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  • 1. Andersson, C.
    et al.
    Hansson, M.
    Power, U.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Mammalian cell production of a respiratory syncytial virus (RSV) candidate vaccine recovered using a product-specific affinity column2001In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 34, p. 25-32Article in journal (Refereed)
    Abstract [en]

    The recombinant production of a respiratory syncytial virus (RSV) candidate vaccine BBG2Na in baby hamster kidney cells (BHK-21 cells) was investigated. BBG2Na consists of a serum-albumin-binding region (BB) fused to a 101-amino-acid fragment of the RSV G-protein. Semliki Forest virus-based expression vectors encoding both intracellular and secreted forms of BBG2Na were constructed and found to be functional. Affinity recovery of BBG2Na employing human serum albumin columns was found to be inefficient due to the abundance of BSA in the applied samples. Instead, a strategy using a tailor-made affinity ligand based on a combinatorially engineered Staphylococcus aureus protein A domain, showing specific binding to the G-protein part of the product, was evaluated. In conclusion, a strategy for production and successful recovery of BBG2Na in mammalian cells was created, through the development of a product-specific affinity column.

  • 2.
    Eriksson, Cecilia
    et al.
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Schwenk, Jochen M.
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Sjöberg, Anna
    KTH, School of Biotechnology (BIO).
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Affibody molecule-mediated depletion of HSA and IgG using different buffer compositions: a 15 min protocol for parallel processing of 1-48 samples2010In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 56, p. 49-57Article in journal (Refereed)
    Abstract [en]

    High-abundant plasma proteins pose a challenge in a large number of proteomics-based technologies. Depletion of these high-abundant proteins has proven to be a fruitful strategy to circumvent masking of lower-abundant proteins that could serve as valuable biomarker candidates. However, current strategies often do not meet the throughput requirements of large-scale proteomic studies. In the present paper, a flexible and parallelized method for the depletion of high-abundant proteins is described, allowing the removal of the two most abundant proteins from 48 blood-derived samples in less than 15 min using Affibody molecules as affinity ligands. A sample-processing platform like this should be suitable for a number of proteomics technologies; its flexibility in buffer composition allows for different types of downstream applications.

  • 3.
    Falk, Ronny
    et al.
    KTH, Superseded Departments, Biotechnology.
    Agaton, Charlotta
    KTH, Superseded Departments, Biotechnology.
    Kiesler, E.
    Jin, S.
    Wieslander, L.
    Visa, N.
    Hober, Sophia
    KTH, Superseded Departments, Biotechnology.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    An improved dual-expression concept, generating high-quality antibodies for proteomics research2003In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 38, p. 231-239Article in journal (Refereed)
    Abstract [en]

    A novel, improved dual bacterial-expression system, designed for large-scale generation of high-quality polyclonal antibody preparations intended for proteomics research, is presented. The concept involves parallel expression of cDNA-encoded proteins, as a fusion with two different tags in two separate vector systems. Both systems enable convenient blotting procedures for expression screening on crude bacterial cell cultures and single-step affinity purification under denaturing conditions. One of the fusion proteins is used to elicit antibodies, and the second fusion protein is used in an immobilized form as an affinity ligand to enrich antibodies with selective reactivity to the cDNA-encoded part, common for the two fusion proteins. To evaluate the system, four cDNA clones from putative nuclear proteins from the non-biting midge Chironomus tentans were expressed. Antibodies to these cDNA-encoded proteins were generated, enriched and used in blotting and immunofluorescence procedures to determine expression patterns for the native proteins corresponding to the cDNAs. The four antibody preparations showed specific reactivity to the corresponding recombinant cDNA-encoded proteins, and three of the four antibodies gave specific staining in Western-blot analysis of nuclear cell extracts. Furthermore, two of the antibody preparations gave specific staining in immunofluorescence analysis of C. tentans cells. We conclude that the dual-vector concept presented offers a highly stringent strategy for the generation of monospecific polyclonal antibodies, which are useful in proteomics research.

