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  • 1.
    Anfelt, Josefine
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Kaczmarzyk, Danuta
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Shabestary, Kiyan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Renberg, Björn
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Rockberg, Johan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Nielsen, Jens
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. Tech Univ Denmark.
    Hudson, Elton P.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Genetic and nutrient modulation of acetyl-CoA levels in Synechocystis for n-butanol production2015In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 14, article id 167Article in journal (Refereed)
    Abstract [en]

    Background: There is a strong interest in using photosynthetic cyanobacteria as production hosts for biofuels and chemicals. Recent work has shown the benefit of pathway engineering, enzyme tolerance, and co-factor usage for improving yields of fermentation products. Results: An n-butanol pathway was inserted into a Synechocystis mutant deficient in polyhydroxybutyrate synthesis. We found that nitrogen starvation increased specific butanol productivity up to threefold, but cessation of cell growth limited total n-butanol titers. Metabolite profiling showed that acetyl-CoA increased twofold during nitrogen starvation. Introduction of a phosphoketolase increased acetyl-CoA levels sixfold at nitrogen replete conditions and increased butanol titers from 22 to 37 mg/L at day 8. Flux balance analysis of photoautotrophic metabolism showed that a Calvin-Benson-Bassham-Phosphoketolase pathway had higher theoretical butanol productivity than CBB-Embden-Meyerhof-Parnas and a reduced butanol ATP demand. Conclusion: These results demonstrate that phosphoketolase overexpression and modulation of nitrogen levels are two attractive routes toward increased production of acetyl-CoA derived products in cyanobacteria and could be implemented with complementary metabolic engineering strategies.

  • 2.
    Engfeldt, Torun
    et al.
    KTH, School of Biotechnology (BIO).
    Renberg, Björn
    KTH, School of Biotechnology (BIO).
    Brumer, Harry
    KTH, School of Biotechnology (BIO).
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO).
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO).
    Chemical Synthesis of Triple-Labelled Three-Helix Bundle Binding Proteins for Specific Fluorescent Detection of Unlabelled Protein2005In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 6, no 6, p. 1043-1050Article in journal (Refereed)
    Abstract [en]

    Site-specifically triple-labelled three-helix bundle affinity proteins (affibody molecules) have been produced by total chemical Synthesis. The 58 aa affinity proteins were assembled on an automated peptide synthesizer, followed by manual on-resin incorporation of three different reporter groups. An orthogonal protection strategy was developed for the site-specific introduction of 5-(2-aminethylamino)-1-nophthalenesulfonic acid (EDANS) and 6(7-nitrobenzofurazon-4-yiamino)-hexanoic acid (NBDX), constituting a donor/acceptor pair for fluorescence resonance energy transfer (FRET), and a biotin moiety, used for surface immobilization. Circular dichroism and biosensor studies of the synthetic proteins and their recombinant counterparts revealed that the synthetic proteins were folded and retained their binding specificities. The biotin-conjugated protein could be immobilized onto a streptavidin surface without loss of activity. The synthetic, doubly fluorescent-labelled affinity proteins were shown to function as fluorescent biosensors in an assay for the specific detection of unlabelled human IgG and IgA.

  • 3.
    Renberg, Björn
    KTH, School of Biotechnology (BIO).
    Fluorescence-based ligand assays for protein detection using affibody affinity proteins2006Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The detection and quantification of biomolecules, and proteins in particular, are of great interest since these molecules are of fundamental importance to our well-being. Body fluids, as for instance human blood, are well suited for sampling of protein levels. However, the complexity of the fluids and the low abundance of many of the interesting biomolecules makes detection and quantification difficult. This has spurred an interest into the development of many protein detection methods, and of these, ligand assays have proven particularly suitable. In this thesis, different types of ligand assays for protein detection have been developed using affibody molecules as ligands.

