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  • 1.
    Alm, Tove
    KTH, School of Biotechnology (BIO), Proteomics.
    Interaction engineered three-helix bundle domains for protein recovery and detection2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    HTML clipboard The great advances in DNA technology, e.g. sequencing and recombinant DNA techniques, have given us the genetic information and the tools needed to effectively produce recombinant proteins. Recombinant proteins are valuable means in biotechnological applications and are also emerging as alternatives in therapeutic applications. Traditionally, monoclonal antibodies have been the natural choice for biotechnological and therapeutic applications due to their ability to bind a huge range of different molecules and their natural good affinity. However, the large size of antibodies (150 kDa) limits tissue penetration and the recombinant expression is complicated. Therefore, alternative binders with smaller sizes have been derived from antibodies and alternative scaffolds.

    In this thesis, two structurally similar domains, Zbasic and ABDz1, have been used as purification tags in different contexts. They are both three-helical bundles and derived from bacterial surface domains, but share no sequence homology. Furthermore, by redesign of the scaffold used for ABDz1, a molecule intended for drug targeting with extended in-vivo half-life has been engineered. In Papers I and II, the poly-cationic tag Zbasic is explored and evaluated. Paper I describes the successful investigation of Zbasic as a purification handle under denaturating conditions. Moreover, Zbasic is evaluated as an interaction domain in matrixassisted refolding. Two different proteins were successfully refolded using the same setup without individual optimization. In Paper II, Zbasic is further explored as a purification handle under non-native conditions in a multi-parallel setup. In total, 22 proteins with varying characteristics are successfully purified using a multi-parallel protein purification protocol and a robotic system. Without modifications, the system can purify up to 60 proteins without manual handling. Paper I and II clearly demonstrate that Zbasic can be used as an interaction domain in matrix-assisted refolding and that it offers a good alternative to the commonly used His6-tag under denaturating conditions. In paper III, the small bifunctional ABDz1 is selected from a phage display library. Endowed with two different binding interfaces, ABDz1 is capable of binding both the HSA-sepharose and the protein A-derived MabSelect SuRe-matrix. The bifunctionality of the domain is exploited in an orthogonal affinity setup. Three target proteins are successfully purified using the HSA-matrix and the MabSelect SuRe-matrix. Furthermore, the purity of the target proteins is effectively improved by combining the two chromatographic steps. Thus, paper III shows that the small ABDz1 can be used as an effective purification handle and dual affinity tag without target specific optimization. Paper IV describes the selection and affinity maturation of small bispecific drug-targeting molecules. First generation binders against tumor necrosis factor-α are selected using phage display. Thereafter on-cell surface display and flow cytometry is used to select second-generation binders. The binding to tumor necrosis factor-α is improved up to 30 times as compared to the best first generation binder, and a 6-fold improvement of the binding strength was possible with retained HSA affinity. Paper III and IV clearly demonstrate that dual interaction surfaces can successfully be grafted on a small proteinaceous domain, and that the strategy in paper IV can be used for dual selection of bifunctional binders.

  • 2.
    Andersen, Malin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Computational and experimental approaches to regulatory genetic variation2007Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    Genetic variation is a strong risk factor for many human diseases, including diabetes, cancer, cardiovascular disease, depression, autoimmunity and asthma. Most of the disease genes identified so far alter the amino acid sequences of encoded proteins. However, a significant number of genetic variants affecting complex diseases may alter the regulation of gene transcription. The map of the regulatory elements in the human genome is still to a large extent unknown, and it remains a challenge to separate the functional regulatory genetic variations from linked neutral variations.

    The objective of this thesis was to develop methods for the identification of genetic variation with a potential to affect the transcriptional regulation of human genes, and to analyze potential regulatory polymorphisms in the CD36 glycoprotein, a candidate gene for cardiovascular disease.

    An in silico tool for the prediction of regulatory polymorphisms in human genes was implemented and is available at www.cisreg.ca/RAVEN. The tool was evaluated using experimentally verified regulatory single nucleotide polymorphisms (SNPs) collected from the scientific literature, and tested in combination with experimental detection of allele specific expression of target genes (allelic imbalance). Regulatory SNPs were shown to be located in evolutionary conserved regions more often than background SNPs, but predicted transcription factor binding sites were unable to enrich for regulatory SNPs unless additional information linking transcription factors with the target genes were available.

    The in silico tool was applied to the CD36 glycoprotein, a candidate gene for cardiovascular disease. Potential regulatory SNPs in the alternative promoters of this gene were identified and evaluated in vitro and in vivo using a clinical study for coronary artery disease. We observed association to the plasma concentrations of inflammation markers (serum amyloid A protein and C-reactive protein) in myocardial infarction patients, which highlights the need for further analyses of potential regulatory polymorphisms in this gene.

    Taken together, this thesis describes an in silico approach to identify putative regulatory polymorphisms which can be useful for directing limited laboratory resources to the polymorphisms most likely to have a phenotypic effect.

  • 3.
    Anfelt, Josefine
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Kaczmarzyk, Danuta
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Shabestary, Kiyan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Renberg, Björn
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Rockberg, Johan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Nielsen, Jens
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. Tech Univ Denmark.
    Hudson, Elton P.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Genetic and nutrient modulation of acetyl-CoA levels in Synechocystis for n-butanol production2015In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 14, article id 167Article in journal (Refereed)
    Abstract [en]

    Background: There is a strong interest in using photosynthetic cyanobacteria as production hosts for biofuels and chemicals. Recent work has shown the benefit of pathway engineering, enzyme tolerance, and co-factor usage for improving yields of fermentation products. Results: An n-butanol pathway was inserted into a Synechocystis mutant deficient in polyhydroxybutyrate synthesis. We found that nitrogen starvation increased specific butanol productivity up to threefold, but cessation of cell growth limited total n-butanol titers. Metabolite profiling showed that acetyl-CoA increased twofold during nitrogen starvation. Introduction of a phosphoketolase increased acetyl-CoA levels sixfold at nitrogen replete conditions and increased butanol titers from 22 to 37 mg/L at day 8. Flux balance analysis of photoautotrophic metabolism showed that a Calvin-Benson-Bassham-Phosphoketolase pathway had higher theoretical butanol productivity than CBB-Embden-Meyerhof-Parnas and a reduced butanol ATP demand. Conclusion: These results demonstrate that phosphoketolase overexpression and modulation of nitrogen levels are two attractive routes toward increased production of acetyl-CoA derived products in cyanobacteria and could be implemented with complementary metabolic engineering strategies.

  • 4.
    Ayoglu, Burcu
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nilsson, Peter
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Multiplexed antigen bead arrays for the assessment of antibody selectivity and epitope mapping2018In: Epitope Mapping Protocols, Humana Press Inc. , 2018, p. 239-248Chapter in book (Refereed)
    Abstract [en]

    With the increasing number of binding reagents for affinity-based investigations of the human proteome, high-throughput tools for the characterization of the used reagents become essential. For the analysis of binding selectivity, bead-based antigen arrays offer a miniaturized and parallelized assay platform to meet such needs, as they enable two-dimensional multiplexing to analyze up to 384 samples against up to 500 analytes in a single round of analysis. In this chapter, we describe our protocols for the generation of multiplex bead arrays built on immobilized protein fragments, as well as biotinylated peptides. Combined together, these two versions of antigen arrays offer a versatile approach for multiplexed characterization of antibody binding selectivity, off-target interactions, as well as mapping for the amino acids of epitopes involved in antibody binding.

  • 5. Balzani, D.
    et al.
    Holzapfel, Gerhard
    KTH, School of Engineering Sciences (SCI), Solid Mechanics (Dept.), Biomechanics. Graz University of Technology, Institute of Biomechanics, Center of Biomedical Engineering.
    Brinkhues, S.
    Modeling of damage in soft biological tissues and application to arterial walls2011In: Computational Plasticity XI - Fundamentals and Applications, 2011, p. 764-775Conference paper (Refereed)
    Abstract [en]

    A new material model is proposed for the description of stress-softening observed in cyclic tension tests performed on soft biological tissues. The modeling framework is based on the concept of internal variables introducing a scalar-valued variable for the representation of fiber damage. Remanent strains in fiber direction can be represented as a result of microscopic damage of the fiber crosslinks. Particular internal variables are defined able to capture the nature of soft biological tissues that no damage occurs in the physiological loading domain. A specific model is adjusted to experimental data taking into account the supra-physiological loading regime. For the description of the physiological domain polyconvex functions are used which also take into account fiber dispersion in a phenomenological approach. The applicability of the model in numerical simulations is shown by a representative example where the damage distribution in an arterial cross-section is analyzed.

  • 6.
    Cengic, Ivana
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hudson, Elton P.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Surface Display of Small Affinity Proteins on Synechocystis sp Strain PCC 6803 Mediated by Fusion to the Major Type IV Pilin PilA12018In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 200, no 16, article id e00270-18Article in journal (Refereed)
    Abstract [en]

    Functional surface display of small affinity proteins, namely, affibodies (6.5 kDa), was evaluated for the model cyanobacterium Synechocystis sp. strain PCC 6803 through anchoring to native surface structures. These structures included confirmed or putative subunits of the type IV pili, the S-layer protein, and the heterologous Escherichia coli autotransporter antigen 43 system. The most stable display system was determined to be through C-terminal fusion to PilA1, the major type IV pilus subunit in Synechocystis, in a strain unable to retract these pili (Delta pilT1). Type IV pilus synthesis was upheld, albeit reduced, when fusion proteins were incorporated. However, pilus-mediated functions, such as motility and transformational competency, were negatively affected. Display of affibodies on Synechocystis and the complementary anti-idiotypic affibodies on E. coli or Staphylococcus carnosus was able to mediate interspecies cell-cell binding by affibody complex formation. The same strategy, however, was not able to drive cell-cell binding and aggregation of Synechocystis-only mixtures. Successful affibody tagging of the putative minor pilin PilA4 showed that it locates to the type IV pili in Synechocystis and that its extracellular availability depends on PilA1. In addition, affibody tagging of the S-layer protein indicated that the domains responsible for the anchoring and secretion of this protein are located at the N and C termini, respectively. This study can serve as a basis for future surface display of proteins on Synechocystis for biotechnological applications. IMPORTANCE Cyanobacteria are gaining interest for their potential as autotrophic cell factories. Development of efficient surface display strategies could improve their suitability for large-scale applications by providing options for designed microbial consortia, cell immobilization, and biomass harvesting. Here, surface display of small affinity proteins was realized by fusing them to the major subunit of the native type IV pili in Synechocystis sp. strain PCC 6803. The display of complementary affinity proteins allowed specific cell-cell binding between Synechocystis and Escherichia coli or Staphylococcus carnosus. Additionally, successful tagging of the putative pilin PilA4 helped determine its localization to the type IV pili. Analogous tagging of the S-layer protein shed light on the regions involved in its secretion and surface anchoring.

