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  • 251. Gustafsson, Kerstin
    et al.
    Blidberg, Eva
    Elfgren, I. K.
    Hellström, Anna
    Kylin, Henrik
    Gorokhova, E.
    Direct and indirect effects of the fungicide azoxystrobin in outdoor brackish water microcosms2010In: Ecotoxicology, ISSN 0963-9292, E-ISSN 1573-3017, Vol. 19, no 2, p. 431-444Article in journal (Refereed)
    Abstract [en]

    The effects of the strobilurin fungicide azoxystrobin were studied in brackish water microcosms, with natural plankton communities and sediment. Two experiments were conducted: Experiment 1 (nominal conc. 0, 15 and 60 mu g/L, 24-L outdoor microcosms for 21 days) and a second, follow-up, Experiment 2 (nominal conc. 0, 3, 7.5, 15 mu g/L, 4-L indoor microcosms for 12 days). The microcosms represent a simplified brackish water community found in shallow semi-enclosed coastal areas in agricultural districts in the Baltic Sea region. Measured water concentrations of the fungicide (Experiment 1) were, on average, 83 and 62% of nominal concentrations directly after application, and 25 and 30% after 21 days, for the low and high dose treatments, respectively, corresponding to mean DT50-values of 15.1 and 25.8 days, for low and high dose treatments, respectively. In Experiment 1, direct toxic effects on calanoid copepods at both test concentrations were observed. Similarly, in Experiment 2, the copepod abundance was significantly reduced at all tested concentrations. There were also significant secondary effects on zooplankton and phytoplankton community structure, standing stocks and primary production. Very few ecotoxicological studies have investigated effects of plant protection products on Baltic organisms in general and effects on community structure and function specifically. Our results show that azoxystrobin is toxic to brackish water copepods at considerably lower concentrations than previously reported from single species tests on freshwater crustaceans, and that direct toxic effects on this ecologically important group may lead to cascade effects altering lower food webs and ecosystem functioning.

  • 252. Gustavsson, Elin
    et al.
    Ek, Sara
    Steen, Johanna
    KTH, School of Biotechnology (BIO), Proteomics.
    Kristensson, Malin
    Älgenäs, Cajsa
    KTH, School of Biotechnology (BIO), Proteomics.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Wingren, Christer
    Ottosson, Jenny
    KTH, School of Biotechnology (BIO), Proteomics.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Borrebaeck, Carl A. K.
    Surrogate antigens as targets for proteome-wide binder selection2011In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 28, no 4, p. 302-311Article in journal (Refereed)
    Abstract [en]

    In the last decade, many initiatives have been taken to develop antibodies for proteome-wide studies, as well as characterization and validation of clinically relevant disease biomarkers. Phage display offers many advantages compared to conventional antibody generation by immunization and hybridoma technology, since it is an unlimited resource of affinity reagents without batch-to-batch variation and is amendable for high throughput. One of the major bottlenecks to proteome-wide binder selection is the limited supply of suitable target antigens representative of the human proteome. Here, we provide proof of principle of using easily accessible, cancer-associated protein epitope signature tags (PrESTs), routinely generated within the Human Protein Atlas project, as surrogate antigens in phage selectionsfor the retrieval of target specific binders. These binders were subsequently tested in western blot, immunohistochemistry and protein microarray application to demonstrate their functionality.

  • 253.
    Gustavsson, Martin
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Influence of recombinant passenger properties and process conditions on surface expression using the AIDA-I autotransporter2013Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Surface expression has attracted much recent interest, and it has been suggested for a variety of applications. Two such applications are whole-cell biocatalysis and the creation of live vaccines. For successful implementation of these applications there is a need for flexible surface expression systems that can yield a high level of expression with a variety of recombinant fusion proteins. The aim of this work was thus to create a surface expression system that would fulfil these requirements.

     

    A novel surface expression system based on the AIDA-I autotransporter was created with the key qualities being are good, protein-independent detection of the expression through the presence of two epitope tags flanking the recombinant protein, and full modularity of the different components of the expression cassette. To evaluate the flexibility of this construct, 8 different model proteins with potential use as live-vaccines or biocatalysts were expressed and their surface expression levels were analysed.

     

    Positive signals were detected for all of the studied proteins using antibody labelling followed by flow cytometric analysis, showing the functionality of the expression system. The ratio of the signal from the two epitope tags indicated that several of the studied proteins were present mainly in proteolytically degraded forms, which was confirmed by Western blot analysis of the outer membrane protein fraction. This proteolysis was suggested to be due to protein-dependent stalling of translocation intermediates in the periplasm, with indications that larger size and higher cysteine content had a negative impact on expression levels. Process design with reduced cultivation pH and temperature was used to increase total surface expression yield of one of the model proteins by 400 %, with a simultaneous reduction of proteolysis by a third. While not sufficient to completely remove proteolysis, this shows that process design can be used to greatly increase surface expression. Thus, it is recommended that future work combine this with engineering of the bacterial strain or the expression system in order to overcome the observed proteolysis and maximise the yield of surface expressed protein.

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  • 254.
    Gustavsson, Martin
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Surface expression using the AIDA autotransporter:  Towards live vaccines and whole-cell biocatalysis2011Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    The area of surface expression has gathered a lot of interest from research groups all over the world and much work is performed in the area. Autotransporters have been used for surface expression in Gram-negative bacteria. One of the more commonly used autotransporters is the Adhesin Involved in Diffuse Adherence (AIDA) of pathogenic Escherichia coli. The surface expression of enzymes and vaccine epitopes offer several advantages. Surface expressed enzymes gain similar properties to immobilised enzymes, mainly simplified handling and separation using centrifugation. Surface expressed vaccine epitopes can have longer half-lives inside the animal that is to be immunized and surface groups on the host cell can act as adjuvants, increasing the immune response and leading to a better immunisation.

      

    However, while much basic research is directed towards mechanisms of surface expression using autotransporters there are few reports regarding production of surface expressed protein. Thus the aim of this work was the optimisation of the yield and productivity of surface expressed protein. Protein Z, an IgG-binding domain of Staphylococcal protein A, was used as a model protein for the investigation of which cultivation parameters influenced surface expression. The choice of cultivation medium gave the largest impact on expression, which was attributed to effects based on the induction of the native promoter of AIDA. The AIDA system was then used for the expression of two Salmonella surface proteins, SefA and H:gm, with potential for use as vaccine epitopes. SefA was verified located on the cell surface, and H:gm was found in the outer membrane of the host cell, though only in proteolytically truncated forms lacking the His6-tag used for detection. This proteolysis persisted in E. coli strains deficient for the outer membrane protease OmpT and was concluded to be dependent on other proteases. The removal of proteolysis and further optimisation of the yield of surface-expressed protein are important goals of further work.

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  • 255.
    Gustavsson, Martin
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Bäcklund, Emma
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Optimisation of surface expression using the AIDA autotransporter2011In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 10Article in journal (Refereed)
    Abstract [en]

    Background: Bacterial surface display is of interest in many applications, including live vaccine development, screening of protein libraries and the development of whole cell biocatalysts. The goal of this work was to understand which parameters result in production of large quantities of cells that at the same time express desired levels of the chosen protein on the cell surface. For this purpose, staphylococcal protein Z was expressed using the AIDA autotransporter in Escherichia coli.

    Results: The use of an OmpT-negative E. coli mutant resulted in successful expression of the protein on the surface, while a clear degradation pattern was found in the wild type. The expression in the mutant resulted also in a more narrow distribution of the surface anchored protein within the population. Medium optimisation showed that minimal medium with glucose gave more than four times as high expression as LB-medium. Glucose limited fed-batch was used to increase the cell productivity and the highest protein levels were found at the highest feed rates. A maintained high surface expression up to cell dry weights of 18 g l(-1) could also be achieved by repeated glucose additions in batch cultivation where production was eventually reduced by low oxygen levels. In spite of this, the distribution in the bacterial population of the surface protein was narrower using the batch technique.

