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  • 51.
    Sievertzon, Maria
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Wirta, Valtteri
    KTH, Skolan för bioteknologi (BIO).
    Mercer, Alex
    Frisén, Jonas
    Lundeberg, Joakim
    KTH, Skolan för bioteknologi (BIO), Genteknologi.
    Epidermal growth factor (EGF) withdrawal masks gene expression differences in the study of pituitary adenylate cyclase-activating polypeptide (PACAP) activation of primary neural stem cell proliferation2005Inngår i: BMC neuroscience (Online), ISSN 1471-2202, E-ISSN 1471-2202, Vol. 6, s. 55-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background: The recently discovered adult neural stem cells, which maintain continuous generation of new neuronal and glial cells throughout adulthood, are a promising and expandable source of cells for use in cell replacement therapies within the central nervous system. These cells could either be induced to proliferate and differentiate endogenously, or expanded and differentiated in culture before being transplanted into the damaged site of the brain. In order to achieve these goals effective strategies to isolate, expand and differentiate neural stem cells into the desired specific phenotypes must be developed. However, little is known as yet about the factors and mechanisms influencing these processes. It has recently been reported that pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neural stem cell proliferation both in vivo and in vitro.

    Results: We used cDNA microarrays with the aim of analysing the transcriptional changes underlying PACAP induced proliferation of neural stem cells. The primary neural stem/progenitor cells used were neurospheres, generated from the lateral ventricle wall of the adult mouse brain. The results were compared to both differentiation and proliferation controls, which revealed an unexpected and significant differential expression relating to withdrawal of epidermal growth factor (EGF) from the neurosphere growth medium. The effect of EGF removal was so pronounced that it masked the changes in gene expression patterns produced by the addition of PACAP.

    Conclusion: Experimental models aiming at transcriptional analysis of induced proliferation in primary neural stem cells need to take into consideration the significant effect on transcription caused by removal of EGF. Alternatively, EGF-free culture conditions need to be developed.

  • 52.
    Sievertzon, Maria
    et al.
    KTH, Skolan för bioteknologi (BIO), Centra, KTH Genome Center.
    Wirta, Valtteri
    KTH, Skolan för bioteknologi (BIO), Centra, KTH Genome Center.
    Mercer, Alex
    Meletis, Konstantinos
    Erlandsson, Rikard
    Wikström, Lilian
    Frisén, Jonas
    Lundeberg, Joakim
    KTH, Skolan för bioteknologi (BIO), Centra, KTH Genome Center.
    Transcriptome analysis in primary neural stem cells using a tag cDNA amplification method2005Inngår i: BMC neuroscience (Online), ISSN 1471-2202, E-ISSN 1471-2202, Vol. 6, nr 28, s. 13-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background: Neural stem cells ( NSCs) can be isolated from the adult mammalian brain and expanded in culture, in the form of cellular aggregates called neurospheres. Neurospheres provide an in vitro model for studying NSC behaviour and give information on the factors and mechanisms that govern their proliferation and differentiation. They are also a promising source for cell replacement therapies of the central nervous system. Neurospheres are complex structures consisting of several cell types of varying degrees of differentiation. One way of characterising neurospheres is to analyse their gene expression profiles. The value of such studies is however uncertain since they are heterogeneous structures and different populations of neurospheres may vary significantly in their gene expression.

    Results: To address this issue, we have used cDNA microarrays and a recently reported tag cDNA amplification method to analyse the gene expression profiles of neurospheres originating from separate isolations of the lateral ventricle wall of adult mice and passaged to varying degrees. Separate isolations as well as consecutive passages yield a high variability in gene expression while parallel cultures yield the lowest variability.

    Conclusions: We demonstrate a low technical amplification variability using the employed amplification strategy and conclude that neurospheres from the same isolation and passage are sufficiently similar to be used for comparative gene expression analysis.

  • 53.
    Sjöberg, Ronald
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinity Proteomics. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Andersson, Eni
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinity Proteomics. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Hellström, Cecilia
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinity Proteomics. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Mattsson, Cecilia
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinity Proteomics. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinity Proteomics. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Nilsson, Peter
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinity Proteomics. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Ayoglu, Burcu
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinity Proteomics. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    High-density antigen microarrays for the assessment of antibody selectivity and off-target binding2018Inngår i: Epitope Mapping Protocols, Humana Press Inc. , 2018, s. 231-238Kapittel i bok, del av antologi (Fagfellevurdert)
    Abstract [en]

    With the increasing availability of collections of antibodies, their evaluation in terms of binding selectivity becomes an important but challenging task. Planar antigen microarrays are very suitable tools to address this task and provide a powerful proteomics platform for the characterization of the binding selectivity of antibodies toward thousands of antigens in parallel. In this chapter, we describe our in-house developed procedures for the generation of high-density planar antigen microarrays with over 21,000 features. We also provide the details of the assay protocol, which we routinely use for the assessment of binding selectivity of the polyclonal antibodies generated within the Human Protein Atlas. © Springer Science+Business Media, LLC, part of Springer Nature 2018.