  • 4.
    Falk, Ronny
    et al.
    KTH, Superseded Departments, Biotechnology.
    Gräslund, Susanne
    KTH, Superseded Departments, Biotechnology.
    Brundell, Eva
    Höög, Christer
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    A high-stringency proteomics concept aimed for generation of antibodies specific for cDNAencoded proteins2002In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 35, no 2, p. 75-82Article in journal (Refereed)
    Abstract [en]

    A novel dual bacterial expression system, designed for high-throughput generation of antibodies specific for cDNA-encoded proteins, is presented. The concept involves parallel expression of cDNA-encoded proteins, in two vector systems, as fusions with two different tags, both enabling single-step affinity purification under denaturing conditions. One of the fusion tags includes a portion with documented immunopotentiating effect to stimulate antibody production, and the generated fusion proteins are used to elicit antibodies. The second fusion protein is used in an immobilized form as an affinity ligand to enrich, from the generated antisera, antibodies with selective reactivity to the cDNA-encoded part. To evaluate the system, five cDNA clones from a mouse testis cDNA library were expressed, and antibodies to these cDNA-encoded proteins were generated, enriched and used in blotting procedures to determine expression patterns for the native proteins corresponding to the cDNAs. The five antibody preparations showed specific reactivity to the corresponding recombinant cDNA-encoded proteins, and three of the five antibodies gave specific staining in Western-blot screening of various cell types and tissue homogenates. When the same five cDNAs were processed and analysed using a single-vector method, antibodies with a more non-specific staining were generated. We thus conclude that the presented dual-vector method offers a highly stringent strategy for generation of monospecific polyclonal antibodies.

  • 5.
    Friedman, Mikaela
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Lindström, Sara
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Engineering and characterization of a bispecific HER2 × EGFR-binding affibody molecule2009In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 54, p. 121-131Article in journal (Refereed)
    Abstract [en]

    HER2 (human epidermal-growth-factor receptor-2; ErbB2) and EGFR (epidermal-growth-factor receptor) are overexpressed in various forms of cancer, and the co-expression of both HER2 and EGFR has been reported in a number of studies. The simultaneous targeting of HER2 and EGFR has been discussed as a strategy with which to potentially increase efficiency and selectivity in molecular imaging and therapy of certain cancers. In an effort to generate a molecule capable of bispecifically targeting HER2 and EGFR, a gene fragment encoding a bivalent HER2-binding affibody molecule was genetically fused in-frame with a bivalent EGFR-binding affibody molecule via a (G(4)S)(3) [(Gly(4)-Ser)(3)]-encoding gene fragment. The encoded 30 kDa affibody construct (Z(HER2))(2)-(G(4)S)(3)-(Z(EGFR))(2), with potential for bs (bispecific) binding to HER2 and EGFR, was expressed in Escherichia coli and characterized in terms of its binding capabilities. The retained ability to bind HER2 and EGFR separately was demonstrated using both biosensor technology and flow-cytometric analysis, the latter using HER2- and EGFR-overexpressing cells. Furthermore, simultaneous binding to HER2 and EGFR was demonstrated in: (i) a sandwich format employing real-time biospecific interaction analysis where the bs affibody molecule bound immobilized EGFR and soluble HER2; (ii) immunofluorescence microscopy, where the bs affibody molecule bound EGFR-overexpressing cells and soluble HER2; and (iii) a cell-cell interaction analysis where the bs affibody molecule bound HER2-overexpressing SKBR-3 cells and EGFR-overexpressing A-431 cells. This is, to our knowledge, the first reported bs affinity protein with potential ability for the simultaneous targeting of HER2 and EGFR. The potential future use of this and similar constructs, capable of bs targeting of receptors to increase the efficacy and selectivity in imaging and therapy, is discussed.

  • 6.
    Friedman, Mikaela
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Engineered affinity proteins for tumour-targeting applications2009In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 53, p. 1-29Article, review/survey (Refereed)
    Abstract [en]