    In a first study, a homogeneous competitive detection assay was investigated, based on antiidiotypic affibody molecule pairs and fluorescence resonance energy transfer (FRET) as reporting system. The individual members of two anti-idiotypic affibody pairs, each consisting of a target binding (idiotypic) and an anti-idiotypic affibody ligand, were labeled with a donor fluorophore and an acceptor fluorophore, respectively. Incubation with the two target proteins IgA and Taq DNA polymerase resulted in a concentration dependent decrease in the FRET signal, allowing for target protein detection and quantification. For Taq DNA polymerase, detection in 25% human plasma was also possible in the same concentration span as in buffer.

    In a second study, a homogeneous, non-competitive detection system was described. Affibody molecules of 58 amino acids directed against IgA and IgG were produced with chemical synthesis, and two fluorophores capable of FRET were site-specifically introduced. Binding of target protein induced a concentration-dependent change in the relative emission of the two fluorophores, which formed the basis for the detection system.

    In two studies, affibody molecules were evaluated and shown to function well as capture ligands on microarrays. Synthetic affibody molecules directed against Taq DNA polymerase and IgA were modified by the introduction of immobilization tags. Specific immobilization via a C-terminal cysteine or a biotin moiety, or random immobilization via amino groups, were studied in protein microarray experiments and SPR-based biosensor studies. The experiments showed that all immobilization chemistries resulted in functional capture molecules. A short spacer was also introduced, situated between the affibody and the cysteine and biotin moieties, which was shown to improve binding for all constructs. Multidomain affibody constructs of up to four N- to C-terminally linked domains were shown to increase the amount of bound target, compared to monomeric affibody ligands. Six dimeric affibody constructs directed against IgA, IgG, IgE, Taq DNA polymerase, TNF-α and insulin, respectively, showed low limits of detections for their targets and little or no cross-reactivity with the other target proteins. Dimeric affibody molecules directed against IgA and TNF-α were also shown to function in a sandwich format with antibodies for detection of targets in buffer and in human serum and plasma. Successful discrimination between normal and IgA-deficient sera showed that affibody molecules could be used for specific detection of protein in highly complex backgrounds on microarrays.

  • 4.
    Renberg, Björn
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Hedhammar, My
    Mimicking silk spinning in a microchip2014In: Sensors and actuators. B, Chemical, ISSN 0925-4005, E-ISSN 1873-3077, Vol. 195, p. 404-408Article in journal (Refereed)
    Abstract [en]

    Nature's high performance material, spider silk, is formed during the passage of a protein solution through a spinning duct. Herein we present a microfluidic device with dual laminar mobile phases where silk formation can be mimicked and investigated. Recombinant miniature spidroins, with or without the pH-switching N-terminal domain, were used to investigate spinning conditions into silk-like fibers using this setup.

  • 5.
    Renberg, Björn
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Nordin, Jon
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Merca, Anna
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Feldwisch, Joachim
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Affibody molecules in protein capture microarrays: Evaluation of multidomain ligands and different detection formats2007In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 6, no 1, p. 171-179Article in journal (Refereed)
    Abstract [en]

    The importance of the ligand presentation format for the production of protein capture microarrays was evaluated using different Affibody molecules, produced either as single 6 kDa monomers or genetically linked head-to-tail multimers containing up to four domains. The performances in terms of selectivity and sensitivity of the monomeric and the multidomain Affibody molecules were compared by immobilization of the ligands on microarray slides, followed by incubation with fluorescent-labeled target protein. An increase in signal intensities for the multimers was demonstrated, with the most pronounced difference observed between monomers and dimers. A protein microarray containing six different dimeric Affibody ligands with specificity for IgA, IgE, IgG, TNF-alpha, insulin, or Taq DNA polymerase was characterized for direct detection of fluorescent-labeled analytes. No cross-reactivity was observed and the limits of detection were 600 fM for IgA, 20 pM for IgE, 70 fM for IgG, 20 pM for TNF-alpha, 60 pM for insulin, and 10 pM for Taq DNA polymerase. Also, different sandwich formats for detection of unlabeled protein were evaluated and used for selective detection of IgA or TNF-alpha in human serum or plasma samples, respectively. Finally, the presence of IgA was determined using detection of directly Cy5-labeled normal or IgA-deficient serum samples.