  • 7. Chen, G.
    et al.
    Ning, Z.
    Ågren, Hans
    KTH, School of Biotechnology (BIO), Theoretical Chemistry and Biology.
    Nanostructured solar cells2016In: Nanomaterials, ISSN 2079-4991, Vol. 6, no 8, article id 145Article in journal (Refereed)
    Abstract [en]

    We are glad to announce the Special Issue “Nanostructured Solar Cells”, published in Nanomaterials. This issue consists of eight articles, two communications, and one review paper, covering major important aspects of nanostructured solar cells of varying types. From fundamental physicochemical investigations to technological advances, and from single junction solar cells (silicon solar cell, dye sensitized solar cell, quantum dots sensitized solar cell, and small molecule organic solar cell) to tandem multi-junction solar cells, all aspects are included and discussed in this issue to advance the use of nanotechnology to improve the performance of solar cells with reduced fabrication costs.

  • 8.
    Dincbas-Renqvist, Vildan
    et al.
    KTH, Superseded Departments, Biotechnology.
    Lendel, Christofer
    KTH, School of Biotechnology (BIO).
    Dogan, Jakob
    KTH, Superseded Departments, Biotechnology.
    Wahlberg, Elisabet
    KTH, Superseded Departments, Biotechnology.
    Härd, Torleif
    Göteborgs Universitet.
    Thermodynamics of folding, stabilization, and binding in an engineered protein-protein complex2004In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 126, no 36, p. 11220-11230Article in journal (Refereed)
    Abstract [en]

    We analyzed the thermodynamics of a complex protein-protein binding interaction using the (engineered) Z(SPA-1) affibody and it's Z domain binding partner as a model. Free Z(SPA-1) exists in an equilibrium between a molten-globule-like (MG) state and a completely unfolded state, wheras a well-ordered structure is observed in the Z:Z(SPA-1) complex. The thermodynamics of the MG state unfolding equilibrium can be separated from the thermodynamics of binding and stabilization by combined analysis of isothermal titration calorimetry data and a separate van't Hoff analysis of thermal unfolding. We find that (i) the unfolding equilibrium of free Z(SPA-1) has only a small influence on effective binding affinity, that (ii) the Z:Z(SPA-1) interface is inconspicuous and structure-based energetics calculations suggest that it should be capable of supporting strong binding, but that (iii) the conformational stabilization of the MG state to a well-ordered structure in the Z:Z(SPA-1) complex is associated with a large change in conformational entropy that opposes binding.

  • 9.
    Dogan, Jakob
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Structural and thermodynamical basis for molecular recognition between engineered binding proteins2006Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    The structural determination of interacting proteins, both as individual proteins and in their complex, complemented by thermodynamical studies are vital in order to gain in-depth insights of the phenomena leading to the highly selective protein-protein interactions characteristic of numerous life processes. This thesis describes an investigation of the structural and thermodynamical basis for molecular recognition in two different protein-protein complexes, formed between so-called affibody proteins and their respective targets. Affibody proteins are a class of engineered binding proteins, which can be functionally selected for binding to a given target protein from large collections (libraries) constructed via combinatorial engineering of 13 surface-located positions of the 58-residue three-helix bundle Z domain derived from Staphylococcal protein (SPA).

    In a first study, an affibody:target protein pair consisting of the ZSPA-1 affibody and the parental Z domain, with a dissociation constant (Kd) of approximately 1 µM, was investigated. ZSPA-1 was in its free state shown to display molten globule-like characteristics. The enthalpy change on binding between Z and ZSPA-1 as measured by isothermal titration calorimetry, was found to be a non-linear function of temperature. This nonlinearity was found to be due to the temperature dependent folded-unfolded equilibrium of ZSPA-1 upon binding to the Z domain and, the energetics of the unfolding equilibrium of the molten globule state of ZSPA-1 could be separated from the binding thermodynamics. Further dissection of the binding entropy revealed that a significant reduction in conformational entropy resulting from the stabilization of the molten globule state of ZSPA-1 upon complex formation could be a major reason for the moderate binding affinity.

    A second studied affibody:target complex (Kd ~ 0.1 µM) consisted of the ZTaq affibody protein originally selected for binding to Taq DNA polymerase and the anti-ZTaq affibody protein, selected for selective binding to the ZTaq affibody protein, thus constituting an "anti-idiotypic" affinity protein pair. The structure of the ZTaq:anti-ZTaq affibody complex as well as the free state structures of ZTaq and anti-ZTaq were determined using NMR spectroscopy. Both ZTaq and anti-ZTaq are well defined three helix bundles in their free state and do not display the same molten globule-like behaviour of ZSPA-1. The interaction surface was found to involve all of the varied positions in helices 1 and 2 of the anti-ZTaq, the majority of the corresponding side chains in ZTaq, and also several non-mutated residues. The total buried surface area was determined to about 1670 Å2 which is well inside the range of what is typical for many protein-protein complexes, including antibody:antigen complexes. Structural rearrangements, primarily at the side chain level, were observed to take place upon binding. There are similarities between the ZTaq:anti-ZTaq and the Z:ZSPA-1 structure, for instance, the binding interface area in both complexes has a large fraction of non-polar content, the buried surface area is of similar size, and certain residues have the same positioning. However, the relative orientation between the subunits in ZTaq:anti-ZTaq is markedly different from that observed in Z:ZSPA-1. The thermodynamics of ZTaq:anti-ZTaq association were investigated by isothermal titration calorimetry. A dissection of the entropic contributions showed that a large and favourable desolvation entropy of non-polar surface is associated with the binding reaction which is in good agreement with hydrophobic nature of the binding interface, but as in the case for the Z:ZSPA-1 complex a significant loss in conformational entropy opposes complex formation.

    A comparison with complexes involving affibody proteins or SPA domains suggests that affibody proteins inherit intrinsic binding properties from the original SPA surface. The structural and biophysical data suggest that although extensive mutations are carried out in the Z domain to obtain affibody proteins, this does not necessarily affect the structural integrity or lead to a significant destabilization.

  • 10.
    Dogan, Jakob
    et al.
    KTH, School of Biotechnology (BIO).
    Lendel, Christofer
    KTH, School of Biotechnology (BIO).
    Härd, Torleif
    Göteborgs Universitet.
    NMR assignments of the free and bound-state protein components of an anti-idiotypic affibody complex2006In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 36, p. (Electronic publication ahead of print Feb. 6; doi:10.1007/s10858-005-5350-8)Article in journal (Refereed)
  • 11.
    Dogan, Jakob
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Lendel, Christofer
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Härd, Torleif
    Thermodynamics of folding and binding in an affibody:affibody complex2006In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 359, no 5, p. 1305-1315Article in journal (Refereed)
    Abstract [en]

    Affibody binding proteins are selected from phage-displayed libraries of variants of the 58 residue Z domain. Z(Taq) is an affibody originally selected as a binder to Taq DNA polymerase. The anti-Z(Taq) affibody was selected as a binder to Z(Taq) and the Z(Taq):anti-Z(Taq) complex is formed with a dissociation constant K-d = 0.1 mu M. We have determined the structure of the Z(Taq):anti-Z(Taq) complex as well as the free state structures of Z(Taq) and anti-Z(Taq) using NMR. Here we complement the structural data with thermodynamic studies of Z(Taq) and anti-Z(Taq) folding and complex formation. Both affibody proteins show cooperative two-state thermal denaturation at melting temperatures T-M similar to 56 degrees C. Z(Taq):anti-Z(Taq) complex formation at 25 degrees C in 50 mM NaCl and 20 mM phosphate buffer (pH 6.4) is enthalpy driven with Delta H degrees(bind) = -9.0(+/- 0.1) kcal mol(-1). The heat capacity change Delta C-P degrees,(bind) = -0.43(+/- 0.01) kcal mol(-1) K-1 is in accordance with the predominantly non-polar character of the binding surface, as judged from calculations based on changes in accessible surface areas. A further dissection of the small binding entropy at 25 degrees C (-T Delta S degrees(bind) = -0.6(+/- 0.1) kcal mol(-1)) suggests that a favourable desolvation of non-polar surface is almost completely balanced by unfavourable conformational entropy changes and loss of rotational and translational entropy. Such effects can therefore be limiting for strong binding also when interacting protein components are stable and homogeneously folded. The combined structure and thermodynamics data suggest that protein properties are not likely to be a serious limitation for the development of engineered binding proteins based on the Z domain.

  • 12.
    Dånmark, Staffan
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Finne-Wistrand, Anna
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Schander, K.
    Hakkarainen, Minna
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Arvidson, K.
    Mustafa, K.
    Albertsson, Ann-Christine
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    In vitro and in vivo degradation profile of aliphatic polyesters subjected to electron beam sterilization2011In: ACTA BIOMATERIALIA, ISSN 1742-7061, Vol. 7, no 5, p. 2035-2046Article in journal (Refereed)
    Abstract [en]

    Degradation characteristics in response to electron beam sterilization of designed and biodegradable aliphatic polyester scaffolds are relevant for clinically successful synthetic graft tissue regeneration Scaffold degradation in vitro and in vivo were documented and correlated to the macroscopic structure and chemical design of the original polymer The materials tested were of inherently diverse hydrophobicity and crystallinity poly(L-lactide) (poly(LLA)) and random copolymers from L-lactide and epsilon-caprolactone or 1.5-dioxepan-2-one, fabricated into porous and non-porous scaffolds After sterilization, the samples underwent hydrolysis in vitro for up to a year In vivo, scaffolds were surgically implanted into rat calvarial defects and retrieved for analysis after 28 and 91 days In vitro, poly(L-lactide-co-1, 5-dioxepan-2-one) (poly(LLA-co-DXO)) samples degraded most rapidly during hydrolysis, due to the pronounced chain-shortening reaction caused by the sterilization. This was indicated by the rapid decrease in both mass and molecular weight of poly(LLA-co-DXO). Poly(L-lactide-co-epsilon-caprolactone) (poly(LLA-co-CL)) samples were also strongly affected by sterilization, but mass loss was more gradual; molecular weight decreased rapidly during hydrolysis Least affected by sterilization were the poly(LLA) samples, which subsequently showed low mass loss rate and molecular weight decrease during hydrolysis. Mechanical stability varied greatly. poly(LLA-co-CL) withstood mechanical testing for up to 182 days, while poly(LLA) and poly(LLA-co-DXO) samples quickly became too brittle Poly(LLA-co-DXO) samples unexpectedly degraded more rapidly in vitro than in vivo. After sterilization by electron beam irradiation, the three biodegradable polymers present widely diverse degradation profiles, both in vitro and in vivo. Each exhibits the potential to be tailored to meet diverse clinical tissue engineering requirements