    Targeting of tumour-associated antigens is an expanding treatment modality in clinical oncology as an alternative to, or in combination with, conventional treatments, such as chemotherapy, external-radiation therapy and surgery. Targeting of antigens that are unique or more highly expressed in tumours than in normal tissues can be used to increase the specificity and reduce the cytotoxic effect on normal tissues. Several targeting agents have been studied for clinical use, where monoclonal antibodies have been the ones most widely used. More than 20 monoclonal antibodies are approved for therapy today and the largest field is oncology. Advances in genetic engineering and in vitro selection technology has enabled the feasible high-throughput generation of monoclonal antibodies, antibody derivatives [e.g. scFvs, Fab molecules, dAbs (single-domain antibodies), diabodies and minibodies] and more recently also non-immunoglobulin scaffold proteins. Several of these affinity proteins have been investigated for both in vivo diagnostics and therapy. Affinity proteins in tumour-targeted therapy can affect tumour progression by altering signal transduction or by delivering a payload of toxin, drug or radionuclide. The ErbB receptor family has been extensively studied as biomarkers in tumour targeting, primarily for therapy using monoclonal antibodies. Two receptors in the ErbB family, EGFR (epidermal growth factor receptor) and HER2 (epidermal growth factor receptor 2), are over-expressed in various malignancies and associated with poor patient prognosis and are therefore interesting targets for solid turnours. In the present review, strategies are described for tumour targeting of solid turnours using affinity proteins to deliver radionuclides, either for molecular imaging or radiotherapy. Antibodies, antibody derivatives and non-immunoglobulin scaffold proteins are discussed with a certain focus on the affibody (Affibody (R)) molecule.

  • 7.
    Grönwall, Caroline
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Snelders, Eveline
    Department of Oncology and Pathology, Cancer Center Karolinska (CCK), Karolinska Hospital.
    Jarelöv Palm, Anna
    Eriksson, Fredrik
    Department of Oncology and Pathology, Cancer Center Karolinska (CCK), Karolinska Hospital.
    Herne, Nina
    Affibody AB, Bromma.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Generation of Affibody (R) ligands binding interieukin-2 receptor alpha/CD252008In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 50, no 2, p. 97-112Article in journal (Refereed)
    Abstract [en]

    Affibody (R) molecules specific for human IL-2R alpha, the IL-2 (interieukin-2) receptor a subunit, also known as CD25, were selected by phage-display technology from a combinatorial protein library based on the 58-residue Protein A-derived Z domain. The IL-2R system plays a major role in T-cell activation and the regulation of cellular immune responses. Moreover, CD25 has been found to be overexpressed in organ rejections, a number of autoimmune diseases and T-cell malignancies. The phage-display selection using Fc-fused target protein generated 16 unique Affibody (R) molecules targeting CD25. The two most promising binders were characterized in more detail using biosensor analysis and demonstrated strong and selective binding to CD25. Kinetic biosensor analysis revealed that the two monomeric Affibody (R) molecules bound to CD25 with apparent affinities of 130 and 240 nM respectively. The Affibody (R) molecules were, on biosensor analysis, found to compete for the same binding site as the natural ligand IL-2 and the IL-2 blocking monoclonal antibody 2A3. Hence the Affibody (R) molecules were assumed to have an overlapping binding site with IL-2 and antibodies targeting the IL-2 blocking Tac epitope (for example, the monoclonal antibodies Daclizumab and Basiliximab, both of which have been approved for therapeutic use). Furthermore, immunofluorescence microscopy and flow-cytometric analysis of CD25-expressing cells demonstrated that the selected Affibody (R) molecules bound to CD4(+) CD25(+) PMBCs (peripheral-blood mononuclear cells), the IL-2-dependent cell line NK92 and phytohaemagglutinin-activated PMBCs. The potential use of the CD25-binding Affibody (R) molecules as targeting agents for medical imaging and for therapeutic applications is discussed.

  • 8. Hansson, M.
    et al.
    Nygren, Per-Åke
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Design and production of recombinant subunit vaccines2000In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 32, p. 95-107Article, review/survey (Refereed)
    Abstract [en]

    The development of subunit vaccines is presently the main strategy being evaluated for prevention of infectious diseases. The use of recombinant-DNA techniques has facilitated the development of new principles for design and production of subunit vaccines. First of all, the properties of a target protein immunogen can be improved by the use of gene-fusion technology or by the creation of specific changes, to generate 'second-generation protein vaccines', Properties that can be modified include protein solubility, protein stability, in vivo half-lives, etc. In addition, for subunit protein vaccine candidates, the immunogenic properties can be significantly augmented by the addition of immunopotentiating tags or by means of targeting to immunoreactive sites. The recombinant subunit vaccine can furthermore be adapted by gene-fusion technology, to be efficiently incorporated into immunopotentiating adjuvant systems. Also in passive vaccination strategies, i.e. the use of antibodies or antibody fragments for prevention of infectious diseases, the recombinant strategies have become increasingly important. Humanized antibodies and antibody fusion. proteins represent common present anti-infectious-disease agents. The selected examples will indicate that recombinant strategies will indeed have an impact on the design, selection and production of recombinant proteins to be used in the prevention of infectious diseases.

