1 |
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Genetic and biochemical characterization of a novel monoterpene e-lactone hydrolase from Rhodococcus erythropolis DCL14
A monoterpene ε-lactone hydrolase (MLH) from Rhodococcus erythropolis DCL14, catalyzing the ring opening of lactones which are formed during degradation of several monocyclic monoterpenes, including carvone and menthol, was purified to apparent homogeneity. It is a monomeric enzyme of 31 kDa that is active with (4R)-4-isopropenyl-7-methyl-2-oxo-oxepanone and (6R)-6-isopropenyl-3-methyl-2-oxo-oxepanone, lactones derived from (4R)-dihydrocarvone, and 7-isopropyl-4-methyl-2-oxo-oxepanone, the lactone derived from menthone. Both enantiomers of 4-, 5-, 6-, and 7-methyl-2-oxo-oxepanone were converted at equal rates, suggesting that the enzyme is not stereoselective. Maximal enzyme activity was measured at pR 9.5 and 30°C. Determination of the N-terminal amino acid sequence of purified MLH enabled cloning of the corresponding gene by a combination of PCR and colony screening. The gene, designated mlhB (monoterpene lactone hydrolysis), showed up to 43% similarity to members of the GDXG family of lipolytic enzymes. Sequencing of the adjacent regions revealed two other open reading frames, one encoding a protein with similarity to the short-chain dehydrogenase reductase family and the second encoding a protein with similarity to acyl coenzyme A dehydrogenases. Both enzymes are possibly also involved in the monoterpene degradation pathways of this microorganism. Molecular Sequence Numbers: EMBL: AJ292535; GENBANK: AJ292535;
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[PDF]
[Abstract]
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2 |
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Identification of a novel protein antigen encoded by a Mycobacterium tuberculosis-specific RD1 region gene
A genomic DNA region, designated RD1, that is present in virulent and clinical strains of Mycobacterium tuberculosis and M. bovis, has been shown to be deleted in bacillus Calmette Guerin (BCG). The DNA segments corresponding to three open reading frames (ORFs: ORF-10, ORF-14 and ORF-15) of the RD1 region, that are deleted in BCG strains, were amplified from M. tuberculosis genomic DNA by polymerase chain reaction (PCR), subcloned into pGEX-4T vector system and expressed in Escherichia coli as fusion proteins with glutathione-S-transferase (GST). The recombinant proteins appeared as major cellular proteins in SDS-PAGE gels at the expected molecular mass. The identity of each fusion protein was confirmed by reactivity with anti-GST antibodies in Western immunoblots. When pooled human sera from 11 tuberculosis (TB) patients were used as the source of antibodies, only GST- ORF-14 fusion protein reacted in Western immunoblots. The protein corresponding to ORF-14 was then purified to near homogeneity and isolated free of its fusion partner (GST) by treating the purified GST-ORF-14 fusion protein with thrombin protease. In Western immunoblots, the purified ORF-14 protein reacted with antibodies in 26 of 57 human sera (46%) from TB patients while no reactivity was seen with 11 sera from M. bovis BCG-vaccinated healthy subjects. Interestingly, sera from nine of 15 (60%) long-term contacts of TB patients also had antibodies reactive to the ORF-14 protein. These results suggest that the ORF-14 protein in combination with other immunodominant proteins could be useful in the serodiagnosis of individuals infected with M. tuberculosis.
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[Abstract]
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3 |
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Expression of the Pycnoporus cinnabarinus laccase gene in Aspergillus niger and characterization of the recombinant enzyme
article |
2002
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Author: |
Record, E.
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Punt, P.J.
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Chamkha, M.
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Labat, M.
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Hondel, C.A.M.J.J. van den
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Asther, M.