  • 13.
    Fagerberg, Linn
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Strömberg, Sara
    El-Obeid, Adila
    Gry, Marcus
    KTH, School of Biotechnology (BIO), Proteomics.
    Nilsson, Kenneth
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Ponten, Fredrik
    Adplund, Anna
    The Global Protein Expression Pattern in Human Cell LinesManuscript (preprint) (Other academic)
    Abstract [en]

    Human cancer cell lines grown in vitro are frequently used to decipher basic cell biological phenomena but also to specifically study different forms of cancer. Here we present the first large-scale study of protein expression patterns in cell lines using an antibody-based proteomics approach. We analyzed the expression pattern of 5436 proteins in 45 different cell lines using hierarchical clustering, principal component analysis and two-group comparisons for the identification of differentially expressed proteins. The results show that protein profiles of cell lines, as determined using immunohistochemistry, allow for a hierarchical clustering that overall reflects tumor tissues of origin. Hematological cell lines appear to retain their protein profiles to a higher degree than cell lines established from solid tumors, resulting in a clustering that well reflects progenitor cell types. The discrepancy may reflect different levels of in vitro induced alterations in adherent and suspension grown cell lines, respectively. In addition, multiple myeloma cells and cells of myeloid origin were found to share a protein profile, relative the protein profile of lymphoid leukemia and lymphoma cells, possibly reflecting their common dependency of bone marrow microenvironment.

     

  • 14.
    Fugelstad, Johanna
    KTH, School of Biotechnology (BIO), Glycoscience.
    Cellulose Biosynthesis in Oomycetes2008Licentiate thesis, comprehensive summary (Other scientific)
    Abstract [en]

    Oomycetes have long been considered as a separate class within the kingdom Fungi, but they are in fact closer to brown algae. They are currently classified in the Stramenopile eukaryotic kingdom, which includes heterokont algae and water molds. The major cell wall polysaccharides in Oomycetes are b-(1à3) and b-(1à6)-glucans, as well as cellulose, which has never been reported in any fungal species. Chitin - the major cell wall polysaccharide in fungi - occurs in minor amounts in the walls of some Oomycetes. Some Oomycete species are pathogens of great economical importance. For example, species of the genus Phytophthora are well studied plant pathogens that cause considerable economical losses in agriculture. Saprolegniosis, a fish disease caused by species from the genus Saprolegnia, is a major problem in the aquaculture industry and represents a threat to populations of salmonids in natural habitats. Currently, there are no chemicals available that are at the same time efficient Oomycete inhibitors, environmentally friendly and safe for human consumption of treated fishes. The biosynthesis of cellulose in Oomycetes is poorly understood, even though this biochemical pathway represents a potential target for new Oomycete inhibitors. In this work, cellulose biosynthesis was investigated in two selected Oomycetes, the plant pathogen Phytophthora infestans and the fish pathogen Saprolegnia monoica.

    A new Oomycete CesA gene family was identified. It contains four homologues designated as CesA1, CesA2, CesA3 and CesA4. The gene products of CesA1, 2 and 4 contain Pleckstrin Homology domains located at the N-terminus. This represents a novel feature, unique to the Oomycete CesA genes. CesA3 is the dominantly expressed CesA homologue in the mycelium of both S. monoica and P. infestans, while CesA1 and CesA2 are up-regulated in virulent life stages of P. infestans. CesA4 was expressed only in minute amounts in all investigated types of cells. Gene silencing by RNA interference of the whole CesA gene family in P. infestans lead to decreased amounts of cellulose in the cell wall. The inhibitors of cellulose synthesis DCB and Congo Red had an up-regulating effect on SmCesA gene expression, which was accompanied by an increased b-glucan synthase activity in vitro. In addition, these inhibitors slowed down the growth of the mycelium from S. monoica. Zoospores from P. infestans treated with DCB were unable to infect potato leaves and showed aberrant cell wall morphologies similar to those obtained by silencing the CesA gene family.

    Altogether these results show that at least some of the CesA1-4 genes are involved in cellulose biosynthesis and that the synthesis of cellulose is crucial for infection of potato by P. infestans

  • 15.
    Grimm, Sebastian
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Ribosome display for selection and evolution of affibody molecules2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Affinity proteins are invaluable tools in biotechnological and medical applications. This thesis is about combinatorial protein engineering principles for the generation of novel affinity proteins to purify mouse immunoglobulin, detect a potential cancer marker protein or inhibit a cell proliferation pathway.

    In a first study, ribosome display was for the first time applied to the selection of so-called affibody molecules, including the design of a ribosome display gene cassette, initial test enrichment experiments and the selection of binders against murine IgG1. One of the selected binders (ZMAB25) showed a highly selective binding profile to murine IgG1, which was exploited in the recovery of two different mouse monoclonal IgG1 antibodies from a bovine immunoglobulin-containing background. Ribosome display was further applied to the selection of affibody molecules binding to SATB1, a suggested marker protein for metastasizing adenocarcinoma. The study also included the selection of VHH antibody fragments from a naïve gene repertoire displayed on phage. Binders from both classes of protein scaffolds could be isolated that selectively recognized SATB1 but not its close homologue SATB2, and were used to detect endogenous SATB1 in Jurkat cells by immunofluorescence microscopy. The well-established phage display technology was used to select affibody molecules binding to H-Ras and Raf-1, both involved in the mitogen-activated protein kinase (MAPK) pathway and playing a central role in the control of cell proliferation, survival and differentiation. An isolated affibody molecule denoted ZRAF322 was found to selectively bind to Raf-1 and inhibit the interaction between H-Ras and Raf-1 in vitro. In a continued effort, ribosome display was applied to the affinity maturation of the ZRAF322 variant in a novel approach, based on repetitive cycles of diversification by error-prone PCR of the entire affibody gene and ribosome display selection, mimicking the principles of natural evolution. The method involved a monitoring of the progress of evolution and variants of ZRAF322 with 13- to 26-fold improved affinities were obtained, that contained different combinations of single or double amino acid substitutions in either previously randomized or framework positions. Implications of the substitutions for binder stability and selectivity were also investigated, showing that a higher affinity could be associated with a lower thermal melting point and that affinity-improved variants showed uncompromised binding selectivity to the hRaf-1 target.

  • 16.
    Grimm, Sebastian
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Proteomics.
    Yu, Feifan
    KTH, School of Biotechnology (BIO), Proteomics.
    Shibasaki, Seiji
    Vernet, Erik
    KTH, School of Biotechnology (BIO), Proteomics.
    Skogs, Marie
    KTH, School of Biotechnology (BIO), Proteomics.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Selection and characterisation of affibody molecules inhibiting the interaction between Ras and Raf in vitro2010In: NEW BIOTECHNOL, ISSN 1871-6784, Vol. 27, no 6, p. 766-773Article in journal (Refereed)
    Abstract [en]

    Development of molecules with the ability to selectively inhibit particular protein-protein interactions is important in providing tools for understanding cell biology In this work, we describe efforts to select small Ras- and Raf-specific three-helix bundle affibody binding proteins capable of inhibiting the interaction between H-Ras and Raf-1, from a combinatorial library displayed on bacteriophage Target-specific variants with typically high nanomolar or low micromolar affinities (K-D) could be selected successfully against both proteins, as shown by dot blot, ELISA and real-time biospecific interaction analyses Affibody molecule variants selected against H-Ras were shown to bind epitopes overlapping each other at a site that differed from that at which H-Ras interacts with Raf-1 In contrast, an affibody molecule isolated during selection against Raf-1 was shown to effectively inhibit the interaction between H-Ras and Raf-1 in a dose-dependent manner Possible intracellular applications of the selected affibody molecules are discussed

  • 17.
    Gullfot, Fredrika
    KTH, School of Biotechnology (BIO), Glycoscience.
    Synthesis of xyloglucan oligo- and polysaccharides with glycosynthase technology2009Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    Xyloglucans are polysaccharides found as storage polymers in seeds and tubers, and as cross-linking glycans in the cell wall of plants. Their structure is complex with intricate branching patterns, which contribute to the physical properties of the polysaccharide including its binding to and interaction with other glycans such as cellulose.

    Xyloglucan is widely used in bulk quantities in the food, textile and paper making industries. With an increasing interest in technically more advanced applications of xyloglucan, such as novel biocomposites, there is a need to understand and control the properties and interactions of xyloglucan with other compounds, to decipher the relationship between xyloglucan structure and function, and in particular the effect of different branching patterns. However, due to the structural heterogeneity of the polysaccharide as obtained from natural sources, relevant studies have not been possible to perform in practise. This fact has stimulated an interest in synthetic methods to obtain xyloglucan mimics and analogs with well-defined structure and decoration patterns.

    Glycosynthases are hydrolytically inactive mutant glycosidases that catalyse the formation of glycosidic linkages between glycosyl fluoride donors and glycoside acceptors. Since its first conception in 1998, the technology is emerging as a useful tool in the synthesis of large, complex polysaccharides. This thesis presents the generation and characterisation of glycosynthases based on xyloglucanase scaffolds for the synthesis of well-defined homogenous xyloglucan oligo- and polysaccharides with regular substitution patterns.

  • 18.
    Hammarström, Martin
    KTH, School of Biotechnology (BIO).
    Protein production and purification in structural genomics2006Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    The number of gene products available for structural and functional study is increasing at an unprecedented rate as a result of the successful whole genome sequencing projects. Systematic structure determination of proteins on a genomic scale, called structural genomics, can significantly contribute to the field of protein science and to functional annotation of newly identified genes.

