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  • 1. Algenas, Cajsa
    et al.
    Agaton, Charlotta
    Fagerberg, Linn
    Asplund, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Bjorling, Lisa
    Bjorling, Erik
    Kampf, Caroline
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Lundberg, Emma
    Nilsson, Peter
    Persson, Anja
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Pontén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Wernerus, Henrik
    Uhlen, Mathias
    Takanen, Jenny Ottosson
    Hober, Sophia
    Antibody performance in western blot applications is context-dependent2014In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 9, no 3, p. 435-445Article in journal (Refereed)
    Abstract [en]

    An important concern for the use of antibodies in various applications, such as western blot (WB) or immunohistochemistry (IHC), is specificity. This calls for systematic validations using well-designed conditions. Here, we have analyzed 13000 antibodies using western blot with lysates from human cell lines, tissues, and plasma. Standardized stratification showed that 45% of the antibodies yielded supportive staining, and the rest either no staining (12%) or protein bands of wrong size (43%). A comparative study of WB and IHC showed that the performance of antibodies is application-specific, although a correlation between no WB staining and weak IHC staining could be seen. To investigate the influence of protein abundance on the apparent specificity of the antibody, new WB analyses were performed for 1369 genes that gave unsupportive WBs in the initial screening using cell lysates with overexpressed full-length proteins. Then, more than 82% of the antibodies yielded a specific band corresponding to the full-length protein. Hence, the vast majority of the antibodies (90%) used in this study specifically recognize the target protein when present at sufficiently high levels. This demonstrates the context- and application-dependence of antibody validation and emphasizes that caution is needed when annotating binding reagents as specific or cross-reactive. WB is one of the most commonly used methods for validation of antibodies. Our data implicate that solely using one platform for antibody validation might give misleading information and therefore at least one additional method should be used to verify the achieved data.

  • 2.
    Camsund, Daniel
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    Jaramillo, Alfonso
    Genetically engineered light sensors for control of bacterial gene expression2011In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 6, no 7, p. 826-836Article, review/survey (Refereed)
    Abstract [en]

    Light of different wavelengths can serve as a transient, noninvasive means of regulating gene expression for biotechnological purposes. Implementation of advanced gene regulatory circuits will require orthogonal transcriptional systems that can be simultaneously controlled and that can produce several different control states. Fully genetically encoded light sensors take advantage of the favorable characteristics of light, do not need the supplementation of any chemical inducers or co-factors, and have been demonstrated to control gene expression in Escherichia coli. Herein, we review engineered light-sensor systems with potential for in vivo regulation of gene expression in bacteria, and highlight different means of extending the range of available light input and transcriptional output signals. Furthermore, we discuss advances in multiplexing different light sensors for achieving multichromatic control of gene expression and indicate developments that could facilitate the construction of efficient systems for light-regulated, multistate control of gene expression.

  • 3.
    Falk, Ronny
    et al.
    KTH, Molekylär Bioteknologi.
    Ramström, Margareta
    KTH, Proteomik.
    Eriksson, Cecilia
    KTH, Proteomik.
    Uhlén, Mathias
    KTH, Proteomik.
    Wernérus, Henrik
    KTH, Proteomik.
    Hober, Sophia
    KTH, Proteomik.
    Targeted protein pullout from human tissue samples using competitive elution2011In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 6, no 1, p. 28-37Article in journal (Refereed)
    Abstract [en]

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

  • 4.
    Forster, Anthony C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Synthetic biology challenges long-held hypotheses in translation, codon bias and transcription2012In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 7, no 7, p. 835-845Article, review/survey (Refereed)
    Abstract [en]

    Synthetic biology is a powerful experimental approach, not only for developing new biotechnology applications, but also for testing hypotheses in basic biological science. Here, examples from our research using the best model system, Escherichia coli, are reviewed. New evidence drawn from synthetic biology has overturned several long-standing hypotheses regarding the mechanisms of transcription and translation: (i) all native aminoacyl-tRNAs are not equally efficient in translation at equivalent concentrations; (ii) accommodation is not always rate limiting in translation, and may not be for any aminoacyl-tRNA; (iii) proline is the only N-alkyl-amino acid in the genetic code not because of special suitability for protein structure, but because of its comparatively high nucleophilicity; (iv) the usages of most sense codons in E. coli do not correlate with cognate tRNA abundances and (v) class II transcriptional pausing and termination by T7 RNA polymerase cannot be assumed to occur in vivo based on in vitro data. Implications of these conclusions for the biotechnology field are discussed.

