from dimerization and inactivation. In contrast, cyMDH3 still dimerizes but does not form oligomers any longer. From analyses of single and double cysteine mutants and structural modeling of cyMDH3, we conclude that the presence of C2 and C336 allows for multiple cross-links in the higher molecular mass complexes comprising disulfides within the dimer as well as between monomers of two different dimers. Furthermore, nuclear localization of cyMDH isoforms was significantly increased under oxidizing conditions in isolated Arabidopsis protoplasts, in particular of isoform cyMDH3. The unique cyMDH3 C2–C2-linked dimer is, therefore, a good candidate as a redox-sensor taking over moonlighting functions upon disturbances of energy metabolism, as shown previously for the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) where oxidative modification of the sensitive catalytic cysteine residues induces nuclear translocation.

scholarly journals Structure and Specific Activity of Macrophage-Stimulating Lipopeptides from Mycoplasma hyorhinis

1998 ◽  
Vol 66 (10) ◽  
pp. 4804-4810 ◽  
Author(s):  
Peter F. Mühlradt ◽  
Michael Kiess ◽  
Holger Meyer ◽  
Roderich Süssmuth ◽  
Günther Jung
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Specific Activity ◽  
Maximal Activity ◽  
Peritoneal Macrophages ◽  
Composition Analysis ◽  
Mycoplasma Hyorhinis ◽  
Inflammatory Reactions ◽  
Nitric Oxide Release

ABSTRACT Mycoplasmas are potent macrophage stimulators. We describe the isolation of macrophage-stimulatory lipopeptidesS-[2,3-bisacyl(C16:0/C18:0)oxypropyl]cysteinyl-GQTDNNSSQSQQPGSGTTNT andS-[2,3-bisacyl(C16:0/C18:0)oxypropyl]cysteinyl-GQTN derived from the Mycoplasma hyorhinis variable lipoproteins VlpA and VlpC, respectively. These lipopeptides were characterized by amino acid sequence and composition analysis and by mass spectrometry. The lipopeptidesS-[2,3-bis(palmitoyloxy)propyl]cysteinyl-GQTNT andS-[2,3-bis(palmitoyloxy)propyl]cysteinyl-SKKKK and the N-palmitoylated derivative of the latter were synthesized, and their macrophage-stimulatory activities were compared in a nitric oxide release assay with peritoneal macrophages from C3H/HeJ mice. The lipopeptides with the free amino terminus showed half-maximal activity at 3 pM regardless of their amino acid sequence; i.e., they were as active as the previously isolated M. fermentans-derived lipopeptide MALP-2. The macrophage-stimulatory activity of the additionally N-palmitoylated lipopeptide or of the murein lipoprotein from Escherichia coli, however, was lower by orders of magnitude. It is concluded that the lack of N-acyl groups in mycoplasmal lipoproteins explains their exceptionally high in vitro macrophage-stimulatory capacity. Certain features that lipopolysaccharide endotoxin and mycoplasmal lipopeptides have in common are discussed. Lipoproteins and lipopeptides are likely to be the main causative agents of inflammatory reactions to mycoplasmas. This may be relevant in the context of mycoplasmas as arthritogenic pathogens and their association with AIDS.

Formulation of ‘ready-to-use’ human clinical doses of 177Lu-labeled bisphosphonate amide of DOTA using moderate specific activity 177Lu and its preliminary evaluation in human patient

2020 ◽  
Vol 108 (8) ◽  
pp. 661-672
Author(s):  
Sudipta Chakraborty ◽  
Priyalata Shetty ◽  
Rubel Chakravarty ◽  
K. V. Vimalnath ◽  
Chandan Kumar ◽  
...  
Keyword(s):  
Specific Activity ◽  
Specific Binding ◽  
Preliminary Investigation ◽  
Skeletal Metastases ◽  
Preliminary Evaluation ◽  
Binding Studies ◽  
Human Patient ◽  
Biodistribution Studies ◽  
Target Organs