  • 54.
    Song, Yajing
    KTH, Skolan för bioteknologi (BIO), Genteknologi.
    Advances in DNA Detection2014Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    DNA detection technologies have an increasing importance in our everyday lives, with applications ranging from microbial diagnostics to forensic analysis, food safety evaluation, and environmental monitoring. Currently, as the associated costs decrease, DNA diagnostic techniques are routinely used in research laboratories, in clinical and forensic practice.

    The first aim of this thesis is to unravel the potential of DNA detection on cellulose filter paper and further investigate the filter paper as a viable candidate for DNA array support. In Paper I, we studied the method of functionalizing the surface of filter paper and the possibility to detect DNA on the active paper using fluorescence. In Paper II, we addressed visual detection with magnetic beads and increased the detection throughput on the active filter paper, which required no instrumentation. Second, in pursuit of a rapid, sensitive and specific pathogen diagnosis in bloodstream infection (BSI), we explored the possibility of rare DNA detection in the presence of a high amount of background DNA by an enzymatic reaction, which can remove background DNA while enriching the rare DNA fraction. In order to overcome the challenge of the second objective, we developed a chemical fragmentation method to increase the efficiency of enzymatic digestion and hybridization. In addition, DNA library preparation for massively parallel sequencing may benefit from the chemical fragmentation. Paper III and Paper IV introduce this work.

    The findings in Paper I showed that XG-NH2 and PDITC can functionalize the cellulose filter paper and that the activated filter papers can covalently bind oligonucleotides modified with amino groups, while preserving the base pairing ability of the oligonucleotides. In Paper II, visual detection of DNA on active paper was achieved without instrumentation, based on the natural colour of magnetic beads. Furthermore, the possibility to increase the throughput of DNA detection on active paper was demonstrated by successful multiplex detection. In Paper III, the developed chemical fragmentation was verified to be suitable for DNA library preparation in massively parallel sequencing. The fragmentation technique is simple to perform, cost-effective and amenable to automation. In Paper IV, a limited amount of E.coli DNA was detected amid a much larger amount of human background DNA in a BSI model, which comprises of human and E.coli amplicons with an abundance ratio of 108. Human β-actin amplicons were suppressed 105-fold, whereas the E.coli amplicons remained unaffected. The model system was applied to and improved with clinical plasma and blood samples from septic patients.

  • 55.
    Song, Yajing
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Advances in DNA Detection on Paper Chips2013Licentiatavhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    DNA detection has an increasing importance in our everyday lives, with applications ranging from microbial diagnostics to forensic analysis. Currently, as the associated costs decrease, DNA diagnostic techniques are routinely used not only in research laboratories, but also in clinical and forensic practice.

    The present thesis aims to unravel the potential of cellulose filter paper to be a viable candidate for DNA array support. There are two papers in this study. In Paper I, we studied the method of functionalizing the surface of filter paper and the possibility to detect DNA on acitve paper using fluorescence. In Paper II, we investigated visualization and throughput of DNA detection with magnetic beads on active filter papers, an assay which requires no instrumentation (scanner).

    The findings in Paper I show that XG-NH2 and PDITC can functionalize the cellulose filter paper and that the activated filter papers can covalently bind oligonucleotides modified with amino groups to detect DNA. The detection limit of the assay is approximately 0.2 pmol. In Paper II, visualization of DNA detection on active paper is achieved without instrumentation, based on the natural color of magnetic beads. Furthermore, successful multiplex detection supports the potential to increase the throughput of DNA detection on active papers.

    In summary, these studies show that active cellulose filter paper is a good DNA array support candidate as it provides a user-friendly and cost-efficient DNA detection assay. The methods described in Paper I and II are possible sources of development to a point-of-care device for on-site analysis of DNA contents in a sample.