    This thesis covers different aspects of protein production in Eschericiha coli for structural studies in the context of structural genomics. Protocols have been downscaled and standardized to allow for a rapid assessment of the production characteristics for multiple proteins in parallel under a number of different conditions. Foremost, the ability of different proteins and peptide tags to affect the solubility of the recombinant protein when produced as fusion proteins has been systematically studied. Large differences in the success-rate for production of soluble protein in E. coli were found depending on the fusion partner used, with a more than two-fold increase in the number of proteins produced as soluble when comparing the best and the poorest fusion tags. For different constructs with a histidine tag, commonly used to facilitate protein purification, large differences in yield depending on the design of the expression vector were found. When comparing different fusion proteins produced from identical expression vectors, fusions to the GB1 domain were found to result in the highest yield of purified target protein, on average 25 % higher than any of the other fusions.

    The suitability for further structural studies was tested at an intermediate scale for proteins that were identified as soluble in the expression screening. For this purpose, protocols for rapid purification and biophysical characterization using nuclear magnetic resonance and circular dichroism spectroscopy were developed and tested on 19 proteins, of which four were structured.

  • 19.
    Hammarström, Martin
    et al.
    KTH, School of Biotechnology (BIO).
    Woestenenk, Esmeralda
    KTH, School of Biotechnology (BIO).
    Hellgren, Niklas
    KTH, School of Biotechnology (BIO).
    Härd, Torleif
    Berglund, Helena
    KTH, School of Biotechnology (BIO).
    Effect of N-terminal solubility enhancing fusion proteins on yield of purified target protein2006In: Journal of Structural and Functional Genomics, ISSN 1345-711X, E-ISSN 1570-0267, Vol. 7, no 1, p. 1-14Article in journal (Refereed)
    Abstract [en]

    We have studied the effect of solubilising N-terminal fusion proteins on the yield of target protein after removal of the fusion partner and subsequent purification using immobilised metal ion affinity chromatography. We compared the yield of 45 human proteins produced from four different expression vectors: three having an N-terminal solubilising fusion protein (the GB1-domain, thioredoxin, or glutathione S-transferase) followed by a protease cleavage site and a His tag, and one vector having only an N-terminal His tag. We have previously observed a positive effect on solubility for proteins produced as fusion proteins compared to proteins produced with only a His tag in Escherichia coli. We find this effect to be less pronounced when we compare the yields of purified target protein after removal of the solubilising fusion although large target-dependent variations are seen. On average, the GB1+His fusion gives significantly higher final yields of protein than the thioredoxin+His fusion or the His tag, whereas GST+His gives lower yields. We also note a strong correlation between solubility and target protein size, and a correlation between solubility and the presence of peptide fragments that are predicted to be natively disordered.

  • 20.
    Hober, Sophia
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Lindbo, Sarah
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Nilvebrant, Johan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Bispecific applications of non-immunoglobulin scaffold binders2019In: Methods, ISSN 1046-2023, E-ISSN 1095-9130, Vol. 154, p. 143-152Article in journal (Refereed)
    Abstract [en]

    Non-immunoglobulin scaffolds represent a proven group of small affinity proteins that can be engineered in vitro to similar affinity and potency as monoclonal antibodies. Several novel candidate biotherapeutics that exploit the potential advantages scaffold proteins hold over larger and more complex antibodies have been developed over the past decade. The ease of using small and robust binding proteins as flexible and modular building blocks has led to the development of a wide range of innovative approaches to combine them in various bi- and multispecific formats. This progress is expected to aid the ongoing challenge of identifying niche applications where clear differentiation from antibody-based molecules will be key to success. Given the many engineering options that are available for non-immunoglobulin scaffold proteins, they have potential to not only complement but probably also surpass antibodies in certain applications.

  • 21.
    Hu, Francis Jingxin
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Utilizing Solid Phase Cloning, Surface Display And Epitope Information for Antibody Generation and Characterization2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Antibodies have become indispensable tools in diagnostics, research and as therapeutics. There are several strategies to generate monoclonal antibodies (mAbs) in order to avoid the drawbacks of polyclonal antibodies (pAbs) for therapeutic use. Moreover, the growing interest in precision medicine requires a well-characterized target and antibody to predict the responsiveness of a treatment. This thesis describes the use of epitope information and display technologies to generate and characterize antibodies. In Paper I, we evaluated if the epitope information of a well-characterized pAb could be used to generate mAbs with retained binding characteristics. In Paper II, the epitope on the complement protein C5 towards Eculizumab was mapped with surface display, the results of which explained the non-responsiveness of Eculizumab treatment among a patient group due to a mutated C5 gene. With this in mind, we showed efficacy in treatment of the mutated C5 variants using a drug binding to another site on C5, suggesting that our approach can be used to guide treatment in precision medicine. In Paper III, a Gram-positive bacterial display platform was evaluated to complement existing platforms for selection of human scFv libraries. When combined with phage display, a thorough library screening and isolation of nano-molar binders was possible. In Paper IV, a solid phase method for directed mutagenesis was developed to generate functional affinity maturation libraries by simultaneous targeting of all six CDRs. The method was also used to create numerous individual mutants to map the paratope of the parent scFv. The paratope information was used to create directed libraries and deep sequencing of the affinity maturation libraries confirmed the viability of the combination approach. Taken together, precise epitope/paratope information together with display technologies have the potential to generate attractive therapeutic antibodies and direct treatment in precision medicine.

  • 22.
    Hu, Francis Jingxin
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Lundqvist, Magnus
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Rockberg, Johan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    SPUX - A Solid Phase Uracil Excision Method for Antibody Affinity Maturation and Paratope MappingManuscript (preprint) (Other academic)
    Abstract [en]

    Mutagenesis libraries are the heart of combinatorial protein engineering where proteins such as antibodies are evolved for improved functionality. Despite recent improvements in gene synthesis and selection methodologies, current methods still fail to provide practical means for synthesis of complete antibody scFv and screening of theoretical diversities, hence forcing the user to focused diversity screening and assembly of shorter oligos to avoid synthesis errors and maximize library functionality. Here we demonstrate a way to generate highly functional tailored mutagenesis libraries for efficient antibody affinity maturation using a rapid cell-free solid phase cloning method with single strand diversity oligonucleotides. For this we are utilizing a combination of a high-fidelity polymerase for PCR-based incorporation of Uracil into a wild-type template, bead-based solid-phase technology for elution of single strand DNA, oligonucleotide annealing, extension and automation, and an uracil excision enzyme cocktail for in vitro degradation of template DNA to minimize background. Our method allowed for fast (8 hours) mutagenesis and automated cloning of a complete set of 50 position specific alanine-mutations for mapping of the paratope of a scFv antibody in a single robot run. We further exemplify our method by generating and stratifying a set of antibody scFv affinity maturation libraries with targeted diversity into critical or nonessential paratope positions, as well as by a complete randomization in all positions. The libraries were subjected to bacterial surface display selections and output was followed by Illumina deep sequencing and binding analysis by SPR. The functional quality of our libraries were high, with a yield of >99% functional diversity in the case for two of our libraries. We were further able to target all positions in all loops with diversity, and we could show the ability to target all six loops with diversity at the same time. The comparison of different library focus showed us that scFv libraries with diversity targeted to non-essential enhancing paratope positions more quickly rendered enrichment of improved binders compared to random diversity or paratope-targeted libraries. Surprisingly several of the improved binders from the random library had beneficial mutations in the same positions targeted by the smaller focused non-essential enhancing residue focused library indicating a possible benefit of focusing diversity to these spots. We believe our method for construction of libraries with site directed mutagenesis to be a viable way for generation of functional and diverse genetic libraries, particularly suitable for affinity maturation and paratope mapping of antibodies.

  • 23.
    Hu, Francis Jingxin
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Volk, Anna-Luisa
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Persson, Helena
    Department of Immunotechnology, Lund University, Medicon Village (Bldg 406), 223 81 Lund, Sweden..
    Säll, Anna
    Department of Immunotechnology, Lund University, Medicon Village (Bldg 406), 223 81 Lund, Sweden..
    Borrebaeck, Carl
    Department of Immunotechnology, Lund University, Medicon Village (Bldg 406), 223 81 Lund, Sweden..
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Rockberg, Johan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Phage and Gram-positive bacterial display of human antibody repertoires enables isolation of functional high affinity bindersIn: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347Article in journal (Other academic)
    Abstract [en]

    Surface display couples genotype with a surface exposed phenotype and thereby allows for screening of gene-encoded protein libraries for desired characteristics. Of the various display systems, phage display is by far the most popular, mainly thanks to its ability to harbor large library sizes. Here, we describe the first use of a grampositive host for display of a library of human antibody genes. The method allows for swift generation of binders by combining phage and gram-positive display, for its ease of use for screening, sorting and ranking by flow cytometry. We demonstrate the utility of this method by identifying specific low nanomolar scFv towards human HER2. The ranking and performance of the scFv isolated by flow sorting in surface immobilized form was retained when expressed as soluble scFv and analyzed by biolayer interferometry as well as after expression as full-length antibodies in mammalian cells. We also show the possibility to use gram-positive display to directly improve the affinity of the identified binders via an affinity maturation step using random mutagenesis and flow sorting. We believe this combined approach has the potential for a more complete scan of the antibody repertoire and for swift affinity maturation of human antibody formats.

  • 24.
    Jarmander, Johan
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Gustavsson, Martin
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Do, Thi-Huyen
    Samuelson, Patrik
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    A dual tag system for facilitated detection of surface expressed proteins in Escherichia coli2012In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 11, article id 118Article in journal (Refereed)
    Abstract [en]

    Background: The discovery of the autotransporter family has provided a mechanism for surface expression of proteins in laboratory strains of Escherichia coli. We have previously reported the use of the AIDA-I autotransport system to express the Salmonella enterica serovar Enteritidis proteins SefA and H: gm. The SefA protein was successfully exposed to the medium, but the orientation of H:gm in the outer membrane could not be determined due to proteolytic cleavage of the N-terminal detection-tag. The goal of the present work was therefore to construct a vector containing elements that facilitates analysis of surface expression, especially for proteins that are sensitive to proteolysis or otherwise difficult to express. Results: The surface expression system pAIDA1 was created with two detection tags flanking the passenger protein. Successful expression of SefA and H:gm on the surface of E. coli was confirmed with fluorescently labeled antibodies specific for the N-terminal His(6)-tag and the C-terminal Myc-tag. While both tags were detected during SefA expression, only the Myc-tag could be detected for H: gm. The negative signal indicates a proteolytic cleavage of this protein that removes the His(6)-tag facing the medium. Conclusions: Expression levels from pAIDA1 were comparable to or higher than those achieved with the formerly used vector. The presence of the Myc- but not of the His(6)-tag on the cell surface during H:gm expression allowed us to confirm the hypothesis that this fusion protein was present on the surface and oriented towards the cell exterior. Western blot analysis revealed degradation products of the same molecular weight for SefA and H:gm. The size of these fragments suggests that both fusion proteins have been cleaved at a specific site close to the C-terminal end of the passenger. This proteolysis was concluded to take place either in the outer membrane or in the periplasm. Since H:gm was cleaved to a much greater extent then the three times smaller SefA, it is proposed that the longer translocation time for the larger H:gm makes it more susceptible to proteolysis.

