Change search
Refine search result
1 - 10 of 10
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Rows per page
  • 5
  • 10
  • 20
  • 50
  • 100
  • 250
Sort
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
Select
The maximal number of hits you can export is 250. When you want to export more records please use the Create feeds function.
  • 1.
    Kronqvist, Nina
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Staphylococcal surface display in directed evolution2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Engineered affinity proteins have together with naturally derived antibodies becomeindispensable tools in many areas of life-science and with the increasing number ofapplications, the need for high-throughput methods for generation of such different affinityproteins is evident. Today, combinatorial protein engineering is the most successful strategy toisolate novel non-immunoglobulin affinity proteins. In this approach, generally termed directedevolution, high-complexity combinatorial libraries are created from which affinity proteins areisolated using an appropriate selection method, thus circumventing the need for detailedknowledge of the protein structure or the binding mechanism, often necessary in more rationalapproaches. Since the introduction of the phage display technology that pioneered the field ofcombinatorial engineering, several alternative selection systems have been developed for thispurpose.This thesis describes the development of a novel selection system based onstaphylococcal surface display and its implementation in directed evolution approaches. In thefirst study, the transformation efficiency to the gram-positive bacteria Staphylococcus carnosus wassuccessfully improved around 10,000-fold to a level that would allow cell surface display ofcomplex combinatorial protein libraries. In two separate studies, the staphylococcal displaysystem was investigated for the applicability in both de novo selection and affinity maturation ofaffibody molecules. First, using a pre-selection strategy with one round of phage display, ahigh-complexity affibody library was displayed on staphylococcal cells. Using fluorescenceactivatedcell sorting, binders with sub-nanomolar affinity to tumor necrosis factor-alpha(TNF-α) were isolated. Second, a combined approach using phage display for de novo selectionof first-generation affibody binders and staphylococcal display in a subsequent affinitymaturation selection was applied to generate binders with low nanomolar affinity to the humanepidermal growth factor receptor-3 (ErbB3). Moreover, in an additional study, thestaphylococcal surface display system was improved by the introduction of a protease 3Ccleavage sequence in the displayed fusion products in order to facilitate straightforwardproduction of soluble proteins for further downstream characterization.Altogether, the presented studies demonstrate that the staphylococcal selection systemindeed is a powerful tool for selection and characterization of novel affinity proteins and couldbecome an attractive alternative to existing selection techniques.

  • 2.
    Kronqvist, Nina
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Severa, Denise
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Wernérus, Henrik
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Simplified characterization through site-specific protease-mediated release of affinity proteins selected by staphylococcal display2008In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 278, no 1, p. 128-136Article in journal (Refereed)
    Abstract [en]

    The production of candidate affinity proteins in a soluble form, for downstream characterization, is often a time-consuming step in combinatorial protein engineering methods. Here, a novel approach for efficient production of candidate clones is described based on direct cleavage of the affinity protein from the surface of Staphylococcus carnosus, followed by affinity purification. To find a suitable strategy, three new fusion protein constructs were created, introducing a protease site for specific cleavage and purification tags for affinity chromatography purifications into the staphylococcal display vector. The three modified strains were evaluated in terms of transformation frequency, surface expression level and protease cleavage efficiency. A protocol for efficient affinity purification of protease-released affinity proteins using the introduced fusion-tags was successfully used, and the functionality of protease-treated and purified proteins was verified in a biosensor assay. To evaluate the devised method, a previously selected HER2-specific affibody was produced applying the new principle and was used to analyze HER2 expression on human breast cancer cells.

  • 3.
    Kronqvist, Nina
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Wernérus, Henrik
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Jonsson, Andreas
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    A novel affinity protein selection system based on staphylococcal cell surface display and flow cytometry2008In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 21, no 4, p. 247-255Article in journal (Refereed)
    Abstract [en]

    Here we describe the first reported use of a Gram-positive bacterial system for the selection of affinity proteins from large combinatorial libraries displayed on the surface of Staphylococcus carnosus. An affibody library of 3 x 109 variants, based on a 58 residue domain from staphylococcal protein A, was pre-enriched for binding to human tumor necrosis factor-alpha (TNF-alpha) using one cycle of phage display and thereafter transferred to the staphylococcal host (106 variants). The staphylococcal-displayed library was subjected to three rounds of flow-cytometric sorting, and the selected clones were screened and ranked by on-cell analysis for binding to TNF-alpha and further characterized using biosensor analysis and circular dichroism spectroscopy. The successful sorting yielded three different high-affinity binders (ranging from 95 pM to 2.2 nM) and constitutes the first selection of a novel affinity protein using Gram-positive bacterial display. The method combines the simplicity of working with a bacterial host with the advantages of displaying recombinant proteins on robust Gram-positive bacteria as well as using powerful flow cytometry in the selection and characterization process.

