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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.
    Berglund, Lisa
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Björling, Erik
    KTH, Skolan för bioteknologi (BIO).
    Gry, Marcus
    KTH, Skolan för bioteknologi (BIO).
    Asplund, Anna
    Uppsala Univ, Rudbeck laboratory.
    Al-Khalili Szigyarto, Cristina
    KTH, Skolan för bioteknologi (BIO).
    Persson, Anja
    KTH, Skolan för bioteknologi (BIO).
    Ottoson, Jenny
    KTH, Skolan för bioteknologi (BIO).
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO).
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO).
    Sivertsson, Åsa
    KTH, Skolan för bioteknologi (BIO).
    Wester, Kenneth
    Uppsala Univ, Rudbeck laboratory.
    Kampf, Caroline
    Uppsala Univ, Rudbeck laboratory.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO).
    Pontén, Fredrik
    Uppsala Univ, Rudbeck laboratory.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO).
    Generation of validated antibodies towards the human proteomeArtikkel i tidsskrift (Annet vitenskapelig)
    Abstract [en]

    Here we show the results from a large effort to generate antibodies towards the human proteome. A high-throughput strategy was developed based on cloning and expression of antigens as recombitant protein epitope signature tags (PrESTs) Affinity purified polyclonal antibodies were generated, followed by validation by protein microarrays, Western blotting and microarray-based immunohistochemistry. PrESTs were selected based on sequence uniqueness relative the proteome and a bioinformatics analysis showed that unique antigens can be found for at least 85% of the proteome using this general strategy. The success rate from antigen selection to validated antibodies was 31%, and from protein to antibody 55%. Interestingly, membrane-bound and soluble proteins performed equally and PrEST lengths between 75 and 125 amino acids were found to give the highest yield of validated antibodies. Multiple antigens were selected for many genes and the results suggest that specific antibodies can be systematically generated to most human proteibs.

  • 3.
    Berglund, Lisa
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Björling, Erik
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Oksvold, Per
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Fagerberg, Linn
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Al-Khalili Szigyarto, Cristina
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Persson, Anja
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ottosson, Jenny
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Lundberg, Emma
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Sivertsson, Åsa
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    et al.,
    A genecentric human protein atlas for expression profiles based on antibodies2008Inngår i: Molecular & Cellular Proteomics, ISSN 1535-9476, Vol. 7, nr 10, s. 2019-2027Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    An attractive path forward in proteomics is to experimentally annotate the human protein complement of the genome in a genecentric manner. Using antibodies, it might be possible to design protein-specific probes for a representative protein from every protein-coding gene and to subsequently use the antibodies for systematical analysis of cellular distribution and subcellular localization of proteins in normal and disease tissues. A new version (4.0) of the Human Protein Atlas has been developed in a genecentric manner with the inclusion of all human genes and splice variants predicted from genome efforts together with a visualization of each protein with characteristics such as predicted membrane regions, signal peptide, and protein domains and new plots showing the uniqueness (sequence similarity) of every fraction of each protein toward all other human proteins. The new version is based on tissue profiles generated from 6120 antibodies with more than five million immunohistochemistry-based images covering 5067 human genes, corresponding to similar to 25% of the human genome. Version 4.0 includes a putative list of members in various protein classes, both functional classes, such as kinases, transcription factors, G-protein-coupled receptors, etc., and project-related classes, such as candidate genes for cancer or cardiovascular diseases. The exact antigen sequence for the internally generated antibodies has also been released together with a visualization of the application-specific validation performed for each antibody, including a protein array assay, Western blot analysis, immunohistochemistry, and, for a large fraction, immunofluorescence-based confocal microscopy. New search functionalities have been added to allow complex queries regarding protein expression profiles, protein classes, and chromosome location. The new version of the protein atlas thus is a resource for many areas of biomedical research, including protein science and biomarker discovery.