AbstractRadiolabeled macrocyclic bisphosphonate ligands have recently been demonstrated to be highly efficacious in treatment of patients with painful bone metastases. Herein, we report a robust protocol for formulation of therapeutically relevant doses of 177Lu-labeled bisphosphonate amide of DOTA (BPAMD) using moderate specific activity 177Lu produced by direct (n,γ) route and its preliminary investigation in human patients. Doses (2.8 ± 0.2 GBq) were formulated with high radiochemical purity (98.3 ± 0.4 %) using a protocol optimized after extensive radiochemical studies. In vitro binding studies with mineralized osteosarcoma cells demonstrated specific binding of the radiotracer. Biodistribution studies in healthy Wistar rats demonstrated rapid skeletal accumulation with fast clearance from the non-target organs. In a patient administered with 555 MBq dose of 177Lu-BPAMD, intense radiotracer uptake was observed in the metastatic skeletal lesions with insignificant uptake in any other major non-targeted organs. Preliminary clinical investigations carried out after administration of 2.6 GBq of 177Lu-BPAMD revealed significant reduction in pain after 1 week without any adverse effects. The developed protocol for formulation of 177Lu-BPAMD doses using moderate specific activity carrier added 177Lu has been found to be effective and warrants wider investigations in patients with painful skeletal metastases.

Molecular characterization, heterologous expression and resistance analysis of OsLEA3-1 from Oryza sativa

Biologia ◽  
2014 ◽  
Vol 69 (5) ◽  
Author(s):  
Tingzhang Hu ◽  
Junnian Yang ◽  
Yongwei Yang ◽  
Yingmei Wu
Keyword(s):  
Fusion Protein ◽  
Heterologous Expression ◽  
Abiotic Stresses ◽  
Structure Prediction ◽  
Bioinformatics Analysis ◽  
Secondary Structure Prediction ◽  
Mature Embryo ◽  
Lactate Dehydrogenase Activity ◽  
E Coli

AbstractLate embryogenesis abundant (LEA) proteins in organisms are closely associated with resistance to abiotic stresses. Here we characterized a rice LEA protein, OsLEA3-1, by bioinformatics analysis and heterologous expression in Escherichia coli. Bioinformatics analysis showed that OsLEA3-1 contains a 603-bp open reading frame encoding a putative polypeptide of 200 amino acids, which contains a “LEA_4” motif at positions 5–48 and belongs to a typical group 3 LEA. OsLEA3-1 polypeptide is rich in Ala, Lys, and Thr, but depleted in Cys, Pro, and Trp residues; and is strongly hydrophilic. Secondary structure prediction showed that OsLEA3-1 polypeptide contained an α-helical domain in positions 4-195 but not any β-sheet domain. OsLEA3-1 gene can express in shoot and root of germinating seeds, seedling, panicles, mature embryo, seed, and callus; and was also up-regulated by ultraviolet (UV), heat, cold, salt, and emergency drought. OsLEA3-1 gene was introduced into E. coli. A fusion protein of about 28.03 kDa was expressed in recombinant E. coli cells after the induction by isopropylthio-β-D-galactoside. Compared with control E. coli cells harbouring pET30a, the accumulation of the OsLEA3-1 fusion protein increased the tolerance of the E. coli recombinants under diverse abiotic stresses: high salinity, metal ions, hyperosmotic, heat, and UV radiation. The OsLEA3-1 has the ability to protect the lactate dehydrogenase activity under heating, drying, and MnCl2 treatment in vitro. The findings suggested that the OsLEA3-1 gene may contribute to the ability of adapting to stressful environments of plants.

scholarly journals A kidney-selective biopolymer for targeted drug delivery

2017 ◽  
Vol 312 (1) ◽  
pp. F54-F64 ◽  
Author(s):  
Gene L. Bidwell ◽  
Fakhri Mahdi ◽  
Qingmei Shao ◽  
Omar C. Logue ◽  
Jamarius P. Waller ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Renal Tissue ◽  
Cysteine Residue ◽  
High Dose ◽  
Binding Studies ◽  
High Accumulation ◽  
Targeting Peptide ◽  
Species Specific