  • 56.
    Steen, Johanna
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ottosson, Jenny
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    The correlation between antigen solubility and immunogenicityManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    In antigen design, several characteristics contribute to the final success of the antigen to elicit the desired immunogenicity. However, it is difficult to screen theses attributes due to the variability within the host immune receptor repertoire. Herein, with the massive numeral of immunizations performed within a proteome-wide endeavor to produce affinity reagents to human proteins, the correlation between the solubility of the antigens and the immunogenicity was investigated. We showed that increased solubility of the antigen resulted in higher success rate in provoking the immune defense as well as higher antibody titers. We have also shown, that the increased antibody titers after affinity purifications indeed reflectthe concentration of target specific antibodies within the serum. Finally, the amino acid composition of soluble antigens was determined to be over-represented in polar residues.

  • 57.
    Sundström, Heléne
    KTH, Skolan för bioteknologi (BIO).
    Analytical tools for monitoring and control of fermentation processes2007Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The overall objective of this work has been to adopt new developments and techniques in the area of measurement, modelling and control of fermentation processes. Flow cytometry and software sensors are techniques which were considered ready for application and the focus was set on developing tools for research aiming at understanding the relationship between measured variables and process quality parameters. In this study fed-batch cultivations have been performed with two different strains of Escherichia coli (E.coli) K12 W3110 with and without a gene for the recombinant protein promegapoietin.

    Inclusion body formation was followed during the process with flow cytometric detection by labelling the inclusion bodies with first an antibody against the protein promegapoietin and then a second fluorescent anti-antibody. The approach to label inclusion bodies directly in disintegrated and diluted cell slurry could be adopted as a method to follow protein production during the process, although the labelling procedure with incubation times and washings was somewhat time-consuming (1.5 h). The labelling of inclusion bodies inside the cells to follow protein production was feasible to perform, although an unexplained decrease in the relative fluorescence intensity occurred late in process. However, it is difficult to translate this qualitative measurement into a quantitative one, since a quantitative protein analysis should give data proportional to the volume, while the labelling of the spheric inclusion bodies gives a signal corresponding to the area of the body, and calibration is not possible. The methods were shown to be useful for monitoring inclusion body formation, but it seems difficult to get quantitative information from the analysis.

    Population heterogeneity analysis was performed, by using flow cytometry, on a cell population, which lost 80-90% viability according to viable count analysis. It was possible to show that the apparent cell death was due to cells incapable of dividing on agar plates after induction. These cells continued to produce the induced recombinant protein. It was shown that almost all cells in the population (≈97%) contained PMP, and furthermore total protein analysis of the medium indicated that only about 1% of the population had lysed. This confirms that the "non-viable" cells according to viable count by cfu analysis produced product.

    The software sensors XNH3 and µNH3, which utilises base titration data to estimate biomass and specific growth rate was shown to correlate well with the off-line analyses during cultivation of E. coli W3110 using minimal medium. In rich medium the µNH3 sensor was shown to give a signal that may be used as a fingerprint of the process, at least from the time of induction. The software sensor KLaC* was shown to respond to foaming in culture that probably was caused by increased air bubble dispersion. The RO/S coefficient, which describes the oxygen to substrate consumption, was shown to give a distinct response to stress caused by lowered pH and addition of the inducing agent IPTG.

    The software sensor for biomass was applied to a highly automated 6-unit multi-bioreactor system intended for fast process development. In this way also specific rates of substrate and oxygen consumption became available without manual sampling.

  • 58.
    Sundström, Heléne
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Enfors, Sven-Olof
    KTH, Skolan för bioteknologi (BIO).
    A bioreactor system for high throughput process developmentManuskript (Annet vitenskapelig)
  • 59.
    Sundström, Heléne
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Enfors, Sven-Olof
    KTH, Skolan för bioteknologi (BIO).
    Software sensors for fermentation processes2008Inngår i: Bioprocess and biosystems engineering (Print), ISSN 1615-7591, E-ISSN 1615-7605, Vol. 31, nr 2, s. 145-152Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Four software sensors based on standard on-line data from fermentation processes and simple mathematical models were used to monitor a number of state variables in Escherichia coli fed-batch processes: the biomass concentration, the specific growth rate, the oxygen transfer capacity of the bioreactor, and the new R-O/S sensor which is the ratio between oxygen and energy substrate consumption. The R-O/S variable grows continuously in a fed-batch culture with constant glucose feed, which reflects the increasing maintenance demand at declining specific growth rate. The R-O/S sensor also responded to rapid pH shift-downs reflecting the increasing demand for maintenance energy. It is suggested that this sensor may be used to monitor the extent of physiological stress that demands energy for survival.