  • 4.
    Boström, Tove
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Danielsson, Frida
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Lundberg, Emma
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Johansson, Henrik J.
    Karlinska Institute, Cancer Proteomics Mass Spectrometry, Dep. of Oncology-Pathology.
    Tegel, Hanna
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Lehtiö, Janne
    Karolinska Institute, Cancer Proteomics Mass Spectrometry, Dep. of Oncology-Pathology.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Ottosson Takanen, Jenny
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Investigating the correlation of protein and mRNA levels in human cell lines using quantitative proteomics and transcriptomicsManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    An important topic of discussion in proteomics is the degree of correlation of RNA and protein levels in cells, tissues and organs. In this study, the difference in protein and mRNA levels for a number of selected gene targets were investigated across six human cell lines using quantitative proteomics and next generation sequencing-based transcriptomics. The copy numbers of 32 proteins were determined using an absolute quantitative proteomics approach (PrEST-SILAC), where heavy isotope-labeled protein fragments were used as internal standards. A cross evaluation of protein copy numbers determined by mass spectrometry and staining profiles using immunohistochemistry showed good correlation. The mRNA levels were determined using RNA sequencing based on digital counting of sequencing reads and the levels determined as FPKM values. Comparison of the relative variations in mRNA and protein levels for individual genes across the six cell lines showed correlation between protein and mRNA levels, including six genes with high variability in expression levels in the six cell lines resulting in an average correlation of 0.9 (Spearman's rank coefficient). In summary, the analysis of the selected protein targets supports the conclusion that the translation rate across cell lines correlates for a particular gene, suggesting that individual protein levels can be predicted from the respective mRNA levels by defining the relation between protein and mRNA, specific for each human gene.

  • 5.
    Boström, Tove
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Ottosson Takanen, Jenny
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Antibodies as means for selective mass spectrometry2015Inngår i: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376XArtikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    For protein analysis of biological samples, two major strategies are used today; mass spectrometry (MS) and antibody-based methods. Each strategy offers advantages and drawbacks. However, combining the two using an immunoenrichment step with MS analysis brings together the benefits of each method resulting in increased sensitivity, faster analysis and possibility of higher degrees of multiplexing. The immunoenrichment can be performed either on protein or peptide level and quantification standards can be added in order to enable determination of the absolute protein concentration in the sample. The combination of immunoenrichment and MS holds great promise for the future in both proteomics and clinical diagnostics. This review describes different setups of immunoenrichment coupled to mass spectrometry and how these can be utilized in various applications.

  • 6. Colwill, Karen
    et al.
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Sundberg, Mårten
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Sjöberg, Ronald
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Sivertsson, Åsa
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Schwenk, Jochen M
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ottosson Takanen, Jenny
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Gräslund, Susanne
    et, al.
    A roadmap to generate renewable protein binders to the human proteome2011Inngår i: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 8, nr 7, s. 551-8Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Despite the wealth of commercially available antibodies to human proteins, research is often hindered by their inconsistent validation, their poor performance and the inadequate coverage of the proteome. These issues could be addressed by systematic, genome-wide efforts to generate and validate renewable protein binders. We report a multicenter study to assess the potential of hybridoma and phage-display technologies in a coordinated large-scale antibody generation and validation effort. We produced over 1,000 antibodies targeting 20 SH2 domain proteins and evaluated them for potency and specificity by enzyme-linked immunosorbent assay (ELISA), protein microarray and surface plasmon resonance (SPR). We also tested selected antibodies in immunoprecipitation, immunoblotting and immunofluorescence assays. Our results show that high-affinity, high-specificity renewable antibodies generated by different technologies can be produced quickly and efficiently. We believe that this work serves as a foundation and template for future larger-scale studies to create renewable protein binders.

  • 7. 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.

  • 8.
    Hedhammar, My
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Stenvall, Maria
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Lönneborg, Rosa
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Nord, Olof
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Sjölin, Olle
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Brismar, Hjalmar
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Uhlén, Matthias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ottosson, Jenny
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    A Novel flow cytometry-based method for analysis of expression levels in Escherichia coli, giving information about precipitated and soluble protein2005Inngår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 119, nr 2, s. 133-146Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A high throughput method for screening of protein expression is described. By using a flow cytometer, levels of both soluble and precipitated protein can simultaneously be assessed in vivo. Protein fragments were fused to the N-terminus of enhanced GFP and the cell samples were analysed using a flow cytometer. Data concerning whole cell fluorescence and light scattering was collected. The whole cell fluorescence is probing intracellular concentrations of soluble fusion proteins. Concurrently, forward scattered light gives data about inclusion body formation, valuable information in process optimisation. To evaluate the method, the cells were disrupted, separated into soluble and non-soluble fractions and analysed by gel electrophoresis. A clear correlation between fluorescence and soluble target protein was shown. Interestingly, the distribution of the cells regarding forward scatter (standard deviation) correlates with the amount of inclusion bodies formed. Finally, the newly developed method was used to evaluate two different purification tags, His(6) and Z(basic), and their effect on the expression pattern.