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Keywords: |
Nutrition · Amino Acid Sequence · Base Sequence · Basidiomycota · Blotting, Northern · Blotting, Western · Cloning, Molecular · DNA, Complementary · Hydrogen-Ion Concentration · Isoelectric Point · Kinetics · Laccase · Molecular Weight · Oxidoreductases · Recombinant Proteins · Temperature · Aspergillus · Aspergillus niger · Prokaryota · Pycnoporus cinnabarinus
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Pycnoporus cinnabarinus laccase lac1 gene was overexpressed in Aspergillus niger, a well-known fungal host producing a large amount of homologous or heterologous enzymes for industrial applications. The corresponding cDNA was placed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter as a strong and constitutive promoter. The laccase signal peptide or the glucoamylase preprosequence of A. niger was used to target the secretion. Both signal peptides directed the secretion of laccase into the culture medium as an active protein, but the A. niger preprosequence allowed an 80-fold increase in laccase production. The identity of the recombinant protein was further confirmed by immunodetection using Western blot analysis and N-terminal sequencing. The molecular mass of the mature laccase was 70 kDa as expected, similar to that of the native form, suggesting no hyperglycosylation. The recombinant laccase was purified in a three-step procedure including a fractionated precipitation using ammonium sulfate, and a concentration by ultrafiltration followed by a Mono Q column. All the characteristics of the recombinant laccase are in agreement with those of the native laccase. This is the first report of the production of a white-rot laccase in A. niger. Molecular Sequence Numbers: GENBANK: AF170093; Chemicals/CAS: DNA, Complementary; Laccase, EC 1.10.3.2; Oxidoreductases, EC 1.-; Recombinant Proteins
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[Abstract]
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4 |
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The construction of new vehicles for the cloning of transcription termination signals
Chemicals/CAS: DNA Restriction Enzymes, EC 3.1.21; DNA, Recombinant; Plasmids; Tryptophan, 73-22-3
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[Abstract]
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5 |
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Species specificity of human interleukin-3 demonstrated by cloning and expression of the homologous rhesus monkey (Macaca mulatta) gene
To enable preclinical studies on homologous interleukin-3 (IL-3) in primate species, we isolated the gene encoding Rhesus monkey IL-3 (RhIL-3). The nucleotide sequence of the RhIL-3 gene displayed 92.9% homology with that of the human IL-3 (hIL-3) gene. The isolated RhIL-3 gene encodes a 143-amino acid (aa) precursor polypeptide, nine C-terminal residues shorter than the human protein. Protein homology was found to be 89.5% for the signal peptide (19 aa) and 80.5% for the mature protein (124 aa). Comparison of the human and RhIL-3 coding sequences showed that the majority of substitutions had occurred at amino acid replacement sites indicating a rapid evolution of the IL-3 protein. After expression of a genomic fragment in COS cells, RhIL-3 cDNA was constructed, which enabled large-scale production of the RhIL-3 polypeptide. RhIL-3 produced by Bacillus licheniformis and purified to homogeneity appeared to be approximately 100-fold more effective in stimulating Rhesus monkey hematopoietic progenitors than hIL-3, whereas RhIL-3 and hIL-3 showed comparable stimulatory activity on normal as well as malignant human hematopoietic cells. Thus, the rapid evolution of hIL-3 has resulted in a unidirectional species specificity, which most likely restricts the in vivo effects of hIL-3 in Macaca species.
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[Abstract]
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6 |
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Human plasminogen activator inhibitor-1 gene. Promoter and structural gene nucleotide sequences
We have determined the nucleotide sequence of the human plasminogen activator inhibitor-1 (PAI-1) gene and significant stretches of DNA which extend into its 5'- and 3'-flanking DNA regions; a total sequence of 15,867 base pairs (bp) is presented. The sequenced 5'-flanking DNA (1,520 bp) contains the essential eukaryotic cis-type proximal regulatory elements CCAAT and TATAA; the more distal 5'-flanking DNA region, as well as some introns, contain sequence elements which share identities with known eukaryotic enhancer elements. A major finding is the identification of a large region of shared nucleotides (comprising of about 520 bp) between the 5'-flanking DNAs of PAI-1 and tissue-type plasminogen activator genes. The length of the PAI-1 5'-untranslated region was found to be 145 bp as determined by nuclease analysis. The remaining PAI-1 structural gene consists of amino acid coding regions (containing a total of 1,206 bp, coding for the 23 amino acids of the signal peptide and 379 amino acids of the mature PAI-1 protein), 8 intron regions (a total of 8,978 bp), and a long 3'-untranslated region of about 1,800 bp which contains several polyadenylation sites. Two types of repetitive DNA elements are located within the PAI-1 structural gene and flanking DNAs: we have found 12 Alu elements and 5 repeats of a long poly (Pur) element. These Alu-Pur elements may represent a subset of the more abundant Alu family of repetitive sequence elements. Molecular Sequence Numbers: GENBANK: J03764; Chemicals/CAS: plasminogen activator inhibitor, 105844-41-5; Cosmids; Glycoproteins; Plasminogen Activators, EC 3.4.21.-; Plasminogen Inactivators