  • 6.
    Renberg, Björn
    et al.
    KTH, Superseded Departments, Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Eklund, Malin
    KTH, Superseded Departments, Biotechnology.
    Eriksson Karlström, Amelie
    KTH, Superseded Departments, Biotechnology.
    Fluorescence resonance energy transfer-based detection of analytes using antiidiotypic affinity protein pairs2004In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 334, no 1, p. 72-80Article in journal (Refereed)
    Abstract [en]

    A new method for specific detection of proteins based on fluorescence resonance energy transfer (FRET) using affinity proteins (affibodies) derived from combinatorial engineering of Staphylococcal protein A has been developed. Antiidiotypic affibody pairs were used in a homogeneous competitive binding assay, where the idiotypic, target-specific affibody was labeled with fluorescein and the antiidiotypic affibody was labeled with tetramethylrhodamine. Intermolecular FRET between the two fluorescent probes was observed in the antiidiotypic affibody complex, but upon addition of target protein the antiidiotypic affibody was displaced, which was monitored by a shift in the relative emission of the donor and acceptor fluorophores. The feasibility of the system was demonstrated by the detection of IgA and Taq DNA polymerase with high specificity, using two different antiidiotypic affibody pairs. Detection of Taq DNA polymerase in 25% human plasma was successfully carried out, demonstrating that the method can be used for analysis of proteins in samples of complex composition.

  • 7.
    Renberg, Björn
    et al.
    KTH, School of Biotechnology (BIO).
    Shiroyama, Ikue
    KTH, School of Biotechnology (BIO).
    Engfeldt, Torun
    KTH, School of Biotechnology (BIO).
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO).
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO).
    Affibody protein capture microarrays: synthesis and evaluation of random and directed immobilization of affibody molecules2005In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 341, no 2, p. 334-343Article in journal (Refereed)
    Abstract [en]

    Affibody molecules, 58-amino acid three-helix bundle proteins directed to different targets by combinatorial engineering of staphylococcal protein A, were used as capture ligands on protein microarrays. An evaluation of slide types and immobilization strategies was performed to find suitable conditions for microarray production. Two affibody molecules, ZTaq and ZIgA, binding Taq DNA polymerase and human IgA, respectively, were synthesized by solid phase peptide synthesis using an orthogonal protection scheme, allowing incorporation of selective immobilization handles. The resulting affibody variants were used for random surface immobilization (through amino groups) or oriented surface immobilization (through cysteine or biotin coupled to the side chain of Lys58). Evaluation of the immobilization techniques was carried out using both a real-time surface plasmon resonance biosensor system and a microarray system using fluorescent detection of Cy3-labeled target protein. The results from the biosensor analyses showed that directed immobilization strategies significantly improved the specific binding activity of affibody molecules. However, in the microarray system, random immobilization onto carboxymethyl dextran slides and oriented immobilization onto thiol dextran slides resulted in equally good signal intensities, whereas biotin-mediated immobilization onto streptavidin-coated slides produced slides with lower signal intensities and higher background staining. For the best slides, the limit of detection was 3 pM for IgA and 30 pM for Taq DNA polymerase.

  • 8. Sato, Kae
    et al.
    Tachihara, Atsuki
    Renberg, Björn
    KTH, School of Biotechnology (BIO), Proteomics.
    Mawatari, Kazuma
    Sato, Kiichi
    Tanaka, Yuki
    Jarvius, Jonas
    Nilsson, Mats
    Kitamori, Takehiko
    Microbead-based rolling circle amplification in a microchip for sensitive DNA detection2010In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 10, no 10, p. 1262-1266Article in journal (Refereed)
    Abstract [en]

    The sensitive detection and quantification of DNA targets in the food industry and in environmental and clinical settings are issues of utmost importance in ensuring contamination-free food, monitoring the environment, and battling disease. Selective probes coupled with powerful amplification techniques are therefore of major interest. In this study, we set out to create an integrated microchemical chip that benefits from microfluidic chip technology in terms of sensitivity and a strong detection methodology provided jointly by padlock probes and rolling circle amplification (RCA). Here, we have integrated padlock probes and RCA into a microchip. The chip uses solid phase capture in a microchannel to enable washing cycles and decrease analytical area, and employs on-bead RCA for single-molecule amplification and detection. We investigated the effects of reagent concentration and amount of padlock probes, and demonstrated the feasibility of detecting Salmonella.