    Conclusions: A number of parameters in recombinant protein production were seen to influence the surface expression of the model protein with respect both to the productivity and to the display on the individual cell. The choice of medium and the cell design to remove proteolytic cleavage were however the most important. Both fed-batch and batch processing can be successfully used, but prolonged batch processing is probably only possible if the chosen strain has a low acetic acid production.

  • 256.
    Gustavsson, Martin
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Hörnström, David
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Lundh, Susanna
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Belotserkovsky, Jaroslav
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Biocatalysis on the surface of Escherichia coli: melanin pigmentation of the cell exterior2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 36117Article in journal (Refereed)
    Abstract [en]

    Today, it is considered state-of-the-art to engineer living organisms for various biotechnology applications. Even though this has led to numerous scientific breakthroughs, the enclosed interior of bacterial cells still restricts interactions with enzymes, pathways and products due to the mass-transfer barrier formed by the cell envelope. To promote accessibility, we propose engineering of biocatalytic reactions and subsequent product deposition directly on the bacterial surface. As a proof-of-concept, we used the AIDA autotransporter vehicle for Escherichia coli surface expression of tyrosinase and fully oxidized externally added tyrosine to the biopolymer melanin. This resulted in a color change and creation of a black cell exterior. The capture of ninety percent of a pharmaceutical wastewater pollutant followed by regeneration of the cell bound melanin matrix through a simple pH change, shows the superior function and facilitated processing provided by the surface methodology. The broad adsorption spectrum of melanin could also allow removal of other micropollutants.

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  • 257.
    Gustavsson, Martin
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Lee, Sang Yup
    Prospects of microbial cell factories developed through systems metabolic engineering2016In: Microbial Biotechnology, ISSN 1751-7907, E-ISSN 1751-7915, Vol. 9, no 5, p. 610-617Article in journal (Refereed)
    Abstract [en]

    While academic-level studies on metabolic engineering of microorganisms for production of chemicals and fuels are ever growing, a significantly lower number of such production processes have reached commercial-scale. In this work, we review the challenges associated with moving from laboratory-scale demonstration of microbial chemical or fuel production to actual commercialization, focusing on key requirements on the production organism that need to be considered during the metabolic engineering process. Metabolic engineering strategies should take into account techno-economic factors such as the choice of feedstock, the product yield, productivity and titre, and the cost effectiveness of midstream and downstream processes. Also, it is important to develop an industrial strain through metabolic engineering for pathway construction and flux optimization together with increasing tolerance to products and inhibitors present in the feedstock, and ensuring genetic stability and strain robustness under actual fermentation conditions.

  • 258.
    Gustavsson, Martin
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Muraleedharan, Madhu Nair
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Surface Expression of omega-Transaminase in Escherichia coli2014In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 80, no 7, p. 2293-2298Article in journal (Refereed)
    Abstract [en]

    Chiral amines are important for the chemical and pharmaceutical industries, and there is rapidly growing interest to use transaminases for their synthesis. Since the cost of the enzyme is an important factor for process economy, the use of whole-cell biocatalysts is attractive, since expensive purification and immobilization steps can be avoided. Display of the protein on the cell surface provides a possible way to reduce the mass transfer limitations of such biocatalysts. However, transaminases need to dimerize in order to become active, and furthermore, they require the cofactor pyridoxal phosphate; consequently, successful transaminase surface expression has not been reported thus far. In this work, we produced an Arthrobacter citreus omega-transaminase in Escherichia coli using a surface display vector based on the autotransporter adhesin involved in diffuse adherence (AIDA-I), which has previously been used for display of dimeric proteins. The correct localization of the transaminase in the E. coli outer membrane and its orientation toward the cell exterior were verified. Furthermore, transaminase activity was detected exclusively in the outer membrane protein fraction, showing that successful dimerization had occurred. The transaminase was found to be present in both full-length and proteolytically degraded forms. The removal of this proteolysis is considered to be the main obstacle to achieving sufficient whole-cell transaminase activity.

  • 259.
    Hagrot, Erika
    KTH, School of Biotechnology (BIO).
    Development of a culture system for modeling of pH effects in CHO cells2011Independent thesis Advanced level (professional degree), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    pH is a key parameter in the optimization of animal cell processes, and has be linked to specific patterns of consumption and production of extracellular metabolites. However, the effect of extracellular pH on intracellular metabolism has not been fully elucidated. Metabolic flux analysis is a mathematical method that can be used to generate the intracellular flux distributions in cells, e.g. as a function of some environmental parameter. In this work, the overall objective was to develop a culture system and experimental protocol for cultivation of CHO cells, which can be used to generate data for analysis of the relationship between extracellular pH and intracellular fluxes in CHO cells by metabolic flux analysis. First, shake-flask culture of an IgG-producing cell line was performed to select an academic and chemically-defined medium with known composition. This was followed by subsequent adaptation of the cells. It was found that the originally selected medium had to be supplemented with a commercial medium to produce acceptable growth and viability. Shake-flask culture was also performed to evaluate the effect of the biological buffer HEPES on cell growth and viability, and the pH-stability during culture. HEPES-concentrations in the investigated range (7.5-45 mM) did not show an apparent effect on cell growth or viability. The higher concentrations gave slightly better buffering capacity at inoculation, however were not sufficient to keep pH stable during culture. As a result, the idea of using shake flask culture and similar techniques for cultivation of cells at various pH set-points was dismissed. Instead, a culture system and protocol based on a 100 mL Spinner flask with pH-regulation was custom-designed for the project. Features of the final design included continuous monitoring of pH and DO, stable temperature at 37

    °C, adjustable agitation rate, as well as the option to incorporate inflow of air, O2 and CO2. In addition, the possibility to disconnect the flask unit to perform medium exchange and sample collection away from the reactor site (i.e. in a laminar flow workbench) was integrated into the design and protocol. The system was demonstrated for pseudo-perfusion culture with the adapted IgG-producing cell line at pH 7.0 during 24 days. Optimized regulation settings were identified. It was shown that the system could support viable cell densities of up to 11 MVC/mL and high viability (> 90 %). During the final phase of culture, stable growth, at specific growth rates of approximately 0.7 Day-1, was achieved. The specific rates of consumption and production of the key metabolites glucose, glutamine, lactate and NH4+, as well as 20 amino acids were analyzed. A majority of the rates were in accordance with CHO cell metabolism. The expected consumption of a majority of the essential amino acids and main carbon sources glucose and glutamine were confirmed, as well as the associated production of by-products lactate and NH4+. The system and protocol developed in this work can be used in future experiments to generate data describing metabolic profiles as a function of various pH-set points. This data may then be used in metabolic flux analysis to further elucidate the metabolism behind pH effects in CHO cells.

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  • 260.
    Hagrot, Erika
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Macroscopic models of Chinese hamster ovary cell cultures2019Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Biopharmaceuticals treat a range of diseases, and is a growing sector within the pharmaceutical industry. A majority of these complex molecules are produced by genetically modified mammalian cells in large-scale cell cultures. Biopharmaceutical process development is costly and labor intensive, and has often been based on time-consuming empirical methods and trial-and-error. Mathematical modeling has great potential to speed up this work. A central question however, engaging researchers from various fields, is how to translate these complex biological systems into feasible and useful models.

    For biopharmaceutical production, macroscopic kinetic flux modeling has been proposed. This model type is derived from typical data obtained in the industry, and has been able to simulate cell growth and the uptake/secretion of important metabolites. Often, however, their scope is limited to specific culture conditions due to e.g. the lack of information on reaction kinetics, limited data sets, and simplifications to achieve calculability.