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[PDF]
[Abstract]
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7 |
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Molecular cloning and characterization of the alkaline ceramidase from Pseudomonas aeruginosa PA01
Ceramidase (CDase) hydrolyzes the amide bond in ceramides to yield free fatty acid and sphingosine. From a 3-L Pseudomonas aeruginosa PA01 culture, 70 μg of extracellular alkaline, Ca2+-dependent CDase, was purified to homogeneity, the N-terminal sequence was determined, and the CDase gene was cloned. The CDase gene encodes a 670 amino acid protein with a 26 amino acid signal peptide. CDase was expressed in five prokaryotic and eukaryotic expression systems. Small amounts of recombinant active extracellular CDase were expressed by Pseudomonas putida KT2440. In Pichia pastoris GS115 low amounts of recombinant extracellular glycosylated CDase were expressed. High levels of intracellular CDase were expressed by Escherichia coli DH5α and E. coli BL21 cells under control of the lac-promoter and T7-promoter, respectively. From a 3-L E. coli DH5α culture, 280 μg of pure CDase was obtained after a three-step purification protocol. Under control of the T7-promotor CDase, without its signal peptide, was produced in inclusion bodies in E. coli BL21 cells. After refolding, 1.8mg of pure active CDase was obtained from a 2.4-L culture after ammonium sulfate precipitation and gel filtration. Both the recombinant and wild-type CDases have a pH optimum of 8.5. The recombinant enzyme was partially characterized. This is the first report of a high yield CDase production system allowing detailed characterization of the enzyme at the molecular level. © 2003 Elsevier Science (USA). All rights reserved.
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[Abstract]
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8 |
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Identification and characterization of a family of secretion-related small GTPase-encoding genes from the filamentous fungus Aspergillus niger : a putative SEC4 homologue is not essential for growth
article |
2001
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Author: |
Punt, P.J.
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Seiboth, B.
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Weenink, X.O.
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Zeijl, C. van
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Lenders, M.
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Konetschny, C.
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Ram, A.F.J.
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Montijn, R.
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Kubicek, C.P.
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Hondel, C.A.M.J.J. van den
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Keywords: |
Biology · Amino Acid Sequence · Aspergillus niger · Cloning, Molecular · Fungal Proteins · Genes, Essential · Genes, Fungal · Genetic Complementation Test · Glucose · Molecular Sequence Data · Multigene Family · Mutation · Nucleic Acid Hybridization · Polymerase Chain Reaction · Polysaccharides · rab GTP-Binding Proteins · Saccharomyces cerevisiae · Saccharomyces cerevisiae Proteins · Sequence Alignment · Sequence Homology, Amino Acid · Aspergillus niger · Fungi · Mammalia · Saccharomyces cerevisiae
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DNA fragments containing genetic information for five secretion-related small GTPases of Aspergillus niger (srgA-E) were isolated and identified as members of different Rab/Ypt subfamilies. This isolation and the search for similar sequences in fungal genomic and EST databases showed that, in contrast to Saccharomyces cerevisiae, filamentous fungi also possess homologues of mammalian Rab2 GTPases. Multiple transcripts with unusually long 5′ and 3′ untranslated regions were found for all srg genes. Their level of expression was independent of the type of carbon source used for growth. Although the transcripts of srgA and srgB were abundant to the same extent throughout the cultivation, that of the other genes peaked during the early growth phase and then declined. Two genes, srgA and srgB, were characterized further. The protein encoded by srgA exhibited relatively low identity (58%) to its closest S. cerevisiae homologue SEC4, whereas the protein encoded by srgB showed 73% identity with S. cerevisiae YPT1. In contrast to other SEC4 homologues, srgA was unable to complement an S. cerevisiae sec4 mutant, and its disruption was not lethal in A. niger. SrgA mutants displayed a twofold increase in their hyphal diameter, unusual apical branching and strongly reduced protein secretion during growth on glucose. Molecular Sequence Numbers: A44334, AJ224685, AJ278657, AJ278658, AJ278659, AJ278660, AJ278661, AJ278662, AJ404733, AJ404734, P07560, S32965, X52099, X52100, X52469, X52475, X59598, X72833, X72834, X76173, X76174, X76175, Z22220, Z98598; Chemicals/CAS: Fungal Proteins; Glucose, 50-99-7; maltodextrin, 9050-36-6; Polysaccharides; rab GTP-Binding Proteins, EC 3.6.1.-; Saccharomyces cerevisiae Proteins; SEC4 protein, S cerevisiae, EC 3.6.1.-.