  • 25.
    Lendel, Christofer
    KTH, School of Biotechnology (BIO).
    Molecular principles of protein stability and protein-protein interactions2005Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    Proteins with highly specific binding properties constitute the basis for many important applications in biotechnology and medicine. Immunoglobulins have so far been the obvious choice but recent advances in protein engineering have provided several novel constructs that indeed challenge antibodies. One class of such binding proteins is based on the 58 residues three-helix bundle Z domain from staphylococcal protein A (SPA). These so-called affibodies are selected from libraries containing Z domain variants with 13 randomised positions at the immunoglobulin Fc-binding surface. This thesis aims to describe the principles for molecular recognition in two protein-protein complexes involving affibody proteins. The first complex is formed by the ZSPA-1 affibody binding to its own ancestor, the Z domain (Kd ~1 μM). The second complex consists of two affibodies: ZTaq, originally selected to bind Taq DNA polymerase, and anti-ZTaq, an anti-idiotypic binder to ZTaq with a Kd ~0.1 μM. The basis for the study is the determination of the three-dimensional structures using NMR spectroscopy supported by biophysical characterization of the uncomplexed proteins and investigation of binding thermodynamics using isothermal titration calorimetry. The free ZSPA-1 affibody is a molten globule-like protein with reduced stability compared to the original scaffold. However, upon target binding it folds into a well-defined structure with an interface topology resembling that displayed by the immunoglobulin Fc fragment when bound to the Z domain. At the same time, structural rearrangements occur in the Z domain in a similar way as in the Fc-binding process. The complex interface buries 1632 Å2 total surface area and 10 out of 13 varied residues in ZSPA-1 are directly involved in inter-molecular contacts. Further characterization of the molten globule state of ZSPA-1 revealed a native-like overall structure with increased dynamics in the randomised regions (helices 1 and 2). These features were reduced when replacing some of the mutated residues with the corresponding wild-type Z domain residues. The nature of the free ZSPA-1 affects the thermodynamics of the complex formation. The contribution from the unfolding equilibrium of the molten globule was successfully separated from the binding thermodynamics. Further decomposition of the binding entropy suggests that the conformational entropy penalty associated with stabilizing the molten globule state of ZSPA-1 upon binding seriously reduces the binding affinity. The ZTaq:anti-ZTaq complex buries in total 1672 Å2 surface area and all varied positions in anti-ZTaq are directly involved in binding. The main differences between the Z:ZSPA-1 and the ZTaq:anti-ZTaq complexes are the relative subunit orientation and certain specific interactions. However, there are also similarities, such as the hydrophobic interface character and the role of certain key residues, which are also found in the SPA:Fc interaction. Structural rearrangements upon binding are also common features of these complexes. Even though neither ZTaq nor anti-ZTaq shows the molten globule behaviour seen for ZSPA-1, there are indications of dynamic events that might affect the binding affinity. This study provides not only a molecular basis for affibody-target recognition, but also contributions to the understanding of the mechanisms regulating protein stability and protein-protein interactions in general.

  • 26.
    Lendel, Christofer
    et al.
    KTH, Superseded Departments, Biotechnology.
    Dincbas-Renqvist, Vildan
    KTH, Superseded Departments, Biotechnology.
    Flores, Alexander
    KTH, Superseded Departments, Biotechnology.
    Wahlberg, Elisabet
    KTH, Superseded Departments, Biotechnology.
    Dogan, Jakob
    KTH, Superseded Departments, Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Härd, Torleif
    Biophysical characterization of ZSPA-1-A phage-display selected binder to protein A2004In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 13, no 8, p. 2078-2088Article in journal (Refereed)
    Abstract [en]

    Affibodies are a novel class of binding proteins selected from phagemid libraries of the Z domain from staphylococcal protein A. The Z(SPA-1) affibody was selected as a binder to protein A, and it binds the parental Z domain with micromolar affinity. In earlier work we determined the structure of the Z:Z(SPA-1) complex and noted that Z(SPA-1) in the free state exhibits several properties characteristic of a molten globule. Here we present a more detailed biophysical investigation of Z(SPA-1) and four Z(SPA-1) mutants with the objective to understand these properties. The characterization includes thermal and chemical denaturation profiles, ANS binding assays, size exclusion chromatography, isothermal titration calorimetry, and an investigation of structure and dynamics by NMR. The NMR characterization of Z(SPA-1) was facilitated by the finding that trimethylamine N-oxide (TMAO) stabilizes the molten globule conformation in favor of the fully unfolded state. All data taken together lead us to conclude the following: (1) The topology of the molten globule conformation of free Z(SPA-1) is similar to that of the fully folded structure in the Z-bound state; (2) the extensive mutations in helices 1 and 2 destabilize these without affecting the intrinsic stability of helix 3; (3) stabilization and reduced aggregation can be achieved by replacing mutated residues in Z(SPA-1) with the corresponding wild-type Z residues. This stabilization is better correlated to changes in helix propensity than to an expected increase in polar versus nonpolar surface area of the fully folded state.

  • 27.
    Lendel, Christofer
    et al.
    KTH, School of Biotechnology (BIO).
    Dogan, Jakob
    KTH, School of Biotechnology (BIO).
    Härd, Torleif
    Göteborgs Universitet.
    Structural basis for molecular recognition in an affibody:affibody complex2006In: Asia-Pacific Journal of Molecular Biology and Biotechnology, ISSN 0128-7451, Vol. 359, no 5, p. 1293-1304Article in journal (Refereed)
    Abstract [en]

    Affibody molecules constitute a class of engineered binding proteins based on the 58-residue three-helix bundle Z domain derived from staphylococcal protein A (SPA). Affibody proteins are selected as binders to target proteins by phage display of combinatorial libraries in which typically 13 side-chains on the surface of helices 1 and 2 in the Z domain have been randomized. The Z(Taq):anti-Z(Taq) affibody-affibody complex, consisting of Z(Taq), originally selected as a binder to Taq DNA polymerase, and anti-Z(Taq), selected as binder to Z(Taq), is formed with a dissociation constant K-d similar to 100 nM. We have determined high-precision solution structures of free Z(Taq) and anti-Z(Taq), and the Z(Taq):anti-Z(Taq) complex under identical experimental conditions (25 degrees C in 50 mM NaCl with 20 mM potassium phosphate buffer at pH 6.4). The complex is formed with helices 1 and 2 of anti-Z(Taq) in perpendicular contact with helices 1 and 2 of Z(Taq). The interaction surface is large (similar to 1670 angstrom(2)) and unusually non-polar (70 %) compared to other protein-protein complexes. It involves all varied residues on anti-Z(Taq), most corresponding (Taq DNA polymerase binding) side-chains on Z(Taq), and several additional side-chain and backbone contacts. Other notable features include a substantial rearrangement (induced fit) of aromatic side-chains in Z(Taq) upon binding, a close contact between glycine residues in the two subunits that might involve aliphatic glycine H alpha to backbone carbonyl hydrogen bonds, and four hydrogen bonds made by the two guanidinium (NH2)-H-eta groups of an arginine side-chain. Comparisons of the present structure with other data for affibody proteins and the Z domain suggest that intrinsic binding properties of the originating SPA surface might be inherited by the affibody binders. A thermodynamic characterization of Z(Taq) and anti-Z(Taq) is presented in an accompanying paper.

  • 28.
    Lendel, Christofer
    et al.
    KTH, Superseded Departments, Biotechnology.
    Wahlberg, Elisabet
    KTH, Superseded Departments, Biotechnology.
    Berglund, Helena
    KTH, Superseded Departments, Biotechnology.
    Eklund, Malin
    KTH, Superseded Departments, Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Härd, Torleif
    KTH, Superseded Departments, Biotechnology.
    1H, 13C and 15N resonance assignments of an affibody-target complex2002In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 24, no 3, p. 271-272Article in journal (Refereed)
  • 29.
    Lindbo, Sarah
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Gunneriusson, Elin
    Affibody AB.
    Ekblad, Caroline
    Affibody AB.
    Fant, Gunilla
    Affibody AB.
    Hober, Sophia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Design, construction and characterization of an ABD-based library with improved stabilityManuscript (preprint) (Other academic)
    Abstract [en]

    Recombinant affinity proteins binding specifically to other molecules are important tools for many clinical and industrial applications. Small robust protein scaffolds have proven to be well suited as frameworks for generation of novel affinity binders due to their stability. Here we used the albumin-binding domain (ABD) of protein G from Streptococcus G148 as scaffold to design a new combinatorial library capable of generating stable binders to various target proteins with high affinity and specificity. To create a robust framework able to generate highly stable binders, mutations in the non-binding region were evaluated and residues providing increased stability were introduced into the scaffold. By combining rational design with combinatorial protein engineering we also evaluated the surface exposed amino acids at the albumin-binding interface and identified 11 residues suitable for randomization. The potency of the novel scaffold library was assessed by screening for binders using phage display against three distinct targets; complement factor 4, (C4), insulin and interleukin-6 (IL-6). Binders in the nanomolar range with melting temperature above 57°C were selected for all three targets. Notably, the identified IL-6 binders were characterized by extreme thermal stability with variants demonstrating organized structures even at 90°C. This demonstrates that stable binders with distinct specificities can be generated and thus proves the high potential of the library.

  • 30.
    Ljubimova, Darja
    KTH, School of Engineering Sciences (SCI), Mechanics, Structural Mechanics.
    Numerical modelling of the human eye accommodation2005Licentiate thesis, comprehensive summary (Other scientific)
    Abstract [en]

    This thesis addresses the biomechanics of the human eye accommodation. It deals with the development of numerical model of a 29-year-old eye, incorporating the vitreous body as a part of accommodative apparatus. A more complete understanding of the mechanism of accommodation becomes increasingly important as new types of lens implants and surgical procedures to correct both accommodative loss in aphakia as well as in the aging process are being explored. It is necessary to conduct the experimental and analytical studies to gain a better comprehension on ophthalmologic processes. The accommodation has been investigated through numerical simulations based on finite element analysis. The extensive literature survey was the platform for establishing relevant modelling procedures. The calculations were carried out using the commercial general-purpose finite element software ABAQUS. All materials were modelled as being linearly elastic and the interiors on the lens and vitreous were assumed to be incompressible. Present research seeks to investigate the validity of some fundamental assumptions about the construction and functioning of human accommodation system. The model is rather general and involves the synthesis of disparate sets of geometric and mechanical data from a variety of published sources. Different configurations of the structural model can easily be simulated by appropriate adjustments of parameters. The results of this study are broadly in agreement with published observations. The model behaviour is consistent with classical Helmholtz theory. It is shown that such a modelling exercise captures at least some physiological aspects of human accommodation. The proposed procedures and developed inverse methodology can be a useful tool to derive previously not documented parameters and test the consistency of different sets of experimental measurements. The obtained results can be used to draw some recommendations in pursuit of the clinical imperatives of ophthalmologists.