    In this thesis, the macroscopic kinetic model type is the starting point, but the goal is to capture a variety of culture conditions, as will be necessary for future applications in process optimization. The effects of varied availability of amino acids in the culture medium on cell growth, uptake/secretion of metabolites, and product secretion were studied in cell cultures.

    In Paper I, the established methodology of Metatool was tested: (i) a simplified metabolic network of approx. 30 reactions was defined; (ii) all possible so-called elementary flux modes (EFMs) through the network were identified using an established mathematical algorithm; and (iii) the effect on each flux was modelled by a simplified generalized kinetic equation. A limitation was identified; the Metatool algorithm could only handle simple networks, and therefore several reactions had to be discarded. In this paper, a new strategy for the kinetics was developed. A pool of alternative kinetic equations was created, from which a smaller set could be given higher weight as determined via data-fitting. This improved the simulations.

    The identification of EFMs was further studied in papers II–IV. In Paper II, a new algorithm was developed based on the column generation optimization technique, that in addition to the network also accounts for the data from one of the parallel cultures. The method identifies a subset of the EFMs that can optimally fit the data, even in more complex metabolic networks.

    In Paper III, a kinetic model based on EFM subsets in a 100 reaction network was generated, which further improved the simulations. Finally, in Paper IV, the algorithm was extended to EFM identification in a genome-scale network. Despite the high complexity, small subsets of EFMs relevant to the experimental data could be e ciently identified.

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  • 261.
    Hagrot, Erika
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Oddsdóttir, Hildur Aesa
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.), Optimization and Systems Theory.
    Forsgren, Anders
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.), Optimization and Systems Theory.
    Chotteau, Véronique
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Identification of experimentally relevant elementary flux mode subsets in a genome-scale metabolic network of CHO cell metabolism using column generationManuscript (preprint) (Other academic)
    Abstract [en]

    An elementary flux mode (EFM) is a stoichiometrically balanced pathway through a metabolic network that links extracellular substrates to products. For large and complex networks, finding all such pathways is a computational challenge due to the combinatorial explosion of possible modes. Herein, we show how a new algorithm based on the column generation optimization technique can be applied to efficiently identify small sets of pathways in a genome-scale metabolic network. We examine the metabolism of Chinese hamster ovary (CHO) cells by identifying pathways that are relevant to data obtained in a pseudo-perfusion cell culture experiment. Based on the identified pathways, we examine the intracellular metabolism behind the uptake and utilization of extracellular medium components for the biomass and mAb synthesis, and the generation of extracellular metabolic by-products.

  • 262. Hakkaart, Xavier DV
    et al.
    Pronk, Jack T
    van Maris, Antonius JA
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    A Simulator-Assisted Workshop for Teaching Chemostat Cultivation in Academic Classes on Microbial Physiology2017In: Journal of Microbiology & Biology Education, Vol. 18, no 3Article in journal (Refereed)
    Abstract [en]

    Understanding microbial growth and metabolism is a key learning objective of microbiology and biotechnology courses, essential for understanding microbial ecology, microbial biotechnology and medical microbiology. Chemostat cultivation, a key research tool in microbial physiology that enables quantitative analysis of growth and metabolism under tightly defined conditions, provides a powerful platform to teach key features of microbial growth and metabolism. Substrate-limited chemostat cultivation can be mathematically described by four equations. These encompass mass balances for biomass and substrate, an empirical relation that describes distribution of consumed substrate over growth and maintenance energy requirements (Pirt equation), and a Monod-type equation that describes the relation between substrate concentration and substrate-consumption rate. The authors felt that the abstract nature of these mathematical equations and a lack of visualization contributed to a suboptimal operative understanding of quantitative microbial physiology among students who followed their Microbial Physiology B.Sc. courses. The studio-classroom workshop presented here was developed to improve student understanding of quantitative physiology by a set of question-guided simulations. Simulations are run on Chemostatus, a specially developed MATLAB-based program, which visualizes key parameters of simulated chemostat cultures as they proceed from dynamic growth conditions to steady state. In practice, the workshop stimulated active discussion between students and with their teachers. Moreover, its introduction coincided with increased average exam scores for questions on quantitative microbial physiology. The workshop can be easily implemented in formal microbial physiology courses or used by individuals seeking to test and improve their understanding of quantitative microbial physiology and/or chemostat cultivation.

  • 263.
    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.

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  • 264.
    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.

  • 265.
    Hamsten, Carl
    KTH, School of Biotechnology (BIO), Proteomics.
    Protein based approaches to understand and prevent contagious bovine pleuropneumonia2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Contagious bovine pleuropneumonia (CBPP) is a severe infectious disease caused by Mycoplasma mycoides subsp. mycoides small colony type (M. mycoides SC) and is a vast problem in Africa. Current CBPP prevention is based on attenuated live strain vaccines, but these are limited by factors such as short-term immunity, cold-chain dependence and retained virulence. CBPP can be diagnosed using post-mortem examination, identification of the agent using culture and PCR based methods as well as serological diagnostic methods, but the latter are generally not sensitive enough and there is also demand for an inexpensive, pen side field test.The research presented in this thesis was focused on using recombinantly expressed surface proteins from M. mycoides SC to characterize humoral immune responses to CBPP. Thereby candidate proteins to be used in development of serological diagnostic methods and possibly subunit vaccines could be identified. As a first step, five putative variable surface proteins of M. mycoides SC were expressed and purified from E. coli in Paper I. These proteins were analyzed using immunoblotting techniques and results showed that one protein, MSC_0364, was variably expressed on the surface of M. mycoides SC in vitro. Paper II presents expanded efforts including cloning and expression of 64 recombinant surface proteins and an assay for high throughput analysis of protein-specific IgG, IgA and IgM titers in hundreds of sera using a bead-based screening assay. The assay was evaluated by protein-specific inhibition experiments, comparisons to Western blotting and monitoring of immune responses over time in a study with sera taken from eight animals over 293 days from a previous vaccine trial.Papers III and IV present applications using the recombinant proteins and bead-based screening assay wherein proteins for diagnostic and vaccine development were identified. In Paper III, the assay was used to screen 61 proteins using well-characterized serum samples from cattle with CBPP and healthy controls, resulting in selection of eight proteins suitable for diagnostic use. These proteins were combined and evaluated in a proof-of-concept ELISA with a discriminative power that enabled 96% correct classification of sera from CBPP-affected and CBPP-free bovines. Paper IV reports the results and protein-specific analyses of a vaccine trial using the recombinant putative variable surface proteins presented in Paper I as a subunit vaccine. The vaccine conferred no protection, but a weak vaccine response could not be excluded as the cause of failure. In an effort to identity other protein candidates to be used in a subunit vaccine, protein-specific analysis of humoral immune responses elicited by the currently approved live strain vaccine, T1/44, were investigated. Here, five proteins with high IgG titers associated to immunity were identified: LppQ, MSC_02714, MSC_0136, MSC_0079 and MSC_0431. These proteins may be important in the development of a novel subunit vaccine against CBPP.