  • 60.
    Sundström, Heléne
    et al.
    KTH, Tidigare Institutioner, Bioteknologi.
    Wållberg, Fredrik
    KTH, Tidigare Institutioner, Bioteknologi.
    Ledung, E.
    Department of Biology and Chemical Engineering, Mälardalen University.
    Norrman, B.
    Department of Biology and Chemical Engineering, Mälardalen University.
    Hewitt, C. J.
    Department of Chemical Engineering (Biochemical Engineering), University of Birmingham.
    Enfors, Sven-Olof
    KTH, Tidigare Institutioner, Bioteknologi.
    Segregation to non-dividing cells in recombinant Escherichia coli fed-batch fermentation processes2004Inngår i: Biotechnology letters, ISSN 0141-5492, E-ISSN 1573-6776, Vol. 26, nr 19, s. 1533-1539Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In Escherichia coli fermentation processes, a drastic drop in viable cell count as measured by the number of colony forming units per ml (c.f.u. ml(-1)) is often observed. This phenomenon was investigated in a process for the production of the recombinant fusion protein, promegapoietin (PMP). After induction, the number of c.f.u. ml(-1) dropped to similar to10% of its maximum though the biomass concentration continued to increase. Flow cytometric analysis of viability and intracellular concentration of PMP showed that almost all cells were alive and contributed to the production. Thus, the drop in the number of c.f.u. ml(-1) probably reflects a loss of cell division capability rather than cell death.

  • 61. Tong, Jianhua
    et al.
    Sommer, Gerhard
    Regitnig, Peter
    Holzapfel, Gerhard A.
    KTH, Skolan för teknikvetenskap (SCI), Hållfasthetslära (Inst.).
    Dissection Properties and Mechanical Strength of Tissue Components in Human Carotid Bifurcations2011Inngår i: Annals of Biomedical Engineering, ISSN 0090-6964, E-ISSN 1573-9686, Vol. 39, nr 6, s. 1703-1719Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Carotid artery dissections can be triggered by several factors. The underlying biomechanical phenomena and properties are unclear. This study investigates the dissection properties of 62 human carotid bifurcations using two experimental methods: direct tension and peeling tests. Direct tension tests study the mechanical strength of the tissue components in radial direction, while peeling tests quantify the fracture energy required to propagate a dissection in a tissue. Results show that the interface between the healthy adventitia and media has the highest radial failure stress (132 +/- A 20 kPa, mean +/- A SD, n = 25), whereas the lowest value occurs between the diseased intima and media (104 +/- A 24 kPa, n = 18). The radial tissue strength at the bifurcation is the highest compared with locations that are away from the central region of the bifurcation. Force/width values required to separate the individual layers and to dissect the media in the circumferential direction are always lower than related values in the axial direction, suggesting anisotropic dissection properties. Dissection energies per reference area generated during the peeling tests are also lower for strips in the circumferential direction than for axial strips, and they vary significantly with the location, as shown for the media. Histological investigations demonstrate that interfacial ruptures mainly occur in the media in both types of tests and are 2-5 elastic lamellae away from the external and internal elastic laminae. A remarkably "rougher" dissection surface is generated during axial peeling tests when compared with tests performed in the circumferential direction.

  • 62.
    Volk, Anna-Luisa
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Hu, Francis Jingxin
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH).
    Rockberg, Johan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH).
    Epitope mapping of antibodies using bacterial cell surface display of gene fragment libraries2018Inngår i: Epitope Mapping Protocols, Humana Press, 2018, s. 141-157Kapittel i bok, del av antologi (Fagfellevurdert)
    Abstract [en]

    The unique property of specific high affinity binding to more or less any target of interest has made antibodies tremendously useful in numerous applications. Hence, knowledge of the precise binding site (epitope) of antibodies on the target protein is one of the most important features for understanding its performance and determining its reliability in immunoassays. Here, we describe an updated protocol for high-resolution method for mapping epitopes of antibodies based on bacterial surface expression of antigen fragments followed by antibody-based flow cytometric analysis. Epitopes are determined by DNA sequencing of the sorted antibody-binding cells followed by sequence alignment back to the antigen sequence. The method described here has been useful for the mapping of both monoclonal and polyclonal antibodies with varying sizes of epitopes.