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[Abstract]
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9 |
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Genetic analysis of acidocin B : a novel bacteriocin produced by Lactobacillus acidophilus
article |
1995
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Author: |
Leer, R.J.
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Vossen, J.M.B.M. van der
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Giezen, M. van
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Noort, J.M. van
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Pouwels, P.H.
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Keywords: |
Biology · Amino Acid Sequence · Bacteriocins · Base Sequence · Chromatography, High Pressure Liquid · Cloning, Molecular · Clostridium · Conserved Sequence · DNA, Bacterial · Genes, Bacterial · Lactobacillus · Lactobacillus acidophilus · Molecular Sequence Data · Multigene Family · Open Reading Frames · Plasmids · Protein Sorting Signals · Restriction Mapping · Support, Non-U.S. Gov't · Transformation, Bacterial
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The genes encoding the production of acidocin B, a bacteriocin produced by Lactobacillus acidophilus strain M46 which is active against Listeria monocytogenes, Clostridium sporogenes, Brochothrix thermosphacta, Lactobacillus fermentum and Lactobacillus delbrueckii subsp. bulgaricus, but inactive against most other Lactobacillus species, were previously localized on a 4 kb Xbal-HindIII fragment of plasmid pCV461. In the present work, DNA sequence analysis revealed the presence of three consecutive ORFs, which potentially code for hydrophobic peptides composed of 60, 91 and 114 amino acids, respectively, and a fourth ORF of opposite polarity which could potentially encode a peptide of 59 amino acids. The middle ORF (ORF-2; acdB) was identified as the gene encoding acidocin B by comparing the amino acid composition of highly purified acidocin B with the deduced amino acid sequence of ORF-2. Our results suggest that acidocin B is synthesized as a precursor which is processed at a site which conforms to the '-3, -1' rules of von Heijne to yield active acidocin B (59 amino acids). The presence of an immunity-protein-encoding gene on the 4 kb Xbal-BamHI fragment was deduced from the capacity of a plasmid vector harbouring this fragment to confer immunity upon transformation of L. fermentum NCK127. One of the three non-assigned ORFs may encode this immunity protein.
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[Abstract]
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10 |
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Expression of cytochromes P450 in mammalian cells.
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11 |
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Functional expression in Lactobacillus plantarum of xylP encoding the isoprimeverose transporter of Lactobacillus pentosus
The xylP gene of Lactobacillus pentosus, the first gene of the xylPQR operon, was recently found to be involved in isoprimeverose metabolism. By expression of xylP on a multicopy plasmid in Lactobacillus plantarum 80, a strain which lacks active isoprimeverose and D-xylose transport activities, it was shown that xylP encodes a transporter. Functional expression of the XylP transporter was shown by uptake of isoprimeverose in L. plantarum 80 cells, and this transport was driven by the proton motive force generated by malolactic fermentation. XylP was unable to catalyze transport of D-xylose.
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[Abstract]
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12 |
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Lactobacilli as live vaccine delivery vectors: Progress and prospects
Evidence is accumulating that lactobacilli influence the immune response in a strain-dependent manner. This immunomodulatory capacity is important for the development of the immune response, and also identifies Lactobacillus as a potent oral vaccine carrier. Most of our current knowledge of the use of lactobacilli for vaccination purposes has been obtained with tetanus toxin fragment C (TTFC) as the model antigen. This knowledge, together with our ever-increasing understanding of the immune system and recent developments in cloning and expression techniques, should enable the utilisation of antigens other than TTFC and has made the development of lactobacilli as live vaccines a realistic prospect. Chemicals/CAS: Bacterial Vaccines; Peptide Fragments; Recombinant Proteins; tetanus toxin fragment C; Tetanus Toxin; Vaccines, Synthetic
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[Abstract]
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13 |
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Assignment of the human tissue-type plasminogen activator gene (PLAT) to chromosome 8
Using 1.2kb 3'-terminal Pst-I fragment of a full length tissue-type plasminogen activator (t-PA) cDNA clone (ptPA-8FL) and a set of rodent human somatic cell hydrids, the corresponding human gene PLAT was localized on chromosome 8. Chemicals/CAS: plasminogen activator, 9039-53-6; DNA Restriction Enzymes, EC 3.1.21; DNA, 9007-49-2; Genetic Markers; Tissue Plasminogen Activator, EC 3.4.21.68
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[Abstract]
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14 |
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An epitope delivery system for use with recombinant mycobacteria
article |
1998
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Author: |
Hetzel, C.