  • 9. Jonasson, P.
    et al.
    Liljeqvist, S.
    Nygren, Per-Åke
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Genetic design for facilitated production and recovery of recombinant proteins in Escherichia coli2002In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 35, p. 91-105Article, review/survey (Refereed)
    Abstract [en]

    Genetic strategies have been used for more than two decades to improve bacterial bioprocesses and to simplify recovery procedures. Such strategies include the design of efficient expression vectors and the improvement of bacterial production strains in different ways, e.g. by deletion of protease genes or engineering for overexpression of rare-codon tRNAs, foldases or chaperones. Gene multimerization is another such principle that has proved beneficial to improve production yields. Genetic strategies have furthermore been exploited to facilitate recovery processes by adapting the product for a particular purification principle. In this area, affinity fusions have been commonly used, but other principles, such as modified isoelectric point (pI) or hydrophobic properties have also been successfully investigated. A recent drastic step forward in the use of gene technology to improve recovery processes for recombinant proteins is the introduction of combinatorial protein engineering to generate tailor-made product-specific affinity ligands. This strategy, which allows efficient recovery of a recombinant protein in its native form, is likely to be increasingly used also in industrial-scale bioprocesses, since novel protein ligands have been described that can be sanitized using common industrial cleaning-in-p lace procedures. The examples presented in this review make it evident that genetic strategies will be of utmost importance in the future for facilitating production and recovery of recombinant proteins.

  • 10.
    Jonsson, Andreas
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Wållberg, Helena
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Herne, N.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Frejd, F. Y.
    Generation of tumour-necrosis-factor-alpha-specific affibody molecules capable of blocking receptor binding in vitro2009In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 54, p. 93-103Article in journal (Refereed)
    Abstract [en]

    Affibody molecules specific for human TNF-alpha (tumour necrosis factor-alpha) were selected by phage-display technology from a library based on the 58-residue Protein A-derived Z domain. TNF-alpha is a proinflammatory cytokine involved in several inflammatory diseases and, to this day, four TNF-alpha-blocking protein pharmaceuticals have been approved for clinical use. The phage selection generated 18 unique cysteine-free affibody sequences of which 12 were chosen, after sequence cluster analysis, for characterization as proteins. Biosensor binding studies of the 12 Escherichia coli-produced and IMAC (immobilized-metal-ion affinity chromatography)-purified affibody molecules revealed three variants that demonstrated the strongest binding to human TNF-alpha. These three affibody molecules were subjected to kinetic binding analysis and also tested for their binding to mouse, rat and pig TNF-alpha. For Z(TNF alpha:185), subnanomolar affinity (K-D = 0.1-0.5 nM) for human TNF-alpha was demonstrated, as well as significant binding to TNF-alpha from the other species. Furthermore, the binding site was found to overlap with the binding site for the TNF-alpha receptor, since this interaction could be efficiently blocked by the Z(TNF-alpha:185) affibody. When investigating six dimeric affibody constructs with different linker lengths, and one trimeric construct, it was found that the inhibition of the TNF-alpha binding to its receptor could be further improved by using dinners with extended linkers and/or a trimeric affibody construct. The potential implication of the results for the future design of affibody-based reagents for the diagnosis of inflammation is discussed.