  • 31.
    Ljubimova, Darja
    et al.
    KTH, School of Engineering Sciences (SCI), Mechanics, Structural Mechanics.
    Eriksson, Anders
    KTH, School of Engineering Sciences (SCI), Mechanics, Structural Mechanics.
    Bauer, Svetlana
    Numerical study of the effect of vitreous support on eye accommodation2005In: Acta of Bioengineering and Biomechanics, ISSN 1509-409X, Vol. 7, no 2, p. 3-15Article in journal (Refereed)
    Abstract [en]

    The aim of the current work was to extend previously created finite element models of accommodation such as the one by BURD [2] by addition of vitreous. The zonule consisted of anterior and central sets and vitreous was modelled as a linear clastic incompressible body. An inverse method was used to find some important, previously not documented, aspects. The model was found to behave according to the expectations, with results consistent with classical Helmholtz theory.

  • 32.
    Lundin, Sverker
    KTH, School of Biotechnology (BIO), Gene Technology.
    Methods to Prepare DNA for Efficient Massive Sequencing2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Massive sequencing has transformed the field of genome biology due to the continuous introduction and evolution of new methods. In recent years, the technologies available to read through genomes have undergone an unprecedented rate of development in terms of cost-reduction. Generating sequence data has essentially ceased to be a bottleneck for analyzing genomes instead to be replaced by limitations in sample preparation and data analysis. In this work, new strategies are presented to increase both the throughput of library generation prior to sequencing, and the informational content of libraries to aid post-sequencing data processing. The protocols developed aim to enable new possibilities for genome research concerning project scale and sequence complexity.

    The first two papers that underpin this thesis deal with scaling library production by means of automation. Automated library preparation is first described for the 454 sequencing system based on a generic solid-phase polyethylene-glycol precipitation protocol for automated DNA handling. This was one of the first descriptions of automated sample handling for producing next generation sequencing libraries, and substantially improved sample throughput. Building on these results, the use of a double precipitation strategy to replace the manual agarose gel excision step for Illumina sequencing is presented. This protocol considerably improved the scalability of library construction for Illumina sequencing. The third and fourth papers present advanced strategies for library tagging in order to multiplex the information available in each library. First, a dual tagging strategy for massive sequencing is described in which two sets of tags are added to a library to trace back the origins of up to 4992 amplicons using 122 tags. The tagging strategy takes advantage of the previously automated pipeline and was used for the simultaneous sequencing of 3700 amplicons. Following that, an enzymatic protocol was developed to degrade long range PCR-amplicons and forming triple-tagged libraries containing information of sample origin, clonal origin and local positioning for the short-read sequences. Through tagging, this protocol makes it possible to analyze a longer continuous sequence region than would be possible based on the read length of the sequencing system alone. The fifth study investigates commonly used enzymes for constructing libraries for massive sequencing. We analyze restriction enzymes capable of digesting unknown sequences located some distance from their recognition sequence. Some of these enzymes have previously been extensively used for massive nucleic acid analysis. In this first high throughput study of such enzymes, we investigated their restriction specificity in terms of the distance from the recognition site and their sequence dependence. The phenomenon of slippage is characterized and shown to vary significantly between enzymes. The results obtained should favor future protocol development and enzymatic understanding.

    Through these papers, this work aspire to aid the development of methods for massive sequencing in terms of scale, quality and knowledge; thereby contributing to the general applicability of the new paradigm of sequencing instruments.

  • 33.
    Lundin, Sverker
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Gruselius, Joel
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nystedt, Björn
    Science for Life Laboratory, Stockholm University, Department of Biochemistry and Biophysics, Stockholm.
    Lexow, Preben
    Käller, Max
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hierarchical molecular tagging to resolve long continuous sequences by massively parallel sequencing2013In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 3, p. 1186-Article in journal (Refereed)
    Abstract [en]

    Here we demonstrate the use of short-read massive sequencing systems to in effect achieve longer read lengths through hierarchical molecular tagging. We show how indexed and PCR-amplified targeted libraries are degraded, sub-sampled and arrested at timed intervals to achieve pools of differing average length, each of which is indexed with a new tag. By this process, indices of sample origin, molecular origin, and degree of degradation is incorporated in order to achieve a nested hierarchical structure, later to be utilized in the data processing to order the reads over a longer distance than the sequencing system originally allows. With this protocol we show how continuous regions beyond 3000 bp can be decoded by an Illumina sequencing system, and we illustrate the potential applications by calling variants of the lambda genome, analysing TP53 in cancer cell lines, and targeting a variable canine mitochondrial region.

  • 34.
    Lundin, Sverker
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Jemt, Anders
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Finn, Terje-Hegge
    Foam, Napoleon
    Pettersson, Erik
    Käller, Max
    Wirta, Valtteri
    Lexow, Preben
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Endonuclease specificity and sequence dependence of Type IIS restriction enzymes2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 1, article id e0117059Article in journal (Refereed)
    Abstract [en]

    Restriction enzymes that recognize specific sequences but cleave unknown sequence outside the recognition site are extensively utilized tools in molecular biology. Despite this, systematic functional categorization of cleavage performance has largely been lacking. We established a simple and automatable model system to assay cleavage distance variation (termed slippage) and the sequence dependence thereof. We coupled this to massively parallel sequencing in order to provide sensitive and accurate measurement. With this system 14 enzymes were assayed (AcuI, BbvI, BpmI, BpuEI, BseRI, BsgI, Eco57I, Eco57MI, EcoP15I, FauI, FokI, GsuI, MmeI and SmuI). We report significant variation of slippage ranging from 1-54%, variations in sequence context dependence, as well as variation between isoschizomers. We believe this largely overlooked property of enzymes with shifted cleavage would benefit from further large scale classification and engineering efforts seeking to improve performance. The gained insights of in-vitro performance may also aid the in-vivo understanding of these enzymes.

  • 35.
    Lundin, Sverker
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Stranneheim, Henrik
    KTH, School of Biotechnology (BIO), Gene Technology.
    Pettersson, Erik
    KTH, School of Biotechnology (BIO), Gene Technology.
    Klevebring, Daniel
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Increased Throughput by Parallelization of Library Preparation for Massive Sequencing2010In: PLOS ONE, ISSN 1932-6203, Vol. 5, no 3, p. e10029-Article in journal (Refereed)
    Abstract [en]

    Background: Massively parallel sequencing systems continue to improve on data output, while leaving labor-intensive library preparations a potential bottleneck. Efforts are currently under way to relieve the crucial and time-consuming work to prepare DNA for high-throughput sequencing. Methodology/Principal Findings: In this study, we demonstrate an automated parallel library preparation protocol using generic carboxylic acid-coated superparamagnetic beads and polyethylene glycol precipitation as a reproducible and flexible method for DNA fragment length separation. With this approach the library preparation for DNA sequencing can easily be adjusted to a desired fragment length. The automated protocol, here demonstrated using the GS FLX Titanium instrument, was compared to the standard manual library preparation, showing higher yield, throughput and great reproducibility. In addition, 12 libraries were prepared and uniquely tagged in parallel, and the distribution of sequence reads between these indexed samples could be improved using quantitative PCR-assisted pooling. Conclusions/Significance: We present a novel automated procedure that makes it possible to prepare 36 indexed libraries per person and day, which can be increased to up to 96 libraries processed simultaneously. The yield, speed and robust performance of the protocol constitute a substantial improvement to present manual methods, without the need of extensive equipment investments. The described procedure enables a considerable efficiency increase for small to midsize sequencing centers.

  • 36.
    Löfdahl, Per-Åke
    KTH, School of Engineering Sciences (SCI), Physics.
    Improved solubility of TEV protease by directed evolution2006In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 121, no 3, p. 291-298Article in journal (Refereed)
    Abstract [en]

    The efficiency and high specificity of tobacco etch virus (TEV) protease has made it widely used for cleavage of recombinant fusion proteins. However, the production of TEV protease in E. coli is hampered by low solubility. We have subjected the gene encoding TEV protease to directed evolution to improve the yield of soluble protein. Libraries of mutated genes obtained by error-prone PCR and gene shuffling were introduced into the Gateway cloning system for facilitated transfer between vectors for screening, purification, or other applications. Fluorescence based in vivo solubility screening was carried out by cloning the libraries into a plasmid encoding a C-terminal GFP fusion. Mutant genes giving rise to high GFP fluorescence intensity indicating high levels of soluble TEV–GFP were subsequently transferred to a vector providing a C-terminal histidine tag for expression, purification, and activity tests of mutated TEV. We identified a mutant, TEVSH, in which three amino acid substitutions result in a five-fold increase in the yield of purified protease with retained activity.

  • 37. Meletis, Konstantinos
    et al.
    Wirta, Valtteri
    KTH, School of Biotechnology (BIO).
    Hede, Sanna-Maria
    Nistér, Monica
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO).
    Frisén, Jonas
    p53 suppresses the self-renewal of adult neural stem cells2006In: Development, ISSN 0950-1991, E-ISSN 1477-9129, Vol. 133, no 2, p. 363-369Article in journal (Refereed)
    Abstract [en]

    There is increasing evidence that tumors are heterogeneous and that a subset of cells act as cancer stem cells. Several proto-oncogenes and tumor suppressors control key aspects of stem cell function, suggesting that similar mechanisms control normal and cancer stem cell properties. We show here that the prototypical tumor suppressor p53, which plays an important role in brain tumor initiation and growth, is expressed in the neural stem cell lineage in the adult brain. p53 negatively regulates proliferation and survival, and thereby self-renewal, of neural stem cells. Analysis of the neural stem cell transcriptome identified the dysregulation of several cell cycle regulators in the absence of p53, most notably a pronounced downregulation of p21 expression. These data implicate p53 as a suppressor of tissue and cancer stem cell self-renewal.