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Janssen, R.
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Ely, S.J.
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Kristensen, N.M.
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Bunting, K.
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Cooper, J.B.
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Lamb, J.R.
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Young, D.B.
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Thole, J.E.R.
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Keywords: |
Health · Mycobacterium antigen · Antigen expression · Immunogenicity · Mycobacterium tuberculosis · Mycobacterium vaccae · Nonhuman · Protein targeting · Vaccine production · Amino Acid Sequence · Animals · Antigens, Bacterial · Antigens, Dermatophagoides · Bacterial Proteins · CD4-Positive T-Lymphocytes · CD8-Positive T-Lymphocytes · Cloning, Molecular · Epitopes, T-Lymphocyte · Female · Gene Expression · Glycoproteins · Interferon Type II · Mice · Mice, Inbred C57BL · Molecular Sequence Data · Mycobacterium · Mycobacterium bovis · Recombinant Fusion Proteins · Recombination, Genetic · Superoxide Dismutase
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We have developed a novel epitope delivery system based on the insertion of peptides within a permissive loop of a bacterial superoxide dismutase molecule. This system allowed high-level expression of heterologous peptides in two mycobacterial vaccine strains, Mycobacterium bovis bacille Calmette- Guerin (BCG) and Mycobacterium vaccae. The broader application of the system was analyzed by preparation of constructs containing peptide epitopes from a range of infectious agents and allergens. We report detailed characterization of the immunogenicity of one such construct, in which an epitope from the Der p1 house dust mite allergen was expressed in M. vaccae. The construct was able to stimulate T-cell hybridomas specific for Der p1, and it induced peptide-specific gamma interferon responses when used to immunize naive mice. This novel expression system demonstrates new possibilities for the use of mycobacteria as vaccine delivery vehicles. Chemicals/CAS: Antigens, Bacterial; Antigens, Dermatophagoides; Bacterial Proteins; Epitopes, T-Lymphocyte; Glycoproteins; Interferon Type II, 82115-62-6; Recombinant Fusion Proteins; SodA protein, Bacteria; Superoxide Dismutase, EC 1.15.1.1
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[PDF]
[Abstract]
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15 |
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The promoter of the rice gene GOS2 is active in various different monocot tissues and binds rice nuclear factor ASF-1
article |
1992
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Author: |
Pater, Sylvia B. de
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Mark, F. van der
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Rueb, S.
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Katagiri, F.
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Chua, N.H.
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Schilperoort, R.A.
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Hensgens, L.A.M.
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Keywords: |
Beta glucuronidase · GOS2 protein, Oryza sativa · Splicing factor 2 · Vegetable protein · Biosynthesis · Comparative study · Genetics · Metabolism · Molecular cloning · Molecular genetics · Promoter region · Rice · Amino Acid Sequence · Base Sequence · Cloning, Molecular · Comparative Study · Genes, Plant · Genetic Engineering · Glucuronidase · Molecular Sequence Data · Nuclear Proteins · Oryza sativa · Plant Proteins · Plants · Promoter Regions (Genetics) · Recombinant Proteins · Restriction Mapping · Sequence Homology, Amino Acid · Support, Non-U.S. Gov't · Tissue Distribution
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A single copy gene has been isolated, termed GOS2, from rice. Sequence comparison revealed highly similar genes in mammals and yeast, indicating that GOS2 encodes an evolutionary conserved protein. GOS2 mRNA was detected in all tissues examined. When the upstream region was translationally fused to the reporter gene gusA it was found to drive expression in a variety of rice tissues and in cell suspensions of other monocot species following introduction by particle bombardment. Therefore, the GOS2 promoter is potentially useful for genetic engineering of monocots. A DNA-binding activity from rice, termed rice ASF-1, with similar binding specificity as the cloned tobacco transcription factor TGA-1a, was found to bind to a TGACG sequence motif in the GOS2 promoter. Possible roles for rice ASF-1 in the transcriptional activation of the GOS2 promoter are discussed. Chemicals/CAS: beta glucuronidase, 9001-45-0; Glucuronidase, EC 3.2.1.31; GOS2 protein, Oryza sativa; Nuclear Proteins; Plant Proteins; Recombinant Proteins; splicing factor 2
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[Abstract]
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16 |
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Organization and characterization of three genes involved in D-xylose catabolism in Lactobacillus pentosus
article |
1991
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Author: |
Lokman, B.C.