  • 11.
    Larsson, Gen
    KTH, School of Biotechnology (BIO).
    Evaluation of inducible promoters on the secretion of a ZZ-proinsulin fusion protein in Escherichia coli2003In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 38Article in journal (Refereed)
  • 12.
    Larsson, Gen
    et al.
    KTH, School of Biotechnology (BIO).
    Boström, Maria
    KTH, School of Biotechnology (BIO).
    Evaluation of inducible promoters on the secretion of a zz-proinsulin fusion protein in Escherichia coli2003In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 38, p. 87-93Article in journal (Refereed)
  • 13.
    Li, Jingjing
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Vernet, Erik
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Larsson, Barbro
    Höidén-Guthenberg, Ingmarie
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Selection of affibody molecules to the ligand-binding site of the insulin-like growth factor-1 receptor2010In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 55, p. 99-109Article in journal (Refereed)
    Abstract [en]

    Affibody molecules binding to the site of hormone interaction in IGF-IR (insulin-like growth factor-I receptor) were successfully selected by phage-display technology employing a competitive-elution strategy during biopanning, whereby release of receptor-bound phagemids was accomplished by competition with IGFI (insulin-like growth factor-I). In non-competitive selections, the elution of receptor-bound phagemids was performed by imidazole or low-pH incubation, which also resulted in the isolation of affibody molecules that could bind to the receptor. An ELISA-based assay showed that the affibody molecules generated by IGF-I competition during elution, in addition to affibody molecules generated in the noncompetitive selections, could compete with IGF-I for binding to the receptor. The affinities of the isolated variants to IGF-IR-overexpressing MCF-7 cells were determined and ranged from high nanomolar to 2.3 nM. The most promising variant, Z(4;40), was shown to recognize IGF- IR efficiently in several different contexts: in analyses based on flow cytometry, fluorescence microscopy and receptor pull-down from cell extracts. In addition, when Z, was added to the medium of MCF-7 cells that were dependent on IGF-I for efficient growth, it was found to have a dose-dependent growth-inhibitory effect on the cells. Applications of affibody-based reagents for quantitative and qualitative analyses of IGF- I R status, as well as applications of affibody-based reagents for therapy, are discussed.

  • 14.
    Lundberg, Emma
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Selection and characterization of Affibody (R) ligands to the transcription factor c-Jun2009In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 52, p. 17-27Article in journal (Refereed)
    Abstract [en]

    c-Jun is a highly oncogenic transcription factor involved in the development of different types of cancer. In the present study we have generated c-Jun-binding-affinity proteins from a phage-displayed library of so-called 'Affibody (R) ligands', developed by combinatorial engineering of a non-immunoglobulin-based scaffold protein. Homodimeric c-Jun protein was recombinantly produced in Escherichia coli and, prior to selection, the quality of the target protein was investigated by binding analyses, which indicated specific binding to a double-stranded DNA hairpin construct containing a c-Jun response element, but not to a control sequence. Isolated Affibody (R) variants from the phage selection were expressed in E. coli, purified by affinity chromatography and their interaction with c-Jun was analysed. In biosensor analyses, one Affibody (R) ligand, denoted Z(cJun518) was shown to interact with immobilized c-Jun protein with an apparent dissociation constant of 5 mu M. By constructing a head-to-tail homodimeric version of Z(cJun518), its apparent affinity for c-Jun could be increased threefold, suggesting co-operativity effects in the binding to the immobilized c-Jun protein. Further characterization of the Z(cJun518) Affibody (R) molecule demonstrated, in both affinity-capture and Western-blotting experiments, its ability to interact selectively with c-Jun, even when the c-Jun target was present in a complex protein background consisting of a bacterial cell lysate. Z(cJun518) could also be used to stain the c-Jun-overexpressing cell line C8161 visualized by confocal fluorescence microscopy. Results from competition experiments indicated that the binding epitope on c-Jun for the Z(cJun518) Affibody (R) molecule was separate from the binding sites of both a polyclonal antibody raised against the unstructured N-terminal domain and a double-stranded DNA hairpin containing a c-Jun response element. The potential intracellular use of Affibody (R) ligands directed against transcription factors and other oncogenic factors is discussed.

