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  • 1.
    Alm, Tove
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Steen, Johanna
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ottosson, Jenny
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    High-throughput protein purification under denaturating conditions by the use of cation exchange chromatography2007Inngår i: Biotechnology Journal, ISSN 1860-6768, Vol. 2, s. 709-716Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A high-throughput protein purification strategy using the polycationic Z(basic) tag has been developed. In order for the strategy to be useful both for soluble and less soluble proteins, a denaturating agent, urea, was used in all purification steps. First, four target proteins were genetically fused to the purification tag, Z(basic). These protein constructs were purified by cation exchange chromatography and eluted using a salt gradient. From the data achieved, a purification strategy was planned including stepwise elution to enable parallel protein purification using a laboratory robot. A protocol that includes all steps, equilibration of the chromatography resin, load of sample, wash, and elution, all without any manual handling steps, was handled by the laboratory robot. The program allows automated purification giving milligram amounts of pure recombinant protein of up to 60 cell lysates. In this study 22 different protein constructs, with different characteristics regarding pI and solubility, were successfully purified by the laboratory robot. The data show that Z(basic) can be used as a general purification tag also under denaturating conditions. Moreover, the strategy enables purification of proteins with different pI and solubility using ion exchange chromatography (IEXC). The procedure is highly reproducible and allows for high protein yield and purity and is therefore a good complement to the commonly used His(6)-tag.

  • 2. Gustavsson, Elin
    et al.
    Ek, Sara
    Steen, Johanna
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Kristensson, Malin
    Älgenäs, Cajsa
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Wingren, Christer
    Ottosson, Jenny
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Borrebaeck, Carl A. K.
    Surrogate antigens as targets for proteome-wide binder selection2011Inngår i: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 28, nr 4, s. 302-311Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 3.
    Steen, Johanna
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Characterization of Antigenic Properties and High Throughput Protein Purification2010Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    To understand the cellular processes, knowledge of the localization and function of proteins are essential. There are several high throughput ventures examining the human proteome. However, there are some bottlenecks in these ventures. For example the production and expression of soluble proteins for analysis. Another obstacle for affinity proteomics is the generation of high quality antibodies, invaluable tools in biotechnological applications. The objective in this thesis was to facilitate protein purification and sample preparation before analysis and downstream applications. We also aimed to attain more information on what constitutes an ideal immunogen, and on how different immune systems respond to a common amino acid sequence.

     

    In one of the projects an automated purification set-up was developed to ensure high recovery of up to milligram amounts of protein with high purity. The system allowed up to 60 recombinant proteins to be purified under both native and denaturing conditions. In another project, the same developed set-up was additionally shown to work with an alternative chromatography resin with small adjustments. Instead of immobilized metal ion affinity chromatography, used in the first project, ion exchange chromatography was applied under denaturing conditions, with good results. To further automate the production line in high throughput projects, an automated sample preparation was set up for mass spectrometry and e.g. gel electrophoresis analysis. We showed that a crude cell lysate could be used as input in the magnetic bead based system, and totally absent from manual handling, the output was purified and buffer exchanged samples ready for mass spectrometry analysis, as well as a fraction of sample that could be used for complementary analyses, for example gel electrophoresis to determine the protein concentration and purity.

     

    The other objective was – as noted – to gain better comprehension of antibody generation to foreign proteins, and to shed more light over how to design a good antigen. First was a solubility assay developed that determined the remaining fraction of soluble protein after reduction of the concentration of denaturing agent. The assay was performed in a 96 deep well plate, and only instrumentation available in a standard laboratory was necessary. The fact that the assay could be automated on a pipetting robot, increased the throughput and reduced the necessary manual handling. Obtained information on antigen solubility was correlated to the cognate antibody titers. At average the antibody yield was higher when a soluble antigen was used for immunization. Also, the probability of failing in eliciting an immune response was increased if an insoluble antigen was used. However, the antibody titers in each solubility class were highly diverse, and thus also some insoluble antigens were found that provoked the immune system. To further examine the differences between different B cell repertoires, a massive epitope mapping was performed with more than 400 different antisera reacting to the same amino acid sequence. Antigenic hot spot regions were discovered, as well as regions depleted in antibody recognition. However, in one third of the antisera the most abundant antigenic region did not elicit any binding of antibodies. This further validates the conclusion that good antigen design is essential, however is it not certain the outcome of immunizations can ever be determined a priori due to the variability between hosts. An alternative to immunization is selection of affinity reagents by phage display. In the last project an initial parallelized set-up selected antibody fragments that showed high specificity and were compatible with several biotechnological applications, making the set-up a promising alternative to conventional immunization in proteome-wide endeavors.