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  • 266.
    Hamsten, Carl
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Neiman, Maja
    KTH, School of Biotechnology (BIO), Proteomics.
    Schwenk, Jochen M.
    KTH, School of Biotechnology (BIO), Proteomics.
    Hamsten, Marica
    KTH, School of Biotechnology (BIO), Proteomics.
    March, John B.
    Persson, Anja
    KTH, School of Biotechnology (BIO), Proteomics.
    Recombinant surface proteomics as a tool to analyze humoral immune responses in bovines infected by Mycoplasma mycoides subsp. mycoides SC2009In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 8, no 11, p. 2544-2554Article in journal (Refereed)
    Abstract [en]

    A systematic approach to characterize the surface proteome of Mycoplasma mycoides subspecies mycoides small colony type (M. mycoides SC), the causing agent of contagious bovine pleuropneumonia (CBPP) in cattle, is presented. Humoral immune responses in 242 CBPP affected cattle and controls were monitored against one third of the surface proteins of M. mycoides SC in a high-throughput magnetic bead based assay. First, 64 surface proteins were selected from the genome sequence of M. mycoides SC and expressed as recombinant proteins in E. coli. Binding of antibodies to each individual protein could then be analyzed simultaneously in minute sample volumes with the Luminex suspension array technology. The assay was optimized on Namibian CBPP positive sera and Swedish negative controls to allow detection and 20-fold mean signal separation between CBPP positive and negative sera. Signals were proven to be protein-specific by inhibition experiments and results agreed with western blot experiments. The assay's potential to monitor IgG, IgM and IgA responses over time was shown in a proof-of-concept study with 116 sera from 8 animals in a CBPP vaccine study. In conclusion, a toolbox with recombinant proteins and a flexible suspension array assay that allows multiplex analysis of humoral immune responses to M mycoides SC, has been created.

  • 267.
    Hamsten, Carl
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Tjipura-Zaire, Georgina
    McAuliffe, Laura
    Hübschle, Otto
    Scacchia, Massimo
    Ayling, Roger D.
    Persson, Anja
    KTH, School of Biotechnology (BIO), Proteomics.
    Protein-Specific Analysis of Humoral Immune Responses in a Clinical Trial for Vaccines against Contagious Bovine Pleuropneumonia2010In: Clinical and Vaccine Immunology, ISSN 1556-6811, E-ISSN 1556-679X, Vol. 17, no 5, p. 853-861Article in journal (Refereed)
    Abstract [en]

    Specific humoral immune responses in a clinical trial on cattle for vaccines against contagious bovine pleuropneumonia (CBPP) were investigated. The trial included a subunit vaccine consisting of five recombinant putative variable surface proteins of the infectious agent Mycoplasma mycoides subspecies mycoides small colony type (M. mycoides SC) compared to the currently approved attenuated vaccine strain T1/44 and untreated controls. Humoral immune responses to 65 individual recombinant surface proteins of M. mycoides SC were monitored by a recently developed bead based array assay. Responses to the subunit vaccine components were found to be weak. Animals vaccinated with this vaccine were not protected and had CBPP lesions similar to the untreated controls. In correlating protein-specific humoral responses to T1/44 induced immunity, five proteins associated with a protective immune response were identified,namely LppQ and those of ORFs MSC_0271, MSC_0136, MSC_0079 and MSC_0431. The five proteins may be important candidates in the development of a novel subunit vaccine against CBPP.

  • 268.
    Hamsten, Carl
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Westberg, Joakim
    Bölske, Göran
    Ayling, Roger
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Persson, Anja
    KTH, School of Biotechnology (BIO), Proteomics.
    Expression and immunogenicity of six putative variable surface proteins in Mycoplasma mycoides subsp. mycoides SC.2008In: Microbiology, ISSN 1350-0872, E-ISSN 1465-2080, Vol. 154, p. 539-549Article in journal (Refereed)
    Abstract [en]

    Variable surface protein Vmm and five Vmm-type proteins from Mycoplasma mycoides subsp. mycoides SC were analysed to determine whether these proteins are expressed in vivo in animals affected by contagious bovine pleuropneumonia (CBPP) and in vitro. Recombinant versions of these proteins were constructed and expressed in Escherichia coli after mutation of the TGA Trp codons to TGG. These proteins were then analysed by dot and Western blotting with sera from CBPP-affected cattle. Furthermore, affinity-purified polyclonal antibodies to the recombinant proteins were used in Western and colony blotting to look for expression of the putative Vmm-type proteins in cultured M. mycoides SC. This study demonstrates that immunoglobulins in CBPP sera recognize all putative Vmm-type proteins tested, indicating that these proteins or their homologues are expressed by mycoplasmas during natural infections. Vmm and one of the putative Vmm-type proteins showed variable expression in vitro.

  • 269. Hansen, Henning Gram
    et al.
    Nilsson, Claes Nymand
    Lund, Anne Mathilde
    Kol, Stefan
    Grav, Lise Marie
    Lundqvist, Magnus
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Rockberg, Johan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Lee, Gyun Min
    Andersen, Mikael Rordam
    Kildegaard, Helene Faustrup
    Versatile microscale screening platform for improving recombinant protein productivity in Chinese hamster ovary cells2015In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 18016Article in journal (Refereed)
    Abstract [en]

    Chinese hamster ovary (CHO) cells are widely used as cell factories for the production of biopharmaceuticals. In contrast to the highly optimized production processes for monoclonal antibody (mAb)-based biopharmaceuticals, improving productivity of non-mAb therapeutic glycoproteins is more likely to reduce production costs significantly. The aim of this study was to establish a versatile target gene screening platform for improving productivity for primarily non-mAb glycoproteins with complete interchangeability of model proteins and target genes using transient expression. The platform consists of four techniques compatible with 96-well microplates: lipid-based transient transfection, cell cultivation in microplates, cell counting and antibody-independent product titer determination based on split-GFP complementation. We were able to demonstrate growth profiles and volumetric productivity of CHO cells in 96-half-deepwell microplates comparable with those obtained in shake flasks. In addition, we demonstrate that split-GFP complementation can be used to accurately measure relative titers of therapeutic glycoproteins. Using this platform, we were able to detect target gene-specific increase in titer and specific productivity of two non-mAb glycoproteins. In conclusion, the platform provides a novel miniaturized and parallelisable solution for screening target genes and holds the potential to unravel genes that can enhance the secretory capacity of CHO cells.

  • 270.
    Hao, Nanjing
    et al.
    Department of Chemistry, University of Massachusetts, USA.
    Neranon, Kitjanit
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Ramström, Olof
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Yan, Mingdi
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry. Department of Chemistry, University of Massachusetts, USA.
    Glyconanomaterials for biosensing applications2016In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 76, no 15, p. 113-130Article in journal (Refereed)
    Abstract [en]

    Nanomaterials constitute a class of structures that have unique physiochemical properties and are excellent scaffolds for presenting carbohydrates, important biomolecules that mediate a wide variety of important biological events. The fabrication of carbohydrate-presenting nanomaterials, glyconanomaterials, is of high interest and utility, combining the features of nanoscale objects with biomolecular recognition. The structures can also produce strong multivalent effects, where the nanomaterial scaffold greatly enhances the relatively weak affinities of single carbohydrate ligands to the corresponding receptors, and effectively amplifies the carbohydrate-mediated interactions. Glyconanomaterials are thus an appealing platform for biosensing applications. In this review, we discuss the chemistry for conjugation of carbohydrates to nanomaterials, summarize strategies, and tabulate examples of applying glyconanomaterials in in vitro and in vivo sensing applications of proteins, microbes, and cells. The limitations and future perspectives of these emerging glyconanomaterials sensing systems are furthermore discussed.