  • 15.
    Löfdahl, Per-Åke
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Affinity maturation of a TNF-α binding affibodymolecule by Darwinian survival selection2010In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 55, p. 111-120Article in journal (Refereed)
    Abstract [en]

    The introduction of different methodologies for construction and screening ofcomplex protein libraries has provided powerful means in protein engineeringfor development of molecules with desired traits. A challenge faced in manysituations is to adapt a given methodology for efficient and rapid identification ofthe most interesting variants present in a library. In the present study, theconcept of Darwinian selection based on a growth advantage for clones havingthe desired trait has been investigated. Using a β-lactamase-based ProteinFragment Complementation Assay (PCA), an affinity maturation of a TNF-αbinding affibody molecule of an initial 2 nM affinity for the target has beenperformed. Initial characterization of the PCA system, based on the affinitydriven reconstitution of β-lactamase activity in the periplasm of cells harbouringa library member showing affinity for a co-expressed target protein, showed thatthe system was responsive to promoter induction level, interaction affinity andapplied selection pressure. Using combinatorial protein engineering principles, a107 library of second generation affibody molecules was constructed andsubjected to selection of improved variants by library growth in liquid culture.The results showed that after a pre-selection step on semi-solid media toeliminate non-binding variants, present in majority, two rounds of selection inliquid culture resulted in an enrichment for binders showing up ten-fold higheraffinity to the TNF-α target than the ancestral variant. Biosensor analysesshowed that the major factor for the improved affinity could be attributed toreduced off-rate constants.

  • 16.
    Nilvebrant, Johan
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Kuku, Gamze
    Björkelund, Hanna
    Nestor, Marika
    Selection and in vitro characterization of human CD44v6-binding antibody fragments2012In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 59, no 5, p. 367-380Article in journal (Refereed)
    Abstract [en]

    The cluster of differentiation (CD) 44v6 antigen has been suggested to be involved in tumor formation, invasion, and metastasis formation, and has been observed in a majority of primary and metastatic squamous cell carcinomas of the head and neck. Probes specifically binding to this region may be utilized as tools for the challenging tasks of early detection and targeted treatments of small residual disease. In this project, an epitope-guided phage display selection of human fragment antigen-binding (Fab) fragments with affinity to the v6 sequence was performed. A selected set of Fab fragments was shown to specifically recognize increasingly complex forms of the target sequence, both in the form of a short synthetic or recombinant peptide and in the context of a purified extracellular domain of CD44. The binding was independent of known v6-sequence variation and posttranslational modifications that are common in the CD44 protein family. Furthermore, real-time interaction measurements on antibody fragments labeled with 125I showed specific and high-affinity binding to the antigen present on cultured head and neck squamous cell carcinoma cells. There was no cross-reactivity toward cells that lack the target protein. As hypothesized, characterization of the interaction between Fab fragments and the targets using the mathematical tool Interaction Map revealed more heterogeneous interactions on cells than with pure proteins analyzed by surface plasmon resonance. One main candidate Fab fragment with optimal affinity for all forms of the target sequence was identified. The flexible recombinant source of the Fab fragments might aid the development of tailored molecules adapted for therapeutic or diagnostic applications in the future.

  • 17. Nordstrom, T.
    et al.
    Ronaghi, M.
    Forsberg, L.
    de Faire, U.
    Morgenstern, R.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Direct analysis of single-nucleotide polymorphism on double-stranded DNA by pyrosequencing2000In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 31, p. 107-112Article in journal (Refereed)
    Abstract [en]

    Pyrosequencing, a new method for DNA sequencing, is gaining widespread use for many different types of DNA analysis. The method takes advantage of four coupled enzymes in a single tube assay to monitor DNA synthesis in real time using a luminometric detection system. Here, we demonstrate the use of pyrosequencing for direct analysis of single-nucleotide polymorphism on double-stranded PCR product. Pyrosequencing data on the human glutathione peroxidase gene (GPXI) from several individuals were analysed and three different allelic variants were determined and confirmed. The possibility of further simplifying the sequencing and template-preparation steps is discussed.

  • 18.
    Ramström, Margareta
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Zuberovic, Aida
    Analytical Chemistry, Department of Physical and Analytical Chemistry, Uppsala University.
    Grönwall, Caroline
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Hanrieder, Jörg
    Analytical Chemistry, Department of Physical and Analytical Chemistry, Uppsala University.
    Bergquist, Jonas
    Analytical Chemistry, Department of Physical and Analytical Chemistry, Uppsala University.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Development of affinity columns for the removal of high-abundance proteins in cerebrospinal fluid2009In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 52, no 2, p. 159-166Article in journal (Refereed)
    Abstract [en]