  • 9. Neubauer, A.
    et al.
    Golson, R.
    Ukkonen, K.
    Krause, M.
    Tegel, Hanna
    KTH, Skolan för bioteknologi (BIO), Biokemi (stängd 20130101).
    Ottosson, Jenny
    KTH, Skolan för bioteknologi (BIO), Biokemi (stängd 20130101).
    Wittrup Larsen, Marianne
    KTH, Skolan för bioteknologi (BIO), Biokemi (stängd 20130101).
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi (stängd 20130101).
    Neubauer, P.
    Vasala, A.
    Controlling nutrient release in cell cultivation2009Inngår i: Genetic Engineering and Biotechnology News, ISSN 1935-472X, Vol. 29, nr 11, s. 50-51Artikkel i tidsskrift (Fagfellevurdert)
  • 10.
    Nilsson, Peter
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Larsson, Karin
    KTH, Skolan för bioteknologi (BIO).
    Persson, Anja
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO).
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Björling, Erik
    KTH, Skolan för bioteknologi (BIO).
    Ottoson, Jenny
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ödling, Jenny
    KTH, Skolan för bioteknologi (BIO).
    Sundberg, Mårten
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Al-Khalili Szigyarto, Cristina
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Paavilainen, Linda
    Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
    Andersson, Ann-Catrin
    Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
    Kampf, Caroline
    Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
    Wester, Kenneth
    Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
    Pontén, Fredrik
    Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
    Towards a human proteome atlas: High-throughput generation of mono-specific antibodies for tissue profiling2005Inngår i: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 5, s. 4327-4337Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A great need exists for the systematic generation of specific antibodies to explore the human proteome. Here, we show that antibodies specific to human proteins can be generated in a high-throughput manner involving stringent affinity purification using recombinant protein epitope signature tags (PrESTs) as immunogens and affinity-ligands. The specificity of the generated affinity reagents, here called mono-specific antibodies (msAb), were validated with a novel protein microarray assay. The success rate for 464 antibodies generated towards human proteins was more than 90% as judged by the protein array assay. The antibodies were used for parallel profiling of patient biopsies using tissue microarrays generated from 48 human tissues. Comparative analysis with well-characterized monoclonal antibodies showed identical or similar specificity and expression patterns. The results suggest that a comprehensive atlas containing extensive protein expression and subcellular localization data of the human proteome can be generated in an efficient manner with mono-specific antibodies.

  • 11.
    Ottosson, J.
    et al.
    KTH.
    Steen, J.
    Tegel, Hanna
    KTH.
    Konrad, A.
    KTH.
    Halimi, A.
    KTH.
    Wrethagen, U.
    KTH.
    Xu, L. Lan
    KTH.
    Pettersson, K.
    KTH.
    Widehammar, J.
    KTH.
    Dahlgren, L. -G
    KTH.
    Hober, Sophia
    KTH.
    High throughput protein production and purification in the Human Protein Atlas program2006Inngår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 5, nr 10, s. S40-S40Artikkel i tidsskrift (Annet vitenskapelig)
  • 12.
    Ottosson, J.
    et al.
    KTH.
    Wernerus, H.
    KTH.
    Nilsson, P.
    KTH.
    Tegel, Hanna
    KTH.
    Larsson, K.
    KTH.
    Uhlén, Mathias
    KTH.
    Hober, S.
    KTH.
    High throughput antibody generation and validation for antibody proteomics2005Inngår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 4, nr 8, s. S64-S64Artikkel i tidsskrift (Annet vitenskapelig)
  • 13.
    Ottosson, Jenny
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Enthalpy and Entropy in Enzyme Catalysis: A Study of Lipase Enantioselectivity2001Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Biocatalysis has become a popular technique in organic synthesis due to high activity and selectivity of enzyme catalyzed reactions. Enantioselectivity is a particularly attractive enzyme property, which is utilized for the production of enantiopure substances. Determination of the temperature dependence of enzyme enantioselectivity allows for thermodynamic analyses that reveal the contribution of differential activation enthalpy, ΔR-SΔH, and entropy, ΔR-SΔS. In the present investigation the influence of substrate structure, variations on enzyme structure and of reaction media on the enantioselectivity of Candida Antarctica lipase B has been studied.

