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  • 1. Eriksson, T.
    et al.
    Stals, I.
    Collen, A.
    Tjerneld, F.
    Claeyssens, M.
    Stalbrand, H.
    Brumer, Harry
    KTH, Superseded Departments, Biotechnology.
    Heterogeneity of homologously expressed Hypocrea jecorina (Trichoderma reesei) Cel7B catalytic module2004In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 271, no 7, p. 1266-1276Article in journal (Refereed)
    Abstract [en]

    The catalytic module of Hypocrea jecorina (previously Trichoderma reesei) Cel7B was homologously expressed by transformation of strain QM9414. Post-translational modifications in purified Cel7B preparations were analysed by enzymatic digestions, high performance chromatography, mass spectrometry and site-directed mutagenesis. Of the five potential sites found in the wild-type enzyme, only Asn56 and Asn182 were found to be N-glycosylated. GlcNAc(2)Man(5) was identified as the predominant N-glycan, although lesser amounts of GlcNAc(2)Man(7) and glycans carrying a mannophosphodiester bond were also detected. Repartition of neutral and charged glycan structures over the two glycosylation sites mainly accounts for the observed microheterogeneity of the protein. However, partial deamidation of Asn259 and a partially occupied O-glycosylation site give rise to further complexity in enzyme preparations.

  • 2. Gudmundsson, G H
    et al.
    Agerberth, B
    Odeberg, Jacob
    KTH, Superseded Departments, Biotechnology.
    Bergman, T
    Olsson, B
    Salcedo, R
    The human gene FALL39 and processing of the cathelin precursor to the antibacterial peptide LL-37 in granulocytes.1996In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 238, no 2, p. 325-32Article in journal (Refereed)
    Abstract [en]

    The peptide FA-LL-37, previously termed FALL-39, was originally predicted from on ORF of a cDNA clone isolated from a human bone marrow library. This peptide was synthesized and found to have antibacterial activity. We have now characterized and sequenced the complete gene for FA-LL-37, termed FALL39. It is a compact gene of 1963 bp with four exons. Exons 1-3 code for a signal sequence and the cathelin region. Exon 4 contains the information for the mature antibacterial peptide. Our results indicate that FALL39 is the only member of the cathelin gene family present in the human genome. Potential binding sites for acute-phase-response factors are identified in the promoter and in intron 2. A possible role for the cytokine interleukin-6 in the regulation of FALL 39 is discussed. Anti-(FA-LL-37) IgG located the peptide in granulocytes and we isolated the mature peptide from these cells after degranulation. Structural analysis determined the mature peptide to be LL-37. To obtain LL-37 for antibacterial assays, synthetic FA-LL-37 was degraded with dipeptidyl-peptidase I. This analysis showed that mature LL-37 is a potent antibacterial peptide.

  • 3. Him, J. L. K.
    et al.
    Pelosi, L.
    Chanzy, H.
    Putaux, J. L.
    Bulone, Vincent
    KTH, Superseded Departments, Biotechnology.
    Biosynthesis of (1 -> 3)-beta-D-glucan (callose) by detergent extracts of a microsomal fraction from Arabidopsis thaliana2001In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 268, no 17, p. 4628-4638Article in journal (Refereed)
    Abstract [en]

    The aim of this work was to develop a biochemical approach to study (1-->3)-beta -D-glucan (callose) biosynthesis using suspension cultures of Arabidopsis thaliana. Optimal conditions for in vitro synthesis of callose corresponded to an assay mixture containing 50 mM Mops buffer, pH6.8, 1 mM UDP-glucose, 8 mM Ca2+ and 20 mM cellobiose. The enzyme was Ca2+-dependent, and addition of Mg2+ to the reaction mixture did not favour cellulose biosynthesis. Enzyme kinetics suggested the existence of positive. homotropic cooperativity of (1-->3)-beta -D-glucan synthase for the substrate UDP-glucose, in agreement with the hypothesis that callose synthase consists of a multimeric complex containing several catalytic subunits. Detergents belonging to different families were tested for their ability to extract and preserve membrane-bound (1-->3)-beta -D-glucan synthase activity. Cryo-transmission electron microscopy experiments showed that n-octyl-beta -D-glucopyranoside allowed the production of micelle-like structures, whereas vesicles were obtained with Chaps and Zwittergent 3-12. The morphology and size of the (1-->3)-beta -D-glucans synthesized in vitro by fractions obtained with different detergents were affected by the nature of the detergent tested. These data suggest that the general organization of the glucan synthase complexes and the properties of them in vitro products are influenced by the detergent used for protein extraction. The reaction products synthesized by different detergent extracts were characterized by infrared spectroscopy, methylation analysis, C-13-N-MR spectroscopy, electron microscopy and X-ray diffraction. These products were identified as linear (1-->3)-beta -D-glucans having a degree of polymerization higher than 100, a microfibrillar structure, and a low degree of crystallinity.