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  • 271.
    Harahap, Fumi
    et al.
    KTH, School of Industrial Engineering and Management (ITM), Energy Technology, Energy and Climate Studies, ECS.
    Silveira, Semida
    KTH, School of Industrial Engineering and Management (ITM), Energy Technology, Energy and Climate Studies, ECS.
    Khatiwada, Dilip
    KTH, School of Industrial Engineering and Management (ITM), Energy Technology, Energy and Climate Studies, ECS.
    Integrated biorefinery vs. stand alone biodieselproduction in Indonesia – an economic analysis2017In: European Biomass Conference and Exhibition Proceedings, 2017Conference paper (Other academic)
    Abstract [en]

    Biofuel policy instruments have largely steered the expansion of the biodiesel industry in Indonesia,promoting investments and creating fuel markets. Despite the growth, biodiesel use has not yet reached thedeployment targets set by the government. Low profitability and dysfunctional markets forces some plants to operatefar below the installed production capacity, which results in a deficit of biodiesel supply for domestic markets. At thesame time, biodiesel is being exported. The current production configuration of biodiesel in a standalone biodieselplant is perceived to be unprofitable without government subsidy. Therefore, we propose a comparative economicanalysis for biodiesel production in Indonesia using two configurations: the standalone production system typicallyused at present, and an integrated bio-refinery plant. The results show that the biodiesel production cost in thebiorefinery is 13% higher compared to the production cost in a standalone plant. However, due to higher revenuesgenerated in the biorefinery (16% higher than standalone system), biorefinery concept offers more profits to theindustry. Under current economic conditions, the integrated biorefinery concept brings advantages throughimprovement of efficiency in the biodiesel production system and higher production of other valuable products suchas electricity.

  • 272.
    Hassan, Noor
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Geiger, Barbara
    Gandini, Rosaria
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Patel, Bharat K. C.
    Kittl, Roman
    Haltrich, Dietmar
    Nguyen, Thu-Ha
    Divne, Christina
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Tan, Tien Chye
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Engineering a thermostable Halothermothrix orenii beta-glucosidase for improved galacto-oligosaccharide synthesis2016In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 100, no 8, p. 3533-3543Article in journal (Refereed)
    Abstract [en]

    Lactose is produced in large amounts as a by-product from the dairy industry. This inexpensive disaccharide can be converted to more useful value-added products such as galacto-oligosaccharides (GOSs) by transgalactosylation reactions with retaining beta-galactosidases (BGALs) being normally used for this purpose. Hydrolysis is always competing with the transglycosylation reaction, and hence, the yields of GOSs can be too low for industrial use. We have reported that a beta-glucosidase from Halothermothrix orenii (HoBGLA) shows promising characteristics for lactose conversion and GOS synthesis. Here, we engineered HoBGLA to investigate the possibility to further improve lactose conversion and GOS production. Five variants that targeted the glycone (-1) and aglycone (+1) subsites (N222F, N294T, F417S, F417Y, and Y296F) were designed and expressed. All variants show significantly impaired catalytic activity with cellobiose and lactose as substrates. Particularly, F417S is hydrolytically crippled with cellobiose as substrate with a 1000-fold decrease in apparent k(cat), but to a lesser extent affected when catalyzing hydrolysis of lactose (47-fold lower k(cat)). This large selective effect on cellobiose hydrolysis is manifested as a change in substrate selectivity from cellobiose to lactose. The least affected variant is F417Y, which retains the capacity to hydrolyze both cellobiose and lactose with the same relative substrate selectivity as the wild type, but with similar to 10-fold lower turnover numbers. Thin-layer chromatography results show that this effect is accompanied by synthesis of a particular GOS product in higher yields by Y296F and F417S compared with the other variants, whereas the variant F417Y produces a higher yield of total GOSs.

  • 273.
    Hassanzadeh, Masoumeh
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Wood Chemistry and Pulp Technology.
    Li, Jiebing
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    TUNICATES FOR BIO MATERIALS PRODUCTION: EFFECTS OF DIFFERENT FACTORS ON CELLULOSE AND PROTEIN COMPOSITION2014In: PAPERS OF THE 22ND EUROPEAN BIOMASS CONFERENCE: SETTING THE COURSE FOR A BIOBASED ECONOMY, 2014, p. 1116-1123Conference paper (Refereed)
    Abstract [en]

    Tunicates, a group of marine animals, is gaining a lot of interests in case of medical, food market, water pollution, cellulosic nanomaterial, and biofuel production issues due to their consisting of chemical compounds such as cellulose, amino-sugars, and proteins or protein-polysaccharide complexes. In this work, two dominant species of Scandinavian tunicates have been investigated by extraction and characterization of their cellulose, and amino acids. Samples in different sizes, ages, place of growing (Distance to ocean's surface), and different chemical pretreatment, have been evaluated in their compositions to see the best conditions for extraction of cellulose and protein. For pure cellulose and bioethanol productions, the samples growing near to the ocean surface at the best harvesting time (after completion of metamorphosis), recommended to be explored. The highest amount of protein in tunicate body has been found in the internal organs with a total amino acid content of around 52 %. In addition, the larger and elder the sample is, the higher amount of protein it contains. Hence, for feed supplementing point of view, the internal organs of tunicates with higher size and age are favored to be considered. Eventually, a combination of both H3PO4 and Ba (OH)(2) might lead to a significantly high cellulose percentage (66.5%) and a high protein removal percentage (protein content of 6%) when aiming at cellulose extraction.

  • 274. Helgstrand, M
    et al.
    Allard, Peter
    KTH, Superseded Departments, Biotechnology.
    QSim, a program for NMR simulations2004In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 30, no 1, p. 71-80Article in journal (Refereed)
    Abstract [en]

    We present QSim, a program for simulation of NMR experiments. Pulse sequences are implemented and analyzed in QSim using a mouse driven interface. QSim can handle almost any modern NMR experiment, using multiple channels, shaped pulses, mixing, decoupling, phase-cycling and pulsed field gradients. Any number of spins with any spin quantum number can, in theory, be used in simulations. Relaxation is accounted for during all steps of pulse sequences and relaxation interference effects are supported. Chemical kinetics between any numbers of states can be simulated. Both classical and quantum mechanical calculations can be performed. The result of a simulation can be presented either as magnetization as a function of time or as a processed spectrum.

  • 275.
    Hellmér, Elin
    KTH, School of Architecture and the Built Environment (ABE), Sustainable development, Environmental science and Engineering, Industrial Ecology.
    CLIMATE PERFORMANCE OF BIOFUELS: PRODUCED FROM FOREST RESIDUE HARVESTED IN SWEDEN2016Independent thesis Basic level (degree of Bachelor), 10 credits / 15 HE creditsStudent thesis
    Abstract [en]

    Biofuels produced from forest residues are much discussed in a Swedish context, among other things due to concerns for climate change. However, the undertaking of climate performance calculations is not an exact science. To examine whether climate concerns may be met by biofuels produced from forest residues, a literature review was carried out, analysing studies across methodologies.

    The scope for the literature review was limited to climate performance calculations for biofuels produced from forest residues harvested in Sweden. Five articles have been chosen for presentation, whereof one was carried out according to ISO 14040:2006 methodology, two according to climate performance calculations as stipulated by RED, two according to cumulative radiative forcing (CRF) and one according to a bottom up model using data from demo plants in Sweden (one study covers both ISO and RED methodology). All five studies presented in this paper suggest that climate performance of biofuels produced from forest residue (harvested in Sweden) show significantly better climate performance than fossil fuels.

    The local, environmental effects as well as future potential for harvesting of forest residue were also explored. A synthesis report on local, environmental effects suggests that the local, environmental effects are small. Furthermore, it is concludes that the effects on SOC are minor. Lastly, it is suggested that there is potential of increased harvesting of forest residue in Sweden in the magnitude of 30 TWh. 

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  • 276.
    Hendil-Forssell, Peter
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Rational engineering of esterases for improved amidase specificity in amide synthesis and hydrolysis2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Biocatalysis is an ever evolving field that uses enzymes or microorganisms for chemical synthesis. By utilizing enzymes that generally have evolved for specific reactions under mild conditions and temperatures, biocatalysis can be a more environmentally friendly option compared to traditional chemistry.