  • 38.
    Nguyen Gia, Tuan
    et al.
    University of Turku, Finland.
    Jiang, Mingzhe
    University of Turku, Finland.
    Rahmani, Amir-Mohammad
    KTH, School of Information and Communication Technology (ICT), Industrial and Medical Electronics. University of Turku, Finland.
    Westerlund, Tomi
    University of Turku, Finland.
    Mankodiya, Kunal
    University of Rhode Island, USA.
    Liljeberg, Pasi
    University of Turku, Finland.
    Tenhunen, Hannu
    KTH, School of Information and Communication Technology (ICT), Industrial and Medical Electronics. University of Turku, Finland.
    Fog Computing in Body Sensor Networks: An Energy Efficient Approach2015Conference paper (Refereed)
    Abstract [en]

    Internet of Things based systems provides a viable and organized approach to improve health and wellbeing of mankind. Particularly, health monitoring systems based on wireless body sensor networks become attainable due to increasing number of elderly people that needs healthcare services frequently. In such system, power consumption of a sensor node is an important issue. In order to handle the issue, a smart gateway with fog computing capabilities is presented. Fog computing includes several services such as distributed database management, Electrocardiography (ECG) feature extraction, user graphical interface with access management and push notations. With fog computing, the burden of a cloud server can be reduced and more than 50% of power consumption can be saved at a sensor node. Additionally, through fog computing, the system ensures that the obtained health data can be visualized and diagnosed in real-time even though there is a disconnection between the gateway and cloud server.

  • 39. Nhan, Nguyen Thanh
    et al.
    Gonzalez de Valdivia, Ernesto
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Gustavsson, Martin
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Hai, Truong Nam
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Surface display of Salmonella epitopes in Escherichia coli and Staphylococcus carnosus2011In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 10, p. 22-Article in journal (Refereed)
    Abstract [en]

    Background: Salmonella enterica serotype Enteritidis (SE) is considered to be one of the most potent pathogenic Salmonella serotypes causing food-borne disease in humans. Since a live bacterial vaccine based on surface display of antigens has many advantages over traditional vaccines, we have studied the surface display of the SE antigenic proteins, H: gm and SefA in Escherichia coli by the beta-autotransporter system, AIDA. This procedure was compared to protein translocation in Staphylococcus carnosus, using a staphylococci hybrid vector earlier developed for surface display of other vaccine epitopes. Results: Both SefA and H: gm were translocated to the outer membrane in Escherichia coli. SefA was expressed to full length but H: gm was shorter than expected, probably due to a proteolytic cleavage of the N-terminal during passage either through the periplasm or over the membrane. FACS analysis confirmed that SefA was facing the extracellular environment, but this could not be conclusively established for H: gm since the N-terminal detection tag (His(6)) was cleaved off. Polyclonal salmonella antibodies confirmed the sustained antibody-antigen binding towards both proteins. The surface expression data from Staphylococcus carnosus suggested that the H: gm and SefA proteins were transported to the cell wall since the detection marker was displayed by FACS analysis. Conclusion: Apart from the accumulated knowledge and the existence of a wealth of equipment and techniques, the results indicate the selection of E. coli for further studies for surface expression of salmonella antigens. Surface expression of the full length protein facing the cell environment was positively proven by standard analysis, and the FACS signal comparison to expression in Staphylococcus carnosus shows that the distribution of the surface protein on each cell was comparatively very narrow in E. coli, the E. coli outer membrane molecules can serve as an adjuvant for the surface antigenic proteins and multimeric forms of the SefA protein were detected which would probably be positive for the realisation of a strong antigenic property. The detection of specific and similar proteolytic cleavage patterns for both the proteins provides a further starting point for the investigation and development of the Escherichia coli AIDA autotransporter efficiency.

  • 40.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Affinity ligands from biological combinatorial libraries2011In: Methods of biochemical analysis, ISSN 0076-6941, E-ISSN 1934-4325, Vol. 54, p. 269-78Article in journal (Refereed)
  • 41.
    Nyrén, Pål
    KTH, School of Biotechnology (BIO).
    Method enabling detection of single-stranded DNA ligation activity2017In: Archives of industrial biotechnology, Vol. 1, no 1, p. 35-40Article in journal (Refereed)
    Abstract [en]

    The enzyme DNA ligase is an essential tool in many different applications in the field of biotechnology. For these applications the DNA quality can be crucial for the final result. In this paper we present a new method that can be used for specific studies of DNA ligases and for simultaneous analyzes of DNA quality. More specifically, a simple, fast and sensitive method for detection of single-stranded DNA (ssDNA) ligase activity is presented. The new method is based on real-time detection of nucleotide incorporation over the ligation site catalyzed by a DNA polymerase. The ratio between the incorporation signal after and before the ligation site gives an estimate of the ligation efficiency. To further increase the robustness of the assay the exact amount of unligated product can be easily estimated in a separate assay using a 3’-end blocked oligonucleotide functioning as template. Produced circular DNA can be cleaned from unligated DNA by exonuclease I treatment and the quality of the circle can be easily analyzed by a simple sequencing procedure. If the ssDNA used for the ligation reaction contains truncated fragments this will be observed by uneven signals during the analysis. The new method can be a useful tool for researchers working with both basic and applied projects in the field of biotechnology.

  • 42.
    Oskarsson, Mattias
    KTH, School of Biotechnology (BIO), Gene Technology.
    Analysis of the origin and spread of the domestic dog using Y-chromosome DNA and mtDNA sequence data2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The domestic dog was probably the first domesticated animal, and the only one to spread to all continents in ancient times. The dog is one of the most phenotypically diverse animals, a result of human selection throughout dog history. Studies of the genetic origins and early spread of domestic dogs is important to gather information about biological and cultural mechanisms behind domestication but also to investigate early human history. The step from a hunter and gatherer lifestyle to farming is one of the most important steps in human history. In this thesis I will present work aimed at understanding both domestic dog origins and dispersal. In order to be able to investigate dog origins based on a second haploid chromosome we identified 14,437 bp of Y-chromosomal DNA sequence. Based on this we show that dogs in Asia south of Yangtze River (ASY) has the highest genetic diversity and was founded from a large number of wolf founders confirming earlier mtDNA results. Early dog dispersal is tightly coupled to human history with the dog brought along as a cultural item. We have for the first time investigated the dog dispersal into Polynesia and Australia and our data can be used as evidence for a more complex settlement of Polynesia than earlier indicated from archaeological and linguistic studies. Analysis of Y-chromosome SNPs in Australian dingoes confirms earlier mtDNA genetic studies that the dingo is part of the domestic dog phylogeny and was founded from a small population of domestic dogs. We have also for the first time investigated the dog population on Madagascar and our data strongly indicates a mainland African origin for the Madagascan dogs. Finally, we have investigated the American dog population sampled from throughout the continent and also for the first time included putative indigenous breed dogs such as Chihuahua and Pero Sín Pelo del Peru, and the free-ranging Carolina dogs from southern USA. Our data clearly indicates a primarily Old World origin for the indigenous breed dogs and also for the free-ranging Carolina dogs in USA. We can also for the first time present evidence for continuity between the ancient and extant dog population with e.g. exclusive sharing of a haplotype between a modern sample of Chihuahua and an ancient Mexican sample.

  • 43. Papapetridis, Ioannis
    et al.
    Goudriaan, Maaike
    Vitali, María Vázquez
    Keijzer, Nikita A
    Broek, Marcel
    van Maris, Antonius
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. Delft University of Technology, The Netherlands.
    Pronk, Jack T.
    Optimizing anaerobic growth rate and fermentation kinetics in Saccharomyces cerevisiae strains expressing Calvin-cycle enzymes for improved ethanol yield2018In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 11, no 1, article id 17Article in journal (Refereed)
    Abstract [en]

    Background: Reduction or elimination of by-product formation is of immediate economic relevance in fermentation processes for industrial bioethanol production with the yeast Saccharomyces cerevisiae. Anaerobic cultures of wildtype S. cerevisiae require formation of glycerol to maintain the intracellular NADH/NAD(+) balance. Previously, functional expression of the Calvin-cycle enzymes ribulose-1,5-bisphosphate carboxylase (RuBisCO) and phosphoribulokinase (PRK) in S. cerevisiae was shown to enable reoxidation of NADH with CO2 as electron acceptor. In slow-growing cultures, this engineering strategy strongly decreased the glycerol yield, while increasing the ethanol yield on sugar. The present study explores engineering strategies to improve rates of growth and alcoholic fermentation in yeast strains that functionally express RuBisCO and PRK, while maximizing the positive impact on the ethanol yield. Results: Multi-copy integration of a bacterial-RuBisCO expression cassette was combined with expression of the Escherichia coli GroEL/GroES chaperones and expression of PRK from the anaerobically inducible DAN1 promoter. In anaerobic, glucose-grown bioreactor batch cultures, the resulting S. cerevisiae strain showed a 31% lower glycerol yield and a 31% lower specific growth rate than a non-engineered reference strain. Growth of the engineered strain in anaerobic, glucose-limited chemostat cultures revealed a negative correlation between its specific growth rate and the contribution of the Calvin-cycle enzymes to redox homeostasis. Additional deletion of GPD2, which encodes an isoenzyme of NAD(+)-dependent glycerol-3-phosphate dehydrogenase, combined with overexpression of the structural genes for enzymes of the non-oxidative pentose-phosphate pathway, yielded a CO2-reducing strain that grew at the same rate as a non-engineered reference strain in anaerobic bioreactor batch cultures, while exhibiting a 86% lower glycerol yield and a 15% higher ethanol yield. Conclusions: The metabolic engineering strategy presented here enables an almost complete elimination of glycerol production in anaerobic, glucose-grown batch cultures of S. cerevisiae, with an associated increase in ethanol yield, while retaining near wild-type growth rates and a capacity for glycerol formation under osmotic stress. Using current genome-editing techniques, the required genetic modifications can be introduced in one or a few transformations. Evaluation of this concept in industrial strains and conditions is therefore a realistic next step towards its implementation for improving the efficiency of first-and second-generation bioethanol production.