    The contribution of enthalpy, ΔR-SΔH, and entropy, TΔR-SΔS, to the differential free energy, ΔR-SΔG, of kinetic resolutions of sec-alcohols were of similar magnitude. Generally the two terms were counteracting, meaning that the enantiomer favored by enthalpy was disfavored by entropy. 3-Hexanol was an exception where the preferred enantiomer was favored both by enthalpy and by entropy. Resolution of 1-bromo-2-butanol revealed non-steric interactions to influence both ΔR-SΔH and ΔR-SΔS. Molecular modeling of the spatial freedom of the enzyme-substrate transition state indicated correlation tothe transition state entropy. The acyl chain length was shown to affect enantioselectivity in transesterifications of a sec-alcohol.

    Point mutations in the active site were found to decrease or increase enantioselectivity. The changes were caused by partly compensatory changes in both ΔR-SΔH and ΔR-SΔS. Studies on single and double mutation variants showed that the observed changes were not additive.

    Enantioselectivity was strongly affected by the reaction media. Transesterifications of a sec-alcohol catalyzed by Candida Antarctica lipase B was studied in eight liquidorganic solvents and supercritical carbon dioxide. A correlation of enantioselectivity and the molecular volume of the solvent was found.

    Differential activation enthalpy, ΔR-SΔH, and entropy, ΔR-SΔS, display a compensatory nature. However this compensation is not perfect, which allows for modifications of enantioselectivity. The components of the thermodynamic parameters are highly complex and interdependent but if their roles are elucidated rational design of enantioselective enzymatic processes may be possible.

     

     

  • 14.
    Ottosson, Jenny
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Fransson, Linda
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Hult, Karl
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Substrate entropy in enzyme enantioselectivity: An experimental and molecular modeling study of a lipase2002Inngår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 11, nr 6, s. 1462-1471Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The temperature dependence of the enantioselectivity of Candida antarctica lipase B for 3-hexanol, 2-butanol, 3-methyl-2-butanol, 3,3-dimethyl-2-butanol, and 1-bromo-2-butanol revealed that the differential activation entropy, Delta(R-S)Delta(S)(divided bydivided by)., was as significant as the differential activation enthalpy, Delta(R-S)DeltaH(divided bydivided by), to the enantiomeric ratio, E. 1-Bromo-2-butanol, with isosteric substituents, displayed the largest Delta(R-S)DeltaS(divided bydivided by) 3-Hexanol displayed, contrary to other sec-alcohols, a positive Delta(R-S)DeltaS(divided bydivided by). In other words, for 3-hexanol the preferred R-enantiomer is not only favored by enthalpy but also by entropy. Molecular dynamics (MID) simulations and systematic search calculations of the substrate accessible volume within the active site revealed that the (R)-3-hexanol transition state (TS) accessed a larger volume within the active site than the (S)-3-hexanol TS. This correlates well with the hi-her TS entropy of (R)-3-hexanol. In addition, this enantiomer did also yield a higher number of allowed conformations, N, from the systematic search routines, than did the S-enantiomer. The substrate accessible volume was greater for the enantiomer preferred by entropy also for 2-butanol. For 3,3-dimethyl-2-butanol, however, neither MD-simulations nor systematic search calculations yielded substrate accessible volumes that correlate to TS entropy. Ambiguous results were achieved for 3-methyl-2-butanol.

  • 15.
    Ottosson, Jenny
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Fransson, Linda
    KTH, Tidigare Institutioner                               , Bioteknologi.
    King, Jerry W.
    National Center for Agricultural Utilization Research, ARS/USDA.
    Hult, Karl
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Size as a parameter for solvent effects on Candida antarctica lipase B enantioselectivity2002Inngår i: Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology, ISSN 0167-4838, E-ISSN 1879-2588, Vol. 1594, nr 2, s. 325-334Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Changes in solvent type were shown to yield significant improvement of enzyme enantioselectivity. The resolution of 3-methyl-2-butanol catalyzed by Candida antarctica lipase B, CALB, was studied in eight liquid organic solvents and supercritical carbon dioxide, SCCO2. Studies of the temperature dependence of the enantiomeric ratio allowed determination of the enthalpic (Delta(R-S)Delta H-double dagger) as well as the entropic (Delta(R-S)Delta S-double dagger) contribution to the overall enantioselectivity (Delta(R-S)Delta G(double dagger) = -RTlnE). A correlation of the enantiomeric ratio, E. to the van der Waals volume of the solvent molecules was observed and suggested as one of the parameters that govern solvent effects on enzyme catalysis. An enthalpy-entropy compensation relationship was indicated between the studied liquid solvents. The enzymatic mechanism must be of a somewhat different nature in SCCO2, as this reaction in this medium did not follow the enthalpy-entropy compensation relation.

