Search results for "recombinant"
showing 10 items of 1150 documents
Genetic rearrangements in the pathogenicity locus of Clostridium difficile strain 8864 – implications for transcription, expression and enzymatic act…
1998
The pathogenicity locus (PaLoc) of Clostridium difficile isolate 8864 was investigated to locate genetic rearrangements that would explain the exceptional pathogenicity of this particular isolate. Two major changes were defined: an insertion of 1.1 kb between the two genes tcdA and tcdE, coding for the enterotoxin and an accessory protein of unknown function, respectively, and a deletion of 5.9 kb encompassing the 3' ends of tcdA and tcdC. Transcription of the tcdA-E genes is severely affected by both rearrangements, explaining the demonstrated complete lack of TcdA polypeptide. We present a model of coordinate, growth-related transcription of the tcdA-E genes that confirms our previous fin…
The catalytic activity of the endoplasmic reticulum-resident protein microsomal epoxide hydrolase towards carcinogens is retained on inversion of its…
1996
Diol epoxides formed by the sequential action of cytochrome P-450 and the microsomal epoxide hydrolase (mEH) in the endoplasmic reticulum (ER) represent an important class of ultimate carcinogenic metabolites of polycyclic aromatic hydrocarbons. The role of the membrane orientation of cytochrome P-450 and mEH relative to each other in this catalytic cascade is not known. Cytochrome P-450 is known to have a type I topology. According to the algorithm of Hartman, Rapoport and Lodish [(1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5786–5790], which allows the prediction of the membrane topology of proteins, mEH should adopt a type II membrane topology. Experimentally, mEH membrane topology has been …
Efficient expression of a Paenibacillus barcinonensis endoglucanase in Saccharomyces cerevisiae.
2011
Abstract The endoglucanase coded by celA (GenBank Access No. Y12512) from Paenibacillus barcinonensis, an enzyme with good characteristics for application on paper manufacture from agricultural fibers, was expressed in Saccharomyces cerevisiae by using different domains of the cell wall protein Pir4 as translational fusion partners, to achieve either secretion or cell wall retention of the recombinant enzyme. Given the presence of five potential N-glycosylation sites in the amino acid sequence coded by celA, the effect of glycosylation on the enzymatic activity of the recombinant enzyme was investigated by expressing the recombinant fusion proteins in both, standard and glycosylation-defici…
A novel cytotoxin from Clostridium difficile serogroup F is a functional hybrid between two other large clostridial cytotoxins.
1999
Abstract The large clostridial cytotoxins (LCTs) constitute a group of high molecular weight clostridial cytotoxins that inactivate cellular small GTP-binding proteins. We demonstrate that a novel LCT (TcdB-1470) from Clostridium difficile strain 1470 is a functional hybrid between “reference” TcdB-10463 andClostridium sordellii TcsL-1522. It bound to the same specific receptor as TcdB-10463 but glucosylated the same GTP-binding proteins as TcsL-1522. All three toxins had equal enzymatic potencies but were equally cytotoxic only when microinjected. When applied extracellularly TcdB-1470 and TcdB-10463 were considerably more potent cytotoxins than TcsL-1522. The small GTP-binding protein R-R…
Identification of Two Mannoproteins Released from Cell Walls of a Saccharomyces cerevisiae mnn1 mnn9 Double Mutant by Reducing Agents
1999
The cell wall of Saccharomyces cerevisiae represents some 30% of the total weight of the cell and is made up of β-glucans, mannose-containing glycoproteins (mannoproteins), and small amounts of chitin (9, 15). The mannoproteins can be divided into three groups according to the linkages that bind them to the structure of the cell wall: (i) noncovalently bound, (ii) covalently bound to the structural glucan, and (iii) disulfide bound to other proteins that are themselves covalently bound to the structural glucan of the cell wall (8). Our work has focused on the disulfide-bound mannoproteins, probably the least well known of the three groups mentioned above. Previous work (25) showed that trea…
Probing suggested catalytic domains of glycosyltransferases by site-directed mutagenesis.