    Amide-type chemistries are important and bond formation avoiding poor atom economy is of high priority in organic chemistry. Biocatalysis could potentially be a solution but restricted substrate scope is a limitation. Esterases/lipases usually display broad substrate scope and catalytic promiscuity but are poor at hydrolyzing amides compared to amidases/proteases. The difference between the two enzyme classes is hypothesized to reside in one key hydrogen bond present in amidases, which facilitates the transition state for nitrogen inversion during catalysis.

    In this thesis the work has been focused on introducing a stabilizing hydrogen bond acceptor in esterases, mimicking that found in amidases, to develop better enzymatic catalysts for amide-based chemistries.

    By two strategies, side-chain or water interaction, variants were created in three esterases that displayed up to 210-times increased relative amidase specificity compared to the wild type. The best variant displayed reduced activation enthalpy corresponding to a weak hydrogen bond. The results show an estimated lower limit on how much the hydrogen bond can be worth to catalysis.

    MsAcT catalyze kinetically controlled N-acylations in water. An enzymatic one-pot one-step cascade was developed for the formation of amides from aldehydes in water that gave 97% conversion. In addition, engineered variants of MsAcT with increased substrate scope could synthesize an amide in water with 81% conversion, where the wild type gave no conversion. Moreover, variants of MsAcT displayed up to 32-fold change in specificity towards amide synthesis and a switch in reaction preference favoring amide over ester synthesis.

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  • 277.
    Hendil-Forssell, Peter
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Syren, Per-Olof
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Exploring water as building bricks in enzyme engineering2015In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 51, no 97, p. 17221-17224Article in journal (Refereed)
    Abstract [en]

    A novel enzyme engineering strategy for accelerated catalysis based on redesigning a water network through protein backbone deshielding is presented. Fundamental insight into the energetic consequences associated with the design is discussed in the light of experimental results and computer simulations. Using water as biobricks provides unique opportunities when transition state stabilisation is not easily attained by traditional enzyme engineering.

  • 278.
    Hendil-Forssell, Peter
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Semlitsch, Stefan
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Engineering the esterase/acyltransferase from Mycobacterium smegmatis: extended substrate scope for amide synthesis in waterManuscript (preprint) (Other academic)
    Abstract [en]

    Some esterases/lipases display high acyl transfer activity, favoring alcoholysis over hydrolysis, which make them valuable catalysts for synthesis reactions in aqueous media. An esterase from Mycobacterium smegmatis, MsAcT, has been characterized as an efficient catalyst for ester synthesis in water. The acyl donor specificity for MsAcT was however found to be very narrow and the enzyme displayed no activity towards esters with larger acyl group than butyrate. With rational engineering, the narrow acyl donor specificity of wild type MsAcT enzyme was altered and variants displaying extended substrate scope were generated. A double mutant, T93A/F154A, could accommodate methyl nonanoate as substrate, i.e. five carbons longer acyl group as compared to wild type, without compromising the acyl transfer capabilities. With similar selectivity towards a broad range of acyl donors (propionate to nonanoate) this is a more applicable catalyst than the wild type. Furthermore, the T93A/F154A variant was an efficient catalyst for synthesis of N-benzylhexanamide in water using methyl hexanoate as acyl donor, which is not a substrate for the wild type enzyme. The conversion reached 81% and the enzyme variant could potentially be used to produce amides in water with a wide variety of acyl donors.

  • 279.
    Hendil-Forssell, Peter
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Semlitsch, Stefan
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Rational engineering of an esterase/acyltransferase for improved amidase specificity in amide synthesis and hydrolysisManuscript (preprint) (Other academic)
    Abstract [en]

    The esterase/acyltransferase from Mycobacterium smegmatis, MsAcT, display high acyltransfer capacity in water media with demonstrations found for both ester and amide syntheses. However, it has recently been discovered that esterases in contrast to amidases lack a key hydrogen bond in the transition state, donated by the scissile NH-group of the substrate. Esterases with improved amidase performance have been achieved with the introduction of amino-acid side chains or water network as hydrogen bond acceptors. Using the esterase from Mycobacterium smegmatis, MsAcT, the influence of this hydrogen bond was studied in both amide hydrolysis and synthesis, using a rational engineering approach. Two positions were selected for mutagenesis and enzyme variants with improved performance in amide synthesis and hydrolysis were generated. Compared to the wild-type, variant F154A had the highest absolute increase in amidase specificity (11-fold) and I194Q had the greatest change in relative amidase versus esterase reaction specificity (160-fold). The relative reaction specificities for amide over ester synthesis followed a similar trend as that of hydrolysis and the best variant was I194Q with a 32-fold increase compared to wt. Based on MD-simulations water seems to play an important role in the transition state as a hydrogen bond bridge between the NH-group of the amide substrate and the enzyme.

  • 280.
    Hendrikse, Natalie M.
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Swedish Orphan Biovitrum AB, Stockholm, SE-112 76, Sweden.
    Holmberg Larsson, Albin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Swedish Orphan Biovitrum AB, Stockholm, SE-112 76, Sweden.
    Svensson Gelius, S.
    Kuprin, S.
    Nordling, E.
    Syrén, Per-Olof
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Exploring the therapeutic potential of modern and ancestral phenylalanine/tyrosine ammonia-lyases as supplementary treatment of hereditary tyrosinemia2020In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 10, no 1, article id 1315Article in journal (Refereed)
    Abstract [en]

    Phenylalanine/tyrosine ammonia-lyases (PAL/TALs) have been approved by the FDA for treatment of phenylketonuria and may harbour potential for complementary treatment of hereditary tyrosinemia Type I. Herein, we explore ancestral sequence reconstruction as an enzyme engineering tool to enhance the therapeutic potential of PAL/TALs. We reconstructed putative ancestors from fungi and compared their catalytic activity and stability to two modern fungal PAL/TALs. Surprisingly, most putative ancestors could be expressed as functional tetramers in Escherichia coli and thus retained their ability to oligomerize. All ancestral enzymes displayed increased thermostability compared to both modern enzymes, however, the increase in thermostability was accompanied by a loss in catalytic turnover. One reconstructed ancestral enzyme in particular could be interesting for further drug development, as its ratio of specific activities is more favourable towards tyrosine and it is more thermostable than both modern enzymes. Moreover, long-term stability assessment showed that this variant retained substantially more activity after prolonged incubation at 25 °C and 37 °C, as well as an increased resistance to incubation at 60 °C. Both of these factors are indicative of an extended shelf-life of biopharmaceuticals. We believe that ancestral sequence reconstruction has potential for enhancing the properties of enzyme therapeutics, especially with respect to stability. This work further illustrates that resurrection of putative ancestral oligomeric proteins is feasible and provides insight into the extent of conservation of a functional oligomerization surface area from ancestor to modern enzyme.