  • 44.
    Papapetridis, Ioannis
    et al.
    Delft Univ Technol, Dept Biotechnol, Maasweg 9, NL-2629 HZ Delft, Netherlands..
    Verhoeven, Maarten D.
    Delft Univ Technol, Dept Biotechnol, Maasweg 9, NL-2629 HZ Delft, Netherlands..
    Wiersma, Sanne J.
    Delft Univ Technol, Dept Biotechnol, Maasweg 9, NL-2629 HZ Delft, Netherlands..
    Goudriaan, Maaike
    Delft Univ Technol, Dept Biotechnol, Maasweg 9, NL-2629 HZ Delft, Netherlands..
    van Maris, Antonius J. A.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. Delft Univ Technol, Dept Biotechnol, Maasweg 9, NL-2629 HZ Delft, Netherlands..
    Pronk, Jack T.
    Delft Univ Technol, Dept Biotechnol, Maasweg 9, NL-2629 HZ Delft, Netherlands..
    Laboratory evolution for forced glucose-xylose co-consumption enables identification of mutations that improve mixed-sugar fermentation by xylose-fermenting Saccharomyces cerevisiae2018In: FEMS yeast research (Print), ISSN 1567-1356, E-ISSN 1567-1364, Vol. 18, no 6, article id foy056Article in journal (Refereed)
    Abstract [en]

    Simultaneous fermentation of glucose and xylose can contribute to improved productivity and robustness of yeast-based processes for bioethanol production from lignocellulosic hydrolysates. This study explores a novel laboratory evolution strategy for identifying mutations that contribute to simultaneous utilisation of these sugars in batch cultures of Saccharomyces cerevisiae. To force simultaneous utilisation of xylose and glucose, the genes encoding glucose-6-phosphate isomerase (PGI1) and ribulose-5-phosphate epimerase (RPE1) were deleted in a xylose-isomerase-based xylose-fermenting strain with a modified oxidative pentose-phosphate pathway. Laboratory evolution of this strain in serial batch cultures on glucose-xylose mixtures yielded mutants that rapidly co-consumed the two sugars. Whole-genome sequencing of evolved strains identified mutations in HXK2, RSP5 and GAL83, whose introduction into a non-evolved xylose-fermenting S. cerevisiae strain improved co-consumption of xylose and glucose under aerobic and anaerobic conditions. Combined deletion of HXK2 and introduction of a GAL83(G673T) allele yielded a strain with a 2.5-fold higher xylose and glucose co-consumption ratio than its xylose-fermenting parental strain. These two modifications decreased the time required for full sugar conversion in anaerobic bioreactor batch cultures, grown on 20 g L-1 glucose and 10 g L-1 xylose, by over 24 h. This study demonstrates that laboratory evolution and genome resequencing of microbial strains engineered for forced co-consumption is a powerful approach for studying and improving simultaneous conversion of mixed substrates.

  • 45. Park, Hyundai
    et al.
    Sysak, Matthew N.
    Chen, Hui-Wen
    Fang, Alexander W.
    Liang, Di
    Liao, Ling
    Koch, Brian R.
    Bovington, Jock
    Tang, Yongbo
    KTH, School of Information and Communication Technology (ICT), Microelectronics and Applied Physics, MAP.
    Wong, Kristi
    Jacob-Mitos, Matt
    Jones, Richard
    Bowers, John E.
    Device and Integration Technology for Silicon Photonic Transmitters2011In: IEEE Journal of Selected Topics in Quantum Electronics, ISSN 1077-260X, E-ISSN 1558-4542, Vol. 17, no 3, p. 671-688Article in journal (Refereed)
    Abstract [en]

    The device and integration technology for silicon photonic transmitters are reviewed in this paper. The hybrid silicon platform enables on-chip lasers to be fabricated with silicon photonic circuits and can be integrated in the CMOS back-end flow. Laser arrays from multiple die bonding and quantum well intermixing techniques are demonstrated to extend the spectral bandwidth from the laser array of the transmitter. Two modulator technologies, silicon modulators and hybrid silicon modulators, are also described.

  • 46.
    Pettersson, Erik
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Zajac, Pawel
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ståhl, Patrik
    KTH, School of Biotechnology (BIO), Gene Technology.
    Jacobsson, Josefin
    Fredriksson, Robert
    Marcus, Claude
    Schiöth, Helgi
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Allelotyping by Massively Parallel Pyrosequencing of SNP-carrying Trinucleotide Threads2008In: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 29, no 2, p. 323-329Article in journal (Refereed)
    Abstract [en]

    Here we present an approach for allelotyping combining the multiplexing features of the trinucleotide threading (TnT) method with pooling of genomic DNA and massively parallel pyrosequencing, enabling reliable allele frequency estimation in large cohorts. The approach offers several benefits as compared to array-based methods and allows undertaking highly complex studies without compromising accuracy, while keeping the workload to a minimum. This proof-of-concept study involves formation of trinucleotide threads, targeting a total of 147 single-nucleotide polymorphisms (SNPs) related to obesity and cancer, for multiplex amplification and allele extraction from a pool of 462 genomes, followed by massively parallel pyrosequencing. Approximately 177k reads were approved, identified, and assigned to SNP-carrying threads rendering representative allele frequencies in the cohort.

  • 47.
    Ramachandraiah, Harisha
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Zelenin, Sergey
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Amasia, Mary
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    In situ isothermal rolling circle amplification (RCA) of DNA and bead based visualization of RCA products on an integrated lab on DVD platform for low cost molecular diagnostics2014In: 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014, 2014, p. 1422-1424Conference paper (Refereed)
    Abstract [en]

    Isothermal rolling circle amplification (RCA) is a simple and versatile isothermal enzymatic nucleic acid amplification techniques, and has been demonstrated as a ideal tool for biomedical research. We demonstrate for the first time the detection of padlock probes mediated and rolling circle amplification of DNA on centrifugal microfluidic system. DNA spotted on DVD substrate is amplified in-situ and novel bead-based detection of the amplified product is demonstrated using a modified commercial DVD reader for "image based" molecular diagnostics.

  • 48. Richter, Karin
    et al.
    Wirta, Valtteri
    KTH, School of Biotechnology (BIO).
    Dahl, Lina
    Bruce, Sara
    KTH, School of Biotechnology (BIO).
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO).
    Carlsson, Leif
    Williams, Cecilia
    KTH, School of Biotechnology (BIO).
    Global gene expression analyses of hematopoietic stem cell-like cell lines with inducible Lhx2 expression2006In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 7, p. 75-Article in journal (Refereed)
    Abstract [en]

    Background: Expression of the LIM-homeobox gene Lhx2 in murine hematopoietic cells allows for the generation of hematopoietic stem cell (HSC)-like cell lines. To address the molecular basis of Lhx2 function, we generated HSC-like cell lines where Lhx2 expression is regulated by a tet-on system and hence dependent on the presence of doxycyclin (dox). These cell lines efficiently down-regulate Lhx2 expression upon dox withdrawal leading to a rapid differentiation into various myeloid cell types.

    Results: Global gene expression of these cell lines cultured in dox was compared to different time points after dox withdrawal using microarray technology. We identified 267 differentially expressed genes. The majority of the genes overlapping with HSC-specific databases were those down-regulated after turning off Lhx2 expression and a majority of the genes overlapping with those defined as late progenitor-specific genes were the up-regulated genes, suggesting that these cell lines represent a relevant model system for normal HSCs also at the level of global gene expression. Moreover, in situ hybridisations of several genes down-regulated after dox withdrawal showed overlapping expression patterns with Lhx2 in various tissues during embryonic development.

    Conclusion: Global gene expression analysis of HSC-like cell lines with inducible Lhx2 expression has identified genes putatively linked to self-renewal / differentiation of HSCs, and function of Lhx2 in organ development and stem / progenitor cells of non-hematopoietic origin.

  • 49.
    Sanchez Garcia, Diana Mirey
    KTH, School of Chemical Science and Engineering (CHE).
    Quantification of Cellular Accumulation and Lipid Binding Properties for Phospholipidosis Inducing Drugs and Bioaccumulationg Environmental Toxicants2016Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Highly accumulating compounds in human body are found ubiquitously around the world, and can originate from the industry or from costumers. Depending on their physicochemical properties, they will tend to accumulate by different mechanisms. For example, cationic amphiphilic drugs (CADs) have been reported to accumulate in great extent in lysosomes, whereas fat tissue is the primary accumulation site for organic pollutants. Therefore, understanding the rules that govern their accumulation is fundamental for the creation of accurate screening methods and understanding the mechanisms of their biological effects. Here we describe the interaction of a group of compounds with cells and cell-free chromatographic systems. The interaction with cells was analyzed through the comparison of accumulation and retention of the studied compounds in lung epithelial cells and adipocytes. For understanding the interaction with lipids in cell-free systems, C18 and IAM PC columns were used to calculate LogD and CHIIAM, which represent the binding to neutral lipids and phospholipids, respectively. High Spearman correlation was found in accumulation and retention among the different cell types (r=0.869 and 0.9441, respectively). It was observed that interaction with phospholipids is strongly correlated to cellular accumulation and retention in both cell lines (r = 0.893 and 0.915, respectively). Finally, we found that one of environmental pollutants (PFOS) presented cellular accumulation and phospholipid binding behavior similar to CADs.

  • 50. Siedler, S.
    et al.
    Kumar Khatri, Narendar
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Zsohár, A.
    Kjærbølling, I.
    Vogt, M.
    Hammar, Petter
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nielsen, C. F.
    Marienhagen, J.
    Sommer, M. O. A.
    Jönsson, Håkan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Development of a Bacterial Biosensor for Rapid Screening of Yeast p-Coumaric Acid Production2017In: ACS Synthetic Biology, E-ISSN 2161-5063, Vol. 6, no 10, p. 1860-1869Article in journal (Refereed)
    Abstract [en]

    Transcription factor-based biosensors are used to identify producer strains, a critical bottleneck in cell factory engineering. Here, we address two challenges with this methodology: transplantation of heterologous transcriptional regulators into new hosts to generate functional biosensors and biosensing of the extracellular product concentration that accurately reflects the effective cell factory production capacity. We describe the effects of different translation initiation rates on the dynamic range of a p-coumaric acid biosensor based on the Bacillus subtilis transcriptional repressor PadR by varying its ribosomal binding site. Furthermore, we demonstrate the functionality of this p-coumaric acid biosensor in Escherichia coli and Corynebacterium glutamicum. Finally, we encapsulate yeast p-coumaric acid-producing cells with E. coli-biosensing cells in picoliter droplets and, in a microfluidic device, rapidly sort droplets containing yeast cells producing high amounts of extracellular p-coumaric acid using the fluorescent E. coli biosensor signal. As additional biosensors become available, such approaches will find broad applications for screening of an extracellular product.

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