  • 63.
    Wahlberg, Elisabet
    KTH, Skolan för bioteknologi (BIO).
    Structure determination and thermodynamic stabilization of an engineered protein-protein complex2006Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The interaction between two 6 kDa proteins has been investigated. The studied complex of micromolar affinity (Kd) consists of the Z domain derived from staphylococcal protein A and the related protein ZSPA-1, belonging to a group of binding proteins denoted affibody molecules generated via combinatorial engineering of the Z domain. Affibody-target protein complexes are good model systems for structural and thermodynamic studies of protein-protein interactions. With the Z:ZSPA-1 pair as a starting point, we determined the solution structure of the complex and carried out a preliminary characterization of ZSPA-1. We found that the complex contains a rather large (ca. 1600 Å2) interaction interface with tight steric and polar/nonpolar complementarity. The structure of ZSPA-1 in the complex is well-ordered in a conformation that is very similar to that of the Z domain. However, the conformation of the free ZSPA-1 is best characterized by comparisons with protein molten globules. It shows a reduced secondary structure content, aggregation propensity, poor thermal stability, and binds the hydrophobic dye ANS. This molten globule state of ZSPA-1 is the native state in the absence of the Z domain, and the ordered state is only adopted following a stabilization that occurs upon binding. A more extensive characterization of ZSPA-1 suggested that the average topology of the Z domain is retained in the molten globule state but that it is represented by a multitude of conformations. Furthermore, the molten globule state is only marginally stable, and a significant fraction of ZSPA-1 exists in a completely unfolded state at room temperature. A complete thermodynamic characterization of the Z:ZSPA-1 pair suggests that the stabilization of the molten globule state to an ordered three helix structure in the complex is associated with a significant conformational entropy penalty that might influence the binding affinity negatively and result in an intermediate-affinity (µM) binding protein. This can be compared to a dissociation constant of 20-70 nM for the complex Z:Fc of IgG where Z uses the same binding surface as in Z:ZSPA-1. Structure analyses of Z in the free and bound state reveal an induced fit response upon complex formation with ZSPA-1 where a conformational change of several side chains in the binding surface increases the accessible surface area with almost 400 Å2 i.e. almost half of the total interaction surface in the complex. Two cysteine residues were introduced at specific positions in ZSPA-1 for five mutants in order to stabilize the conformation of ZSPA-1 by disulfide bridge formation. The mutants were thermodynamically characterized and the binding affinity of one mutant showed an improvement by more than a factor of ten. The improvement of the introduced cysteine bridge correlates with an increase in binding enthalpy rather than with entropy. Further analysis of the binding entropy suggests that the conformational entropy change in fact is reduced but its favorable contribution is opposed by a less favorable desolvation enthalpy change. These studies illustrate the structural and thermodynamic complexity of protein-protein interactions, but also that this complexity can be dissected and understood. In this study, a comprehensive characterization of the ZSPA-1 affibody has gained insight into the intricate mechanisms involved in complex formation. These theories were supported by the design of a ZSPA-1 mutant with improved binding affinity.

  • 64.
    Wahlberg, Elisabet
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Härd, Torleif
    Conformational Stabilization of an Engineered Binding Protein2006Inngår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 128, nr 23, s. 7651-7660Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We analyzed the thermodynamic basis for improvement of a binding protein by disulfide engineering. The Z(SPA-1) affibody binds to its Z domain binding partner with a dissociation constant K-d = 1.6 mu M, and previous analyses suggested that the moderate \affinity is due to the conformational heterogeneity of free Z(SPA-1) rather than to a suboptimal binding interface. Studies of five stabilized Z(SPA-1) double cystein mutants show that it is possible to improve the affinity by an order of magnitude to K-d = 130 nM, which is close to the range (20 to 70 nM) observed with natural Z domain binders, without altering the protein-protein interface obtained by phage display. Analysis of the binding thermodynamics reveals a balance between conformational entropy and desolvation entropy: the expected and favorable reduction of conformational entropy in the best-binding Z(SPA-1) mutant is completely compensated by an unfavorable loss of desolvation entropy. This is consistent with a restriction of possible conformations in the disulfide-containing mutant and a reduction of average water-exposed nonpolar surface area in the free state, resulting in a smaller conformational entropy penalty, but also a smaller change in surface area, for binding of mutant compared to wild-type Z(SPA-1). Instead, higher Z domain binding affinity in a group of eight Z(SPA-1) variants correlates with more favorable binding enthalpy and enthalpy- entropy compensation. These results suggest that protein-protein binding affinity can be improved by stabilizing conformations in which enthalpic effects can be fully explored.