    Various approaches for removal of high-abundance components in body fluids are currently available. While most methods are constructed for plasma depletion, there is a need for body-fluid-specific strategies. The aim of the present study was to design an affinity matrix suitable for the depletion of high-abundance proteins in CSIF (cerebrospinal fluid). Hence, molecules with specific affinity towards proteins present at high concentration in CSIF were desired. Affibody(R) molecules are specific binders of small size that have shown high stability under various conditions and are therefore good candidates for such a matrix. The protein composition in CSF resembles that in plasma. However, 20 % of the proteins are brain-derived and are therefore present in higher proportions in CSF than in plasma, whereas larger plasma-derived proteins are less abundant in CSF. Therefore five high-abundance CSIF proteins were chosen for the design of a CSF-specific depletion setup. Affibody(R) molecules with specificity towards HSA (human serum albumin), IgG, transferrin and transthyretin were combined in an affinity column. In addition, polyclonal antibodies against cystatin C were coupled to chromatographic beads and packed in a separate column. Highly reproducible and efficient removal of the five target proteins was observed. The proportion of depleted proteins were estimated to be 99, 95, 74, 92 and 83 % for HSA, IgG, transferrin, transthyretin and cystatin C respectively. SDS/PAGE analysis was used for monitoring and identifying proteins in native CSF, depleted CSIF samples and the captured fractions. Moreover, shotgun proteomics was used for protein identification in native as well as depleted: CSIF and the achieved data were compared. Enhanced identification of lower abundance components was observed in the depleted fraction, in terms of more detected peptides per protein.

  • 19.
    Wernérus, Henrik
    et al.
    KTH, Superseded Departments, Biotechnology.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Biotechnological applications for surface-engineered bacteria2004In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 40, p. 209-228Article, review/survey (Refereed)
    Abstract [en]

    Display of heterologous proteins on the surface of micro-organisms, enabled by means of recombinant DNA technology, has become an increasingly popular strategy in microbiology, biotechnology and vaccinology. Both Gram-negative and Gram-positive bacteria have been investigated for potential applications. The present review will describe the most commonly used systems for bacterial display, with a focus on the biotechnology applications. Live bacterial vaccine-delivery vehicles have long been investigated through the surface display of foreign antigens and, recently, 'second-generation' vaccine-delivery vehicles have been generated by the addition of mucosal targeting signals, as a means to increase immune responses. Engineered bacteria have also the potential to act as novel microbial biocatalysts with heterologous enzymes immobilized as surface exposed on the bacterial cell surface. They provide the potential for new types of whole-cell diagnostic devices, since single-chain antibodies and other type of tailor-made binding proteins can be displayed on bacteria. Bacteria with increased binding capacity for certain metal ions can be created, and potential environmental or biosensor applications for such recombinant bacteria as biosorbents are being explored. Certain bacteria have also been employed to display various polypeptide libraries for use as devices in in vitro selection applications. Part of the present review has been devoted to a more in-depth description of a promising Gram-positive display system, i.e. Staphylococcus carnosus, and its applications. The review describes the basic principles of the different bacterial display systems and discusses current uses and possible future trends of these emerging technologies.

  • 20.
    Wikman, Maria
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Friedman, Mikaela
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Pinitkiatisakul, S.
    Hemphill, A.
    Lövgren-Bengtsson, K.
    Lunden, A.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Applying biotin-streptavidin binding for iscom (immunostimulating complex) association of recombinant immunogens2005In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 41, p. 163-174Article in journal (Refereed)
    Abstract [en]