Improving drug delivery to the kidney using renal-targeted therapeutics is a promising but underdeveloped area. We aimed to develop a kidney-targeting construct for renal-specific drug delivery. Elastin-like polypeptides (ELPs) are nonimmunogenic protein-based carriers that can stabilize attached small-molecule and peptide therapeutics. We modified ELP at its NH2-terminus with a cyclic, seven-amino acid kidney-targeting peptide (KTP) and at its COOH-terminus with a cysteine residue for tracer conjugation. Comparative in vivo pharmacokinetics and biodistribution in rat and swine models and in vitro cell binding studies using human renal cells were performed. KTP-ELP had a longer plasma half-life than ELP in both animal models and was similarly accumulated in kidneys at levels fivefold higher than untargeted ELP, showing renal levels 15- to over 150-fold higher than in other major organs. Renal fluorescence histology demonstrated high accumulation of KTP-ELP in proximal tubules and vascular endothelium. Furthermore, a 14-day infusion of a high dose of ELP or KTP-ELP did not affect body weight, glomerular filtration rate, or albuminuria, or induce renal tissue damage compared with saline-treated controls. In vitro experiments showed higher binding of KTP-ELP to human podocytes, proximal tubule epithelial, and glomerular microvascular endothelial cells than untargeted ELP. These results show the high renal selectivity of KTP-ELP, support the notion that the construct is not species specific, and demonstrate that it does not induce acute renal toxicity. The plasticity of ELP for attachment of any class of therapeutics unlocks the possibility of applying ELP technology for targeted treatment of renal disease in future studies.

Characterization of the evolutionarily conserved iron–sulfur cluster of sirohydrochlorin ferrochelatase from Arabidopsis thaliana

2012 ◽  
Vol 444 (2) ◽  
pp. 227-237 ◽  
Author(s):  
Kaushik Saha ◽  
Michael E. Webb ◽  
Stephen E. J. Rigby ◽  
Helen K. Leech ◽  
Martin J. Warren ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Quaternary Structure ◽  
Specific Activity ◽  
Close Association ◽  
Physiological Role ◽  
Cysteine Residue ◽  
In Planta ◽  
Iron Sulfur ◽  
Tetrapyrrole Metabolism

Sirohaem is a cofactor of nitrite and sulfite reductases, essential for assimilation of nitrogen and sulfur. Sirohaem is synthesized from the central tetrapyrrole intermediate uroporphyrinogen III by methylation, oxidation and ferrochelation reactions. In Arabidopsis thaliana, the ferrochelation step is catalysed by sirohydrochlorin ferrochelatase (SirB), which, unlike its counterparts in bacteria, contains an [Fe–S] cluster. We determined the cluster to be a [4Fe–4S] type, which quickly oxidizes to a [2Fe–2S] form in the presence of oxygen. We also identified the cluster ligands as four conserved cysteine residues located at the C-terminus. A fifth conserved cysteine residue, Cys135, is not involved in ligating the cluster directly, but influences the oxygen-sensitivity of the [4Fe–4S] form, and possibly the affinity for the substrate metal. Substitution mutants of the enzyme lacking the Fe–S cluster or Cys135 retain the same specific activity in vitro and dimeric quaternary structure as the wild-type enzyme. The mutant variants also rescue a defined Escherichia coli sirohaem-deficient mutant. However, the mutant enzymes cannot complement Arabidopsis plants with a null AtSirB mutation, which exhibits post-germination arrest. These observations suggest an important physiological role for the Fe–S cluster in planta, highlighting the close association of iron, sulfur and tetrapyrrole metabolism.

Cloning and expression ofSpodoptera lituranucleopolyhedrovirusgp41gene inEscherichia coliand preparation of antibody

2005 ◽  
Vol 2 (2) ◽  
pp. 113-117
Author(s):  
Pan Li-Jing ◽  
Li Zhao-Fei ◽  
Yin Chong ◽  
Lv Lei ◽  
Pang Yi
Keyword(s):  
Fusion Protein ◽  
Prokaryotic Expression ◽  
Recombinant Plasmid ◽  
Nitrilotriacetic Acid ◽  
Cloning And Expression ◽  
Reading Frame ◽  
Pcr Product ◽  
Prokaryotic Expression Vector ◽  
Polymerase Chain

AbstractGP41, a major glycoprotein, identified in the occlusion-derived virions (ODV) of baculoviruses, is required for the egress of nucleocapsids from the nucleus in the pathway of budded virion (BV) synthesis. Using the polymerase chain reaction (PCR), the open reading frame (ORF) ofSpodoptera lituranucleopolyhedrovirus (SpltMNPV)gp41gene was obtained from SpltMNPV genomic DNA. The PCR product was cloned into pMD18-T vector to get the recombinant plasmid (pT-gp41). Thegp41gene was recombinedin vitrowith prokaryotic expression vector pQE30 and transformed intoEscherichia coliM15 [pREP4]. The M15 [pREP4] strain, containinggp41recombinant plasmid, expressed a 37.9 kDa 6×His-tag fusion protein after induction with 1 mmol/l isopropylthio-β-d-galactoside (IPTG). The fusion protein was purified with a nickel-nitrilotriacetic acid (Ni–NTA) resin column and used as the immunogen to raise GP41-specific antibody. Western blotting analysis indicated that the antibody was suitable to be used for further analysis of GP41 protein.