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Santen, P. van
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Verdoes, J.C.
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Kruse, J.
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Leer, R.J.
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Posno, M.
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Pouwels, P.H.
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Keywords: |
Biology · Amino acid comparison · D-xylose isomerase · D-xylulose kinase · NMR analysis · Regulatory protein · Xylose · Xylose isomerase · Amino acid sequence · Gene structure · Lactobacillus · Nonhuman · Nucleotide sequence · Priority journal · Sequence homology · Amino Acid Sequence · Bacillus subtilis · Base Sequence · Carbohydrate Epimerases · Cloning, Molecular · DNA, Bacterial · Genes, Bacterial · Lactobacillus · Magnetic Resonance Spectroscopy · Molecular Sequence Data · Multigene Family · Open Reading Frames · Phosphotransferases · Plasmids · Repressor Proteins · Restriction Mapping · Sequence Alignment · Sequence Homology, Nucleic Acid · Streptomyces · Xylose
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A cluster of three genes involved in D-xylose catabolism (viz. xylose genes) in Lactobacillus pentosus has been cloned in Escherichia coli and characterized by nucleotide sequence analysis. The deduced gene products show considerable sequence similarity to a repressor protein involved in the regulation of expression of xylose genes in Bacillus subtilis (58%), to E. coli and B. subtilis D-xylose isomerase (68% and 77%, respectively), and to E. coli D-xylulose kinase (58%). The cloned genes represent functional xylose genes since they are able to complement the inability of a L. casei strain to ferment D-xylose. NMR analysis confirmed that 13C-xylose was converted into 13C-acetate in L. casei cells transformed with L. pentosus xylose genes but not in untransformed L. casei cells. Comparison with the aligned amino acid sequences of D-xylose isomerases of different bacteria suggests that L. pentosus D-xylose isomerase belongs to the same similarity group as B. subtilis and E. coli D-xylose isomerase and not to a second similarity group comprising D-xylose isomerases of Streptomyces violaceoniger, Ampullariella sp. and Actinoplanes. The organization of the L. pentosus xylose genes, 5'-xylR (1167 bp, repressor) - xylA (1350 bp, D-xylose isomerase) - xylB (1506 bp, D-xylulose kinase) - 3' is similar to that in B. subtilis. In contrast to B. subtilis xylR, L. pentosus xylR is transcribed in the same direction as xylA and xylB. Molecular Sequence Numbers: GENBANK: M57384, S55527, S55528, S55529, S55530, S55531, S55532, S63909, S69823, Z14057; Chemicals/CAS: Carbohydrate Epimerases, EC 5.1.3; DNA, Bacterial; Phosphotransferases, EC 2.7; Plasmids; Repressor Proteins; xylose isomerase, EC 5.3.1.5; Xylose; xylulokinase, EC 2.7.1.17
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[Abstract]
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17 |
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Molecular and biochemical characterization of a novel intracellular invertase from Aspergillus niger with transfructosylating activity
A novel subfamily of putative intracellular invertase enzymes (glycoside hydrolase family 32) has previously been identified in fungal genomes. Here, we report phylogenetic, molecular, and biochemical characteristics of SucB, one of two novel intracellular invertases identified in Aspergillus niger. The sucB gene was expressed in Escherichia coli and an invertase-negative strain of Saccharomyces cerevisiae. Enzyme purified from E. coli lysate displayed a molecular mass of 75 kDa, judging from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Its optimum pH and temperature for sucrose hydrolysis were determined to be 5.0 and 37 to 40°C, respectively. In addition to sucrose, the enzyme hydrolyzed 1-kestose, nystose, and raffinose but not inulin and levan. SucB produced 1-kestose and nystose from sucrose and 1-kestose, respectively. With nystose as a substrate, products up to a degree of polymerization of 4 were observed. SucB displayed typical Michaelis-Menten kinetics with substrate inhibition on sucrose (apparent Km, K i, and Vmax of 2.0 ± 0.2 mM, 268.1 ± 18.1 mM, and 6.6 ± 0.2 μmol min-1 mg-1 of protein [total activity], respectively). At sucrose concentrations up to 400 mM, transfructosylation (FTF) activity contributed approximately 20 to 30% to total activity. At higher sucrose concentrations, FTF activity increased to up to 50% of total activity. Disruption of sucB in A. niger resulted in an earlier onset of sporulation on solid medium containing various carbon sources, whereas no alteration of growth in liquid culture medium was observed. SucB thus does not play an essential role in inulin or sucrose catabolism in A. niger but may be needed for the intracellular conversion of sucrose to fructose, glucose, and small oligosaccharides. Copyright © 2007, American Society for Microbiology. All Rights Reserved.