  • 5.
    Forster, Anthony C.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Lee, Sang Yup
    Editorial: NextGen SynBio has arrived...2012In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 7, no 7, p. 827-827Article in journal (Other academic)
  • 6.
    Grönwall, Caroline
    et al.
    KTH, Molekylär Bioteknologi.
    Sjöberg, Anna
    Affibody AB, Bromma.
    Ramström, Margareta
    KTH, Molekylär Bioteknologi.
    Höidén-Guthenberg, Ingmarie
    Affibody AB, Bromma.
    Hober, Sophia
    KTH, Molekylär Bioteknologi.
    Jonasson, Per
    Affibody AB, Bromma.
    Ståhl, Stefan
    KTH, Molekylär Bioteknologi.
    Affibody-mediated transferrin depletion for proteomics applications2007In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 2, no 11, p. 1389-1398Article in journal (Refereed)
    Abstract [en]

    An Affibody® (Affibody) ligand with specific binding to human transferrin was selected by phage display technology from a combinatorial protein library based on the staphylococcal protein A (SpA)-derived Z domain. Strong and selective binding of the selected Affibody ligand to transferrin was demonstrated using biosensor technology and dot blot analysis. Impressive specificity was demonstrated as transferrin was the only protein recovered by affinity chromatography from human plasma. Efficient Affibody-mediated capture of transferrin, combined with IgG- and HSA-depletion, was demonstrated for human plasma and cerebrospinal fluid (CSF). For plasma, 85% of the total transferrin content in the samples was depleted after only two cycles of transferrin removal, and for CSF, 78% efficiency was obtained in single-step depletion. These results clearly suggest a potential for the development of Affibody-based resins for the removal of abundant proteins in proteomics analyses.

  • 7.
    Jansson, Ronnie
    et al.
    Swedish Univ Agr Sci, Dept Anat Physiol & Biochem, Uppsala, Sweden.
    Lau, Cheuk H
    Swedish Univ Agr Sci, Dept Anat Physiol & Biochem, Uppsala, Sweden.; Swedish Univ Agr Sci, Dept Chem & Biotechnol, Uppsala, Sweden.
    Ishida, Takuya
    Univ Tokyo, Grad Sch Agr & Life Sci, Dept Biomat Sci, Tokyo, Japan.
    Ramström, Margareta
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Sandgren, Mats
    Swedish Univ Agr Sci, Dept Chem & Biotechnol, Uppsala, Sweden.
    Hedhammar, My
    Swedish Univ Agr Sci, Dept Anat Physiol & Biochem, Uppsala, Sweden.; KTH Royal Inst Technol, Sch Biotechnol, Div Prot Technol, Stockholm, Sweden .
    Functionalized silk assembled from a recombinant spider silk fusion protein (Z-4RepCT) produced in the methylotrophic yeast Pichia pastoris.2016In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 11, no 5, p. 687-699Article in journal (Refereed)
    Abstract [en]

    Functional biological materials are a growing research area with potential applicability in medicine and biotechnology. Using genetic engineering, the possibility to introduce additional functions into spider silk-based materials has been realized. Recently, a recombinant spider silk fusion protein, Z-4RepCT, was produced intracellularly in Escherichia coli and could after purification self-assemble into silk-like fibers with ability to bind antibodies via the IgG-binding Z domain. In this study, the use of the methylotrophic yeast Pichia pastoris for production of Z-4RepCT has been investigated. Temperature, pH and production time were influencing the amount of soluble Z-4RepCT retrieved from the extracellular fraction. Purification of secreted Z-4RepCT resulted in a mixture of full-length and degraded silk proteins that failed to self-assemble into fibers. A position in the C-terminal domain of 4RepCT was identified as being subjected to proteolytic cleavage by proteases in the Pichia culture supernatant. Moreover, the C-terminal domain was subjected to glycosylation during production in P. pastoris. These observed alterations of the CT domain are suggested to contribute to the failure in fiber assembly. As alternative approach, Z-4RepCT retrieved from the intracellular fraction, which was less degraded, was used and shown to retain ability to assemble into silk-like fibers after enzymatic deglycosylation.