     

  • 4.
    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.

  • 5.
    Steen, Johanna
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Larsson, Karin
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ottosson, Jenny
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Antigenic mapping and characterization of Albumin Binding ProteinManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    The possibility to predict the location of antigenic determinants is a desirable feature in antibody production ventures and for vaccine development. However, antigenic propensity scales available today are poor, and so far it is not possible to predict the best antigen to trigger the immune system. Here, a unique set of 411 antisera towards a common part of allantigens within the Human Protein Atlas project has made it possible to perform massive epitope mapping. This effort generated a true map of the antigenic regions of this common N-terminal tag, and rendered it possible to further investigate what features that generate a good antigen. Investigations on variation in epitope occurrence are often an obstacle when mapping antigens, because of the ethics of using more animals than necessary for antibody production. As a consequence, not much has been done to verify epitopes found and the variance between different immunizations has not been thoroughly investigated. Herein it was shown that the most immunopotentating sites were only detected by the polyclonal antibodies in 70% of the immunizations, demonstrating the need of good antigen design. Detected epitopes also showed that aromatic amino acids, some positively charged aminoacids, and serine and glycine were over-represented in the antigenic hot spot regions. The detected antigenic regions were also shown to have fairly low correlation to several antigenic propensity scales.

  • 6.
    Steen, Johanna
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ramström, Margareta
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ottosson, Jenny
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Automated sample preparation method for mass spectrometry analysis on recombinant proteins2009Inngår i: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1216, nr 20, s. 4457-4464Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A completely automated procedure for the purification and desalting of proteins with a polyhistidine purification tag prior to mass spectrometry analysis is presented. The system is ideal for rapid quality control and optimization studies and it provides researchers with a straightforward, reliable tool for studies of recombinant proteins. Forty-eight samples can be prepared within 4.5 h and only small cultivation and buffer volumes are needed. In this proof of concept, 19,000-35,000 Da recombinant proteins from both crude and clarified cell lysates were successfully prepared for subsequent analysis by electrospray ionization anti matrix-assisted laser desorption/ionization mass spectrometry as well as by gel electrophoresis.

  • 7.
    Steen, Johanna
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ottosson, Jenny
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    High-throughput protein purification using an automated set-up for high-yield affinity chromatography2006Inngår i: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 46, nr 2, s. 173-178Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    One of the key steps in high-throughput protein production is protein purification. A newly developed high-yield protein purification and isolation method for laboratory scale use is presented. This procedure allows fully automated purification of up to 60 cell lysates with milligram yields of pure recombinant protein in 18.5 h. The method is based on affinity chromatography and has been set up on an instrument that utilizes positive pressure for liquid transfer through columns. A protocol is presented that includes all steps of equilibration of the chromatography resin, load of sample, wash, and elution without any manual handling steps. In contrast to most existing high-throughput protein purification procedures, positive pressure is used for liquid transfer rather than vacuum. Positive pressure and individual pumps for each liquid channel contribute to controlled flow rates and eliminate the risk of introducing air in the chromatography resin and therefore ensure stable chromatography conditions. The procedure is highly reproducible and allows for high protein yield and purity.

  • 8.
    Stenvall, Maria
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Steen, Johanna
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ottosson, Jenny
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    High-throughput solubility assay for purified recombinant protein immunogens2005Inngår i: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1752, nr 1, s. 6-10Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A high-throughput assay is described for analysis of the solubility of purified recombinant proteins. The assay is based on affinity purification of proteins in the presence of chaotropic agents followed by a dilution and incubation step to investigate the solubility in the absence of high concentrations of such agents. The assay can be performed in a 96-well format, which makes it well suited for high-throughput applications. For 125 recombinant proteins expressed as part of an antibody-based proteomics effort, experimental solubility data were compared to calculated hydrophobicity values based on the amino acid sequence of each protein. This comparison showed only weak correlation between the theoretical and experimental values, which emphasizes the importance of experimental assays to determine the solubility of recombinant proteins.