  • 16.
    Ottosson, Jenny
    et al.
    KTH, Tidigare Institutioner, Bioteknologi.
    Hult, Karl
    KTH, Tidigare Institutioner, Bioteknologi.
    Influence of acyl chain length on the enantioselectivity of Candida antarctica lipase B and its thermodynamic components in kinetic resolution of sec-alcohols2001Inngår i: Journal of Molecular Catalysis B: Enzymatic, ISSN 1381-1177, E-ISSN 1873-3158, Vol. 11, nr 4-6, s. 1025-1028Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The enantioselectivity, E, of Candida antarctica lipase B (CALB) was found to be strongly influenced by the chain length of the achiral acyl donor employed in the transesterification of 3-methyl-2-butanol. Of the four studied acyl donors, the longest, vinyl octanoate, afforded the highest enantioselectivity. This was true over the temperature range studied, 6-70 degreesC. Measurements of the temperature dependence of E allows for separation of the enthalpic and entropic components of enantioselectivity. Changes in E with chain length were mainly caused by changes in the entropic component except for the reaction with vinyl propionate, which differed from the others also in the enthalpic component. Optimisation of acyl donor adds one more possibility to improve the yield of enantiopurity in the production of optically active compounds apart from optimisation of solvent, temperature, water activity, and choice of enzyme.

  • 17.
    Ottosson, Jenny
    et al.
    KTH, Tidigare Institutioner, Bioteknologi.
    Rotticci-Mulder, C
    Rotticci, D
    Hult, Karl
    KTH, Tidigare Institutioner, Bioteknologi.
    Rational design of enantio selective enzymes requires considerations of entropy2001Inngår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 10, nr 9, s. 1769-1774Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Entropy was shown to play an equally important role as enthalpy for how enantioselectivity changes when redesigning an enzyme. By studying the temperature dependence of the enantiomeric ratio E of an enantioselective enzyme, its differential activation enthalpy (Delta (R-S)DeltaH(double dagger)) and entropy (Delta (R-S)DeltaS(double dagger)) components can be determined. This was done for the resolution of 3-methyl-2-butanol catalyzed by Candida antarctica lipase B and five variants with one or two point mutations. Delta (R-S)DeltaS(double dagger) was in all cases equally significant as Delta (R-S)DeltaH(double dagger) to E. One variant, T103G, displayed an increase in E, the others a decrease. The altered enantioselectivities of the variants were all related to simultaneous changes in Delta (R-S)DeltaH(double dagger) and Delta (R-S)DeltaS(double dagger). Although the changes in Delta (R-S)DeltaH(double dagger) and Delta (R-S)DeltaS(double dagger) were of a compensatory nature the compensation was not perfect, thereby allowing modifications of E. Both the W104H and the T103G variants displayed larger Delta (R-S)DeltaH(double dagger). than wild type but exhibited a decrease or increase, respectively, in E due to their different relative increase in Delta (R-S)DeltaS(double dagger).

  • 18.
    Ottosson, Jenny
    et al.
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Steen, Johanna
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Stenvall, Maria
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Tegel, Hanna
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Uhlén, Mathias
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Hober, Sophia
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    High throughput protein expression and purification for antibody proteomics2004Inngår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 3, nr 10, s. S169-S169Artikkel i tidsskrift (Annet vitenskapelig)
  • 19. Overbeeke, A
    et al.
    Ottosson, Jenny
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Hult, Karl
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Jongejan, A
    Duine, A
    The temperature dependence of enzymatic kinetic resolutions reveals the relative contribution of enthalpy and entropy to enzymatic enantioselectivity1999Inngår i: Biocatalysis and Biotransformation, ISSN 1024-2422, E-ISSN 1029-2446, Vol. 17, nr 1, s. 61-79Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The temperature dependence of the enantioselectivity of several lipase-calalyzed hydrolysis and acylation reactions of racemic esters and alcohols has been determined. From the results we estimated the difference in activation enthalpy (Delta Delta H-# and activation entropy (Delta Delta S-#) for the two enantiomers in the enantioselective reaction step. Contrary to earlier suggestions, we found that the enthalpic and entropic contributions to the enantioselectivity are of similar magnitude. A plot of Delta Delta H-# versus Delta Delta S-#-values of data available in the literature for various enzyme-substrate combinations revealed a tempting correlation between the enthalpic and entropic contributions. This observation would imply enthalpy-entropy compensation to be a general feature of enantioselective enzymatic catalysis. On closer inspection of the data set it was realized that this trend must be considered fortuitous. It originates from the non-random collection of those enzyme-substrate combinations for which the numerical value of the enantiomeric ratio can be measured with a suitable degree of accuracy at ambient temperatures. Indications for the occurrence of genuine enthalpy-entropy compensation, however, have been observed for series of homologous substrates and changes of solvent composition.