  • 65.
    Wahlberg, Elisabet
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Lendel, Christofer
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Helgstrand, Magnus
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Allard, Peter
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Dincbas-Renqvist, Vildan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Hedqvist, Anders
    Berglund, Helena
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Nygren, Per-Åke
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Härd, Torleif
    KTH, Tidigare Institutioner                               , Bioteknologi.
    An affibody in complex with a target protein: Structure and coupled folding.2003Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 100, nr 6, s. 3185-3190Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Combinatorial protein engineering provides powerful means for functional selection of novel binding proteins. One class of engineered binding proteins, denoted affibodies, is based on the three-helix scaffold of the Z domain derived from staphylococcal protein A. The Z(SPA-1) affibody has been selected from a phage-displayed library as a binder to protein A. Z(SPA-1) also binds with micromolar affinity to its own ancestor, the Z domain. We have characterized the Z(SPA-1) affibody in its uncomplexed state and determined the solution structure of a Z:Z(SPA-1) protein-protein complex. Uncomplexed Z(SPA-1) behaves as an aggregation-prone molten globule, but folding occurs on binding, and the original (Z) three-helix bundle scaffold is fully formed in the complex. The structural basis for selection and strong binding is a large interaction interface with tight steric and polar/nonpolar complementarity that directly involves 10 of 13 mutated amino acid residues on Z(SPA-1). We also note similarities in how the surface of the Z domain responds by induced fit to binding of Z(SPA-1) and Ig Fc, respectively, suggesting that the Z(SPA-1) affibody is capable of mimicking the morphology of the natural binding partner for the Z domain.

  • 66. Wang, Guokun
    et al.
    Björk, Sara
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Huang, Mingtao
    Liu, Quanli
    Campbell, Kate
    Nielsen, Jens
    Jönsson, Håkan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Petranovic, Dina
    RNAi expression tuning, microfluidic screening, and genome recombineering for improved protein production in Saccharomyces cerevisiae2019Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 116, nr 19, s. 9324-9332Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The cellular machinery that supports protein synthesis and secretion lies at the foundation of cell factory-centered protein production. Due to the complexity of such cellular machinery, the challenge in generating a superior cell factory is to fully exploit the production potential by finding beneficial targets for optimized strains, which ideally could be used for improved secretion of other proteins. We focused on an approach in the yeast Saccharomyces cerevisiae that allows for attenuation of gene expression, using RNAi combined with high-throughput microfluidic single-cell screening for cells with improved protein secretion. Using direct experimental validation or enrichment analysis-assisted characterization of systematically introduced RNAi perturbations, we could identify targets that improve protein secretion. We found that genes with functions in cellular metabolism (YDC1, AAD4, ADE8, and SDH1), protein modification and degradation (VPS73, KTR2, CNL1, and SSA1), and cell cycle (CDC39), can all impact recombinant protein production when expressed at differentially down regulated levels. By establishing a workflow that incorporates Cas9-mediated recombineering, we demonstrated how we could tune the expression of the identified gene targets for further improved protein production for specific proteins. Our findings offer a high throughput and semirational platform design, which will improve not only the production of a desired protein but even more importantly, shed additional light on connections between protein production and other cellular processes.

  • 67.
    Wirta, Valtteri
    KTH, Skolan för bioteknologi (BIO).
    Mining the transcriptome - methods and applications2006Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Regulation of gene expression occupies a central role in the control of the flow of genetic information from genes to proteins. Regulatory events on multiple levels ensure that the majority of the genes are expressed under controlled circumstances to yield temporally controlled, cell and tissue-specific expression patterns. The combined set of expressed RNA transcripts constitutes the transcriptome of a cell, and can be analysed on a large-scale using both sequencing and microarray-based methods.

    The objective of this work has been to develop tools for analysis of the transcriptomes (methods), and to gain new insights into several aspects of the stem cell transcriptome (applications). During recent years expectations of stem cells as a resource for treatment of various disorders have emerged. The successful use of endogenously stimulated or ex vivo expanded stem cells in the clinic requires an understanding of mechanisms controlling their proliferation and self-renewal.

    This thesis describes the development of tools that facilitate analysis of minute amounts of stem cells, including RNA amplification methods and generation of a cDNA array enriched for genes expressed in neural stem cells. The results demonstrate that the proposed amplification method faithfully preserves the transcript expression pattern. An analysis of the feasibility of a neurosphere assay (in vitro model system for study of neural stem cells) clearly shows that the culturing induces changes that need to be taken into account in design of future comparative studies. An expressed sequence tag analysis of neural stem cells and their in vivo microenvironment is also presented, providing an unbiased large-scale screening of the neural stem cell transcriptome. In addition, molecular mechanisms underlying the control of stem cell self-renewal are investigated. One study identifies the proto-oncogene Trp53 (p53) as a negative regulator of neural stem cell self-renewal, while a second study identifies genes involved in the maintenance of the hematopoietic stem cell phenotype.