  • 4.
    Kronqvist, Nina
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Malm, Magdalena
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Göstring, Lovisa
    Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
    Gunneriusson, Elin
    Nilsson, Martin
    Affibody AB, Stockholm, Sweden.
    Höidén-Guthenberg, Ingmarie
    Affibody AB, Stockholm, Sweden.
    Gedda, Lars
    Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Uppsala Sweden.
    Frejd, Fredrik Y.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Löfblom, John
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Combining phage and staphylococcal surface display for generation of ErbB3-specific Affibody molecules2011In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 24, no 4, p. 385-396Article in journal (Refereed)
    Abstract [en]

    Emerging evidence suggests that the catalytically inactive ErbB3 (HER3) protein plays a fundamental role in normal tyrosine kinase receptor signaling as well as in aberrant functioning of these signaling pathways, resulting in several forms of human cancers. ErbB3 has recently also been implicated in resistance to ErbB2-targeting therapies. Here we report the generation of high-affinity ErbB3-specific Affibody molecules intended for future molecular imaging and biotherapeutic applications. Using a high-complexity phage-displayed Affibody library, a number of ErbB3 binders were isolated and specific cell-binding activity was demonstrated in immunofluorescence microscopic studies. Subsequently, a second-generation library was constructed based on sequences of the candidates from the phage display selection. By exploiting the sensitive affinity discrimination capacity of a novel bacterial surface display technology, the affinity of candidate Affibody molecules was further increased down to subnanomolar affinity. In summary, the demonstrated specific targeting of native ErbB3 receptor on human cancer cell lines as well as competition with the heregulin/ErbB3 interaction indicates that these novel biological agents may become useful tools for diagnostic and therapeutic targeting of ErbB3-expressing cancers. Our studies also highlight the powerful approach of combining the advantages of different display technologies for generation of functional high-affinity protein-based binders. Potential future applications, such as radionuclide-based diagnosis and treatment of human cancers are discussed.

  • 5.
    Kronqvist, Nina
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Malm, Magdalena
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Rockberg, Johan
    KTH, School of Biotechnology (BIO), Proteomics.
    Hjelm, Barbara
    KTH, School of Biotechnology (BIO), Proteomics.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Staphylococcal surface display in combinatorial protein engineering and epitope mapping of antibodies2010In: Recent Patents on Biotechnology, ISSN 1872-2083, Vol. 4, no 3, p. 171-182Article in journal (Refereed)
    Abstract [en]

    The field of combinatorial protein engineering for generation of new affinity proteins started in the mid 80s by the development of phage display. Although phage display is a prime example of a simple yet highly efficient method, manifested by still being the standard technique 25 years later, new alternative technologies are available today. One of the more successful new display technologies is cell display. Here we review the field of cell display for directed evolution purposes, with focus on a recently developed method employing Gram-positive staphylococci as display host. Patents on the most commonly used cell display systems and on different modifications as well as specific applications of these systems are also included. General strategies for selection of new affinity proteins from cell-displayed libraries are discussed, with detailed examples mainly from studies on the staphylococcal display system. In addition, strategies for characterization of recombinant proteins on the staphylococcal cell surface, with an emphasis on an approach for epitope mapping of antibodies, are included.

  • 6.
    Kronqvist, Nina
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Rockberg, Johan
    KTH, School of Biotechnology (BIO), Proteomics.
    Hjelm, Barbara
    KTH, School of Biotechnology (BIO), Proteomics.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    New Ways for Discovery of Biopharmaceuticals: Emerging Techniques using Surface Display on Gram-positive Bacteria for Combinatorial Protein Engineering and Characterization2009In: Bioforum Europe, ISSN 1611-597X, Vol. 13, no 6-7, p. 022-Article in journal (Refereed)
  • 7.
    Löfblom, John
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Kronqvist, Nina
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Wernérus, Henrik
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Optimization of electroporation-mediated transformation: Staphylococcus carnosus as model organism2007In: Journal of Applied Microbiology, ISSN 1364-5072, E-ISSN 1365-2672, Vol. 102, no 3, p. 736-747Article in journal (Refereed)
    Abstract [en]

    The study was conducted with an aim to optimize the transformation efficiency of the Gram-positive bacterium Staphylococcus carnosus to a level that would enable the creation of cell surface displayed combinatorial protein libraries.

