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  • 1. Andersson, C.
    et al.
    Sandberg, L.
    Wernérus, Henrik
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Johansson, M.
    Lovgren-Bengtsson, K.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Improved systems for hydrophobic tagging of recombinant immunogens for efficient iscom incorporation2000Inngår i: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 238, nr 02-jan, s. 181-193Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We have previously reported a strategy for production in Escherichia coli of recombinant immunogens fused to a hydrophobic tag to improve their capacity to associate with an adjuvant formulation [Andersson et al., J. Immunol. Methods 222 (1999) 171]. Here, we describe a further development of the previous strategy and present significant improvements. In the novel system, the target immunogen is produced with an N-terminal affinity tag suitable for affinity purification, and a C-terminal hydrophobic tag, which should enable association through hydrophobic interactions of the immunogen with an adjuvant system, here being immunostimulating complexes (iscoms). Two different hydrophobic tags were evaluated: (i) a tag denoted M, derived from the membrane-spanning region of Staphylococcus aureus protein A (SpA), and (ii) a tag denoted MI consisting of the transmembrane region of hemagglutinin from influenza A virus. Furthermore, two alternative affinity tags were evaluated; the serum albumin-binding protein ABP, derived from streptococcal protein G, and the divalent IgG-binding ZZ-domains derived from SpA. A malaria peptide M5, derived from the central repeat region of the Plasmodium falciparum blood-stage antigen Pf155/RESA, served as model immunogen in this study. Four different fusion proteins, ABP-MS-M, ABP-MS-MI, ZZ-MS-M and ZZ-MS-MI, were thus produced, affinity purified and evaluated in iscom-incorporation experiments. All of the fusion proteins were found in the iscom fractions in analytical ultracentrifugation, indicating iscom incorporation. This was further supported by electron microscopy analysis showing that iscoms were formed. In addition, these iscom preparations were demonstrated to induce MS-specific antibody responses upon immunisation of mice, confirming the successful incorporation into iscoms. The novel system for hydrophobic tagging of immunogens, with optional affinity and hydrophobic tags, gave expression levels that were increased ten to fifty-fold, as compared to the earlier reported system. We believe that the presented strategy would be a convenient way to achieve efficient adjuvant association for recombinant immunogens.

  • 2.
    Berglund, Lisa
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Björling, Erik
    KTH, Skolan för bioteknologi (BIO).
    Gry, Marcus
    KTH, Skolan för bioteknologi (BIO).
    Asplund, Anna
    Uppsala Univ, Rudbeck laboratory.
    Al-Khalili Szigyarto, Cristina
    KTH, Skolan för bioteknologi (BIO).
    Persson, Anja
    KTH, Skolan för bioteknologi (BIO).
    Ottoson, Jenny
    KTH, Skolan för bioteknologi (BIO).
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO).
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO).
    Sivertsson, Åsa
    KTH, Skolan för bioteknologi (BIO).
    Wester, Kenneth
    Uppsala Univ, Rudbeck laboratory.
    Kampf, Caroline
    Uppsala Univ, Rudbeck laboratory.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO).
    Pontén, Fredrik
    Uppsala Univ, Rudbeck laboratory.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO).
    Generation of validated antibodies towards the human proteomeArtikkel i tidsskrift (Annet vitenskapelig)
    Abstract [en]

    Here we show the results from a large effort to generate antibodies towards the human proteome. A high-throughput strategy was developed based on cloning and expression of antigens as recombitant protein epitope signature tags (PrESTs) Affinity purified polyclonal antibodies were generated, followed by validation by protein microarrays, Western blotting and microarray-based immunohistochemistry. PrESTs were selected based on sequence uniqueness relative the proteome and a bioinformatics analysis showed that unique antigens can be found for at least 85% of the proteome using this general strategy. The success rate from antigen selection to validated antibodies was 31%, and from protein to antibody 55%. Interestingly, membrane-bound and soluble proteins performed equally and PrEST lengths between 75 and 125 amino acids were found to give the highest yield of validated antibodies. Multiple antigens were selected for many genes and the results suggest that specific antibodies can be systematically generated to most human proteibs.