  • 20. Ponten, Fredrik
    et al.
    Gry, Marcus
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Fagerberg, Linn
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Lundberg, Emma
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Asplund, Anna
    Berglund, Lisa
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Oksvold, Per
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Björling, Erik
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Kampf, Caroline
    Navani, Sanjay
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ottosson, Jenny
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Persson, Anja
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Wester, Kenneth
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    A global view of protein expression in human cells, tissues, and organs2009Inngår i: Molecular Systems Biology, ISSN 1744-4292, E-ISSN 1744-4292, Vol. 5Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Defining the protein profiles of tissues and organs is critical to understanding the unique characteristics of the various cell types in the human body. In this study, we report on an anatomically comprehensive analysis of 4842 protein profiles in 48 human tissues and 45 human cell lines. A detailed analysis of over 2 million manually annotated, high-resolution, immunohistochemistry- based images showed a high fraction (>65%) of expressed proteins in most cells and tissues, with very few proteins (<2%) detected in any single cell type. Similarly, confocal microscopy in three human cell lines detected expression of more than 70% of the analyzed proteins. Despite this ubiquitous expression, hierarchical clustering analysis, based on global protein expression patterns, shows that the analyzed cells can be still subdivided into groups according to the current concepts of histology and cellular differentiation. This study suggests that tissue specificity is achieved by precise regulation of protein levels in space and time, and that different tissues in the body acquire their unique characteristics by controlling not which proteins are expressed but how much of each is produced. Molecular Systems Biology 5: 337; published online 22 December 2009; doi:10.1038/msb.2009.93

  • 21.
    Pontén, Fredrik
    et al.
    Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University.
    Gry, Marcus
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Björling, Erik
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Berglund, Lisa
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Al-Khalili Szigyarto, Cristina
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Lundberg, Emma
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Andersson-Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Asplund, Anna
    Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Kampf, Caroline
    Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University.
    Nilsson, Kenneth
    Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University.
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ottosson, Jenny
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Persson, Anja
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Wester, Kenneth
    Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ubiquitous protein expression in human cells, tissues and organsManuskript (Annet vitenskapelig)
  • 22.
    Rotticci, Didier
    et al.
    KTH, Tidigare Institutioner, Kemi.
    Ottosson, Jenny
    KTH, Tidigare Institutioner, Bioteknologi.
    Norin, Torbjörn
    KTH, Tidigare Institutioner, Kemi.
    Hult, Karl
    KTH, Tidigare Institutioner, Kemi.
    Candida Antarctica lipase B: A tool for the preparation of optically active alcohols2001Inngår i: Methods in Biotechnology 15: Enzymes in Nonaqueous Solvents, Humana Press, 2001, Vol. 15, s. 261-276Kapittel i bok, del av antologi (Fagfellevurdert)
  • 23.
    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.

  • 24.
    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.

  • 25.
    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.

  • 26.
    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.

  • 27.
    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.

  • 28.
    Sundqvist, Gustav
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Stenvall, Maria
    KTH, Skolan för bioteknologi (BIO).
    Berglund, Helena
    Ottosson, Jenny
    KTH, Skolan för bioteknologi (BIO).
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    A general, robust method for the quality control of intact proteins using LC–ESI-MS2007Inngår i: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 852, nr 1-2, s. 188-194Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A simple and robust method for the routine quality control of intact proteins based on liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI-MS) is presented. A wide range of prokaryotic and eukaryotic proteins expressed recombinantly in Escherichia coli or Pichia pastoris has been analyzed with medium- to high-throughput with on-line desalting from multi-well sample plates. Particular advantages of the method include fast chromatography and short cycle times, the use of inexpensive trapping/desalting columns, low sample carryover, and the ability to analyze proteins with masses ranging from 5 to 100 kDa with greater than 50 ppm accuracy. Moreover, the method can be readily coupled with optimized chemical reduction and alkylation steps to facilitate the analysis of denatured or incorrectly folded proteins (e.g., recombinant proteins sequestered in E. coli inclusion bodies) bearing cysteine residues, which otherwise form intractable multimers and non-specific adducts by disulfide bond formation.