  • 281. Henning, P
    et al.
    Andersson, K M E
    Frykholm, K
    Ali, A
    Magnusson, M K
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Granio, O
    Hong, S S
    Boulanger, P
    Lindholm, L
    Tumor cell targeted gene delivery by adenovirus 5 vectors carrying knobless fibers with antibody-binding domains2005In: Gene Therapy, ISSN 0969-7128, E-ISSN 1476-5462, Vol. 12, no 3, p. 211-224Article in journal (Refereed)
    Abstract [en]

    Most human carcinoma cell lines lack the high-affinity receptors for adenovirus serotype 5 (Ad5) at their surface and are nonpermissive to Ad5. We therefore tested the efficiency of retargeting Ad5 to alternative cellular receptors via immunoglobulin (Ig)-binding domains inserted at the extremity of short-shafted, knobless fibers. The two recombinant Ad5' s constructed, Ad5/R7- Z(wt)- Z(wt) and Ad5/R7-C2-C2, carried tandem Ig-binding domains from Staphylococcal protein A ( abbreviated Z(wt)) and from Streptococcal protein G (C2), respectively. Both viruses bound their specific Ig isotypes with the expected affinity. They transduced human carcinoma cells independently of the CAR pathway, via cell surface receptors targeted by specific monoclonal antibodies, that is, EGF-R on A549, HT29 and SW1116, HER-2/ neu on SK-OV-3 and SK-BR-3, CA242 ( epitope recognized by the monoclonal antibody C242) antigen on HT29 and SW1116, and PSMA ( prostate-specific membrane antigen) expressed on HEK-293 cells, respectively. However, Colo201 and Colo205 cells were neither transduced by targeting CA242 or EGF-R nor were LNCaP cells transduced by targeting PSMA. Our results suggested that one given surface receptor could mediate transduction of certain cells but not others, indicating that factors and steps other than cell surface expression and virus - receptor interaction are additional determinants of Ad5-mediated transduction of tumor cells. Using penton base RGD mutants, we found that one of these limiting steps was virus endocytosis.

  • 282.
    Henrique, Pacini
    et al.
    KTH, School of Industrial Engineering and Management (ITM), Energy Technology, Energy and Climate Studies, ECS.
    Silveira, Semida
    KTH, School of Industrial Engineering and Management (ITM), Energy Technology, Energy and Climate Studies, ECS.
    Consumer choice between Ethanol and Gasoline: Lessons from the Cases of Brazil and Sweden2010In: Conference proceedings 3rd International Scientific Conference on “Energy systems with IT” / [ed] Erik Dahlquist, Jenny Palm, 2010Conference paper (Refereed)
  • 283.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Biotech reviews: keeping up with current developments2011In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 6Article in journal (Other academic)
  • 284.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Global biotech challenges2010In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 5Article in journal (Other academic)
  • 285.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Mutated immunoglobulin-binding protein2002Patent (Other (popular science, discussion, etc.))
    Abstract [en]

    The present invention relates to an immunoglobulin-binding protein, wherein at least one asparagine residue has been mutated to an amino acid other than glutamine or aspartic acid, which mutation confers an increased chemical stability at pH-values of up to about 13-14 compared to the parental molecule. The protein can for example be derived from a protein capable of binding to other regions of the immunoglobulin molecule than the complementarity determining regions (CDR), such as protein A, and preferably the B-domain of Staphylococcal protein A. The invention also relates to a matrix for affinity separation, which comprises an immunoglobulin-binding protein as ligand coupled to a solid support, in which protein ligand at least one asparagine residue has been mutated to an amino acid other than glutamine.

  • 286.
    Hober, Sophia
    et al.
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Forsberg, G
    Palm, G
    Hartmanis, M
    Nilsson, B
    Disulfide exchange folding of insulin-like growth factor I.1992In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 31, no 6Article in journal (Refereed)
    Abstract [en]

    The disulfide exchange folding properties of insulin-like growth factor I (IGF-I) have been analyzed in a redox buffer containing reduced (10 mM) and oxidized (1 mM) glutathione. Under these conditions, the 3 disulfide bridges of the 70 amino acid peptide were not quantitatively formed. Instead, five major forms of IGF-I were detected, and these components were concluded to be in equilibrium as their relative amounts were similar starting from either reduced, native, or a mismatched variant of IGF-I containing two non-native disulfides. The different components in the mixtures were trapped by thiol alkylation using vinylpyridine and subsequently isolated by reverse-phase HPLC. The purified variants were further characterized using plasma desorption mass spectrometry and peptide mapping. Two of the five different forms were identified as native and mismatched IGF-I. One form was a variant with only one disulfide bond, and the other two major components had two disulfides formed. In a separate experiment, early refolding intermediates were trapped by pyridylethylation after only 90 s of refolding in the glutathione buffer, starting from reduced IGF-I. The intermediates were identical to the components observed at equilibrium, but at different relative concentrations. On the basis of the disulfide bond patterns of the different components in the equilibrium mixtures, we conclude that the disulfide between cysteines-47 and -52 in IGF-I is an unfavorable high-energy bond that may exist in the native molecule in a strained configuration.

  • 287.
    Hober, Sophia
    et al.
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Hansson, A
    Uhlén, Mathias
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Nilsson, B
    Folding of insulin-like growth factor I is thermodynamically controlled by insulin-like growth factor binding protein.1994In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 33, no 22Article in journal (Refereed)
    Abstract [en]

    Insulin-like growth factor I (IGF-I) is thermodynamically unable to quantitatively form its native disulfides under reversible redox conditions in vitro [Hober et al. (1992) Biochemistry 31, 1749-1756]. These results prompted the question of how IGF-I may overcome this energetic problem in its folding in vivo. Here, we report that an IGF-I precursor, IGF-I-Ea, shows disulfide-exchange folding properties similar to those of mature IGF-I and, thus, is concluded not to overcome the identified folding problem of mature IGF-I. However, correct disulfide bonds are formed very efficiently when insulin-like growth factor binding protein 1 is added in equimolar amounts to IGF-I to the refolding mixture. On the basis of these results, we propose that one important function of at least one of the six homologous insulin-like growth factor binding proteins is to assist in the formation and maintenance of the native disulfides of IGF-I. To our knowledge, this is the first example where the folding of a mammalian protein or peptide in circulation has been demonstrated to be thermodynamically controlled by its binding protein. Speculatively, this could provide a mechanism to regulate the half-life of IGF-I in vivo by altering the interaction with insulin-like growth factor binding proteins.

  • 288.
    Hober, Sophia
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Johansson, Hans J.
    GE Healthcare.
    Bjorkman, Tomas
    GE Healthcare.
    Protein ligands2002Patent (Other (popular science, discussion, etc.))
  • 289.
    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.

  • 290.
    Hober, Sophia
    et al.
    KTH, Superseded Departments, Biotechnology.
    Nilsson, Björn
    IVA.
    Use of IGF-BP for refolding of IGF1996Patent (Other (popular science, discussion, etc.))
    Abstract [en]

    Processes for refolding of insulin-like growth factor (IGF) comprise contacting IGF in a reduced or misfolded form with insulin-like growth factor binding protein (IGF-BP), and recovering native IGF.

  • 291.
    Hofström, Camilla
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting2013Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Affibody molecules are small (7 kDa) affinity proteins of non-immunoglobulin origin that have been generated to specifically interact with a large number of clinically important molecular targets.

    In this thesis, Affibody molecules have been employed as tracers for radionuclide molecular imaging of HER2- and IGF-1R-expressing tumors, paper I-IV, and for surface knock-down of EGFR, paper V. In paper I, a tag with the amino acid sequence HEHEHE was fused to the N-terminus of a HER2-specific Affibody molecule, (ZHER2), and was shown to enable facile IMAC purification and efficient tri-carbonyl 99mTc-labeling. In vivo evaluation of radioactivity uptake in different organs showed an improved biodistribution, including a 10-fold lower radioactivity uptake in liver, compared to the same construct with a H6-tag. In paper II, it was further shown that an N-terminally placed HEHEHE-tag on ZHER2 provided lower unspecific uptake of radioactivity in liver compared to its H6-tagged counterpart even when radiolabeling was at the C-terminus using alternative chemistries to attach 99mTc, 111In or 125I. In paper III, the H6-tag’s composition and position was varied with regards to charge, hydrophobicity and its C- or N-terminal placement on ZHER2. Among the ten variants investigated, it was found that an N-terminal HEHEHE-tag provided the most favorable overall biodistribution profile and that introduction of hydrophobic and positively charged amino acids provoked liver uptake of radioactivity. In paper IV, the HEHEHE-tag was shown to enable IMAC purification and tri-carbonyl 99mTc-labeling of an IGF-1R-specific Affibody molecule and improved its overall biodistribution when compared to the same construct with a H6-tag. In paper V, the aim was to develop an intracellular receptor-entrapment system to reduce the surface levels of EGFR. An EGFR-specific Affibody molecule was expressed as a fusion to different mutants of an intracellular transport protein in SKOV-3 cells, resulting in a collection of cell lines with 50%, 60%, 80% and 96% reduced surface level of EGFR. Analysis of the proliferation rate of these cell lines showed that a modest reduction (15%) in proliferation occurs between 60% and 80% reduction of the surface level of EGFR.