    We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic peptide or lipid tags to improve their capacity to be incorporated into an adjuvant formulation. In the present study, we have explored the strong interaction between biotin and SA (streptavidin) (K-D approximate to 10(-15) M) to couple recombinant immunogens to iscoms (immunostimulating complexes). Two different concepts were evaluated. In the first concept, a His(6)-tagged SA fusion protein (His(6)-SA) was bound to Ni2+-loaded iscom matrix (iscom without associated protein), and biotinylated immunogens were thereafter associated with the SA-coated iscoms. The immunogens were either biotinylated in vivo on E. coli expression or double biotinylated in vivo and in vitro. In the second concept, the recombinant immunogens were expressed as SA fusion proteins, which were directly bound to a biotinylated iscom matrix. A 53-amino-acid malaria peptide (M), derived from the central repeat region of the Plasmodium faiciparum blood-stage antigen Pf155/RESA, and a 232-amino-acid segment (SRS2') from the central region (from Pro-97 to Lys-328) of the major surface antigen NcSRS2 of the protozoan parasite Neospora caninum, served as model immunogens in the present study. All fusion proteins generated were found to be efficiently expressed and could be recovered to high purity using affinity chromatography. The association between the different immunogen-containing fusion proteins and the corresponding iscom matrix was demonstrated by analytical ultracentrifugation in a sucrose density gradient. However, some fusion proteins were, to a certain extent, also found to associate unspecifically with a regular iscom matrix. Furthermore, selected iscom fractions were demonstrated to induce high-titre antigen-specific antibody responses on immunization of mice. For the particular target immunogen SRS2', the induced antibodies demonstrated reactivity to the native antigen NcSRS2. We believe that the presented concepts offer convenient methods to achieve efficient adjuvant association of recombinant immunogens, and the advantages and disadvantages of the two concepts are discussed.

  • 21. Wikman, Maria
    et al.
    Rowcliffe, Eric
    Friedman, Mikaela
    Henning, Petra
    Lindholm, Leif
    Olofsson, Sigvard
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Selection and characterization of an HIV-1 gp120-binding affibody ligand2006In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 45, p. 93-105Article in journal (Refereed)
    Abstract [en]

    To evaluate the possibility of generating novel proteins binding to highly glycosylated viral proteins, affibody ligands were selected by bactericiphage display technology to the HIV-1 envelope glycoprotein gp120 (glycoprotein 120), from a combinatorial protein library based on the 58-amino-acid-residue staphylococcal Protein A domain. The predominant variant from the bacteriophage selection was produced in Escherichia coli and characterized by biosenscir analyses. Both univalent and bivalent affibody molecules were shown to bind selectively to the gp120 target molecule in a biosensor analysis. The dissociation equilibrium constants (K.) were determined to be approx. 100 nM for the univalent affibody and 10 nM for the bivalent affibody, confirming the stronger gp120 binding of the bivalent affibody ligand. The affibody constructs were further introduced into the AdS (adenovirus type 5) fibre gene, and the recombinant fibres were shown to bind selectively to gp 120 in a biosensor analysis and to gp160 transiently expressed in African-green-monkey (Cercopithecus aethiops) kidney cells. Neither the affibody ligand nor the AdS fibres showed any virus neutralization activity, suggesting that the affibody bound to a non-neutralizing site on gp 120. To investigate the binding site for the affibody ligand on gp120, CD4 (cluster of differentiation 4) and a panel of mAbs (monoclonal antibodies) known to bind to gp 120 were allowed to compete with the affibody ligand in a biosensor study. Two mAbs, 670-30D and 697-30D, were found to compete with gp120 for overlapping binding sites. Although neutralization effects were not achieved in this initial investigation, the successful selection of a gp120-binding affibody ligand indicates that future affibody-based strategies might evolve to complement antibody-based efforts for HIV-I therapy. Strategies for directed selection of affibody ligands binding to neutralizing epitopes and the potential of using adenovirus for gene-therapy-mediated efforts are discussed.

  • 22.
    Wållberg, Helena
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Design and evaluation of radiolabeled tracers for tumor imaging2013In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 60, no 4, p. 365-383Article in journal (Refereed)
    Abstract [en]

    The growing understanding of tumor biology and the identification of tumor-specific genetic and molecular alterations, such as the overexpression of membrane receptors and other proteins, allows for personalization of patient management using targeted therapies. However, this puts stringent demands on the diagnostic tools used to identify patients who are likely to respond to a particular treatment. Radionuclide molecular imaging is a promising noninvasive method to visualize and characterize the expression of such targets. A number of different proteins, from full-length antibodies and their derivatives to small scaffold proteins and peptide receptor-ligands, have been applied to molecular imaging, each demonstrating strengths and weaknesses. Here, we discuss the concept of molecular targeting and, in particular, molecular imaging of cancer-associated targets. Additionally, we describe important biotechnological considerations and desired features when designing and developing tracers for radionuclide molecular imaging.

1 - 22 of 22
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