  • 29.
    Tegel, Hanna
    et al.
    KTH.
    Hedhammar, My
    KTH.
    Uhlén, Mathias
    KTH.
    Ottosson, J.
    KTH.
    Hober, Sophia
    KTH.
    Novel flow cytometry-based method for analysis of protein production in Escherichia coli2005Inngår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 4, nr 8, s. S66-S66Artikkel i tidsskrift (Annet vitenskapelig)
  • 30.
    Tegel, Hanna
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Hedhammar, My
    KTH, Skolan för bioteknologi (BIO).
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO).
    Ottosson, Jenny
    KTH, Skolan för bioteknologi (BIO).
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO).
    Flow cytometry-based analysis of promoter effects on solubility of recombinantly expressed proteins2007Inngår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 131, nr 2, s. S9-S9Artikkel i tidsskrift (Annet vitenskapelig)
  • 31.
    Tegel, Hanna
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Malm, Katarina
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Halldin, Anneli
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Älgenäs, Cajsa
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Ottosson Takanen, Jenny
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    In-depth study of the positive effects of Escherichia coli Rosetta(DE3) on recombinant protein productionManuskript (preprint) (Annet vitenskapelig)
  • 32.
    Tegel, Hanna
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ottosson, Jenny
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Enhancing the protein production levels in Escherichia coli with a strong promoter2011Inngår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 278, nr 5, s. 729-739Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In biotechnology, the use of Escherichia coli for recombinant protein production has a long tradition, although the optimal production conditions for certain proteins are still not evident. The most favorable conditions for protein production vary with the gene product. Temperature and induction conditions represent parameters that affect total protein production, as well as the amount of soluble protein. Furthermore, the choice of promoter and bacterial strain will have large effects on the production of the target protein. In the present study, the effects of three different promoters (T7, trc and lacUV5) on E. coli production of target proteins with different characteristics are presented. The total amount of target protein as well as the amount of soluble protein were analyzed, demonstrating the benefits of using a strong promoter such as T7. To understand the underlying causes, transcription levels have been correlated with the total amount of target protein and protein solubility in vitro has been correlated with the amount of soluble protein that is produced. In addition, the effects of two different E. coli strains, BL21(DE3) and Rosetta(DE3), on the expression pattern were analyzed. It is concluded that the regulation of protein production is a combination of the transcription and translation efficiencies. Other important parameters include the nucleotide-sequence itself and the solubility of the target protein.

  • 33.
    Tegel, Hanna
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Steen, Johanna
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Konrad, Anna
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Nikidin, Hero
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Pettersson, Katarina
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Stenvall, Maria
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Tourle, Samuel
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Wrethagen, Ulla
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Xu, Lan Lan
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Yderland, Louise
    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 production--lessons from scaling up from 10 to 288 recombinant proteins per week2009Inngår i: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 4, nr 1, s. 51-57Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The demand for high-throughput recombinant protein production has markedly increased with the increased activity in the field of proteomics. Within the Human Protein Atlas project recombinantly produced human protein fragments are used for antibody production. Here we describe how the protein expression and purification protocol has been optimized in the project to allow for han- dling of nearly 300 different proteins per week. The number of manual handling steps has been significantly reduced (from 18 to 9) and the protein purification has been completely automated.

  • 34.
    Tegel, Hanna
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Tourle, Samuel
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ottosson, Jenny
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Persson, Anja
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Increased levels of recombinant human proteins with the Escherichia coli strain Rosetta(DE3)2010Inngår i: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 69, nr 2, s. 159-167Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The effect of two Escherichia coli expression strains on the production of recombinant human protein fragments was evaluated. High-throughput protein production projects, such as the Swedish Human Protein Atlas project, are dependent on high protein yield and purity. By changing strain from E. coli BL21(DE3) to E. coli Rosetta(DE3) the overall success rate of the protein production has increased dramatically. The Rosetta(DE3) strain compensates for a number of rare codons. Here, we describe how the protein expression of human gene fragments in E. coli strains BL21(DE3) and Rosetta(DE3) was evaluated in two stages. Initially a test set of 68 recombinant proteins that previously had been expressed in BL21(DE3) was retransformed and expressed in Rosetta(DE3). The test set generated very positive results with an improved expression yield and a significantly better purity of the protein product which prompted us to implement the Rosetta(DE3) strain in the high-throughput protein production. Except for analysis of protein yield and purity the sequences were also analyzed regarding number of rare codons and rare codon clusters. The content of rare codons showed to have a significant effect on the protein purity. Based on the results of this study the atlas project permanently changed expression strain to Rosetta(DE3).