2003
The plant enzyme arbutin synthase isolated from cell suspension cultures of Rauvolfia serpentina and heterologously expressed in Escherichia coli is a member of the NRD1beta family of glycosyltransferases. This enzyme was used to prove, by site-directed mutagenesis, suggested catalytic domains and reaction mechanisms proposed for enzyme-catalyzed glycosylation. Replacement of amino acids far from the NRD domain do not significantly affect arbutin synthase activity. Exchange of amino acids at the NRD site leads to a decrease of enzymatic activity, e.g. substitution of Glu368 by Asp. Glu368, which is a conserved amino acid in glycosyltransferases located at position 2 and is important for enz…
Membrane-insertion fragments of Bcl-xL, Bax, and Bid.
2004
Apoptosis regulators of the Bcl-2 family associate with intracellular membranes from mitochondria and the endoplasmic reticulum, where they perform their function. The activity of these proteins is related to the release of apoptogenic factors, sequestered in the mitochondria, to the cytoplasm, probably through the formation of ion and/or protein transport channels. Most of these proteins contain a C-terminal putative transmembrane (TM) fragment and a pair of hydrophobic alpha helices (alpha5-alpha6) similar to the membrane insertion fragments of the ion-channel domain of diphtheria toxin and colicins. Here, we report on the membrane-insertion properties of different segments from antiapopt…
A role for the immunoglobulin-like domain of the human IL-6 receptor. Intracellular protein transport and shedding.
1999
Interleukin (IL)-6, IL-11 and cililary neurotrophic factor (CNTF) belong to the same family of hematopoietic and neurotrophic cytokines. Their receptor complexes contain a cytokine-binding alpha receptor and the common glycoprotein (gp)130 subunit for signal transduction. The extracellular parts of the alpha-receptor subunits consist of a membrane-proximal cytokine-binding domain and an N-terminal immunoglobulin (Ig)-like domain with unknown function. We examined the role of the Ig-like domain of IL-6R by constructing deletion mutants lacking the Ig domain (IL-6RDeltaIg and soluble IL-6RDeltaIg). IL-6RDeltaIg was shed as effectively as wild-type IL-6R from transfected COS-7 cells upon 4beta…
Expression ofYWP1,a Gene That Encodes a SpecificYarrowia lipolyticaMycelial Cell Wall Protein, inSaccharomyces cerevisiae
1997
Abstract The YWP1 gene encoding a specific mycelial cell wall protein of Yarrowia lipolytica has been cloned and expressed in Saccharomyces cerevisiae using different episomal plasmids. Because the plasmids pYAE35BB and pYAE35ES carrying the YWP1 gene (including the 5′ noncoding promoter sequences) failed to express it, the YWP1 gene was cloned under the control of GAL/CYC or ACT S. cerevisiae promoters. A main band with an apparent molecular mass of 70 kDa was detected by immunoblotting in the cell wall fraction of transformants. Ywp1 processing and incorporation to the cell wall were similar in both Y. lipolytica and S. cerevisiae but not in its final localization in the cell wall. In Y. …
Maturation of barley cysteine endopeptidase expressed in Trichoderma reesei is distorted by incomplete processing
2002
Maturation of barley cysteine endopeptidase B (EPB) in Trichoderma reesei was studied with metabolic inhibitors, Western blotting, and immuno microscopy. The inactive 42-kDa recombinant EPB proprotein, first detected in apical cells, was sequentially processed in a time-dependent manner to a secreted polypeptide of 38.5 kDa, and thereafter, to polypeptides of 37.5, 35.5, and 32 kDa exhibiting enzyme activity both in the hyphae and culture medium. The sizes of the different forms of recombinant EPB were in accordance with molecular masses calculated from the deduced amino acid sequence, assuming cleavage at four putative Kex2p sites present in the 42-kDa proprotein. Both the liquid and the z…