  • 4.
    Kalbin, Georgi
    et al.
    Göteborgs universitet.
    Ohlsson, Anna Birgitta
    KTH, Superseded Departments (pre-2005), Biochemistry and Biotechnology.
    Berglund, Torkel
    KTH, Superseded Departments (pre-2005), Biochemistry and Biotechnology.
    Rydström, Jan
    Göteborgs universitet.
    Strid, Åke
    Göteborgs universitet.
    Ultraviolet-B-radiation-induced changes in nicotinamide and glutathione metabolism and gene expression in plants1997In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 249, no 2, p. 465-472Article in journal (Refereed)
    Abstract [en]

    Pea (Pisum sativum L. cv. Greenfeast) plants were exposed to supplementary ultraviolet-B (UV-B) radiation (biologically effective dose rates normalised to 300 nm UV-B-BE,B-300: 0.18, 0.32 or 1.4 W m(-2)). Leaf nicotinamide, trigonelline, GSH(tot) (total glutathione) and (GSSG (oxidised glutathione) levels remained unchanged after exposure to the lowest dose rates. 1.4 W m(-2) UV-B-BE,B-300 gave rise to 60-fold and 4.5-fold increases in GSSG and GSH(tot), respectively. 3.5-fold and 9.5-fold increases were found in nicotinamide and trigonelline, respectively. cab (Chlorophyll-a/b-binding protein) transcript levels decreased and CHS (chalcone synthase) and PAL (phenylalanine ammonia-lyase) mRNA increased after shorter UV-B exposures (hours) to the higher dose rate of UV-B and after exposure to the intermediate dose rate. CHS and PAL mRNAs also increased after prolonged exposure to the lowest dose rate. cab transcripts completely disappeared. whereas CHS and PAL mRNA levels rose by 60-fold and 17-fold, respectively, after 12 h exposure at the highest dose rate and 12 h of development. Our results indicate that nicotinamide or trigonelline do not function as signalling compounds for CHS and PAL gene expression. Elevated nicotinamide and trigonelline levels occur in response to UV-B, but only at UV-B doses high enough to cause oxidative stress.

  • 5. Nilsson, J
    et al.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics.
    Williams, Y
    Pettersson, L
    Uhlén, M
    Nygren, P A
    Competitive elution of protein A fusion proteins allows specific recovery under mild conditions.1994In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 224, no 1, p. 103-8Article in journal (Refereed)
    Abstract [en]

    A novel system is described for mild elution of fusion proteins by competitive elution. The approach is based on displacement of immobilized fusions containing a monovalent IgG-binding staphylococcal protein A fragment (Z) from an IgG-affinity matrix by a divalent fragment fused to a serum-albumin-binding region derived from streptococcal protein G. Using real-time interaction analysis, the binding (K(aff)) to polyclonal human IgG was found to be 3.3 (+/- 0.4) x 10(8) M-1 for divalent ZZ and 2.0 (+/- 0.1) x 10(7) M-1 for monovalent Z. This more than tenfold difference in binding strength ensures a high efficiency in the elution step. The competitor protein can specifically be removed and recovered from the elution mixture by subsequent passage through a human serum albumin(HSA)-affinity column, leaving only the target fusion protein in the flow-through fraction. Here, we show that a recombinant Klenow fragment of DNA polymerase I expressed in Escherichia coli can be recovered with high yield, and retained activity, from a crude bacterial lysate by IgG-affinity chromatography using mild conditions during both binding and elution.