  • 35.
    Tegel, Hanna
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Yderland, Louise
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Boström, Tove
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Eriksson, Cecilia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ukkonen, Kaisa
    Vasala, Antti
    Neubauer, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ottosson, Jenny
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Parallel production and verification of protein products using a novel high-throughput screening method2011Inngår i: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 6, nr 8, s. 1018-1025Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Protein production and analysis in a parallel fashion is today applied in laboratories worldwide and there is a great need to improve the techniques and systems used for this purpose. In order to save time and money, a fast and reliable screening method for analysis of protein production and also verification of the protein product is desired. Here, a micro-scale protocol for the parallel production and screening of 96 proteins in plate format is described. Protein capture was achieved using immobilized metal affinity chromatography and the product was verified using matrix-assisted laser desorption ionization time-of-flight MS. In order to obtain sufficiently high cell densities and product yield in the small-volume cultivations, the EnBase (R) cultivation technology was applied, which enables cultivation in as small volumes as 150 mu L. Here, the efficiency of the method is demonstrated by producing 96 human, recombinant proteins, both in micro-scale and using a standard full-scale protocol and comparing the results in regard to both protein identity and sample purity. The results obtained are highly comparable to those acquired through employing standard full-scale purification protocols, thus validating this method as a successful initial screening step before protein production at a larger scale.

  • 36.
    Uhlén, Mathias
    et al.
    KTH.
    Al-Khalili Szigyarto, Cristina
    KTH.
    Ottosson, H. Jenny
    KTH.
    Nilsson, E. Peter
    KTH.
    Andersson, K. Ann-Catrin
    Uppsala Univ, Stockholm, Sweden..
    Caroline, K.
    Uppsala Univ, Stockholm, Sweden..
    Fredrik, P.
    Uppsala Univ, Stockholm, Sweden..
    A human protein atlas for normal and disease tissue2005Inngår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 4, nr 8, s. S15-S15Artikkel i tidsskrift (Annet vitenskapelig)
  • 37.
    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.

  • 38.
    Uhlén, Mathias
    et al.
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Fagerberg, Linn
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Hallström, Björn M
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Lindskog, Cecilia
    Oksvold, Per
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Mardinoglu, Adil
    Sivertsson, Åsa
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Kampf, Caroline
    Sjöstedt, Evelina
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Asplund, Anna
    Olsson, IngMarie
    Edlund, Karolina
    Lundberg, Emma
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Navani, Sanjay
    Szigyarto, Cristina Al-Khalili
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Odeberg, Jacob
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Djureinovic, Dijana
    Takanen, Jenny Ottosson
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Alm, Tove
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Edqvist, Per-Henrik
    Berling, Holger
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Tegel, Hanna
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Mulder, Jan
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Nilsson, Peter
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Hamsten, Marica
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    von Feilitzen, Kalle
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Forsberg, Mattias
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Persson, Lukas
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Johansson, Fredric
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Zwahlen, Martin
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    von Heijne, Gunnar
    Nielsen, Jens
    Pontén, Fredrik
    Tissue-based map of the human proteome2015Inngår i: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 347, nr 6220, s. 1260419-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Resolving the molecular details of proteome variation in the different tissues and organs of the human body will greatly increase our knowledge of human biology and disease. Here, we present a map of the human tissue proteome based on an integrated omics approach that involves quantitative transcriptomics at the tissue and organ level, combined with tissue microarray-based immunohistochemistry, to achieve spatial localization of proteins down to the single-cell level. Our tissue-based analysis detected more than 90% of the putative protein-coding genes. We used this approach to explore the human secretome, the membrane proteome, the druggable proteome, the cancer proteome, and the metabolic functions in 32 different tissues and organs. All the data are integrated in an interactive Web-based database that allows exploration of individual proteins, as well as navigation of global expression patterns, in all major tissues and organs in the human body.

  • 39.
    Älgenäs, Cajsa
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Agaton, Charlotta
    Fagerberg, Linn
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Asplund, Anna
    Björling, Lisa
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Björling, Erik
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Kampf, Caroline
    Lundberg, Emma
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Persson, Anja
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Wester, Kenneth
    Pontén, Fredrik
    Wernerus, Henrik
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Ottosson Takanen, Jenny
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Antibody performance in western blot applications is context- dependent2014Inngår i: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 9, nr 3, s. 435-445Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

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

1 - 39 of 39
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