    To facilitate future analysis of neural stem cells, all microarray data generated is publicly available through the ArrayExpress microarray data repository, and the expressed sequence tag data is available through the GenBank.

  • 68.
    Wu, Yu-Tang
    et al.
    Univ Paris 11, Univ Paris Saclay, Inst Integrat Biol Cell, NanoBioPhoton Nanofret Com,CNRS,CEA, Orsay, France..
    Qiu, Xue
    Univ Paris 11, Univ Paris Saclay, Inst Integrat Biol Cell, NanoBioPhoton Nanofret Com,CNRS,CEA, Orsay, France..
    Lindbo, Sarah
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Susumu, Kimihiro
    US Naval Res Lab, Opt Sci Div, Code 5600, Washington, DC USA.;KeyW Corp, Hanover, MD 21076 USA..
    Medintz, Igor L.
    US Naval Res Lab, Ctr Bio Mol Sci & Engn, Code 6900, Washington, DC USA..
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Centra, Centrum för Bioprocessteknik, CBioPT. KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Hildebrandt, Niko
    Univ Paris 11, Univ Paris Saclay, Inst Integrat Biol Cell, NanoBioPhoton Nanofret Com,CNRS,CEA, Orsay, France..
    Quantum Dot-Based FRET Immunoassay for HER2 Using Ultrasmall Affinity Proteins2018Inngår i: Small, ISSN 1613-6810, E-ISSN 1613-6829, Vol. 14, nr 35, artikkel-id 1802266Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Engineered scaffold affinity proteins are used in many biological applications with the aim of replacing natural antibodies. Although their very small sizes are beneficial for multivalent nanoparticle conjugation and efficient Forster resonance energy transfer (FRET), the application of engineered affinity proteins in such nanobiosensing formats has been largely neglected. Here, it is shown that very small (approximate to 6.5 kDa) histidine-tagged albumin-binding domain-derived affinity proteins (ADAPTs) can efficiently self-assemble to zwitterionic ligand-coated quantum dots (QDs). These ADAPT-QD conjugates are significantly smaller than QD-conjugates based on IgG, Fab', or single-domain antibodies. Immediate applicability by the quantification of the human epidermal growth factor receptor 2 (HER2) in serum-containing samples using time-gated Tb-to-QD FRET detection on the clinical benchtop immunoassay analyzer KRYPTOR is demonstrated here. Limits of detection down to 40 x 10(-12)m (approximate to 8 ng mL(-1)) are in a relevant clinical concentration range and outperform previously tested assays with antibodies, antibody fragments, and nanobodies.

  • 69.
    Wållberg, Fredrik
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Sundström, Heléne
    KTH, Skolan för bioteknologi (BIO).
    Ledung, E.
    Department of Biology and Chemical Engineering, Mälardalen University.
    Hewitt, C. J.
    Centre for Formulation Engineering, School of Engineering (Chemical Engineering), University of Birmingham.
    Enfors, Sven-Olof
    KTH, Skolan för bioteknologi (BIO).
    Monitoring and quantification of inclusion body formation in Escherichia coli by multi-parameter flow cytometry2005Inngår i: Biotechnology letters, ISSN 0141-5492, E-ISSN 1573-6776, Vol. 27, nr 13, s. 919-926Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Multi-parameter flow cytometry was used to monitor the formation of promegapoietin (PMP) inclusion bodies during a high cell density Escherichia coli fed-batch fermentation process. Inclusion bodies were labelled with a primary antibody and then with a secondary fluorescent antibody. Using this method it was possible to detect PMP inclusion body formation with a high specificity and it was possible to monitor the increased accumulation of the protein with process time (6-48 mg PMP/g CDW) whilst highlighting population heterogeneity.