  • 8. Malm, Magdalena
    et al.
    Bass, Tarek
    Gudmundsdotter, Lindvi
    Lord, Martin
    Frejd, Fredrik Y.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Stahl, Stefan
    Lofblom, John
    Engineering of a bispecific affibody molecule towards HER2 and HER3 by addition of an albumin-binding domain allows for affinity purification and in vivo half-life extension2014In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 9, no 9, p. 1215-1222Article in journal (Refereed)
    Abstract [en]

    Emerging strategies in cancer biotherapy include the generation and application of bispecific antibodies, targeting two tumor-associated antigens for improved tumor selectivity and potency. Here, an alternative format for bispecific molecules was designed and investigated, in which two Affibody molecules were linked by an albumin-binding domain (ABD). Affibody molecules are small (6 kDa) affinity proteins and this new format allows for engineering of molecules with similar function as full-length bispecific antibodies, but in a dramatically smaller size (around eight-fold smaller). The ABD was intended to function both as a tag for affinity purification as well as for in vivo half-life extension in future preclinical and clinical investigations. Affinity-purified bispecific Affibody molecules, targeting HER2 and HER3, showed simultaneous binding to the three target proteins (HER2, HER3, and albumin) when investigated in biosensor assays. Moreover, simultaneous interactions with the receptors and albumin were demonstrated using flow cytometry on cancer cells. The bispecific Affibody molecules were also able to block ligand-induced phosphorylation of the HER receptors, indicating an anti-proliferative effect. We believe that this compact and flexible format has great potential for developing new potent bispecific affinity proteins in the future, as it combines the benefits of a small size (e.g. improved tissue penetration and reduced cost of goods) with a long circulatory half-life.

  • 9.
    Stjernberg Bejhed, Rebecca
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Solid State Physics.
    Zardán Gómez de la Torre, Teresa
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Nanotechnology and Functional Materials.
    Svedlindh, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Solid State Physics.
    Strömberg, Mattias
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Solid State Physics.
    Optomagnetic bioassay for qualitative biplex detection of bacterial DNA sequences2015In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 10, no 3, p. 469-472Article in journal (Refereed)
    Abstract [en]

    There is an increasing need to develop novel bioassay methods for low-cost, rapid, and easy-to-use multiplex detection of pathogens in various fields ranging from human infectious disease diagnosis, drinking water quality control, to food safety applications. Due to their unique advantages, magnetic and optomagnetic bioassay principles are particularly promising for biodetection platforms that will be used in developing countries. In this paper, an optomagnetic method for rapid and cost-efficient qualitative biplex detection of bacterial DNA sequences is demonstrated. Within less than two hours, the assay gives an answer to whether none, both, or only one of the bacterial DNA sequences is present in the sample. The assay relies on hybridization of oligonucleotide-functionalized magnetic nanobeads of two different sizes to rolling circle amplification (RCA) products originating from two different bacterial targets. The different bead sizes are equipped with different oligonucleotide probes, complementary to only one of the RCA products, and the read-out is carried out in the same sample volume. In an optomagnetic setup, the frequency modulation of transmitted laser light in response to an applied AC magnetic field is measured. The presented methodology is potentially interesting for low-cost screening of pathogens relating to both human and veterinary medicine in resource-poor regions of the world.

  • 10.
    Strömberg, Mattias
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Solid State Physics.
    Zardán Gómez de la Torre, Teresa
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Nanotechnology and Functional Materials.
    Nilsson, Mats
    Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Solna, Sweden.
    Svedlindh, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Solid State Physics.
    Strømme, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Nanotechnology and Functional Materials.
    A magnetic nanobead-based bioassay provides sensitive detection of single- and biplex bacterial DNA using a portable AC susceptometer2014In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 9, no 1, p. 137-145Article in journal (Refereed)
    Abstract [en]

    Bioassays relying on magnetic read-out using probe-tagged magnetic nanobeads are potential platforms for low-cost biodiagnostic devices for pathogen detection. For optimal assay performance it is crucial to apply an easy, efficient and robust bead-probe conjugation protocol. In this paper, sensitive (1.5 pM) singleplex detection of bacterial DNA sequences is demonstrated in a portable AC susceptometer by a magnetic nanobead-based bioassay principle; the volume-amplified magnetic nanobead detection assay (VAM-NDA). Two bead sizes, 100 and 250 nm, are investigated along with a highly efficient, rapid, robust, and stable conjugation chemistry relying on the avidin-biotin interaction for bead-probe attachment. Avidin-biotin conjugation gives easy control of the number of detection probes per bead; thus allowing for systematic investigation of the impact of varying the detection probe surface coverage upon bead immobilization in rolling circle amplified DNA-coils. The existence of an optimal surface coverage is discussed. Biplex VAM-NDA detection is for the first time demonstrated in the susceptometer: Semi-quantitative results are obtained and it is concluded that the concentration of DNA-coils in the incubation volume is of crucial importance for target quantification. The present findings bring the development of commercial biodiagnostic devices relying on the VAM-NDA further towards implementation in point-of-care and outpatient settings.

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