Sarcoplasmic reticulum dysfunction: phospholipid alterations induced by lysosomal phospholipase C

1986 ◽  
Vol 251 (5) ◽  
pp. H1017-H1023
Author(s):  
R. Franson ◽  
D. Gamache ◽  
W. Blackwell ◽  
D. Eisen ◽  
M. L. Hess
Keyword(s):  
Free Radicals ◽  
Sarcoplasmic Reticulum ◽  
Myocardial Ischemia ◽  
Phospholipase C ◽  
Oxygen Free Radicals ◽  
Specific Activity ◽  
Phospholipid Composition ◽  
Maximal Activity ◽  
Global Ischemia

Previous work has demonstrated that myocardial ischemia results in a breakdown of the excitation-contraction coupling system of cardiac muscle associated with lysosomal activation. It has been hypothesized that lysosomal activation during the course of myocardial ischemia is mediated by the production of oxygen free radicals. We have tested the hypothesis that myocardial ischemia results in the activation of lysosomal phospholipase C and disruption of calcium transport in sarcoplasmic reticulum (SR) mediated by oxygen free radicals. Three groups of dogs were studied: sham-operated controls (n = 6); normothermic global ischemia of 30-min duration (n = 6); and 30 min of normothermic global ischemia pretreated with intracoronary superoxide dismutase (SOD, 10 micrograms/ml) plus catalase (25 micrograms/ml). In vitro, isolated SR demonstrated a significant depression of calcium uptake rates and Ca2+-stimulated, Mg2+-dependent ATPase activity at both pH 7.0 and 6.4 with the depression at pH 6.4 greater than 7.0. This depression of SR function was significantly inhibited in hearts pretreated with SOD plus catalase. In sham-operated controls, acid-induced dysfunction was associated with substantial loss of phospholipid phosphorus and major changes in phospholipid composition. SR contained an extremely active, ion-independent sphingomyelinase-phospholipase C (SM-PLC) that had maximal activity at pH 4.5-5.0. This SM-PLC was activated when control SR was incubated at acid pH and the specific activity of SM-PLC was decreased 50% in SR isolated from normothermic global ischemia. Activity remained at control levels in hearts pretreated with SOD plus catalase.(ABSTRACT TRUNCATED AT 250 WORDS)

Production and characterization of recombinant chicken insulin-like growth factor-II from Escherichia coli

1995 ◽  
Vol 14 (1) ◽  
pp. 79-90 ◽  
Author(s):  
Z Upton ◽  
G L Francis ◽  
K Kita ◽  
J C Wallace ◽  
F J Ballard
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Genetically Engineered ◽  
Binding Studies ◽  
Igf Binding Proteins ◽  
Igf Receptor

ABSTRACT Recombinant chicken (c)IGF-II has been produced in Escherichia coli after first modifying a plasmid that coded for a human (h)IGF-II fusion protein. The cIGF-II fusion protein, deposited in bacterial inclusion bodies, was dissolved under reducing conditions, desalted, subjected to anion-exchange chromatography and refolded. Recombinant cIGF-II was then released from the fusion protein using a genetically engineered serine protease and purified to homogeneity by reverse-phase HPLC. In vitro analysis of recombinant cIGF-II revealed differences between cIGF-II and its human counterpart. Recombinant cIGF-II was less potent than hIGF-II in stimulating protein synthesis in rat myoblasts. This appeared to be due to a decreased affinity for the type-1 IGF receptor. The human and chicken peptides were similar, however, in studies assessing binding to the type-2 IGF receptor and to IGF-binding proteins. Moreover, recombinant cIGF-II and hIGF-II were equipotent in both biological and receptor binding studies in chick embryo fibroblasts, suggesting that there may be a difference between mammalian and avian type-1 IGF receptors.

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