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[Abstract]
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18 |
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Natural and recombinant fungal laccases for paper pulp bleaching
article |
2004
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Author: |
Sigoillot, C.
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Record, E.
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Belle, V.
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Robert, J.L.
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Levasseur, A.
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Punt, P.J.
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Hondel, C.A.M.J.J. van den
·
Fournel, A.
·
Sigoillot, J.C.
·
Asther, M.
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Keywords: |
Biology · Biotechnology · fungal enzyme · laccase · recombinant enzyme · fungus · amino terminal sequence · article · Aspergillus niger · Aspergillus oryzae · biotechnology · bleaching · controlled study · delignification · enzyme analysis · enzyme induction · enzyme substrate · Michaelis constant · nonhuman · nucleotide sequence · oxidation reduction reaction · physical chemistry · protein synthesis · pulp processing · Aspergillus niger · Aspergillus oryzae · Biotechnology · Cloning, Molecular · Genes, Fungal · Industrial Microbiology · Laccase · Lignin · Oxidation-Reduction · Paper · Polyporaceae · Recombinant Proteins · Substrate Specificity · Aspergillus · Aspergillus niger · Aspergillus oryzae · Fungi · Pycnoporus cinnabarinus · Triticum aestivum
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Three laccases, a natural form and two recombinant forms obtained from two different expression hosts, were characterized and compared for paper pulp bleaching. Laccase from Pycnoporus cinnabarinus, a well known lignolytic fungus, was selected as a reference for this study. The corresponding recombinant laccases were produced in Aspergillus oryzae and A. niger hosts using the lacI gene from P. cinnabarinus to develop a production process without using the expensive laccase inducers required by the native source. In flasks, production of recombinant enzymes by Aspergilli strains gave yields close to 80 mg l -1. Each protein was purified to homogeneity and characterized, demonstrating that the three hosts produced proteins with similar physico-chemical properties, including electron paramagnetic resonance spectra and N-terminal sequences. However, the recombinant laccases have higher Michaelian (Km) constants, suggesting a decrease in substrate/enzyme affinity in comparison with the natural enzyme. Moreover, the natural laccase exhibited a higher redox potential (around 810 mV), compared with A. niger (760 mV) and A. oryzae (735 mV). Treatment of wheat straw Kraft pulp using laccases expressed in P. cinnabarinus or A. niger with 1-hydroxybenzotriazole as redox mediator achieved a delignification close to 75%, whereas the recombinant laccase from A. oryzae was not able to delignify pulp. These results were confirmed by thioacidolysis. Kinetic and redox potential data and pulp bleaching results were consistent, suggesting that the three enzymes are different and each fungal strain introduces differences during protein processing (folding and/or glycosylation).
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[Abstract]
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19 |
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Cloning, sequence analysis, and characterization of the genes involved in isoprimeverose metabolism in Lactobacillus pentosus
article |
1998
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Author: |
Chaillou, S.
·
Lokman, B.C.
·
Leer, R.J.
·
Posthuma, C.
·
Postma, P.W.
·
Pouwels, P.H.