  • 70.
    Zajac, Pawel
    KTH, Skolan för bioteknologi (BIO), Genteknologi.
    Parallel target selection by trinucleotide threading2009Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    DNA is the code for all life. Via intermediary RNA the information encoded by the genome is relayed to proteins executing the various functions in a cell. Together, this repertoire of inherently linked biological macromolecules determines all characteristics and features of a cell. Technological advancements during the last decades have enabled the pursuit of novel types of studies and the investigation of the cell and its constituents at a progressively higher level of detail. This has shed light on numerous cellular processes and on the underpinnings of several diseases. For the majority of studies focusing on nucleic acids, an amplification step has to be implemented before an analysis, scoring or interrogation method translates the amplified material into relevant biological information. This information can, for instance, be the genotype of particular SNPs or STRs, or the abundance level of a set of interesting transcripts. As such, amplification plays a significant role in nucleic acid assays. Over the years, a number of techniques – most notably PCR – has been devised to meet this amplification need, specifically or randomly multiplying desired regions. However, many of the approaches do not scale up easily rendering comprehensive studies cumbersome, time-consuming and necessitating large quantities of material.Trinucleotide threading (TnT) – forming the red thread throughout this thesis – is a multiplex amplification method, enabling simultaneous targeted amplification of several nucleic acid regions in a specific manner. TnT begins with a controlled linear DNA thread formation, each type of thread corresponding to a segment of interest, by a gap-fill reaction using a restricted trinucleotide set. The whole collection of created threads is subsequently subjected to an exponential PCR amplification employing a single primer pair. The generated material can thereafter be analyzed with a multitude of readout and detection platforms depending on the issue or characteristic under consideration.TnT offers a high level of specificity by harnessing the inherent specificities of a polymerase and a ligase acting on a nucleotide set encompassing three out of the four nucleotide types. Accordingly, several erroneous events have to occur in order to produce artifacts. This necessitates override of a number of control points.The studies constituting this thesis demonstrate integration of the TnT amplification strategy in assays for analysis of various aspects of DNA and RNA. TnT was adapted for expression profiling of intermediately-sized gene sets using both conventional DNA microarrays and massively parallel second generation 454 sequencing for readout. TnT, in conjunction with 454 sequencing, was also employed for allelotyping, defined as determination of allele frequencies in a cohort. In this study, 147 SNPs were simultaneously assayed in a pool comprising genomic DNA of 462 individuals. Finally, TnT was recruited for parallel amplification of STR loci with detection relying on capillary gel electrophoresis. In all investigations, the material generated with TnT was of sufficient quality and quantity to produce reliable and accurate biological information.Taken together, TnT represents a viable multiplex amplification technique permitting parallel amplification of genomic segments, for instance harboring polymorphisms, or of expressed genes. In addition to these, this versatile amplification module can be implemented in assays targeting a range of other features of genomes and transcriptomes.

  • 71.
    Zajac, Pawel
    et al.
    KTH, Skolan för bioteknologi (BIO), Genteknologi.
    Lundeberg, Joakim
    KTH, Skolan för bioteknologi (BIO), Genteknologi.
    Ahmadian, Afshin
    KTH, Skolan för bioteknologi (BIO), Genteknologi.
    Targeted transcript profiling by sequencingManuskript (Annet vitenskapelig)
    Abstract [en]

    In recent years, second generation sequencers have been employed to study various facets of the transcriptome in a comprehensive manner. However, intermediary gene sets featuring differentially expressed genes can reduce the dimensionality of experiments while providing researchers with the most significant data. Trinucleotide threading (TnT) is a multiplex amplification method previously implemented in an assay for expression profiling of moderate gene sets. Here, two additional detection systems were evaluated with a focus on lowering the input material requirements. 32 genes were simultaneously assayed with detection either by direct hybridization of TnT products or by sequencing these using the massively parallel 454 sequencer. Both approaches produced reliable transcript abundance data starting from total RNA from about 200 cells. The direct hybridization readout is beneficial for smaller-scale studies, while more ambitious efforts employing numerous individuals are, together with a sample barcoding and pooling scheme, well suited for the second generation sequencing approach. Moreover, with protocol optimizations the starting material requirements for the sequencing strategy may be further reduced. Accordingly, this study presents a targeted RNA-Seq method.

  • 72.
    Zajac, Pawel
    et al.
    KTH, Skolan för bioteknologi (BIO), Genteknologi.
    Öberg, Christine
    KTH, Skolan för bioteknologi (BIO), Genteknologi.
    Ahmadian, Afshin
    KTH, Skolan för bioteknologi (BIO), Genteknologi.
    Analysis of Short Tandem Repeats by Parallel DNA Threading2009Inngår i: PLoS ONE, ISSN 1932-6203, Vol. 4, nr 11, s. e7823-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The majority of studies employing short tandem repeats (STRs) require investigation of several of these genetic markers. As such, we demonstrate the feasibility of the trinucleotide threading (TnT) approach for scalable analysis of STRs. The TnT method represents a parallel amplification alternative that addresses the obstacles associated with multiplex PCR. In this study, analysis of the STR fragments was performed with capillary gel electrophoresis; however, it should be possible to combine our approach with the massive 454 sequencing platform to considerably increase the number of targeted STRs.

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