  • 9. Shirai, K.
    et al.
    Renberg, Björn
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Sato, K.
    Mawatari, K.
    Kitamori, T.
    Graft linker immobilization for spatial control of antibody immobilization inside fused microchips2009In: Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Chemical and Biological Microsystems Society , 2009, p. 1566-1568Conference paper (Refereed)
    Abstract [en]

    A new method for antibody immobilization to the inside of fused silica microchips coated with a protein repelling polymer (2-methacryloyloxyethyl phosphorylcholine polymer, MPC polymer) is described. Antibody patterning is achieved by using a new photoreactive linker which is composed of three-parts: a carboxyl group (-COOH), a hydrophilic PEG spacer and a photoreactive benzophenone. The linker is grafted with UV which introduces free carboxyl groups to the microchannel surface. Antibodies are immobilized to these areas, by using amide bonding between protein -NH2's and linker's -COOH. Patterned anti-BSA antibodies retained functionality and were capable of antigen specific capture.

  • 10. Shirai, Kentaro
    et al.
    Renberg, Björn
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Sato, Kae
    Mawatari, Kazuma
    Konno, Tomohiro
    Ishihara, Kazuhiko
    Kitamori, Takehiko
    Graft linker immobilization for spatial control of protein immobilization inside fused microchips2009In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 30, no 24, p. 4251-4255Article in journal (Refereed)
    Abstract [en]

    Fused silica glass microchips have several attractive features for lab-on-a-chip applications; they can be machined with excellent precision down to nanospace; are stable; transparent and can be modified with a range of silanization agents to change channel surface properties. For immobilization, however, ligands must be added after bonding, since the harsh bonding conditions using heat or hydrofluoric acid would remove all prior immobilized ligands. For spatial control over immobilization, UV-mediated immobilization offers several advantages; spots can be created in parallel, the feature size can be made small, and spatial control over patterns and positions is excellent. However, UV sensitive groups are often based on hydrophobic chemical moieties, which unfortunately result in greater non-specific binding of biomolecules, especially proteins. Here, we present techniques in which any -CHx (x = 1,2,3) containing surface coating can be used as foundation for grafting a hydrophilic linker with a chemical anchor, a carboxyl group, to which proteins and amine containing molecules can be covalently coupled. Hence, the attractive features of many well-known protein and biomolecule repelling polymer coatings can be utilized while achieving site-specific immobilization only to pre-determined areas within the bonded microchips.

  • 11. Xi, H.
    et al.
    Renberg, Björn
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Sato, K.
    Mawatari, K.
    Nilsson, M.
    Kitamori, T.
    Towards single molecule detection in minute volumes - UV-grafted dna probes with padlock probe detection and RCA amplification2009In: Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Chemical and Biological Microsystems Society , 2009, p. 1554-1556Conference paper (Refereed)
    Abstract [en]

    The technique of Immobilizing DNA in extended nano-space (10-1000 nm) was developed. This technique can be used for rolling circle amplification (RCA) and will finally make single DNA detection and nano-immunoassay possible. Here, we combine UV-mediated immobilization to DNA, examine two different methods for immobilization, and couple to padlock probe and RCA. Detection of fluorescence labeled DNA hybridization to RCA demonstrated that DNA analysis in minute volumes succeeded. Our technique can be served to detect single DNA, and we are developing this technique in extended nano-sapce to define the mechnisms of DNA amplification by record RCA in real time.

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