  • 3.
    Berglund, Lisa
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Björling, Erik
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Oksvold, Per
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Fagerberg, Linn
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Al-Khalili Szigyarto, Cristina
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Persson, Anja
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ottosson, Jenny
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Lundberg, Emma
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Sivertsson, Åsa
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    et al.,
    A genecentric human protein atlas for expression profiles based on antibodies2008Inngår i: Molecular & Cellular Proteomics, ISSN 1535-9476, Vol. 7, nr 10, s. 2019-2027Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    An attractive path forward in proteomics is to experimentally annotate the human protein complement of the genome in a genecentric manner. Using antibodies, it might be possible to design protein-specific probes for a representative protein from every protein-coding gene and to subsequently use the antibodies for systematical analysis of cellular distribution and subcellular localization of proteins in normal and disease tissues. A new version (4.0) of the Human Protein Atlas has been developed in a genecentric manner with the inclusion of all human genes and splice variants predicted from genome efforts together with a visualization of each protein with characteristics such as predicted membrane regions, signal peptide, and protein domains and new plots showing the uniqueness (sequence similarity) of every fraction of each protein toward all other human proteins. The new version is based on tissue profiles generated from 6120 antibodies with more than five million immunohistochemistry-based images covering 5067 human genes, corresponding to similar to 25% of the human genome. Version 4.0 includes a putative list of members in various protein classes, both functional classes, such as kinases, transcription factors, G-protein-coupled receptors, etc., and project-related classes, such as candidate genes for cancer or cardiovascular diseases. The exact antigen sequence for the internally generated antibodies has also been released together with a visualization of the application-specific validation performed for each antibody, including a protein array assay, Western blot analysis, immunohistochemistry, and, for a large fraction, immunofluorescence-based confocal microscopy. New search functionalities have been added to allow complex queries regarding protein expression profiles, protein classes, and chromosome location. The new version of the protein atlas thus is a resource for many areas of biomedical research, including protein science and biomarker discovery.

  • 4.
    Falk, Ronny
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Ramström, Margareta
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Eriksson, Cecilia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Targeted protein pullout from human tissue samples using competitive elution2011Inngår i: Biotechnology Journal, ISSN 1860-6768, Vol. 6, nr 1, s. 28-37Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    One commonly used strategy to gain information on the proteins in a cell is to isolate the proteins of interest by specific binders, often antibodies. Not only the specificity of the capturing antibodies but also the washing and elution conditions are crucial to avoid false-positive protein identifications. Eluting the target protein from the matrix, while avoiding the release of unrelated background proteins, should both provide more correct information on the target protein and its interaction partners, and minimize the effort to perform downstream analyses through the reduced number of eluted proteins. In this study, a novel approach for selective protein pullout is presented. Monospecific antibodies were used to selectively pullout target proteins from a complex biosample. Subsequently, the target proteins were competitively eluted from the affinity media with the recombinant antigen. To deplete the antigen from the eluted sample, I MAC spin columns were utilized to bind the N-terminal His-tag of the antigens. The competitive elution method was applied both to a model system, and for the extraction of a native human target protein. In the model system the recombinant target protein BBC7 was spiked into a protein extract of human liver, whereas an endogenously expressed target protein, cTAGE5, was extracted from the liver extract directly. SDS-PAGE analysis and mass spectrometry confirmed affinity isolation of expected target proteins. More selective elution was obtained using the competitive procedure as compared to elution at low pH. Competitive elution has thus been shown to offer an effective approach for wide-scale pullout experiments where proteins and their interaction partners are to be studied.

  • 5.
    Kronqvist, Nina
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Severa, Denise
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Simplified characterization through site-specific protease-mediated release of affinity proteins selected by staphylococcal display2008Inngår i: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 278, nr 1, s. 128-136Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 6.
    Kronqvist, Nina
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Jonsson, Andreas
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    A novel affinity protein selection system based on staphylococcal cell surface display and flow cytometry2008Inngår i: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 21, nr 4, s. 247-255Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 7.
    Larsson, Karin
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hofström, Camilla
    KTH, Skolan för bioteknologi (BIO).
    Lindskog, C
    Uhlén, Mattias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Gräslund, Torbjörn
    KTH, Skolan för bioteknologi (BIO).
    Hansson, M
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO).
    Angelidou, Pia
    KTH, Skolan för bioteknologi (BIO).
    Hökfelt, T
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO).
    Novel antigen design for the generation of G-protein-coupled receptorantibodies.Manuskript (preprint) (Annet vitenskapelig)
  • 8.
    Larsson, Karin
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO).
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO).
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO).
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO).
    Multiplexed PrEST immunization for high-throughput affinity proteomics2006Inngår i: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 315, s. 110-120Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Monospecific antibodies dfdfdfdf (msAbs) generated through antigen specific purification of polyclonal antisera are valuable tools in proteome analyses. However, proteome wide generation of msAbs would require extensive immunization programs. Therefore, it would be desirable to develop efficient immunization and purification methods to reduce the number of animals needed for such antibody-based research. Here we describe a multiplex immunization strategy for generation of msAbs towards recombinantly produced human protein fragments, denoted PrESTs. Antisera from rabbits immunized with a mixture of two, three, five and up to ten different PrESTs have been purified by a two-step immunoaffinity-based protocol and the efficiency of the purification method was analyzed using a two-color protein array concept. The obtained results showed that almost 80% of the animals immunized with antigens composed of two or three different PrESTs yielded antibodies recognizing all the included PrESTs. Furthermore, the modified two-step purification method effectively eliminated all background binding and produced pure antibody pools against individual PrESTs. This indicates that the multiplexed PrEST immunization strategy described here could become useful for high-throughput antibody-based proteomics initiatives, thus significantly reducing the number of animals needed in addition to providing a more cost-efficient method for production of msAbs.

  • 9. Lehtio, J.
    et al.
    Wernérus, Henrik
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Samuelson, Patrik
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Teeri, Tuula T.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Directed immobilization of recombinant staphylococci on cotton fibers by functional display of a fungal cellulose-binding domain2001Inngår i: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 195, nr 2, s. 197-204Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The immobilization of recombinant staphylococci onto cellulose fibers through surface display of a fungal cellulose-binding domain (CBD) was investigated. Chimeric proteins containing the CBD from Trichoderma reesei cellulase Cel6A were found to be correctly targeted to the cell wall of Staphylococcus carnosus cells. since full-length proteins could be extracted and affinity-purified. Furthermore. surface accessibility of the CBD was verified using a monoclonal antibody and functionality in terms of cellulose-binding was demonstrated in two different assays in which recombinant staphylococci were found to efficiently bind to cotton fibers. The implications of this strategy of directed immobilization Tor the generation of whole-cell microbial tools Fur different applications will be discussed.

  • 10.
    Löfblom, John
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Kronqvist, Nina
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Optimization of electroporation-mediated transformation: Staphylococcus carnosus as model organism2007Inngår i: Journal of Applied Microbiology, ISSN 1364-5072, E-ISSN 1365-2672, Vol. 102, nr 3, s. 736-747Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 11.
    Löfblom, John
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Sandberg, Julia
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Evaluation of staphylococcal cell surface display and flow cytometry for postselectional characterization of affinity proteins in combinatorial protein engineering applications2007Inngår i: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 73, nr 21, s. 6714-6721Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    For efficient generation of high-affinity protein-based binding molecules, fast and reliable downstream characterization platforms are needed. In this work, we have explored the use of staphylococcal cell surface display together with How cytometry for affinity characterization of candidate affibody molecules directly on the cell surface. A model system comprising three closely related affibody molecules with different affinities for immunoglobulin G and an albumin binding domain with affinity for human serum albumin was used to investigate advantages and differences compared to biosensor technology in a side-by-side manner. Equilibrium dissociation constant (K-D) determinations as well as dissociation rate analysis were performed using both methods, and the results show that the on-cell determinations give both KD and dissociation rate values in a very fast and reproducible manner and that the relative affinities are very similar to the biosensor results. Interestingly, the results also show that there are differences between the absolute affinities determined with the two different technologies, and possible explanations for this are discussed. This work demonstrates the advantages of cell surface display for directed evolution of affinity proteins in terms of fast postselectional, on-cell characterization of candidate clones without the need for subcloning and subsequent protein expression and purification but also demonstrates that it is important to be aware that absolute affinities determined using different methods often vary substantially and that such comparisons therefore could be difficult.

  • 12.
    Löfblom, John
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO).
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO).
    Fine affinity discrimination by normalized fluorescence activated cell sorting in staphylococcal surface display2005Inngår i: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 248, nr 2, s. 189-198Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We have investigated a staphylococcal surface display system for its potential future use as a protein library display system ill combinatorial biochemistry. Efficient affinity-based selections require a system capable of fine affinity discrimination of closely related binders to minimize the loss of potentially improved variants. In this Study, a significant breakthrough was achieved to avoid biases due to potential cell-to-cell variations in surface expression levels, since it was found that a generic protein tag, present within the displayed recombinant surface proteins on the cells, could be successfully employed to obtain normalization of the target-binding signal. Four mutated variants of a staphylococcal protein A domain with different affinity to human IgG were successfully expressed on the surface of recombinant Staphylococcus carnosus cells. The system was evaluated for affinity-based cell sorting experiments, where cell-displayed protein A domains with an 8-fold difference in target affinity were mixed at a ratio of 1: 1000 and sorted using FACS. Enrichment factors around 140-fold were obtained from a single round of sorting under normal library sorting conditions when the top 0.1% fraction having the highest antigen binding to Surface expression level ratio was sorted. The results demonstrate that the system would have a potential as a selection system in protein library display applications, and the normalization strategy should indeed make it possible to achieve fine affinity discriminations in future library selections. (c) 2005 Federation of European Microbiological Societies.

  • 13. Mulder, J.
    et al.
    Björling, Erik
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Jonasson, Kalle
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hokfelt, T.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Tissue Profiling of the Mammalian Central Nervous System Using Human Antibody-based Proteomics2009Inngår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 8, nr 7, s. 1612-1622Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A need exists for mapping the protein profiles in the human brain both during normal and disease conditions. Here we studied 800 antibodies generated toward human proteins as part of a Human Protein Atlas program and investigated their suitability for detailed analysis of various levels of a rat brain using immuno-based methods. In this way, the parallel, rather limited analysis of the human brain, restricted to four brain areas (cerebellum, cerebral cortex, hippocampus, and lateral subventricular zone), could be extended in the rat model to 25 selected areas of the brain. Approximately 100 antibodies (12%) revealed a distinct staining pattern and passed validation of specificity using Western blot analysis. These antibodies were applied to coronal sections of the rat brain at 0.7-mm intervals covering the entire brain. We have now produced detailed protein distribution profiles for these antibodies and acquired over 640 images that form the basis of a publicly available portal of an antibody-based Rodent Brain Protein Atlas database (www.proteinatlas.org/rodentbrain). Because of the systematic selection of target genes, the majority of antibodies included in this database are generated against proteins that have not been studied in the brain before. Furthermore optimized tissue processing and colchicine treatment allow a high quality, more extended annotation and detailed analysis of subcellular distributions and protein dynamics. Molecular & Cellular Proteomics 8: 1612-1622, 2009.

  • 14. Mulder, J.
    et al.
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Shi, T. J.
    Ponten, F.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hokfelt, T.
    Systematically generated antibodies against human gene products: High throughput screening on sections from the rat nervous system2007Inngår i: Neuroscience, ISSN 0306-4522, E-ISSN 1873-7544, Vol. 146, nr 4, s. 1689-1703Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Completion of the Human Genome Project and recent developments in proteomics make it possible to systematically generate affinity reagents to a large portion of the proteome. Recently an antibody-based human protein atlas covering many organs including four areas of the brain has been released (www.proteinatlas.org). Due to the heterogeneity, size, and availability of tissue a more thorough analysis of the human brain is associated with considerable difficulties. Here we applied 120 antibodies raised against 112 human gene products to the smaller rat brain, a rodent animal model, where a single section represents a 'superarray' including many brain areas, and consequently allowing analysis of a huge number of cell types and their neurochemicals. Immunoreactive structures were seen in the investigated brain tissue after incubation with 56 antibodies (46.6%), of which 25 (20.8%) showed a clearly discrete staining pattern that was limited to certain areas, or subsets of brain cells. Bioinformatics, pre-adsorption tests and Western blot analysis were applied to identify non-specific antibodies. Eleven antibodies, including such raised against four 'ambiguous' proteins, passed all validation criteria, and the expression pattern and subcellular distribution of these proteins were studied in detail. To further explore the potential of the systematically generated antibodies, all 11 antibodies that passed validation were used to analyze the spinal cord and lumbar dorsal root ganglia after unilateral transection of the sciatic nerve. Discrete staining patterns were observed for four of the proteins, and injury-induced regulation was found for one of them. In conclusion, the study presented here suggests that a significant portion (10%) of the antibodies generated to a human protein can be used to analyze orthologues present in the rodent brain and to produce a protein-based atlas of the rodent brain. It is hoped that this type of antibody-based, high throughput screening of brain tissue from various rodent disease models will provide new information on the brain chemical neuroanatomy and insights in processes underlying neurological pathologies.

  • 15.
    Nilsson, Peter
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Larsson, Karin
    KTH, Skolan för bioteknologi (BIO).
    Persson, Anja
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO).
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Björling, Erik
    KTH, Skolan för bioteknologi (BIO).
    Ottoson, Jenny
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ödling, Jenny
    KTH, Skolan för bioteknologi (BIO).
    Sundberg, Mårten
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Al-Khalili Szigyarto, Cristina
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Paavilainen, Linda
    Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
    Andersson, Ann-Catrin
    Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
    Kampf, Caroline
    Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
    Wester, Kenneth
    Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
    Pontén, Fredrik
    Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
    Towards a human proteome atlas: High-throughput generation of mono-specific antibodies for tissue profiling2005Inngår i: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 5, s. 4327-4337Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A great need exists for the systematic generation of specific antibodies to explore the human proteome. Here, we show that antibodies specific to human proteins can be generated in a high-throughput manner involving stringent affinity purification using recombinant protein epitope signature tags (PrESTs) as immunogens and affinity-ligands. The specificity of the generated affinity reagents, here called mono-specific antibodies (msAb), were validated with a novel protein microarray assay. The success rate for 464 antibodies generated towards human proteins was more than 90% as judged by the protein array assay. The antibodies were used for parallel profiling of patient biopsies using tissue microarrays generated from 48 human tissues. Comparative analysis with well-characterized monoclonal antibodies showed identical or similar specificity and expression patterns. The results suggest that a comprehensive atlas containing extensive protein expression and subcellular localization data of the human proteome can be generated in an efficient manner with mono-specific antibodies.

  • 16.
    Paavilainen, Linda
    et al.
    Uppsala University.
    Wernerus, Henrik
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlen, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Wester, Kenneth
    Uppsala University.
    Ponten, Fredrik
    Uppsala University.
    Evaluation of monospecific antibodies: A comparison study with commercial analogs using immunohistochemistry on tissue microarrays2008Inngår i: Applied immunohistochemistry & molecular morphology (Print), ISSN 1541-2016, E-ISSN 1533-4058, Vol. 16, nr 5, s. 493-502Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Generation of monospecific antibodies (msAbs) (multiepitope) through affinity purification of polyclonal jntiscra is I plausible strategy, for high-throughput production Of affinity reagents toward large sets of proteins. These antibodies are generated using readily accessible gene Sequence information from publicly available databases. The resulting antibodies have the potential to be used in a variety of assays. probing differentially presented and altered proteins with high sensitivity and specificity. In the present study, 48 msAbs were compared with corresponding commercial analogs. Immunohistochemical staining properties were evaluated on tissue microarrays. representing various normal human tissues from 144 different individuals. MaAbs showed similar immunostaining patterns as compared with corresponding commercial analogs in 44 Out of totally 48 (92%) antibody pairs analyzed. Although only few antibody pairs showed major discrepancies. minor dissimilarities were frequently seen. Our results suggest that msAbs are reliable and valuable tools in antibody-based proteomics. enabling analysis of protein expression patterns in cells and tissues, High-throughput strategies employing such antibodies provide a consistent approach in the exploration of the human proleome.

  • 17. Ponten, Fredrik
    et al.
    Gry, Marcus
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Fagerberg, Linn
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Lundberg, Emma
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Asplund, Anna
    Berglund, Lisa
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Oksvold, Per
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Björling, Erik
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Kampf, Caroline
    Navani, Sanjay
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ottosson, Jenny
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Persson, Anja
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Wester, Kenneth
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    A global view of protein expression in human cells, tissues, and organs2009Inngår i: Molecular Systems Biology, ISSN 1744-4292, E-ISSN 1744-4292, Vol. 5Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Defining the protein profiles of tissues and organs is critical to understanding the unique characteristics of the various cell types in the human body. In this study, we report on an anatomically comprehensive analysis of 4842 protein profiles in 48 human tissues and 45 human cell lines. A detailed analysis of over 2 million manually annotated, high-resolution, immunohistochemistry- based images showed a high fraction (>65%) of expressed proteins in most cells and tissues, with very few proteins (<2%) detected in any single cell type. Similarly, confocal microscopy in three human cell lines detected expression of more than 70% of the analyzed proteins. Despite this ubiquitous expression, hierarchical clustering analysis, based on global protein expression patterns, shows that the analyzed cells can be still subdivided into groups according to the current concepts of histology and cellular differentiation. This study suggests that tissue specificity is achieved by precise regulation of protein levels in space and time, and that different tissues in the body acquire their unique characteristics by controlling not which proteins are expressed but how much of each is produced. Molecular Systems Biology 5: 337; published online 22 December 2009; doi:10.1038/msb.2009.93

  • 18.
    Pontén, Fredrik
    et al.
    Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University.
    Gry, Marcus
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Björling, Erik
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Berglund, Lisa
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Al-Khalili Szigyarto, Cristina
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Lundberg, Emma
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Andersson-Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Asplund, Anna
    Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Kampf, Caroline
    Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University.
    Nilsson, Kenneth
    Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University.
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ottosson, Jenny
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Persson, Anja
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Wester, Kenneth
    Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ubiquitous protein expression in human cells, tissues and organsManuskript (Annet vitenskapelig)
  • 19.
    Samuelson, Patrik
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Wernérus, Henrik
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Svedberg, M.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Staphylococcal surface display of metal-binding polyhistidyl peptides2000Inngår i: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 66, nr 3, s. 1243-1248Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Recombinant Staphylococcus xylosus and Staphylococcus carnosus strains were generated with surface-exposed chimeric proteins containing polyhistidyl peptides designed for binding to divalent metal ions. Surface accessibility of the chimeric surface proteins was demonstrated and the chimeric surface proteins were found to be functional in terms of metal binding, since the recombinant staphylococcal cells were shown to have gained Ni2+- and Cd2+-binding capacity, suggesting that such bacteria could find use in bioremediation of heavy metals. This is, to our knowledge, the first time that recombinant, surface-exposed metal-binding peptides have been expressed on gram-positive bacteria. Potential environmental or biosensor applications for such recombinant staphylococci as biosorbents are discussed.

  • 20.
    Steen, Johanna
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Larsson, Karin
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ottosson, Jenny
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Antigenic mapping and characterization of Albumin Binding ProteinManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    The possibility to predict the location of antigenic determinants is a desirable feature in antibody production ventures and for vaccine development. However, antigenic propensity scales available today are poor, and so far it is not possible to predict the best antigen to trigger the immune system. Here, a unique set of 411 antisera towards a common part of allantigens within the Human Protein Atlas project has made it possible to perform massive epitope mapping. This effort generated a true map of the antigenic regions of this common N-terminal tag, and rendered it possible to further investigate what features that generate a good antigen. Investigations on variation in epitope occurrence are often an obstacle when mapping antigens, because of the ethics of using more animals than necessary for antibody production. As a consequence, not much has been done to verify epitopes found and the variance between different immunizations has not been thoroughly investigated. Herein it was shown that the most immunopotentating sites were only detected by the polyclonal antibodies in 70% of the immunizations, demonstrating the need of good antigen design. Detected epitopes also showed that aromatic amino acids, some positively charged aminoacids, and serine and glycine were over-represented in the antigenic hot spot regions. The detected antigenic regions were also shown to have fairly low correlation to several antigenic propensity scales.

  • 21. Ståhl, Stefan
    et al.
    Robert, A.
    Gunneriusson, E.
    Wernérus, Henrik
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Cano, F.
    Liljeqvist, S.
    Hansson, M.
    Nguyen, T. N.
    Samuelson, Patrik
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Staphylococcal surface display and its applications2000Inngår i: International Journal of Medical Microbiology, ISSN 1438-4221, E-ISSN 1618-0607, Vol. 290, nr 7, s. 571-577Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    Novel surface proteins can be introduced onto the bacterial cell surface by recombinant means. Here, we describe the development of such display systems for two food-grade bacteria, Staphylococcus carnosus and Staphylococcus xylosus, and present how such engineered bacteria can be used in different applications. A study will be described in which such staphylococci were employed as vaccine delivery vehicles to elicit protective antibody responses to respiratory syncytial virus (RSV). The use of surface-engineered staphylococci as novel microbial biocatalysts, as a new type of whole-cell diagnostic devices or for adsorption of metal ions with potential environmental or biosensor applications, will also be discussed.

  • 22.
    Uhlén, Mathias
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Björling, Erik
    KTH, Skolan för bioteknologi (BIO).
    Agaton, Charlotta
    KTH, Skolan för bioteknologi (BIO).
    Al-Khalili Szigyarto, Cristina
    KTH, Skolan för bioteknologi (BIO).
    Amini, Bahram
    KTH, Skolan för bioteknologi (BIO).
    Andersen, Elisabet
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Andersson, Ann-Catrin
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Angelidou, Pia
    KTH, Skolan för bioteknologi (BIO).
    Asplund, Anna
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Asplund, Caroline
    KTH, Skolan för bioteknologi (BIO).
    Berglund, Lisa
    KTH, Skolan för bioteknologi (BIO).
    Bergström, Kristina
    KTH, Skolan för bioteknologi (BIO).
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO).
    Cerjan, Dijana
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Ekström, Marica
    KTH, Skolan för bioteknologi (BIO).
    Elobeid, Adila
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Eriksson, Cecilia
    KTH, Skolan för bioteknologi (BIO).
    Fagerberg, Linn
    KTH, Skolan för bioteknologi (BIO).
    Falk, Ronny
    KTH, Skolan för bioteknologi (BIO).
    Fall, Jenny
    KTH, Skolan för bioteknologi (BIO).
    Forsberg, Mattias
    KTH, Skolan för bioteknologi (BIO).
    Gry Björklund, Marcus
    KTH, Skolan för bioteknologi (BIO).
    Gumbel, Kristoffer
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Halimi, Asif
    KTH, Skolan för bioteknologi (BIO).
    Hallin, Inga
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Hamsten, Carl
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Hansson, Marianne
    KTH, Skolan för bioteknologi (BIO).
    Hedhammar, My
    KTH, Skolan för bioteknologi (BIO).
    Hercules, Görel
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Kampf, Caroline
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Larsson, Karin
    KTH, Skolan för bioteknologi (BIO).
    Lindskog, Mats
    KTH, Skolan för bioteknologi (BIO).
    Lodewyckx, Wald
    KTH, Skolan för bioteknologi (BIO).
    Lund, Jan
    KTH, Skolan för bioteknologi (BIO).
    Lundeberg, Joakim
    KTH, Skolan för bioteknologi (BIO).
    Magnusson, Kristina
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Malm, Erik
    KTH, Skolan för bioteknologi (BIO).
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO).
    Ödling, Jenny
    KTH, Skolan för bioteknologi (BIO).
    Oksvold, Per
    KTH, Skolan för bioteknologi (BIO).
    Olsson, Ingmarie
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Öster, Emma
    KTH, Skolan för bioteknologi (BIO).
    Ottosson, Jenny
    KTH, Skolan för bioteknologi (BIO).
    Paavilainen, Linda
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Persson, Anja
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Rimini, Rebecca
    KTH, Skolan för bioteknologi (BIO).
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO).
    Runeson, Marcus
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Sivertsson, Åsa
    KTH, Skolan för bioteknologi (BIO).
    Sköllermo, Anna
    KTH, Skolan för bioteknologi (BIO).
    Steen, Johanna
    KTH, Skolan för bioteknologi (BIO).
    Stenvall, Maria
    KTH, Skolan för bioteknologi (BIO).
    Sterky, Fredrik
    KTH, Skolan för bioteknologi (BIO).
    Strömberg, Sara
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Sundberg, Mårten
    KTH, Skolan för bioteknologi (BIO).
    Tegel, Hanna
    KTH, Skolan för bioteknologi (BIO).
    Tourle, Samuel
    KTH, Skolan för bioteknologi (BIO).
    Wahlund, Eva
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Waldén, Annelie
    KTH, Skolan för bioteknologi (BIO).
    Wan, Jinghong
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Westberg, Joakim
    KTH, Skolan för bioteknologi (BIO).
    Wester, Kenneth
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Wrethagen, Ulla
    KTH, Skolan för bioteknologi (BIO).
    Xu, Lan Lan
    KTH, Skolan för bioteknologi (BIO).
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Pontén, Fredrik
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    A human protein atlas for normal and cancer tissues based on antibody proteomics2005Inngår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 4, nr 12, s. 1920-1932Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, similar to 400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.

  • 23.
    Uhlén, Mathias
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101). KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Oksvold, Per
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Fagerberg, Linn
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Lundberg, Emma
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Jonasson, Kalle
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Forsberg, Mattias
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Zwahlen, Martin
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Kampf, Caroline
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Wester, Kenneth
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Björling, Lisa
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Pontén, Fredrik
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Towards a knowledge-based Human Protein Atlas2010Inngår i: Nature Biotechnology, ISSN 1087-0156, E-ISSN 1546-1696, Vol. 28, nr 12, s. 1248-1250Artikkel i tidsskrift (Fagfellevurdert)
  • 24.
    Wernérus, Henrik
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Lehtio, J.
    Teeri, Tuula T.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Nygren, Per-Åke
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Generation of metal-binding staphylococci through surface display of combinatorially engineered cellulose-binding domains2001Inngår i: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 67, nr 10, s. 4678-4684Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Ni2+-binding staphylococci were generated through surface display of combinatorially engineered variants of a fungal cellulose-binding domain (CBD) from Trichoderma reesei cellulase CeI7A. Novel CBD variants were generated by combinatorial protein engineering through the randomization of 11 amino acid positions, and eight potentially Ni2+-binding CBDs were selected by phage display technology. These new variants were subsequently genetically introduced into chimeric surface proteins for surface display on Staphylococcus carnosus cells. The expressed chimeric proteins were shown to be properly targeted to the cell wall of S. carnosus cells, since full-length proteins could be extracted and affinity purified. Surface accessibility for the chimeric proteins was demonstrated, and furthermore, the engineered CBDs, now devoid of cellulose-binding capacity, were shown to be functional with regard to metal binding, since the recombinant staphylococci had gained Ni2+-binding capacity. Potential environmental applications for such tailor-made metal-binding bacteria as bioadsorbents in biofilters or biosensors are discussed.

  • 25.
    Wernérus, Henrik
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Lehtiö, Janne
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Samuelson, Patrik
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Engineering of staphylococcal surfaces for biotechnological applications2002Inngår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 96, nr 1, s. 67-78Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    Novel surface proteins can be introduced onto bacterial cell surfaces by recombinant means. Here, we describe various applications of two such display systems for the food-grade bacteria Staphylococcus carnosus and Staphylococcus xylosus, respectively. The achievements in the use of such staphylococci as live bacterial vaccine delivery vehicles will be described. Co-display of proteins and peptides with adhesive properties to enable targeting of the bacteria, have significantly improved the vaccine delivery potential. Recently, protective immunity to respiratory syncytial virus (RSV) could be evoked in mice by intranasal immunization using such 'second generation' vaccine delivery systems. Furthermore, antibody fragments and other 'affinity proteins' with capacity to specifically bind a certain protein, e.g. Staphylococcus aureus protein A-based affibodies, have been surface-displayed on staphylococci as initial efforts to create whole-cell diagnostic devices. Surface display of metal-binding peptides, or protein domains into which metal binding properties has been engineered by combinatorial protein engineering, have been exploited to create staphylococcal bioadsorbents for potential environmental or biosensor applications. The use of these staphylococcal surface display systems as alternatives for display of large protein libraries and subsequent affinity selection of relevant binding proteins by fluorescence-activated cell sorting (FACS) will be discussed.

  • 26.
    Wernérus, Henrik
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Samuelson, Patrik
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Fluorescence-activated cell sorting of specific affibody-displaying staphylococci2003Inngår i: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 69, nr 9, s. 5328-5335Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Efficient enrichment of staphylococcal cells displaying specific heterologous affinity ligands on their cell surfaces was demonstrated by using fluorescence-activated cell sorting. Using bacterial surface display of peptide or protein libraries for the purpose of combinatorial protein engineering has previously been investigated by using gram-negative bacteria. Here, the potential for using a gram-positive bacterium was evaluated by employing the well-established surface expression system for Staphylococcus carnosus. Staphylococcus aureus protein A domains with binding specificity to immunoglobulin G or engineered specificity for the G protein of human respiratory syncytial virus were expressed as surface display on S. carnosus cells. The surface accessibility and retained binding specificity of expressed proteins were demonstrated in whole-cell enzyme and flow cytometry assays. Also, affibody-expressing target cells could be sorted essentially quantitatively from a moderate excess of background cells in a single step by using a high-stringency sorting mode. Furthermore, in a simulated library selection experiment, a more-than-25,000-fold enrichment of target cells could be achieved through only two rounds of cell sorting and regrowth. The results obtained indicate that staphylococcal surface display of affibody libraries combined with fluoresence-activated cell sorting might indeed constitute an attractive alternative to existing technology platforms for affinity-based selections.

  • 27.
    Wernérus, Henrik
    et al.
    KTH, Tidigare Institutioner, Bioteknologi.
    Ståhl, Stefan
    KTH, Tidigare Institutioner, Bioteknologi.
    Biotechnological applications for surface-engineered bacteria2004Inngår i: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 40, s. 209-228Artikkel, forskningsoversikt (Fagfellevurdert)
    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.

  • 28.
    Wernérus, Henrik
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Vector engineering to improve a staphylococcal surface display system2002Inngår i: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 212, nr 1, s. 47-54Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A previously developed expression system for surface display of heterologous proteins on the surface of Staphylococcus carnosus employs the secretion signals from a Staphylococcus hyicus lipase and the cell wall anchoring part of Staphylococcus aureus protein A (SpA) to achieve surface display of expressed recombinant proteins. The system has been successfully used in various applications but the vector has not been considered genetically stable enough to allow protein library display applications, which would be of obvious interest. A new set of vectors, differing in size and devoid of a phage f1 origin of replication, were constructed and evaluated in terms of bacterial growth characteristics and vector stability. Furthermore, surface expression of a model surface protein was monitored by an enzymatic whole-cell assay and flow cytometry. The engineered expression vectors demonstrated dramatically improved stability and growth properties and two of the novel vectors demonstrated retained high surface density of the displayed model protein. The flow cytometry was found to be a powerful toot for observing the surface density of displayed heterologous proteins, and would thus be a rational strategy for monitoring the optimisation of any surface display system. The implications of these improved display vectors for future protein library applications are discussed.

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