  • 9.
    Uhlén, Mathias
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Björling, Erik
    KTH, Skolan för bioteknologi (BIO).
    Agaton, Charlotta
    KTH, Skolan för bioteknologi (BIO).
    Al-Khalili Szigyarto, Cristina
    KTH, Skolan för bioteknologi (BIO).
    Amini, Bahram
    KTH, Skolan för bioteknologi (BIO).
    Andersen, Elisabet
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Andersson, Ann-Catrin
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Angelidou, Pia
    KTH, Skolan för bioteknologi (BIO).
    Asplund, Anna
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Asplund, Caroline
    KTH, Skolan för bioteknologi (BIO).
    Berglund, Lisa
    KTH, Skolan för bioteknologi (BIO).
    Bergström, Kristina
    KTH, Skolan för bioteknologi (BIO).
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO).
    Cerjan, Dijana
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Ekström, Marica
    KTH, Skolan för bioteknologi (BIO).
    Elobeid, Adila
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Eriksson, Cecilia
    KTH, Skolan för bioteknologi (BIO).
    Fagerberg, Linn
    KTH, Skolan för bioteknologi (BIO).
    Falk, Ronny
    KTH, Skolan för bioteknologi (BIO).
    Fall, Jenny
    KTH, Skolan för bioteknologi (BIO).
    Forsberg, Mattias
    KTH, Skolan för bioteknologi (BIO).
    Gry Björklund, Marcus
    KTH, Skolan för bioteknologi (BIO).
    Gumbel, Kristoffer
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Halimi, Asif
    KTH, Skolan för bioteknologi (BIO).
    Hallin, Inga
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Hamsten, Carl
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Hansson, Marianne
    KTH, Skolan för bioteknologi (BIO).
    Hedhammar, My
    KTH, Skolan för bioteknologi (BIO).
    Hercules, Görel
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Kampf, Caroline
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Larsson, Karin
    KTH, Skolan för bioteknologi (BIO).
    Lindskog, Mats
    KTH, Skolan för bioteknologi (BIO).
    Lodewyckx, Wald
    KTH, Skolan för bioteknologi (BIO).
    Lund, Jan
    KTH, Skolan för bioteknologi (BIO).
    Lundeberg, Joakim
    KTH, Skolan för bioteknologi (BIO).
    Magnusson, Kristina
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Malm, Erik
    KTH, Skolan för bioteknologi (BIO).
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO).
    Ödling, Jenny
    KTH, Skolan för bioteknologi (BIO).
    Oksvold, Per
    KTH, Skolan för bioteknologi (BIO).
    Olsson, Ingmarie
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Öster, Emma
    KTH, Skolan för bioteknologi (BIO).
    Ottosson, Jenny
    KTH, Skolan för bioteknologi (BIO).
    Paavilainen, Linda
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Persson, Anja
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Rimini, Rebecca
    KTH, Skolan för bioteknologi (BIO).
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO).
    Runeson, Marcus
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Sivertsson, Åsa
    KTH, Skolan för bioteknologi (BIO).
    Sköllermo, Anna
    KTH, Skolan för bioteknologi (BIO).
    Steen, Johanna
    KTH, Skolan för bioteknologi (BIO).
    Stenvall, Maria
    KTH, Skolan för bioteknologi (BIO).
    Sterky, Fredrik
    KTH, Skolan för bioteknologi (BIO).
    Strömberg, Sara
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Sundberg, Mårten
    KTH, Skolan för bioteknologi (BIO).
    Tegel, Hanna
    KTH, Skolan för bioteknologi (BIO).
    Tourle, Samuel
    KTH, Skolan för bioteknologi (BIO).
    Wahlund, Eva
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Waldén, Annelie
    KTH, Skolan för bioteknologi (BIO).
    Wan, Jinghong
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Westberg, Joakim
    KTH, Skolan för bioteknologi (BIO).
    Wester, Kenneth
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Wrethagen, Ulla
    KTH, Skolan för bioteknologi (BIO).
    Xu, Lan Lan
    KTH, Skolan för bioteknologi (BIO).
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Pontén, Fredrik
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    A human protein atlas for normal and cancer tissues based on antibody proteomics2005Inngår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 4, nr 12, s. 1920-1932Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, similar to 400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.

1 - 9 of 9
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Referera
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  • en-GB
  • en-US
  • fi-FI
  • nn-NO
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