  • 6. Nord, K.
    et al.
    Nord, O.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Kelley, B.
    Ljungqvist, C.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Recombinant human factor VIII-specific affinity ligands selected from phage-displayed combinatorial libraries of protein A2001In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 268, no 15, p. 4269-4277Article in journal (Refereed)
    Abstract [en]

    Factor VIII-specific affibodies were selected from phage displayed libraries constructed by combinatorial mutagenesis of an alpha helical bacterial receptor domain derived from staphylococcal protein A. Bead-immobilized recombinant human factor VIII (rVIII) (80 and 90 kDa chains) protein was used during competitive biopannings in the presence of free 80-kDa chain protein, resulting in the selection of several binders that showed dissociation constants (K-d) in the range 100-200 nm as determined by biosensor analyses. One variant (Z(rVIII:3), 90-kDa chain specific) was further characterized in small-scale affinity chromatography experiments, and showed efficient and selective recovery of biologically active rVIII from Chinese hamster ovary cell supernatant-derived feed stocks. The purity of the enriched rVIII was comparable with rVIII material purified by immunoaffinity chromatography using a 90-kDa chain-specific monoclonal antibody. Interestingly, epitope mapping showed that the monoclonal antibody and the affibody ligand competed for the same or at least overlapping epitopes on rVIII. In addition, the Z(rVIII:3) variant was produced by peptide synthesis with a C-terminal cysteine to enable directed coupling to solid supports. This 59-residue protein was analyzed by circular dichroism and showed a secondary structure content similar to that of the parental Z domain used as scaffold. In biosensor studies. the synthetic affibody was immobilized recruiting the C-terminal cysteine residue, and demonstrated to bind both recombinantly produced and plasma-derived factor VIII, From a secondary library, constructed by re-randomization of relevant positions identified after alignment of the first-generation variants, a panel of affinity-improved second-generation affibodies were selected of which one clone showed a dissociation constant (K-d) for rVIII of 5 nM. Several of these variants also showed higher apparent binding efficiencies towards rVIII when analyzed as immobilized ligands in biosensor experiments. Taken together, the results suggest that affibody ligands produced by bacterial or synthetic routes could be of interest as an alternative to monoclonal antibodies in purification processes or as diagnostic or monitoring tools.

  • 7. Ronnmark, J.
    et al.
    Gronlund, H.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Human immunoglobulin A (IgA)-specific ligands from combinatorial engineering of protein A2002In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 269, no 11, p. 2647-2655Article in journal (Refereed)
    Abstract [en]

    Affinity reagents capable of selective recognition of the different human immunoglobulin isotypes are important detection and purification tools in biotechnology. Here we describe the development and characterization of affinity proteins (affibodies) showing selective binding to human IgA. From protein libraries constructed by combinatorial mutagenesis of a 58-amino-acid, three-helix bundle domain derived from the IgG-binding staphylococcal protein A, variants showing IgA binding were selected by using phage display technology and IgA monoclonal antibodies (myeloma) as target molecules. Characterization of selected clones by biosensor technology showed that five out of eight investigated affibody variants were capable of IgA binding, with dissociation constants (K (d) ) in the range between 0.5 and 3 mum. One variant (Z(IgA1) ) showing the strongest binding affinity was further analyzed, and showed that human IgA subclasses (IgA(1) and IgA(2) ) as well as secretory IgA were recognized with similar efficiencies. No detectable cross-reactivity towards human IgG, IgM, IgD or IgE was observed. The potential use of the Z(IgA1) affibody as a ligand in affinity chromatography applications was first demonstrated by selective recovery of IgA protein from a spiked Escherichia coli total cell lysate, using an affinity column containing a divalent head-to-tail Z(IgA1) affibody dimer construct as a ligand. In addition, efficient affinity recovery of IgA from unconditioned human plasma was also demonstrated.

1 - 7 of 7
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