    Methods and Results: We have thoroughly investigated a number of different parameters for: (i) the preparation of electrocompetent cells; (ii) the treatment of cells before electroporation; (iii) the electroporation step itself; and (iv) improved recovery of transformed cells. Furthermore, a method for heat-induced inactivation of the host cell restriction system was devised to allow efficient transformation of the staphylococci with DNA prepared from other species, such as Escherichia coli. Previously described protocols for S. carnosus, giving transformation frequencies of approximately 10(2) transformants per transformation could be improved to reproducible procedures giving around 10(6) transformants for a single electroporation event, using plasmid DNA prepared from either S. carnosus or E. coli. The transformed staphylococcal cells were analysed using flow cytometry to verify that the entire cell population retained the introduced plasmid DNA and expressed the recombinant protein in a functional form on the cell surface at the same level as the positive control population.

    Conclusions: The results demonstrate that the transformation frequency for S. carnosus could be dramatically increased through optimization of the entire electroporation process, and that the restriction barrier for interspecies DNA transfer, could be inactivated by heat treatment of the cells prior to electroporation.

    Significance and Impact of the Study: The generation of large combinatorial protein libraries, displayed on the surface of S. carnosus can be envisioned in the near future, thus dramatically improving the selection compared with the traditional biopanning procedure used in phage display.

  • 8.
    Malm, Magdalena
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Kronqvist, Nina
    KTH, School of Biotechnology (BIO), Protein Technology.
    Lindberg, Hanna
    KTH, School of Biotechnology (BIO), Protein Technology.
    Gudmundsdotter, Lindvi
    Affibody AB, Stockholm, Sweden.
    Bass, Tarek
    KTH, School of Biotechnology (BIO), Protein Technology.
    Frejd, Fredrik Y.
    Höidén-Guthenberg, Ingmarie
    Affibody AB, Stockholm, Sweden.
    Varasteh, Zohreh
    Departmaent of Medical Chemistry, Preclinical PET Platform, Uppasala University, Uppsala, Sweden.
    Orlova, Anna
    Departmaent of Medical Chemistry, Preclinical PET Platform, Uppasala University, Uppsala, Sweden.
    Tolmachev, Vladimir
    Unit of Biomedical Radiation Sciences, Uppsala University, Uppsala, Sweden.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Inhibiting HER3-Mediated Tumor Cell Growth with Affibody Molecules Engineered to Low Picomolar Affinity by Position-Directed Error-Prone PCR-Like Diversification2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 5, p. e62791-Article in journal (Refereed)
    Abstract [en]

    The HER3 receptor is implicated in the progression of various cancers as well as in resistance to several currently used drugs, and is hence a potential target for development of new therapies. We have previously generated Affibody molecules that inhibit heregulin-induced signaling of the HER3 pathways. The aim of this study was to improve the affinity of the binders to hopefully increase receptor inhibition efficacy and enable a high receptor-mediated uptake in tumors. We explored a novel strategy for affinity maturation of Affibody molecules that is based on alanine scanning followed by design of library diversification to mimic the result from an error-prone PCR reaction, but with full control over mutated positions and thus less biases. Using bacterial surface display and flow-cytometric sorting of the maturation library, the affinity for HER3 was improved more than 30-fold down to 21 PM. The affinity is among the higher that has been reported for Affibody molecules and we believe that the maturation strategy should be generally applicable for improvement of affinity proteins. The new binders also demonstrated an improved thermal stability as well as complete refolding after denaturation. Moreover, inhibition of ligand-induced proliferation of HER3-positive breast cancer cells was improved more than two orders of magnitude compared to the previously best-performing clone. Radiolabeled Affibody molecules showed specific targeting of a number of HER3-positive cell lines in vitro as well as targeting of HER3 in in vivo mouse models and represent promising candidates for future development of targeted therapies and diagnostics.

  • 9. Orlova, A.
    et al.
    Malm, Malin
    KTH, School of Biotechnology (BIO), Protein Technology.
    Lindberg, Hanna
    KTH, School of Biotechnology (BIO), Protein Technology.
    Varasteh, Z.
    Selvaraju, R. K.
    Kronqvist, Nina
    KTH, School of Biotechnology (BIO), Protein Technology.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Tolmachev, V.
    Feasibility of radionuclide imaging of HER3-expressing tumours using technetium-99m labeled affibody molecules2013In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 40, p. S185-S186Article in journal (Other academic)
  • 10. Orlova, Anna
    et al.
    Malm, Magdalena
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Lindberg, Hanna
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Varasteh, Zohreh
    Rosestedt, Maria
    Tolmachev, Vadimir
    Kronqvist, Nina
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Löfblom, John
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Feasibility of radionuclide imaging of HER3-expressing tumors using affibody molecules2013In: Journal of labelled compounds & radiopharmaceuticals, ISSN 0362-4803, E-ISSN 1099-1344, Vol. 56, p. S11-S11Article in journal (Other academic)
1 - 10 of 10
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf