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  • 51.
    Kronqvist, Nina
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hjelm, Barbara
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    New Ways for Discovery of Biopharmaceuticals: Emerging Techniques using Surface Display on Gram-positive Bacteria for Combinatorial Protein Engineering and Characterization2009Inngår i: Bioforum Europe, ISSN 1611-597X, Vol. 13, nr 6-7, s. 022-Artikkel i tidsskrift (Fagfellevurdert)
  • 52. Kuktaite, Ramune
    et al.
    Plivelic, Tomas S.
    Cerenius, Yngve
    Hedenqvist, Mikael S.
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknik.
    Gallstedt, Mikael
    Marttila, Salla
    Ignell, Rickard
    Popineau, Yves
    Tranquet, Oliver
    Shewry, Peter R.
    Johansson, Eva
    Structure and Morphology of Wheat Gluten Films: From Polymeric Protein Aggregates toward Superstructure Arrangements2011Inngår i: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 12, nr 5, s. 1438-1448Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Evaluation of structure and morphology of extruded wheat gluten (WG) films showed WG protein assemblies elucidated on a range of length scales from nano (4.4 angstrom and 9 to 10 angstrom, up to 70 angstrom) to micro (10 mu m). The presence of NaOH in WG films induced a tetragonal structure with unit cell parameters, a = 51.85 angstrom and c = 40.65 angstrom, whereas NH4OH resulted in a bidimensional hexagonal close-packed (HCP) structure with a lattice parameter of 70 angstrom. In the WG films with NH4OH, a highly polymerized protein pattern with intimately mixed glutenins and gliadins bounded through SH/SS interchange reactions was found. A large content of beta-sheet structures was also found in these films, and the film structure was oriented in the extrusion direction. In conclusion, this study highlights complexities of the supramolecular structures and conformations of wheat gluten polymeric proteins in biofilms not previously reported for biobased materials.

  • 53.
    Langer, Krzysztof
    et al.
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Jönsson, Håkan
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Rapid production and recovery of cell spheroids by automated droplet microfluidics2019Manuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    Droplet microfluidics enables high throughput cell processing, analysis and screening by miniaturizing the reaction vessels to nano- or pico-liter water-in oil droplets, but like many other microfluidic formats, droplet microfluidics have not been interfaced with or automated by laboratory robotics. Here we demonstrate automation of droplet microfluidics based on an inexpensive liquid handling robot for the automated production of human scaffold-free cell spheroids, using pipette actuation and interfacing the pipetting tip with a droplet generating microfluidic chip. In this chip we produce highly mono-disperse 290μm droplets with diameter CV of 1.7%. By encapsulating cells in these droplets, we produce cell spheroids in droplets and recover them to standard formats at a throughput of 85000 spheroids per microfluidic circuit per hour. The viability of the cells in spheroids remains high after recovery only decreased by 4% starting from 96% after 16 hours incubation in nanoliter droplets. Scaffold-free cell spheroids and 3D tissue constructs recapitulate many aspects of functional human tissue more accurately than 2D or single cell cultures, but assembly methods for spheroids, e.g. hanging drop micro-plates, has had limited throughput. The increased throughput and decreased cost of our method enables spheroid production at the scale needed for lead discovery drug screening and approaches the cost where these micro tissues could be used as building blocks for organ scale regenerative medicine.

  • 54.
    Langer, Krzysztof
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi.
    Jönsson, Håkan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi.
    Rapid production and recovery of cell spheroids by automated droplet microfluidics2019Inngår i: bioRxivArtikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Droplet microfluidics enables high throughput cell processing, analysis and screening by miniaturizing the reaction vessels to nano- or pico-liter water-in oil droplets, but like many other microfluidic formats, droplet microfluidics have not been interfaced with or automated by laboratory robotics. Here we demonstrate automation of droplet microfluidics based on an inexpensive liquid handling robot for the automated production of human scaffold-free cell spheroids, using pipette actuation and interfacing the pipetting tip with a droplet generating microfluidic chip. In this chip we produce highly mono-disperse 290μm droplets with diameter CV of 1.7%. By encapsulating cells in these droplets, we produce cell spheroids in droplets and recover them to standard formats at a throughput of 85000 spheroids per microfluidic circuit per hour. The viability of the cells in spheroids remains high after recovery only decreased by 4% starting from 96% after 16 hours incubation in nanoliter droplets. Scaffold-free cell spheroids and 3D tissue constructs recapitulate many aspects of functional human tissue more accurately than 2D or single cell cultures, but assembly methods for spheroids, e.g. hanging drop micro-plates, has had limited throughput. The increased throughput and decreased cost of our method enables spheroid production at the scale needed for lead discovery drug screening and approaches the cost where these micro tissues could be used as building blocks for organ scale regenerative medicine.

  • 55.
    Larsbrink, Johan
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Thompson, Andrew J.
    Lundqvist, Magnus
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Gardner, Jeffrey G.
    Davies, Gideon J.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. Univ British Columbia, Canada; .
    A complex gene locus enables xyloglucan utilization in the model saprophyte Cellvibrio japonicus2014Inngår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 94, nr 2, s. 418-433Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The degradation of plant biomass by saprophytes is an ecologically important part of the global carbon cycle, which has also inspired a vast diversity of industrial enzyme applications. The xyloglucans (XyGs) constitute a family of ubiquitous and abundant plant cell wall polysaccharides, yet the enzymology of XyG saccharification is poorly studied. Here, we present the identification and molecular characterization of a complex genetic locus that is required for xyloglucan utilization by the model saprophyte Cellvibrio japonicus. In harness, transcriptomics, reverse genetics, enzyme kinetics, and structural biology indicate that the encoded cohort of an -xylosidase, a -galactosidase, and an -l-fucosidase is specifically adapted for efficient, concerted saccharification of dicot (fucogalacto)xyloglucan oligosaccharides following import into the periplasm via an associated TonB-dependent receptor. The data support a biological model of xyloglucan degradation by C. japonicus with striking similarities - and notable differences - to the complex polysaccharide utilization loci of the Bacteroidetes.

  • 56.
    Lindberg, Hanna
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Härd, Torleif
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    A truncated and dimeric format of an Affibody library on bacteria enables FACS-mediated isolation of amyloid-beta aggregation inhibitors with subnanomolar affinity2015Inngår i: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 10, nr 11, s. 1707-1718Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The amyloid hypothesis suggests that accumulation of amyloid β (Aβ) peptides in the brain is involved in development of Alzheimer's disease. We previously generated a small dimeric affinity protein that inhibited Aβ aggregation by sequestering the aggregation prone parts of the peptide. The affinity protein is originally based on the Affibody scaffold, but is evolved to a distinct interaction mechanism involving complex structural rearrangement in both the Aβ peptide and the affinity proteins upon binding. The aim of this study was to decrease the size of the dimeric affinity protein and significantly improve its affinity for the Aβ peptide to increase its potential as a future therapeutic agent. We combined a rational design approach with combinatorial protein engineering to generate two different affinity maturation libraries. The libraries were displayed on staphylococcal cells and high-affinity Aβ-binding molecules were isolated using flow-cytometric sorting. The best performing candidate binds Aβ with a KD value of around 300 pM, corresponding to a 50-fold improvement in affinity relative to the first-generation binder. The new dimeric Affibody molecule was shown to capture Aβ1-42 peptides from spiked E. coli lysate. Altogether, our results demonstrate successful engineering of this complex binder for increased affinity to the Aβ peptide.

  • 57.
    Lindbo, Sarah
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Garousi, Javad
    Uppsala university.
    Mitran, Bogdan
    Uppsala university.
    Vorobyeva, Anzhelika
    Uppsala university.
    Oroujeni, Maryam
    Uppsala university.
    Orlova, Anna
    Uppsala university.
    Hober, Sophia
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH). KTH, Skolan för bioteknologi (BIO), Centra, Centrum för Bioprocessteknik, CBioPT. KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Tolmachev, Vladimir
    Uppsala university.
    Optimized molecular design of ADAPT-based HER2-imaging probes labelled with 111In and 68Ga2018Inngår i: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 15, nr 7, s. 2674-2683Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Radionuclide molecular imaging is a promising tool for visualization of cancer associated molecular abnormalities in vivo and stratification of patients for specific therapies. ADAPT is a new type of small engineered proteins based on the scaffold of an albumin binding domain of protein G. ADAPTs have been utilized to select and develop high affinity binders to different proteinaceous targets. ADAPT6 binds to human epidermal growth factor 2 (HER2) with low nanomolar affinity and can be used for its in vivo visualization. Molecular design of 111In-labeled anti-HER2 ADAPT has been optimized in several earlier studies. In this study, we made a direct comparison of two of the most promising variants, having either a DEAVDANS or a (HE)3DANS sequence at the N-terminus, conjugated with a maleimido derivative of DOTA to a GSSC amino acids sequence at the C-terminus. The variants (designated DOTA-C59-DEAVDANS-ADAPT6-GSSC and DOTA-C61-(HE)3DANS-ADAPT6-GSSC) were stably labeled with 111In for SPECT and 68Ga for PET. Biodistribution of labeled ADAPT variants was evaluated in nude mice bearing human tumor xenografts with different levels of HER2 expression. Both variants enabled clear discrimination between tumors with high and low levels of HER2 expression. 111In-labeled ADAPT6 derivatives provided higher tumor-to-organ ratios compared to 68Ga-labeled counterparts. The best performing variant was DOTA-C61-(HE)3DANS-ADAPT6-GSSC, which provided tumor-to-blood ratios of 208 ± 36 and 109 ± 17 at 3 h for 111In and 68Ga labels, respectively.

  • 58. Liotta, L. A.
    et al.
    Davis, J. B.
    Couch, R. D.
    Fredolini, Claudia
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Zhou, W.
    Petricoin, E.
    Espina, V.
    Clinical proteomics and molecular pathology2017Inngår i: Molecular Pathology: The Molecular Basis of Human Disease, Elsevier Inc. , 2017, s. 183-203Kapittel i bok, del av antologi (Annet vitenskapelig)
    Abstract [en]

    Genomic and proteomic research is launching the next era of cancer molecular medicine. Molecular expression profiles can uncover clues to functionally important molecules in the development of human disease and generate information to subclassify human tumors and tailor a treatment to the individual patient. The next revolution is the synthesis of proteomic information into functional pathways and circuits in cells and tissues. Such synthesis must take into account the dynamic state of protein post-translational modifications; protein-protein or protein-DNA/RNA interactions; cross-talk between signal pathways; and feedback regulation within cells, between cells, and between tissues. This full set of information may be required before we can fully dissect the specific dysregulated pathways driving tumorigenesis. This higher level of functional understanding will be the basis for true rational therapeutic design that specifically targets the molecular lesions underlying human disease. 

  • 59.
    Liu, Hao
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap. ltai, Mohamed; Garousi, Javad; Tolmachev, Vladimir.
    Lindbo, Sarah
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Ding, Haozhong
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Altai, Mohamed
    Garousi, Javad
    Orlova, Anna
    Tolmachev, Vladimir
    Hober, Sophia
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Gräslund, Torbjörn
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Potent and specific fusion toxins consisting of a HER2‑binding, ABD‑derived affinity protein, fused to truncated versions of Pseudomonas exotoxin A2019Inngår i: International Journal of Oncology, ISSN 1019-6439, Vol. 55, nr 1, s. 309-319Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Fusion toxins consisting of an affinity protein fused to toxic polypeptides derived from Pseudomonas exotoxin A (ETA) are promising agents for targeted cancer therapy. In this study, we examined whether fusion toxins consisting of an albumin binding domain-derived affinity protein (ADAPT) interacting with human epidermal growth factor receptor 2 (HER2), coupled to the ETA-derived polypeptides PE38X8 or PE25, with or without an albumin binding domain (ABD) for half-life extension, can be used for specific killing of HER2-expressing cells. The fusion toxins could easily be expressed in a soluble form in Escherichia coli and purified to homogeneity. All constructs had strong affinity for HER2 (K-D 10 to 26 nM) and no tendency for aggregation could be detected. The fusion toxins including the ABD showed strong interaction with human and mouse serum albumin [equilibrium dissociation constant (K-D) 1 to 3 nM and 2 to 10 nM, respectively]. The in vitro investigation of the cytotoxic potential revealed IC50-values in the picomolar range for cells expressing high levels of HER2. The specificity was also demonstrated, by showing that free HER2 receptors on the target cells are required for fusion toxin activity. In mice, the fusion toxins containing the ABD exhibited an appreciably longer time in circulation. The uptake was highest in liver and kidney. Fusion with PE25 was associated with the highest hepatic uptake. Collectively, the results suggest that fusion toxins consisting of ADAPTs and ETA-derivatives are promising agents for targeted cancer therapy.

  • 60. Liu, Xiao
    et al.
    Spicarova, Zuzana
    Rydholm, Susanna
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Li, Juan
    Brismar, Hjalmar
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Aperia, Anita
    Ankyrin B Modulates the Function of Na,K-ATPase/Inositol 1,4,5-Trisphosphate Receptor Signaling Microdomain2008Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, nr 17, s. 11461-11468Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Na, K-ATPase and inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) can form a signaling microdomain that in the presence of ouabain triggers highly regular calcium oscillations. Downstream effects include NF-kappa B activation. Here we report that ankyrin B (Ank-B), expressed in most mammalian cells, plays a pivotal role in the function of the Na, K-ATPase/ IP3R signaling microdomain. In studies performed on a monkey kidney cell line, we show that Ank-B co-precipitates with both Na, K-ATPase and IP3R. We identify the N terminus tail of the Na, K-ATPase catalytic subunit and the N-terminal portion 1-604 of the IP3R as novel binding sites for Ank-B. Knockdown of Ank-B with small interfering RNA reduced the expression of Ank-B to 15-30%. This down-regulation of Ank-B attenuated the interaction between Na, K-ATPase and IP3R, reduced the number of cells responding to pM doses of ouabain with calcium oscillations, altered the calcium oscillatory pattern, and abolished the ouabain effect on NF-kappa B. In contrast, Ank-B down-regulation had no effect on the ion transporting function of Na, K-ATPase and no effect on the distribution and apparent mobility of Na, K-ATPase in the plasma membrane.

  • 61.
    Lopez Navarro, Indira Patricia
    KTH, Skolan för bioteknologi (BIO).
    Utveckling av affinitets-baserade analys av muskel dialys prover från patienter med facioscapulohumeral muskel dystrofi2016Independent thesis Advanced level (degree of Master (Two Years)), 20 poäng / 30 hpOppgave
    Abstract [en]

    Interstitial Fluid is a complex sample, highly abundant in the human body that can give information regardingtissue secretion, intracellular signaling and tissue health status. The composition of the interstitial fluid can giveinformation regarding the processes occurring in muscles and alterations due to pathological changes occurringduring disease progression. Currently this sample has not yet been characterized within rare diseases like musculardystrophies. Facioscapulohumeral Muscular Dytrophy is an inherited progressive myopathy, characterized by thedegeneration and progressive muscular fiber necrosis of muscles from the face, upper arms and lower limbs. It canbe diagnosed; but in an advanced stage where weakness in the muscles have already occur. Meanwhile there is nocurrent understanding of the mechanisms happening in the muscle. In this project an immunoassay protocol wasdeveloped using suspension bead array technology to create an optimal method to analyze the protein content ofthese samples. The technological platform allows antibody-based capturing and detection of protein targets frombiotinylated biological samples. By modifying an existing protocol for analysis of serum and plasma samplesabundance of 63 protein targets was measured in muscle interstitial fluid from healthy individuals and patientsaffected by facioscapulohumeral dystrophy (FSHD), The optimized steps were the sample pre-treatment, the assaybuffer dilution ratio and the incubation time for capturing the protein targets. The findings of this project indicatethat using 1 μl of muscle interstitial fluid sample with minimized dilution factor and 60-fold molar excess biotinrelative to sample protein concentration enables detection of Interstitial fluid protein components. The proteinsdetected are ret finger protein-like 4B (RFPL4B) and albumin in from affected muscle and histone cluster(HIST1H3A) and albumin in non affected muscle.

  • 62.
    Lundqvist, Magnus
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Methods for cell line and protein engineering2018Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Therapeutic proteins are becoming increasingly important. They are desirable, as they typically possess low adverse effects and higher specificity compared to the traditional, small molecule drugs. But they are also more complex and involve different intricate and expensive development and production processes. Through new technologies in protein and cell line development, more efficient and safer drugs can be readily available and at a lower cost. This thesis gives an overview of how protein therapeutics are developed and produced. It explores strategies to improve the efficacy and safety of protein drugs and how to improve production yields. In the present investigation, two papers present new methods for high-throughput cloning and site-directed mutagenesis using solid-phase immobilization of DNA fragments. These methods were designed to generate new drug candidates with swiftness and ease. Three papers show the development of a new cell line screening system that combines droplet microfluidics and the split-GFP reporter system. This combination allows for relative quantification of secreted recombinant proteins between individual cells and provides a tool for the selection of the best-producing clones for final production from a heterologous cell pool. The final paper explores the possibility to produce proteins at a higher cell density by examining how the metabolome and proteome of a perfusion bioreactor evolve as the cell density reaches exceptionally high levels. The consistent goal of all of these studies is to expedite the development and improve the production of therapeutic proteins, to assist the discovery of new drugs and to bring down production and development costs. Engineered proteins can be used to cure previously incurable diseases or give current medications a higher efficacy. Lower production and development costs can make the treatments available to more people.

  • 63.
    Lundqvist, Magnus
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Thalén, Niklas
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Volk, Anna-Luisa
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Hansen, Henning Gram
    Tech Univ Denmark, Novo Nordisk Fdn Ctr Biosustainabil, Lyngby, Denmark..
    von Otter, Eric
    Nanyang Technol Univ, Sch Biol Sci, Singapore 637551, Singapore..
    Nygren, Per-Åke
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap. The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby, Denmark.
    Rockberg, Johan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Chromophore pre-maturation for improved speed and sensitivity of split-GFP monitoring of protein secretion2019Inngår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, artikkel-id 310Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Complementation-dependent fluorescence is a powerful way to study co-localization or interactions between biomolecules. A split-GFP variant, involving the self-associating GFP 1-10 and GFP 11, has previously provided a convenient approach to measure recombinant protein titers in cell supernatants. A limitation of this approach is the slow chromophore formation after complementation. Here, we alleviate this lag in signal generation by allowing the GFP 1-10 chromophore to mature on a solid support containing GFP 11 before applying GFP 1-10 in analyses. The pre-maturated GFP 1-10 provided up to 150-fold faster signal generation compared to the non-maturated version. Moreover, pre-maturated GFP 1-10 significantly improved the ability of discriminating between Chinese hamster ovary (CHO) cell lines secreting GFP 11-tagged erythropoietin protein at varying rates. Its improved kinetics make the pre-maturated GFP 1-10 a suitable reporter molecule for cell biology research in general, especially for ranking individual cell lines based on secretion rates of recombinant proteins.

  • 64.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Affibody molecules binding to the ErbB3 receptorPatent (Annet (populærvitenskap, debatt, mm))
  • 65.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Bacterial display in directed evolution for generation of new biopharmaceuticals2011Inngår i: Biotech International, ISSN 2032-2887, Vol. 23, nr June, s. 26-29Artikkel i tidsskrift (Annet vitenskapelig)
    Abstract [en]

    The successful use of monoclonal antibodies and antibody derivatives for therapeutic and in vivo diagnostic applications has resulted in a rapid progression in the fields surrounding biopharmaceutical drug discovery and combinatorial protein engineering. Consequently, investigations on new and improved technologies for both generation and characterisation of specific antibodies and alternative protein scaffolds are continuously being reported in the literature. This review summarises the recent efforts in development and application of bacterial display platforms for such purposes.

  • 66.
    Löfblom, John
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Frejd, Fredrik Y.
    Uppsala University.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Non-immunoglobulin based protein scaffolds2011Inngår i: Current Opinion in Biotechnology, ISSN 0958-1669, E-ISSN 1879-0429, Vol. 22, nr 6, s. 843-848Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    Non-immunoglobulin based protein scaffolds have been reported as promising alternatives to traditional monoclonal antibodies for over a decade and are often mentioned as part of the next-generation immunotherapeutics. Today, this class of biologics is beginning to demonstrate its potential for therapeutic applications and several are currently in preclinical or clinical development. A common denominator for most of these new scaffolds is the attractive properties that differentiate them from monoclonal antibodies including small size, cysteine-free sequence, flexible pharmacokinetic properties, and ease of generating multispecific molecules. In addition to therapeutic applications, substantial evidence point to superior performance of several of these scaffolds in molecular imaging compared to full-length antibodies. Here we review the most recent progress using alternative protein scaffolds for therapy and medical imaging.

  • 67.
    Magnusson, Anders
    KTH, Skolan för bioteknologi (BIO).
    Rational redesign of Candida antarctica lipase B2005Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    This thesis describes the use of rational redesign to modify the properties of the enzyme Candida antarctica lipase B. Through carefully selected single-point mutations, we were able to introduce substrate-assisted catalysis and to alter the reaction specificity. Other single-point mutations afforded variants with greatly changed substrate selectivity and enantioselectivity.

    Mutation of the catalytic serine changed the hydrolase activity into an aldolase activity. The mutation decreased the activation energy for aldol addition by 4 kJ×mol-1, while the activation energy increased so much for hydrolysis that no hydrolysis activity could be detected. This mutant can catalyze aldol additions that no natural aldolases can catalyze.

    Mutation of the threonine in the oxyanion hole proved the great importance of its hydroxyl group in the transition-state stabilization. The lost transition-state stabilization was partly replaced through substrate-assisted catalysis with substrates carrying a hydroxyl group. The poor selectivity of the wild-type lipase for ethyl 2-hydroxypropanoate (E=1.6) was greatly improved in the mutant (E=22), since only one enantiomer could perform substrate-assisted catalysis.

    The redesign of the size of the stereospecificity pocket was very successful. Mutation of the tryptophan at the bottom of this pocket removed steric interactions with secondary alcohols that have to position a substituent larger than an ethyl in this pocket. This mutation increased the activity 5 500 times towards 5-nonanol and 130 000 times towards (S)-1-phenylethanol. The acceptance of such large substituents (butyl and phenyl) in the redesigned stereospecificity pocket increases the utility of lipases in biocatalysis. The improved activity with (S)-1-phenylethanol strongly contributed to the 8 300 000 times change in enantioselectivity towards 1-phenylethanol; example of such a large change was not found in the literature. The S-selectivity of the mutant is unique for lipases. Its enantioselectivity increases strongly with temperature reaching a useful S-selectivity (E=44) at 69 °C.

    Thermodynamics analysis of the enantioselectivity showed that the mutation in the stereospecificity pocket mainly changed the entropic term, while the enthalpic term was only slightly affected. This pinpoints the importance of entropy in enzyme catalysis and entropy should not be neglected in rational redesign.

  • 68.
    Magnusson, Anders O.
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Rotticci Mulder, Johanna C.
    KTH, Skolan för bioteknologi (BIO).
    Santagostino, Alberto
    KTH, Skolan för bioteknologi (BIO).
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO).
    Creating Space for Large Secondary Alcohols by Rational Redesign of Candida antarctica Lipase B2005Inngår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 6, nr 6, s. 1051-1056Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The active site of Candida antarctica lipase B (CALB) hosts the catalytic triad (Ser-His-Asp), an oxyanion hole and a stereospecificity pocket. During catalysis, the fast-reacting enantiomer of secondary alcohols places its medium-sized substituent in the stereospecificity pocket and its large substituent towards the active-site entrance. The largest group to fit comfortably in the stereospecificity pocket is ethyl, and this restricts the number of secondary alcohols that are good substrates for CALB. In order to overcome this limitation, the size of the stereospecificity pocket was redesigned by changing Trp104. The substrate specificity of the Trp104Ala mutant compared to that of the wild-type lipase increased 270 times towards heptan-4-ol and 5500 times towards nonan-5-ol; this resulted in the high specificity constants 1100 and 830 s(-1)m(-1), respectively. The substrate selectivity changed over 400000 times for nonan-5-ol over propan-2-ol with both Trp104Ala and the Trp104Gln mutations.

  • 69.
    Malm, Magdalena
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Kronqvist, Nina
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Lindberg, Hanna
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Gudmundsdotter, Lindvi
    Affibody AB, Stockholm, Sweden.
    Bass, Tarek
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Frejd, Fredrik Y.
    Höidén-Guthenberg, Ingmarie
    Affibody AB, Stockholm, Sweden.
    Varasteh, Zohreh
    Departmaent of Medical Chemistry, Preclinical PET Platform, Uppasala University, Uppsala, Sweden.
    Orlova, Anna
    Departmaent of Medical Chemistry, Preclinical PET Platform, Uppasala University, Uppsala, Sweden.
    Tolmachev, Vladimir
    Unit of Biomedical Radiation Sciences, Uppsala University, Uppsala, Sweden.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Inhibiting HER3-Mediated Tumor Cell Growth with Affibody Molecules Engineered to Low Picomolar Affinity by Position-Directed Error-Prone PCR-Like Diversification2013Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, nr 5, s. e62791-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

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

  • 70.
    Neiman, Maja
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101). KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Bead based protein profiling in blood2013Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    This thesis is about protein profiling in blood-derived samples using suspension bead ar- rays built with protein affinity reagents, and the evaluation of binding characteristics and potential disease relation of such profiles.

    A central aim of the presented work was to discover and verify disease associated protein profiles in blood-derived samples such as serum or plasma. This was based on immobiliz- ing antigens or antibodies on color-coded beads for a multiplexed analysis. This concept generally allow for a dual multiplexing because hundreds of samples can be screened for hundreds of proteins in a miniaturized and parallelized fashion. At first, protein antigens were used to study humoral immune responses in cattle suffering from a mycoplasma infec- tion (Paper I). Here, the most immunogenic of the applied antigens were identified based on reactivity profiles from the infected cattle, and were combined into an antigen cocktail to serve as a diagnostic assay in a standard ELISA set-up. Next, antibodies and their em- ployment in assays with directly labeled human samples was initiated. This procedure was applied in a study of kidney disorders where screening of plasma resulted in the discovery of a biomarker candidate, fibulin-1 (Paper II). In parallel to the disease related applica- tions, systematic evaluations of the protein profiles were conducted. Protein profiles from 2,300 antibodies were classified on the bases of binding properties in relation to sample heating and stringent washing (Paper III). With a particular focus on heat dependent de- tectability, a method was developed to visualize those proteins that were captured to the beads in an immunoassay by using Western blotting (Paper IV). In conclusion, this thesis presents examples of the possibilities of comparative plasma profiling enabled by protein bead arrays. 

  • 71.
    Neiman, Maja
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101). KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Nygren, Per-Åke
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101). KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M.
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101). KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Validating the selectivity of antibodies used in multiplexed serum profiling via parallel immunocapture analysisManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    The increasing availability of antibodies towards human proteins drives the generation of new and exploratory data, which can be generated by profiling e.g. plasma samples using multiplexed arrays. However, antibody assays can lead to erroneous results due to cross-­‐reactivity to off-­‐targets. Here, we describe an approach in which an antibody-­‐based suspension bead array is combined with subsequent validation of on-­‐ target binding using a coupled affinity purification assay. Based on differential heat treatment of samples, antibody performance was investigated and the results for antibodies directed towards several complement factors provide insights on the detection of proteins in sera from patients with complement deficiency. In conclusion, the combined parallel flow and blot based immunocapture analysis serves an important, first line tool for resolving differently detected serum or plasma protein profiles proposed by antibody arrays. 

  • 72. Nguyen, T. N.
    et al.
    Libon, C.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Subunit vaccine candidates engineered from the central conserved region of the RSV G protein aimed for parenteral or mucosal delivery2013Inngår i: Molecular Vaccines: From Prophylaxis to Therapy-Volume 1, Springer, 2013, s. 103-118Kapittel i bok, del av antologi (Annet vitenskapelig)
    Abstract [en]

    Respiratory syncytial virus (RSV) is a major pathogen causing severe upper and lower respiratory disease in infants and in elderly worldwide. ccording to WHO, an RSV vaccine is urgently needed. Here, we describe the design of various types of subunit vaccine concepts based on molecular engineering aimed to deliver RSV antigens. Gene segment encoding parts of the conserved central region of the RSV G protein (G2Na) were prepared for various expression and delivery formats: (1) prokaryotically expressed and purifi ed G2Na alone or fused to different carrier proteins, one of them, namely, BBG2Na (Alum), has reached clinical trials in the elderly; (2) G protein-derived antigens surface displayed on lived vectors (non pathogenic bacteria) and (3) nucleic acid vectors. These subunit vaccines were administered with or without adjuvant in rodents and in non-human primates by different routes (parenteral or mucosal). We summarise and compare immunogenicity, protective effi cacy and safety conferred by each immunisation format in RSV experimental animal models. Among these, G2Na proved to be the most promising component for an RSV subunit vaccine.

  • 73. Niklasson, Mia
    et al.
    Maddalo, Gianluca
    Sramkova, Zuzana
    Mutlu, Ercan
    Wee, Shimei
    Sekyrova, Petra
    Schmidt, Linnea
    Fritz, Nicolas
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Dehnisch, Ivar
    Kyriatzis, Gregorios
    Krafcikova, Michaela
    Carson, Brittany B.
    Feenstra, Jennifer M.
    Marinescu, Voichita D.
    Segerman, Anna
    Haraldsson, Martin
    Gustavsson, Anna-Lena
    Hammarstrom, Lars G. J.
    Jensen, Annika Jenmalm
    Uhrbom, Lene
    Altelaar, A. F. Maarten
    Linnarsson, Sten
    Uhlen, Per
    Trantirek, Lukas
    Vincent, C. Theresa
    Nelander, Sven
    Enger, Per Oyvind
    Andang, Michael
    Membrane-Depolarizing Channel Blockers Induce Selective Glioma Cell Death by Impairing Nutrient Transport and Unfolded Protein/Amino Acid Responses2017Inngår i: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 77, nr 7, s. 1741-1752Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Glioma-initiating cells (GIC) are considered the underlying cause of recurrences of aggressive glioblastomas, replenishing the tumor population and undermining the efficacy of conventional chemotherapy. Here we report the discovery that inhibiting T-type voltage-gated Ca2+ and KCa channels can effectively induce selective cell death of GIC and increase host survival in an orthotopic mouse model of human glioma. At present, the precise cellular pathways affected by the drugs affecting these channels are unknown. However, using cell-based assays and integrated proteomics, phosphoproteomics, and transcriptomics analyses, we identified the downstreamsignaling events these drugs affect. Changes in plasma membrane depolarization and elevated intracellular Na+, which compromised Na+-dependent nutrient transport, were documented. Deficits in nutrient deficit acted in turn to trigger the unfolded protein response and the amino acid response, leading ultimately to nutrient starvation and GIC cell death. Our results suggest new therapeutic targets to attack aggressive gliomas.

  • 74. Nilsson, D.
    et al.
    Pettersson, M.
    Gustavsson, P.
    Förster, A.
    Hofmeister, W.
    Wincent, J.
    Zachariadis, V.
    Anderlid, B. -M
    Nordgren, A.
    Mäkitie, O.
    Wirta, Valtteri
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Käller, Max
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Vezzi, F.
    Lupski, J. R.
    Nordenskjöld, M.
    Syk Lundberg, E.
    Carvalho, C. M. B.
    Lindstrand, A.
    Whole-Genome Sequencing of Cytogenetically Balanced Chromosome Translocations Identifies Potentially Pathological Gene Disruptions and Highlights the Importance of Microhomology in the Mechanism of Formation2017Inngår i: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 38, nr 2, s. 180-192Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Most balanced translocations are thought to result mechanistically from nonhomologous end joining or, in rare cases of recurrent events, by nonallelic homologous recombination. Here, we use low-coverage mate pair whole-genome sequencing to fine map rearrangement breakpoint junctions in both phenotypically normal and affected translocation carriers. In total, 46 junctions from 22 carriers of balanced translocations were characterized. Genes were disrupted in 48% of the breakpoints; recessive genes in four normal carriers and known dominant intellectual disability genes in three affected carriers. Finally, seven candidate disease genes were disrupted in five carriers with neurocognitive disabilities (SVOPL, SUSD1, TOX, NCALD, SLC4A10) and one XX-male carrier with Tourette syndrome (LYPD6, GPC5). Breakpoint junction analyses revealed microhomology and small templated insertions in a substantive fraction of the analyzed translocations (17.4%; n = 4); an observation that was substantiated by reanalysis of 37 previously published translocation junctions. Microhomology associated with templated insertions is a characteristic seen in the breakpoint junctions of rearrangements mediated by error-prone replication-based repair mechanisms. Our data implicate that a mechanism involving template switching might contribute to the formation of at least 15% of the interchromosomal translocation events.

  • 75. Nogueira, Eugenia
    et al.
    Mangialavori, Irene C.
    Loureiro, Ana
    Azoia, Nuno G.
    Sarria, Marisa P.
    Nogueira, Patricia
    Freitas, Jaime
    Härmark, Johan
    KTH, Skolan för teknik och hälsa (STH), Naturvetenskap och biomedicin, Strukturell bioteknik. Karolinska Inst, Sch Technol & Hlth.
    Shimanovich, Ulyana
    Rollett, Alexandra
    Lacroix, Ghislaine
    Bernardes, Goncalo J. L.
    Guebitz, Georg
    Hebert, Hans
    Moreira, Alexandra
    Carmo, Alexandre M.
    Rossi, Juan Pablo F. C.
    Gomes, Andreia C.
    Preto, Ana
    Cavaco-Paulo, Artur
    Peptide Anchor for Folate-Targeted Liposomal Delivery2015Inngår i: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 16, nr 9, s. 2904-2910Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Specific folate receptors are abundantly overexpressed in chronically activated macrophages and in most cancer cells. Directed folate receptor targeting using liposomes is usually achieved using folate linked to a phospholipid or cholesterol anchor. This link is formed using a large spacer like polyethylene glycol. Here, we report an innovative strategy for targeted liposome delivery that uses a hydrophobic fragment of surfactant protein D linked to folate. Our proposed spacer is a small 4 amino acid residue linker. The peptide conjugate inserts deeply into the lipid bilayer without affecting liposomal integrity, with high stability and specificity. To compare the drug delivery potential of both liposomal targeting systems, we encapsulated the nuclear dye Hoechst 34580. The eventual increase in blue fluorescence would only be detectable upon liposome disruption, leading to specific binding of this dye to DNA. Our delivery system was proven to be more efficient (2-fold) in Caco-2 cells than classic systems where the folate moiety is linked to liposomes by polyethylene glycol.

  • 76.
    Ohlsson, Anna B.
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Djerbi, Soraya
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Winzell, Anders
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Bessueille, Laurence
    Ståldal, Veronika
    Li, Xinguo
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Blomqvist, Kristina
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Bulone, Vincent
    Teeri, Tuula T.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Berglund, Torkel
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Cell suspension cultures of Populus tremula x P. tremuloides exhibit a high level of cellulose synthase gene expression that coincides with increased in vitro cellulose synthase activity.2006Inngår i: Protoplasma, ISSN 0033-183X, E-ISSN 1615-6102, Vol. 228, nr 4, s. 221-9Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Compared to wood, cell suspension cultures provide convenient model systems to study many different cellular processes in plants. Here we have established cell suspension cultures of Populus tremula L. x P. tremuloides Michx. and characterized them by determining the enzymatic activities and/or mRNA expression levels of selected cell wall-specific proteins at the different stages of growth. While enzymes and proteins typically associated with primary cell wall synthesis and expansion were detected in the exponential growth phase of the cultures, the late stationary phase showed high expression of the secondary-cell-wall-associated cellulose synthase genes. Interestingly, detergent extracts of membranes from aging cell suspension cultures exhibited high levels of in vitro cellulose synthesis. The estimated ratio of cellulose to callose was as high as 50 : 50, as opposed to the ratio of 30 : 70 so far achieved with membrane preparations extracted from other systems. The increased cellulose synthase activity was also evidenced by higher levels of Calcofluor white binding in the cell material from the stationary-phase cultures. The ease of handling cell suspension cultures and the improved capacity for in vitro cellulose synthesis suggest that these cultures offer a new basis for studying the mechanism of cellulose biosynthesis.

  • 77.
    Olofsson, Karl
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Carannante, V.
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Stockholm, Sweden..
    Ohlin, Mathias
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Frisk, Thomas
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Kushiro, K.
    Univ Tokyo, Dept Bioengn, Tokyo, Japan..
    Takai, M.
    Univ Tokyo, Dept Bioengn, Tokyo, Japan..
    Lundqvist, A.
    Karolinska Inst, Dept Oncol Pathol, Stockholm, Sweden..
    Önfelt, Björn
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Wiklund, Martin
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Acoustic formation of multicellular tumor spheroids enabling on-chip functional and structural imaging2018Inngår i: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 18, nr 16, s. 2466-2476Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Understanding the complex 3D tumor microenvironment is important in cancer research. This microenvironment can be modelled in vitro by culturing multicellular tumor spheroids (MCTS). Key challenges when using MCTS in applications such as high-throughput drug screening are overcoming imaging and analytical issues encountered during functional and structural investigations. To address these challenges, we use an ultrasonic standing wave (USW) based MCTS culture platform for parallel formation, staining and imaging of 100 whole MCTS. A protein repellent amphiphilic polymer coating enables flexible production of high quality and unanchored MCTS. This enables high-content multimode analysis based on flow cytometry and in situ optical microscopy. We use HepG2 hepatocellular carcinoma, A498 and ACHN renal carcinoma, and LUTC-2 thyroid carcinoma cell lines to demonstrate (i) the importance of the ultrasound-coating combination, (ii) bright field image based automatic characterization of MTCS, (iii) detailed deep tissue confocal imaging of whole MCTS mounted in a refractive index matching solution, and (iv) single cell functional analysis through flow cytometry of single cell suspensions of disintegrated MTCS. The USW MCTS culture platform is customizable and holds great potential for detailed multimode MCTS analysis in a high-content manner.

  • 78. Orlova, Anna
    et al.
    Malm, Magdalena
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Lindberg, Hanna
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Varasteh, Zohreh
    Rosestedt, Maria
    Tolmachev, Vadimir
    Kronqvist, Nina
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Feasibility of radionuclide imaging of HER3-expressing tumors using affibody molecules2013Inngår i: Journal of labelled compounds & radiopharmaceuticals, ISSN 0362-4803, E-ISSN 1099-1344, Vol. 56, s. S11-S11Artikkel i tidsskrift (Annet vitenskapelig)
  • 79. Orlova, Anna
    et al.
    Malm, Magdalena
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Rosestedt, Maria
    Varasteh, Zohreh
    Andersson, Ken
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Selvaraju, Ram Kumar
    Altai, Mohamed
    Honarvar, Hadis
    Strand, Joanna
    Stahl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Tolmachev, Vladimir
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Imaging of HER3-expressing xenografts in mice using a Tc-99m(CO)(3)-HEHEHE-Z(HER3:08699) affibody molecule2014Inngår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 41, nr 7, s. 1450-1459Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Human epidermal growth factor receptor type 3 (HER3) is a transmembrane receptor tyrosine kinase belonging to the HER (ErbB) receptor family. Membranous expression of HER3 is associated with trastuzumab resistance in breast cancer and the transition to androgen independence in prostate cancer. Imaging of HER3 expression in malignant tumors may provide important diagnostic information that can influence patient management. Affibody molecules with low picomolar affinity to HER3 were recently selected. The aim of this study was to investigate the feasibility of HER3 imaging using radiolabeled Affibody molecules. A HER3-binding Affibody molecule, Z(08699), with a HEHEHE-tag on N-terminus was labeled with Tc-99m(CO)(3) using an IsoLink kit. In vitro and in vivo binding specificity and the cellular processing of the labeled binder were evaluated. Biodistribution of Tc-99m(CO)(3)-HEHEHE-Z(08699) was studied over time in mice bearing HER3-expressing xenografts. HEHEHE-Z(08699) was labeled with Tc-99m(CO)(3) with an isolated yield of > 80 % and a purity of > 99 %. Binding of Tc-99m(CO)(3)-HEHEHE-Z(08699) was specific to BT474 and MCF7 (breast cancer), and LS174T (colon cancer) cells. Cellular processing showed rapid binding and relatively quick internalization of the receptor/Affibody molecule complex (70 % of cell-associated radioactivity was internalized after 24 h). The tumor targeting was receptor mediated and the excretion was predominantly renal. Receptor-mediated uptake was also found in the liver, lung, stomach, intestine, and salivary glands. At 4 h pi, tumor-to-blood ratios were 7 +/- 3 for BT474, and 6 +/- 2 for LS174T xenografts. LS174T tumors were visualized by microSPECT 4 h pi. The results of this study suggest the feasibility of HER3-imaging in malignant tumors using Affibody molecules.

  • 80.
    Pathak, Anuj
    et al.
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, SE-17177 Stockholm, Sweden..
    Bergstrand, Jan
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Kvant- och biofotonik.
    Sender, Vicky
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, SE-17177 Stockholm, Sweden..
    Spelmink, Laura
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, SE-17177 Stockholm, Sweden..
    Aschtgen, Marie-Stephanie
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, SE-17177 Stockholm, Sweden..
    Muschiol, Sandra
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, SE-17177 Stockholm, Sweden..
    Widengren, Jerker
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Kvant- och biofotonik.
    Henriques-Normark, Birgitta
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, SE-17177 Stockholm, Sweden.;Nanyang Technol Univ, Lee Kong Chian Sch Med LKC, Singapore 639798, Singapore.;Nanyang Technol Univ, Singapore Ctr Environm Life Sci Engn SCELSE, Singapore 639798, Singapore.;Karolinska Univ Hosp, Dept Clin Microbiol, SE-17176 Stockholm, Sweden..
    Factor H binding proteins protect division septa on encapsulated Streptococcus pneumoniae against complement C3b deposition and amplification2018Inngår i: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 9, artikkel-id 3398Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Streptococcus pneumoniae evades C3-mediated opsonization and effector functions by expressing an immuno-protective polysaccharide capsule and Factor H (FH)-binding proteins. Here we use super-resolution microscopy, mutants and functional analysis to show how these two defense mechanisms are functionally and spatially coordinated on the bacterial cell surface. We show that the pneumococcal capsule is less abundant at the cell wall septum, providing C3/C3b entry to underlying nucleophilic targets. Evasion of C3b deposition at division septa and lateral amplification underneath the capsule requires localization of the FH-binding protein PspC at division sites. Most pneumococcal strains have one PspC protein, but successful lineages in colonization and disease may have two, PspC1 and PspC2, that we show affect virulence differently. We find that spatial localization of these FH-recruiting proteins relative to division septa and capsular layer is instrumental for pneumococci to resist complement-mediated opsonophagocytosis, formation of membrane-attack complexes, and for the function as adhesins.

  • 81.
    Perez-Zabaleta, Mariel
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    (R)-3-Hydroxybutyrate production in a metabolically engineered Escherichia coli2016Inngår i: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 33, s. S117-S117Artikkel i tidsskrift (Fagfellevurdert)
  • 82.
    Persson, Helena
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Preger, C.
    Marcon, E.
    Lengqvist, J.
    Gräslund, S.
    Antibody validation by immunoprecipitation followed by mass spectrometry analysis2017Inngår i: Methods in Molecular Biology, Humana Press Inc. , 2017, s. 175-187Konferansepaper (Fagfellevurdert)
    Abstract [en]

    We describe a mass spectrometry-based approach for validation of antibody specificity. This method allows validation of antibodies or antibody fragments, against their endogenous targets. It can assess if the antibody is able to bind to its native antigen in cell lysates among thousands of other proteins, DNA, RNA, and other cellular components. In addition, it identifies other proteins the antibody is able to immunoprecipitate allowing for the assessment of antibody specificity and selectivity. This method is easily scalable, adaptable to different cell lines and conditions and has been shown to be reproducible between multiple laboratories.

  • 83.
    Petrou, Georgia
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Jansson, Ronnie
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Hogqvist, Mark
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH).
    Erlandsson, Johan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Fiber- och polymerteknologi.
    Wågberg, Lars
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Fiber- och polymerteknologi.
    Hedhammar, My
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Crouzier, Thomas
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Genetically Engineered Mucoadhesive Spider Silk2018Inngår i: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 19, nr 8, s. 3268-3279Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Mucoadhesion is defined as the adhesion of a material to the mucus gel covering the mucous membranes. The mechanisms controlling mucoadhesion include nonspecific electrostatic interactions and specific interactions between the materials and the mucins, the heavily glycosylated proteins that form the mucus gel. Mucoadhesive materials can be used to develop mucosal wound dressings and noninvasive transmucosal drug delivery systems. Spider silk, which is strong, biocompatible, biodegradable, nontoxic, and lightweight would serve as an excellent base for the development of such materials. Here, we investigated two variants of the partial spider silk protein 4RepCT genetically engineered in order to functionalize them with mucoadhesive properties. The pLys-4RepCT variant was functionalized with six cationically charged lysines, aiming to provide nonspecific adhesion from electrostatic interactions with the anionically charged mucins, while the hGal3-4RepCT variant was genetically fused with the Human Galectin-3 Carbohydrate Recognition Domain which specifically binds the mucin glycans Gal beta 1-3GlcNAc and Gal beta 1-4GlcNAc. First, we demonstrated that coatings, fibers, meshes, and foams can be readily made from both silk variants. Measured by the adsorption of both bovine submaxillary mucin and pig gastric mucin, the newly produced silk materials showed enhanced mucin binding properties compared with materials of wild-type (4RepCT) silk. Moreover, we showed that pLys-4RepCT silk coatings bind mucins through electrostatic interactions, while hGal3-4RepCT silk coatings bind mucins through specific glycan-protein interactions. We envision that the two new mucoadhesive silk variants pLys-4RepCT and hGal3-4RepCT, alone or combined with other biofunctional silk proteins, constitute useful new building blocks for a range of silk protein-based materials for mucosal treatments.

  • 84.
    Polyutov, Sergey
    KTH, Skolan för bioteknologi (BIO).
    Electron-nuclear Dynamics in Nonlinear Optics and X-ray spectroscopy2007Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    This thesis is devoted to theoretical studies of the role of nuclear vibrations on nonlinear and linear absorption, pulse propagation, and resonant scattering of light. The molecular parameters needed for the simulations are obtained through suitable quantum chemical calculations, which are compared with available experimental data.

    The first part of the thesis addresses to modeling of ampli ed spontaneous emission (ASE) in organic chromophores recently studied in a series of experiments. To explain the threshold behavior of the ASE spectra we invoke the idea of competition between di erent ASE channels and non-radiative quenching of the lasing levels. We show that the ASE spectrum changes drastically when the pump intensity approaches the threshold level, namely, when the ASE rate approaches the rate of vibrational relaxation or the rate of solute-solvent relaxation in the rst excited state. According to our simulations the ASE intensity experiences oscillations. Temporal self-pulsations of forward and backward propagating ASE pulses occur due to two reasons: i) the interaction of co- and counter-propagating ASE, and ii) the competition between the ampli ed spontaneous emission and o -resonant absorption.

    In the second part of the thesis we explore two-photon absorption taking into account nuclear vibrational degrees of freedom. The theory, applied to the N101 molecule [p-nitro-p'- diphenylamine stilbene], shows that two-step absorption is red shifted relative to one-photon absorption spectrum in agreement with the measurements. The reason for this e ect is the one-photon absorption from the first excited state. Simulations show that two mechanisms are responsible for the population of this state, two-photon absorption and offresonant one-photon absorption by the wing of the spectral line.

    In the third part of the thesis we study multi-photon dynamics of photobleaching by a periodical sequence of short laser pulses. It is found that the photobleaching as well as the uorescence follow double-exponential dynamics.

    The fourth part of the thesis is devoted to the role of the nuclear dynamics in x-ray spectroscopy. Our studies show that the vibronic coupling of close lying core excited states strongly a ects the resonant x-ray Raman scattering from ethylene and benzene molecules. We demonstrate that the manifestation of the non-adiabatic e ects depends strongly on the detuning of photon energy from the top of photoabsorption. The electronic selection rules are shown to break down when the excitation energy is tuned in resonance with the symmetry breaking vibrational modes. Selection rules are then restored for large detuning. We obtained good agreement with experiment. Finally, our multi-mode theory is applied to simulations of the resonant Auger and x-ray absorption spectra of the ethyne molecule.

  • 85. Quince, Christopher
    et al.
    Delmont, Tom O.
    Raguideau, Sebastien
    Alneberg, Johannes
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Darling, Aaron E.
    Collins, Gavin
    Eren, A. Murat
    DESMAN: a new tool for de novo extraction of strains from metagenomes2017Inngår i: Genome Biology, ISSN 1465-6906, E-ISSN 1474-760X, Vol. 18, artikkel-id 181Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We introduce DESMAN for De novo Extraction of Strains from Metagenomes. Large multi-sample metagenomes are being generated but strain variation results in fragmentary co-assemblies. Current algorithms can bin contigs into metagenome-assembled genomes but are unable to resolve strain-level variation. DESMAN identifies variants in core genes and uses co-occurrence across samples to link variants into haplotypes and abundance profiles. These are then searched for against non-core genes to determine the accessory genome of each strain. We validated DESMAN on a complex 50-species 210-genome 96-sample synthetic mock data set and then applied it to the Tara Oceans microbiome.

  • 86.
    Qundos, Ulrika
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Antibody based plasma protein profiling2013Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    This thesis is about protein profiling in serum and plasma using antibody suspension bead arrays for the analysis of biobanked samples and in the context of prostate cancer biomarker discovery. The influence of sample preparation methods on antibody based protein profiles were investigated (Papers I-III) and a prostate cancer candidate biomarker identified and verified (Papers III-V). Furthermore, a perspective on the research area affinity proteomics and its’ employment in biomarker discovery, for improved understanding and potentially improved disease diagnosis, is provided.

    Paper I presents the results of a comparative plasma and serum protein profiling study, with a targeted biomarker discovery approach in the context of metabolic syndrome. The study yielded a higher number of significant findings and a low experimental variability in blood samples prepared as plasma. Paper II investigated the effects from post-centrifugation delays at different temperatures prior sample storage of serum and plasma samples. Minor effects were found on the detected levels of more than 300 predicted or known plasma proteins. In Paper III, the detectability of proteins in plasma was explored by exposing samples to different pre-analytical heat treatments, prior target capture. Heat induced epitope retrieval was observed for approximately half of the targeted proteins, and resulted in the discovery of different candidate markers for prostate cancer. Several antibodies towards the prostate cancer candidate biomarker CNDP1 were generated, epitope mapped and evaluated in a bead based sandwich immunoassay, as presented in Papers IV and V. Furthermore, the developed sandwich immunoassay targeting multiple distinct CNDP1 epitopes in more than 1000 samples, confirmed the association of CNDP1 levels to aggres- sive prostate cancer and more specifically to prostate cancer patients with regional lymph node metastasis (Paper V).

    As an outcome of the present investigations and in parallel to studies within the Biobank profiling research group, valuable lessons from study design and multiplex antibody analysis of plasma within biomarker discovery to experimental, technical and biological verifications have been collected.

  • 87.
    Redin, David
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi.
    Phasing single DNA molecules with barcode linked sequencing2018Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Elucidation of our genetic constituents has in the past decade predominately taken the form of short-read DNA sequencing. Revolutionary technology developments have enabled vast amounts of biological information to be obtained, but from a medical standpoint it has yet to live up to the promise of associating individual genotypes to phenotypic states of wide-spread clinical relevance. The mechanisms by which complex phenotypes arise have been difficult to ascertain and the value of short-read sequencing platforms have been limited in this regard. It has become evident that resolving the full spectrum of genetic heterogeneity requires accurate long range information of individual haplotypes to be distinguished. Long-range haplotyping information can be obtained experimentally by long-read sequencing platforms or through linkage of short sequencing reads by means of a common barcode. This thesis explores these solutions, primarily through the development of novel technologies to phase short sequences of single molecules using DNA barcoding. A new method for high-throughput phasing of single DNA molecules, achieved by the production and utilization of uniquely barcoded beads in emulsion droplets, is described in Paper I. The results confirm that complex libraries of beads featuring mutually exclusive barcodes can be generated through clonal PCR amplification, and that these beads can be used to phase variations of the 16s rRNA gene which reduces the ambiguity of classifying bacterial species for metagenomics. Paper II describes a second methodology (‘Droplet Barcode Sequencing’) which simplifies the concept of barcoding DNA fragments by omitting the need for beads and instead relying on clonal amplification of single barcoding oligonucleotides. This study also increases the amount of information that can be linked, which is showcased by phasing all exons of the HLA-A gene and successfully resolving all the alleles present in a sample pool of eight individuals. Paper III expands on this work and explores the use of a single molecule sequencing platform to provide full-length sequencing coverage of six genes of the HLA family. The results show that while genes shorter than 10 kb can be resolved with a high degree of accuracy, compensating for a relatively high error rate by means of increased coverage can be challenging for larger genomic loci. Finally, Paper IV introduces the use of barcode-linked reads on an unprecedented scale, with a new assay that enables low-cost haplotyping of whole genomes without the need for predetermined capture sequences. This technology is utilized to generate a haplotype-resolved human genome, call large-scale structural variants and perform reference-free assembly of bacterial and human genomes. At a cost of only $19 USD per sample, this technology makes the benefits of long-range haplotyping available to the vast majority of laboratories which currently rely solely on short-read sequencing platforms.

  • 88.
    Redin, David
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi.
    Frick, Tobias
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi.
    Aghelpasand, Hooman
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi.
    Theland, Jennifer
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi.
    Käller, Max
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi.
    Borgström, Erik
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi.
    Olsen, Remi-Andre
    Stockholms Universitet.
    Ahmadian, Afshin
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi.
    Efficient whole genome haplotyping and single molecule phasing with barcode-linked readsManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    The future of human genomics is one that seeks to resolve the entirety of genetic variation through sequencing. The prospect of utilizing genomics for medical purposes require cost-efficient and accurate base calling, long-range haplotyping capability, and reliable calling of structural variants. Short-read sequencing has lead the development towards such a future but has struggled to meet the latter two of these needs. To address this limitation, we developed a technology that preserves the molecular origin of short sequencing reads, with an insignificant increase to sequencing costs. We demonstrate a library preparation method which enables whole genome haplotyping, long-range phasing of single DNA molecules, and de novo genome assembly through barcode-linked reads (BLR). Millions of random barcodes are used to reconstruct megabase-scale phase blocks and call structural variants. We also highlight the versatility of our technology by generating libraries from different organisms using picograms to nanograms of input material.

  • 89.
    Rinne, Sara S.
    et al.
    Uppsala Univ, Dept Med Chem, Uppsala, Sweden..
    Leitao, Charles Dahlsson
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Mitran, Bogdan
    Uppsala Univ, Dept Med Chem, Uppsala, Sweden..
    Bass, Tarek
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Andersson, Ken G.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Tolmachev, Vladimir
    Uppsala Univ, Dept Immunol Genet & Pathol, Uppsala, Sweden..
    Ståhl, Stefan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Löfblom, John
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Orlova, Anna
    Uppsala Univ, Dept Med Chem, Uppsala, Sweden.;Uppsala Univ, Sci Life Lab, Uppsala, Sweden..
    Optimization of HER3 expression imaging using affibody molecules: Influence of chelator for labeling with indium-1112019Inngår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, artikkel-id 655Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Radionuclide molecular imaging of human epidermal growth factor receptor 3 (HER3) expression using affibody molecules could be used for patient stratification for HER3-targeted cancer therapeutics. We hypothesized that the properties of HER3-targeting affibody molecules might be improved through modification of the radiometal-chelator complex. Macrocyclic chelators NOTA (1,4,7-triazacyclononane-N,N',N ''-triacetic acid), NODAGA (1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-triazacyclononane), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid), and DOTAGA (1,4,7,10-tetraazacyclododececane, 1-(glutaric acid)-4,7,10-triacetic acid) were conjugated to the C-terminus of anti-HER3 affibody molecule Z(08698) and conjugates were labeled with indium-111. All conjugates bound specifically and with picomolar affinity to HER3 in vitro. In mice bearing HER3-expressing xenografts, no significant difference in tumor uptake between the conjugates was observed. Presence of the negatively charged In-111-DOTAGA-complex resulted in the lowest hepatic uptake and the highest tumor-to-liver ratio. In conclusion, the choice of chelator influences the biodistribution of indium-111 labeled anti-HER3 affibody molecules. Hepatic uptake of anti-HER3 affibody molecules could be reduced by the increase of negative charge of the radiometal-chelator complex on the C-terminus without significantly influencing the tumor uptake.

  • 90.
    Robinson, Jonathan L.
    et al.
    Chalmers Univ Technol, Dept Biol & Biol Engn, Kemivagen 10, Gothenburg, Sweden.;Chalmers Univ Technol, Wallenberg Ctr Prot Res, Kemivagen 10, Gothenburg, Sweden..
    Feizi, Amir
    Chalmers Univ Technol, Dept Biol & Biol Engn, Kemivagen 10, Gothenburg, Sweden.;Novo Nordisk Res Ctr Oxford, Old Campus Rd, Oxford, England..
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Nielsen, Jens
    KTH, Centra, Science for Life Laboratory, SciLifeLab. Chalmers Univ Technol, Dept Biol & Biol Engn, Kemivagen 10, Gothenburg, Sweden ; Chalmers Univ Technol, Wallenberg Ctr Prot Res, Kemivagen 10, Gothenburg, Sweden ; Tech Univ Denmark, Novo Nordisk Fdn, Ctr Biosustainabil, DK-2800 Lyngby, Denmark.
    A Systematic Investigation of the Malignant Functions and Diagnostic Potential of the Cancer Secretome2019Inngår i: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 26, nr 10, s. 2622-+Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The collection of proteins secreted from a cell-the secretome-is of particular interest in cancer pathophysiology due to its diagnostic potential and role in tumorigenesis. However, cancer secretome studies are often limited to one tissue or cancer type or focus on biomarker prediction without exploring the associated functions. We therefore conducted a pan-cancer analysis of secretome gene expression changes to identify candidate diagnostic biomarkers and to investigate the underlying biological function of these changes. Using transcriptomic data spanning 32 cancer types and 30 healthy tissues, we quantified the relative diagnostic potential of secretome proteins for each cancer. Furthermore, we offer a potential mechanism by which cancer cells relieve secretory pathway stress by decreasing the expression of tissue-specific genes, thereby facilitating the secretion of proteins promoting invasion and proliferation. These results provide a more systematic understanding of the cancer secretome, facilitating its use in diagnostics and its targeting for therapeutic development.

  • 91.
    Rockberg, Johan
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Hjelm, Barbara
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Epitope mapping using gram-positive surface display2010Inngår i: Current Protocols in Immunology, ISSN 1934-3671, nr SUPPL. 90, s. 9.9.1-9.9.17Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    Antibodies have proven to be invaluable tools for a vast number of applications during the last decades, including protein purification and characterization, medical diagnosis and imaging, and treatment using therapeutic antibiotics. No matter what the aims of the application are, the antibodys binding characteristics will still be the main features determining the assays reliability. Here, we describe a protocol for determination of antibody-binding epitopes using an antigen-focused, library-based approach where library members are generated by fragmentation of antigen DNA and presented as cloned peptides on the cell surface of the Gram-positive bacterium Staphylococcus carnosus. The rigid cell structure of this organism allows for multivalent expression and permits rapid library analysis and sorting of antibody-binding cells using flow-sorting devices. Epitopes are determined by DNA sequencing of the sorted cells and alignment back to the antigen sequence. The protocol described here has been shown useful for mapping of both monoclonal and polyclonal binders with varying epitope lengths.

  • 92.
    Rozkov, Aleksei D.
    et al.
    KTH, Tidigare Institutioner, Bioteknologi.
    Enfors, Sven-Olof
    KTH, Tidigare Institutioner, Bioteknologi.
    Analysis and control of proteolysis of recombinant proteins in Escherichia coli.2004Inngår i: Advances in biochemical engineering/biotechnology, ISSN 0724-6145, Vol. 89, s. 163-195Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Proteolysis is one of the reasons for poor production of recombinant proteins in Escherichia coli. Important properties of E. coli proteases, which are relevant for the production of recombinant proteins, are reviewed. Furthermore, various strategies to control the proteolysis of the recombinant proteins are presented. These strategies for control of proteolysis can be applied on various stages of the process: design of more stable protein, a modification of the host cell in respect to proteolytic activity, optimisation of cultivation and downstream processing. However, before implementing these measures the proteolysis rate should be measured in order to calculate a potential benefit of reduced proteolysis rate.

  • 93. Samalova, Marketa
    et al.
    Melida, Hugo
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Vilaplana, Francisco
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Bulone, Vincent
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Soanes, Darren M.
    Talbot, Nicholas J.
    Gurr, Sarah J.
    The beta-1,3-glucanosyltransferases (Gels) affect the structure of the rice blast fungal cell wall during appressorium-mediated plant infection2017Inngår i: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 19, nr 3, artikkel-id UNSP e12659Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The fungal wall is pivotal for cell shape and function, and in interfacial protection during host infection and environmental challenge. Here, we provide the first description of the carbohydrate composition and structure of the cell wall of the rice blast fungus Magnaporthe oryzae. We focus on the family of glucan elongation proteins (Gels) and characterize five putative beta-1,3-glucan glucanosyltransferases that each carry the Glycoside Hydrolase 72 signature. We generated targeted deletion mutants of all Gel isoforms, that is, the GH72(+), which carry a putative carbohydrate-binding module, and the GH72(-)Gels, without this motif. We reveal that M. oryzae GH72(+) GELs are expressed in spores and during both infective and vegetative growth, but each individual Gel enzymes are dispensable for pathogenicity. Further, we demonstrated that Delta gel1 Delta gel3 Delta gel4 null mutant has a modified cell wall in which 1,3-glucans have a higher degree of polymerization and are less branched than the wild-type strain. The mutant showed significant differences in global patterns of gene expression, a hyper-branching phenotype and no sporulation, and thus was unable to cause rice blast lesions (except via wounded tissues). We conclude that Gel proteins play significant roles in structural modification of the fungal cell wall during appressorium-mediated plant infection.

  • 94.
    Shalaly, Nancy Dekki
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Aneiros, Eduardo
    Blank, Michael
    Mueller, Johan
    Nyman, Eva
    Blind, Michael
    Dabrowski, Michael A.
    Andersson, Christin V.
    Sandberg, Kristian
    Positive Modulation of the Glycine Receptor by Means of Glycine Receptor-Binding Aptamers2015Inngår i: Journal of Biomolecular Screening, ISSN 1087-0571, E-ISSN 1552-454X, Vol. 20, nr 9, s. 1112-1123Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    According to the gate control theory of pain, the glycine receptors (GlyRs) are putative targets for development of therapeutic analgesics. A possible approach for novel analgesics is to develop a positive modulator of the glycine-activated Cl- channels. Unfortunately, there has been limited success in developing drug-like small molecules to study the impact of agonists or positive modulators on GlyRs. Eight RNA aptamers with low nanomolar affinity to GlyR1 were generated, and their pharmacological properties analyzed. Cytochemistry using fluorescein-labeled aptamers demonstrated GlyR1-dependent binding to the plasma membrane but also intracellular binding. Using a fluorescent membrane potential assay, we could identify five aptamers to be positive modulators. The positive modulation of one of the aptamers was confirmed by patch-clamp electrophysiology on L(tk) cells expressing GlyR1 and/or GlyR1. This aptamer potentiated whole-cell Cl- currents in the presence of low concentrations of glycine. To our knowledge, this is the first demonstration ever of RNA aptamers acting as positive modulators for an ion channel. We believe that these aptamers are unique and valuable tools for further studies of GlyR biology and possibly also as tools for assay development in identifying small-molecule agonists and positive modulators.

  • 95.
    Sjöberg, Ronald
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    On Generation and Applications of High-Density Protein Microarrays2015Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Affinity proteomics has experienced rapid development over the last two decades and one of the most promising platforms to emerge are the protein microarrays. The combination of affinity reagents and miniaturisation enables assays for simultaneous high throughput and sensitive protein analysis. Due to the combination of these desrable properties, a multitude of protein array platforms for rapid and efficient study of proteomes and protein interactions are in use today. Although the protein microarray field has more than two decades of history to look back on the development of new protein microarray platforms continues to this day and beyond.

    In the paper I in this thesis, a microarray of eluates from dried blood spot samples collected from neonates were designed and utilised for detection of complement factor 3 (C3) deficiency. The data acquired from the microarrays platform were compared to C3 levels obtained through enzyme-linked immunosorbant assay (ELISA), and the microarray assay were found to separate the C3 deficient samples from the controls. The conclusion of this investigation was that the microarray platform would be suitable for high-throughput screening of C3 deficiency in neonates. Paper II outlines the work in developing a multiplex platform for validation of affinity reagents. A set of 398 affinity binders, originating from five research groups, were profiled against 432 antigens and representing both polyclonal rabbit antibodies, monoclonal mouse antibodies, and recombinant single-chain variable fragments. Approximately 50% of the binders were found to preferably recognise their intended target while 10% of the binders did not generate any, or low, signals with their respective targets. For paper III, a reverse phase array (RPPA) platform using fluorescence-based detection of IgA deficiency in over 2.000 samples where validated on a label-free detection system and ELISA. The data from the label-free platform and the RPPA were found agree well with each other while data from ELISA did with neither of them. It was found that the label-free platform proved to be well-suited for detection of IgA in serum. Paper IV describes one of the world’s largest protein microarrays containing 21.120 recombinant protein fragments. We describe some of the possible applications of these large-scale arrays, such as binding profiles for the validation of antibodies with 11.520 and 21.120 recombinant proteins, as well as screening for autoimmunity in human serum samples.

  • 96.
    Sjöstedt, Evelina
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi.
    Towards a deeper understanding of the human brain2018Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Identifying the proteome variation in different parts of the body provides fundamental molecular details, enabling further findings and mapping of tissue specific proteins. By combining quantitative transcriptomics with qualitative antibody based proteomics, the Human Protein Atlas (HPA) project aims to protein profile each human protein-coding gene. Genes with varying expression levels in the different tissue types are categorized as tissue elevated in one tissue compared to others, thus connecting genes to potential tissue specific functions. This thesis focuses on the most complex organ in the human body, the brain. With its billions of neurons specifically organized and interconnected, the ability of not only controlling the body but also responsible for higher cognitive functions, the brain is still not fully understood.