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Keywords: |
disaccharide · article · bacterial gene · bacterial metabolism · carbohydrate metabolism · fermentation · gene control · gene expression regulation · lactobacillus · molecular cloning · nonhuman · nucleotide sequence · priority journal · regulon · repressor gene · Amino Acid Sequence · Bacterial Proteins · Base Sequence · Carrier Proteins · Cell Compartmentation · Cloning, Molecular · Disaccharides · Genes, Bacterial · Glycosides · Lactobacillus · Molecular Sequence Data · Operon · Sequence Analysis, DNA · Sequence Homology, Amino Acid · Substrate Specificity · Symporters · Xylose · Xylosidases · Bacteria (microorganisms) · Lactobacillus pentosus · Posibacteria · Prokaryota
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Two genes, xylP and xylQ, from the xylose regulon of Lactobacillus pentosus were cloned and sequenced. Together with the repressor gene of the regulon, xylR, the xylPQ genes form an operon which is inducible by xylose and which is transcribed from a promoter located 145 bp upstream of xylP. A putative xylR binding site (xylO) and a cre-like element, mediating CcpA- dependent catabolite repression, were found in the promoter region. L. pentosus mutants in which both xylP and xylQ (LPE1) or only xylQ (LPE2) was inactivated retained the ability to ferment xylose but were impaired in their ability to ferment isoprimeverose (α-D-xylopyranosyl, (1,6)-D- glucopyranose). Disruption of xylQ resulted specifically in the loss of a membrane-associated α-xylosidase activity when LPE1 or LPE2 cells were grown on xylose. In the membrane fraction of wild-type bacteria α-xylosidase could catalyze the hydrolysis of isoprimeverose and p-nitrophenyl-α-D- xylopyranoside with apparent K(m) and V(max) values of 0.2 mM and 446 nmol/min/mg of protein, and 1.3 mM and 54 nmol/min/mg of protein, respectively. The enzyme could also hydrolyze the α-xylosidic linkage in xyloglucan oligosaccharides, but neither methyl-α-D-xylopyranoside nor α- glucosides were substrates. Glucose repressed the synthesis of α-xylosidase fivefold, and 80% of this repression was released in an L. pentosus ΔccpA mutant. The α-xylosidase gene was also expressed in the absence of xylose when xylR was disrupted. Chemicals/CAS: alpha-D-xylosidase, EC 3.2.1.-; Bacterial Proteins; Carrier Proteins; Disaccharides; Glycosides; isoprimeverose cation symporter, Lactobacillus; isoprimeverose, 534-98-5; Symporters; Xylose; Xylosidases, EC 3.2.1.-
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[Abstract]
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Site-directed mutagenesis study of the three catalytic residues of the fructosyltransferases of Lactobacillus reuteri 121
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2004
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Author: |
Ozimek, L.K.
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Hijum, S.A.F.T. van
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Koningsveld, G.A. van
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Maarel, M.J.E.C. van der
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Geel-Schutten, G.H. van
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Dijkhuizen, L.
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Keywords: |
Food technology · Catalytic residue · Fructosyltransferase · Inulosucrase · Levansucrase · Mutagenesis · Acid · Base · Glycosidase · Levansucrase · Mutant protein · Stabilizing agent · Sucrase · Transferase · Bacterium mutant · Enzyme activity · Nonhuman · Site directed mutagenesis · Amino Acid Motifs · Amino Acid Sequence · Amino Acid Substitution · Catalysis · Circular Dichroism · Cloning, Molecular · Conserved Sequence · Escherichia coli · Gene Expression · Genes, Bacterial · Hexosyltransferases · Kinetics · Lactobacillus · Molecular Sequence Data · Mutagenesis, Site-Directed · Sequence Homology, Amino Acid · Substrate Specificity · Bacillus subtilis · Bacteria (microorganisms) · Lactobacillus reuteri
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Bacterial fructosyltransferases (FTFs) are retaining-type glycosidases that belong to family 68 of glycoside hydrolases. Recently, the high-resolution 3D structure of the Bacillus subtilis levansucrase has been solved [Meng, G. and Futterer, K., Nat. Struct. Biol. 10 (2003) 935-941]. Based on this structure, the catalytic nucleophile, general acid/base catalyst, and transition state stabilizer were identified. However, a detailed characterization of site-directed mutants of the catalytic nucleophile has not been presented for any FTF enzyme. We have constructed site-directed mutants of the three putative catalytic residues of the Lactobacillus reuteri 121 levansucrase and inulosucrase and characterized the mutant proteins. Changing the putative catalytic nucleophiles D272 (inulosucrase) and D249 (levansucrase) into their amido counterparts resulted in a 1.5-4×105 times reduction of total sucrase activity. © 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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[Abstract]
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