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    Doctoral Thesis Hofstrom 2013
  • 292. Holmquist, M.
    et al.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Creation of a synthetically useful lipase with higher than wild-type enantioselectivity and maintained catalytic activity1999In: Organic Letters, ISSN 1523-7060, E-ISSN 1523-7052, Vol. 1, no 5, p. 763-765Article in journal (Refereed)
    Abstract [en]

    Formula presented Wild type I: 89.9% ee (E=32) Wild type II: 79.8% ee (E=10) Lipase hybrid: 95.4% ee (E=54) We have found that two Geotrichum candidum lipase isozymes have remarkably different abilities to differentiate between enantiomers of ethyl 2-methyldecanoate. By rational recombination of selected portions of the two isozymes, we have created a novel lipase with an enantioselectivity superior to that of the best wild-type parent isozyme. Site-directed mutagenesis identified two key amino acid residues responsible for the improved enantioselectivity without compromised total activity of the reengineered enzyme.

  • 293.
    Holmquist, Mats
    et al.
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Berglund, Per
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Improved lipase enantioselectivity by combinatorial and rational redesign.2000In: Abstract of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 219, no 1, p. U163-U163Article in journal (Refereed)
  • 294.
    Holzapfel, Gerhard A.
    KTH, School of Engineering Sciences (SCI), Solid Mechanics (Dept.). Graz University of Technology, Austria .
    Computational mechanics of multi-layered collagenous soft tissues: State of the art and challenges ahead2009In: Computational Plasticity X - Fundamentals and Applications, 2009Conference paper (Refereed)
    Abstract [en]

    Accurate modeling and analysis of collagenous soft tissues is key in the field of biomechanics. This contribution describes the structural continuum framework and provides two applications.

  • 295. Honarvar, H.
    et al.
    Strand, J.
    Perols, Anna
    KTH, School of Biotechnology (BIO), Protein Technology.
    Orlova, Anna
    Selvaraju, R. K.
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Protein Technology.
    Tolmachev, V.
    Position for site-specific attachment of a DOTA chelator to synthetic affibody molecules has a different influence on the targeting properties of 68Ga-Compared to 111in-labeled conjugates2014In: Molecular Imaging, ISSN 1535-3508, E-ISSN 1536-0121, Vol. 13, no 10Article in journal (Refereed)
    Abstract [en]

    Affibody molecules, small (7 kDa) scaffold proteins, are a promising class of probes for radionuclide molecular imaging. Radiolabeling of Affibody molecules with the positron-emitting nuclide 68Ga would permit the use of positron emission tomography (PET), providing better resolution, sensitivity, and quantification accuracy than single-photon emission computed tomography (SPECT). The synthetic anti-HER2 ZHER2:S1 Affibody molecule was conjugated with DOTA at the N-terminus, in the middle of helix 3, or at the Cterminus. The biodistribution of 68Ga-and 111In-labeled Affibody molecules was directly compared in NMRI nu/nu mice bearing SKOV3 xenografts. The position of the chelator strongly influenced the biodistribution of the tracers, and the influence was more pronounced for 68Ga-labeled Affibody molecules than for the 111In-labeled counterparts. The best 68Ga-labeled variant was 68Ga-[DOTA-A1]-ZHER2:S1, which provided a tumor uptake of 13 ± 1 %ID/g and a tumor to blood ratio of 39 ± 12 at 2 hours after injection. 111In-[DOTA-A1]-ZHER2:S1 and 111In-[DOTA-K58]-ZHER2:S1 were equally good at this time point, providing a tumor uptake of 15 to 16 %ID/g and a tumor to blood ratio in the range of 60 to 80. In conclusion, the selection of the best position for a chelator in Affibody molecules can be used for optimization of their imaging properties. This may be important for the development of Affibody-based and other protein-based imaging probes.

  • 296.
    Hoyer, Wolfgang
    et al.
    Department of Medical Biochemistry, Swedish Nuclear Magnetic Resonance Center, University of Gothenburg.
    Grönwall, Caroline
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Jonsson, Andreas
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Härd, Torleif
    Department of Medical Biochemistry, Swedish Nuclear Magnetic Resonance Center, University of Gothenburg.
    Stabilization of a beta-hairpin in monomeric Alzheimer´s amyloid beta-peptide inhibits amyloid formation2008In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 13, p. 5099-5104Article in journal (Refereed)
    Abstract [en]

    According to the amyloid hypothesis, the pathogenesis of Alzheimer's disease is triggered by the oligomerization and aggregation of the amyloid-β (Aβ) peptide into protein plaques. Formation of the potentially toxic oligomeric and fibrillar Aβ assemblies is accompanied by a conformational change toward a high content of β-structure. Here, we report the solution structure of Aβ(1–40) in complex with the phage-display selected affibody protein ZAβ3, a binding protein of nanomolar affinity. Bound Aβ(1–40) features a β-hairpin comprising residues 17–36, providing the first high-resolution structure of Aβ in β conformation. The positions of the secondary structure elements strongly resemble those observed for fibrillar Aβ. ZAβ3 stabilizes the β-sheet by extending it intermolecularly and by burying both of the mostly nonpolar faces of the Aβ hairpin within a large hydrophobic tunnel-like cavity. Consequently, ZAβ3 acts as a stoichiometric inhibitor of Aβ fibrillation. The selected Aβ conformation allows us to suggest a structural mechanism for amyloid formation based on soluble oligomeric hairpin intermediates.

  • 297.
    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.

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    fulltext
  • 298.
    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.

  • 299.
    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.

  • 300. Hu, Y.
    et al.
    Zhu, Z.
    Nielsen, Jens
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Chalmers University of Technology, Sweden.
    Siewers, V.
    Heterologous transporter expression for improved fatty alcohol secretion in yeast2018In: Metabolic engineering, ISSN 1096-7176, E-ISSN 1096-7184, Vol. 45, p. 51-58Article in journal (Refereed)
    Abstract [en]

    The yeast Saccharomyces cerevisiae is an attractive host for industrial scale production of biofuels including fatty alcohols due to its robustness and tolerance towards harsh fermentation conditions. Many metabolic engineering strategies have been applied to generate high fatty alcohol production strains. However, impaired growth caused by fatty alcohol accumulation and high cost of extraction are factors limiting large-scale production. Here, we demonstrate that the use of heterologous transporters is a promising strategy to increase fatty alcohol production. Among several plant and mammalian transporters tested, human FATP1 was shown to mediate fatty alcohol export in a high fatty alcohol production yeast strain. An approximately five-fold increase of fatty alcohol secretion was achieved. The results indicate that the overall cell fitness benefited from fatty alcohol secretion and that the acyl-CoA synthase activity of FATP1 contributed to increased cell growth as well. This is the first study that enabled an increased cell fitness for fatty alcohol production by heterologous transporter expression in yeast, and this investigation indicates a new potential function of FATP1, which has been known as a free fatty acid importer to date. We furthermore successfully identified the functional domain of FATP1 involved in fatty alcohol export through domain exchange between FATP1 and another transporter, FATP4. This study may facilitate a successful commercialization of fatty alcohol production in yeast and inspire the design of novel cell factories.

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