    In my search for brain important proteins, genes were classified at different stages based on expression levels. In Paper I and II the transcriptome of cerebral cortex was compared with peripheral organs to classify genes with elevated expression in the brain. Brain expression information was expanded by including external data (GTEx and FANTOM5) into the analysis, in Paper III. Thereafter, in Paper IV, the three datasets (HPA, GTEx and FANTOM5) were aligned and combined, enabling a consensus classification with an improved representation of the brain complexity. The most recent classification provided whole body gene expression profiles and out of the 19,670 protein-coding genes, 2,501 were expressed at elevated levels in the brain compared to the other tissue types. Twelve individual regions represented the brain as an organ, and were further analyzed and compared for regional classification of gene expression. One thousand genes showed regional variation in expression level, thus classified as regionally elevated within the brain. Interestingly, less than 500 of the genes classified as brain elevated on the whole body level, and were also regionally elevated in the brain. Many genes with regionally variable expression within the brain showed higher expression in a peripheral organ than in the brain when comparing whole body expression. Based on elevated expression in the brain or brain regions, more than 3,000 genes were suggested to be of high importance to the brain.

    In addition, this high-throughput approach to combine transcriptomics and protein profiles in tissues and cells further generated new knowledge in several different other aspects: better understanding of uncharacterized and “missing proteins” (Paper III), validation of an antibody improving classification of pituitary adenoma (Paper V) and in Paper VI the possibility to explore cancer specific expression in relation to clinical data and normal tissue expression.

    There are multiple diseases of the brain that are poorly understood on both a cellular and molecular level. While my work mainly focused on identifying and understanding the molecular organization of the normal brain, the ultimate goal of mapping and studying the normal expression baseline is to understand the molecular aspects of disease and identify ways to prevent, treat and cure diseases.

  • 97.
    Sjöstedt, Evelina
    et al.
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi.
    Sivertsson, Åsa
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi.
    Norradin, Feria Hikmet
    Department of Immunology, Genetics and Pathology, Uppsala University.
    Katona, Borbala
    Department of Immunology, Genetics and Pathology, Uppsala University.
    Näsström, Åsa
    Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Sweden.
    Vuu, Jimmy
    Department of Immunology, Genetics and Pathology, Uppsala university.
    Kesti, Dennis
    Department of Immunology, Genetics and Pathology, Uppsala University.
    Oksvold, Per
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Edqvist, Per-Henrik
    Department of Immunology, Genetics and Pathology, Uppsala University.
    Olsson, Ingmarie
    Department of Immunology, Genetics and Pathology, Uppsala University.
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi.
    Lindskog, Cecilia
    Department of Immunology, Genetics and Pathology, Uppsala University.
    Integration of Transcriptomics and Antibody-Based Proteomics for Exploration of Proteins Expressed in Specialized Tissues2018Inngår i: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 17, nr 12, s. 4127-4137Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A large portion of human proteins are referred to as missing proteins, defined as protein-coding genes that lack experimental data on the protein level due to factors such as temporal expression, expression in tissues that are difficult to sample, or they actually do not encode functional proteins. In the present investigation, an integrated omics approach was used for identification and exploration of missing proteins. Transcriptomics data from three different sourcesthe Human Protein Atlas (HPA), the GTEx consortium, and the FANTOM5 consortiumwere used as a starting point to identify genes selectively expressed in specialized tissues. Complementing the analysis with profiling on more specific tissues based on immunohistochemistry allowed for further exploration of cell-type-specific expression patterns. More detailed tissue profiling was performed for >300 genes on complementing tissues. The analysis identified tissue-specific expression of nine proteins previously listed as missing proteins (POU4F1, FRMD1, ARHGEF33, GABRG1, KRTAP2-1, BHLHE22, SPRR4, AVPR1B, and DCLK3), as well as numerous proteins with evidence of existence on the protein level that previously lacked information on spatial resolution and cell-type- specific expression pattern. We here present a comprehensive strategy for identification of missing proteins by combining transcriptomics with antibody-based proteomics. The analyzed proteins provide interesting targets for organ-specific research in health and disease.

  • 98. Stenler, S.
    et al.
    Wiklander, O. P. B.
    Badal-Tejedor, M.
    Turunen, J.
    Nordin, J. Z.
    Hallengärd, D.
    Wahren, B.
    Andaloussi, S. E. L.
    Rutland, Mark W.
    KTH, Skolan för kemivetenskap (CHE), Kemi, Yt- och korrosionsvetenskap.
    Smith, C. I. E.
    Lundin, K. E.
    Blomberg, P.
    Micro-minicircle gene therapy: Implications of size on fermentation, complexation, shearing resistance, and expression2014Inngår i: Molecular Therapy - Nucleic Acids, ISSN 2162-2531, Vol. 3, s. e140-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The minicircle (MC), composed of eukaryotic sequences only, is an interesting approach to increase the safety and efficiency of plasmid-based vectors for gene therapy. In this paper, we investigate micro-MC (miMC) vectors encoding small regulatory RNA. We use a construct encoding a splice-correcting U7 small nuclear RNA, which results in a vector of 650 base pairs (bp), as compared to a conventional 3600 bp plasmid carrying the same expression cassette. Furthermore, we construct miMCs of varying sizes carrying different number of these cassettes. This allows us to evaluate how size influences production, supercoiling, stability and efficiency of the vector. We characterize coiling morphology by atomic force microscopy and measure the resistance to shearing forces caused by an injector device, the Biojector. We compare the behavior of miMCs and plasmids in vitro using lipofection and electroporation, as well as in vivo in mice. We here show that when the size of the miMC is reduced, the formation of dimers and trimers increases. There seems to be a lower size limit for efficient expression. We demonstrate that miMCs are more robust than plasmids when exposed to shearing forces, and that they show extended expression in vivo.

  • 99. Su, Yu-Ching
    et al.
    Hallström, Björn M.
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Bernhard, Sara
    Singh, Birendra
    Riesbeck, Kristian
    Impact of sequence diversity in the Moraxella catarrhalis UspA2/UspA2H head domain on vitronectin binding and antigenic variation2013Inngår i: Microbes and infection, ISSN 1286-4579, E-ISSN 1769-714X, Vol. 15, nr 5, s. 375-387Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The nasopharyngeal pathogen Moraxella catarrhalis recruits vitronectin to subvert complement-mediated killing. Ubiquitous surface protein (UspA) 2 and its hybrid form UspA2H bind vitronectin at the highly diverse N-terminal head domain. Here we characterized the sequence diversity of the head domain in multiple M. catarrhalis clinical isolates (n = 51) with focus on binding of vitronectin. The head domain of the uspA2 genes from 40 isolates were clustered according to an N-terminal sequence motif of UspA2 (NTER2), i.e., NTER2A (55% of uspA2 variants), NTER2B (32.5%), NTER2C (5%), and finally a group without an NTER2 (7.5%). Isolates harbouring the uspA2H gene (n = 11) contained N-terminal GGG repeats. Vitronectin binding to isolates having UspA2 did not correlate to variation in the NTER2 motifs but occurred in UspA2H containing 6 or >= 11 of GGG repeats. Analyses of recombinant UspA2/UspA2H head domains of multiple variants showed UspA2-dependent binding to the C-terminal of vitronectin. Furthermore, polyclonal anti-UspA2 antibodies revealed that the head domain of the majority of Moraxella UspA2/2H was antigenically unrelated, whereas the full length molecules were recognized. In conclusion, the head domains of UspA2/2H have extensive sequence polymorphism without losing vitronectin-binding capacity promoting a general evasion of the host immune system.

  • 100. Summer, D.
    et al.
    Garousi, J.
    Oroujeni, M.
    Mitran, B.
    Andersson, Ken G.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Vorobyeva, A.
    Löfblom, John
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Orlova, A.
    Tolmachev, V.
    Decristoforo, C.
    Cyclic versus Noncyclic Chelating Scaffold for 89Zr-Labeled ZEGFR:2377 Affibody Bioconjugates Targeting Epidermal Growth Factor Receptor Overexpression2018Inngår i: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 15, nr 1, s. 175-185Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Zirconium-89 is an emerging radionuclide for positron emission tomography (PET) especially for biomolecules with slow pharmacokinetics as due to its longer half-life, in comparison to fluorine-18 and gallium-68, imaging at late time points is feasible. Desferrioxamine B (DFO), a linear bifunctional chelator (BFC) is mostly used for this radionuclide so far but shows limitations regarding stability. Our group recently reported on fusarinine C (FSC) with similar zirconium-89 complexing properties but potentially higher stability related to its cyclic structure. This study was designed to compare FSC and DFO head-to-head as bifunctional chelators for 89Zr-radiolabeled EGFR-targeting ZEGFR:2377 affibody bioconjugates. FSC-ZEGFR:2377 and DFO-ZEGFR:2377 were evaluated regarding radiolabeling, in vitro stability, specificity, cell uptake, receptor affinity, biodistribution, and microPET-CT imaging. Both conjugates were efficiently labeled with zirconium-89 at room temperature but radiochemical yields increased substantially at elevated temperature, 85 °C. Both 89Zr-FSC-ZEGFR:2377 and 89Zr-DFO-ZEGFR:2377 revealed remarkable specificity, affinity and slow cell-line dependent internalization. Radiolabeling at 85 °C showed comparable results in A431 tumor xenografted mice with minor differences regarding blood clearance, tumor and liver uptake. In comparison 89Zr-DFO-ZEGFR:2377, radiolabeled at room temperature, showed a significant difference regarding tumor-to-organ ratios. MicroPET-CT imaging studies of 89Zr-FSC-ZEGFR:2377 as well as 89Zr-DFO-ZEGFR:2377 confirmed these findings. In summary we were able to show that FSC is a suitable alternative to DFO for radiolabeling of biomolecules with zirconium-89. Furthermore, our findings indicate that 89Zr-radiolabeling of DFO conjugates at higher temperature reduces off-chelate binding leading to significantly improved tumor-to-organ ratios and therefore enhancing image contrast.

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