Change search
Refine search result
123 1 - 50 of 131
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Rows per page
  • 5
  • 10
  • 20
  • 50
  • 100
  • 250
Sort
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
Select
The maximal number of hits you can export is 250. When you want to export more records please use the 'Create feeds' function.
  • 1. Adler, Belinda
    et al.
    Boström, Tove
    KTH, School of Biotechnology (BIO), Proteomics.
    Ekström, Simon
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Laurell, Thomas
    Miniaturized and Automated High-Throughput Verification of Proteins in the ISET Platform with MALDI MS2012In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 84, no 20, p. 8663-8669Article in journal (Refereed)
    Abstract [en]

    A major bottleneck in high-throughput protein production is the validation step, which is why parallel and automated sample processing methods are highly desirable. Also, a miniaturized sample preparation format is preferred, as the reduction of reagent volumes significantly decreases the analysis cost per sample. We have developed an automated and miniaturized protein sequence verification protocol for recombinant proteins utilizing peptide mass fingerprinting and MS/MS analysis. The integrated selective enrichment target (ISET) platform, previously developed in our group, with its dual functionality, being both a sample preparation platform and a MALDI target plate, is employed. All steps including immobilized metal ion affinity chromatography of protein on cobalt-loaded beads, tryptic digestion, and MALDI MS analysis are performed in an array format, without any sample transfers, on the same ISET chip. The automated configuration reduced the sample preparation time significantly. Starting with crude lysate, a full plate of 48 purified, digested samples prepared for MALDI-MS can be generated in 4 h, with only 30 min of operator involvement. This paper demonstrates the utility of the method by parallel analysis of 45 His-tagged human recombinant proteins.

  • 2.
    Agaton, Charlotta
    et al.
    KTH, Superseded Departments, Biotechnology.
    Falk, Ronny
    KTH, Superseded Departments, Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Hober, Sophia
    KTH, Superseded Departments, Biotechnology.
    Selective enrichment of monospecific polyclonal antibodies for antibody-based proteomics efforts2004In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1043, p. 33-40Article in journal (Refereed)
    Abstract [en]

    A high stringency protocol, suitable for systematic purification of polyclonal antibodies, is described. The procedure is designed to allow the generation of target protein-specific antibodies suitable for functional annotation of proteins. Antibodies were generated by immunization with recombinantly produced affinity-tagged target proteins. To obtain stringent recovery of the antibodies, a two-step affinity chromatography principle was devised to first deplete the affinity tag-specific antibodies followed by a second step for affinity capture of the target protein-specific antibodies. An analytical dot-blot array system was developed to analyze the cross-reactivity of the affinity-purified antibodies. The results suggest that the protocol can be used in a highly parallel and automated manner to generate mono-specific polyclonal antibodies for large-scale, antibody-based proteomics efforts, i.e. affinity proteomics.

  • 3.
    Agaton, Charlotta
    et al.
    KTH, Superseded Departments, Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Hober, Sophia
    KTH, Superseded Departments, Biotechnology.
    Genome-based proteomics2004In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 25, no 9, p. 1280-1288Article in journal (Refereed)
    Abstract [en]

    Protein-protein interactions play crucial roles in various biological pathways and functions. Therefore, the characterization of protein levels and also the network of interactions within an organism would contribute considerably to the understanding of life. The availability of the human genome sequence has created a range of new possibilities for biomedical research. A crucial challenge is to utilize the genetic information for better understanding of protein distribution and function in normal as well as in pathological biological processes. In this review, we have focused on different platforms used for systematic genome-based proteome analyses. These technologies are in many ways complementary and should be seen as various ways to elucidate different functions of the proteome.

  • 4.
    Ahmadian, Afshin
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ehn, M.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Pyrosequencing: History, biochemistry and future2006In: Clinica Chimica Acta, ISSN 0009-8981, E-ISSN 1873-3492, Vol. 363, no 02-jan, p. 83-94Article, review/survey (Refereed)
    Abstract [en]

    Background: Pyrosequencing is a DNA sequencing technology based on the sequencing-by-synthesis principle. Methods: The technique is built on a 4-enzyme real-time monitoring of DNA synthesis by bioluminescence using a cascade that upon nucleotide incorporation ends in a detectable light signal (bioluminescence). The detection system is based on the pyrophosphate released when a nucleotide is introduced in the DNA-strand. Thereby, the signal can be quantitatively connected to the number of bases added. Currently, the technique is limited to analysis of short DNA sequences exemplified by single-nucleotide polymorphism analysis and genotyping. Mutation detection and single-nucleotide polymorphisin genotyping require screening of large samples of materials and therefore the importance of high-throughput DNA analysis techniques is significant. In order to expand the field for pyrosequencing, the read length needs to be improved. Conclusions: Th pyrosequencing system is based on an enzymatic system. There are different current and future applications of this technique.

  • 5.
    Alm, Tove
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Nilvebrant, Johan
    KTH, School of Biotechnology (BIO), Proteomics.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Proteomics.
    Engineering bispecificityinto a single albumin-binding domain aimed for drug-targeting and extended in vivo half-life extension.Manuscript (preprint) (Other academic)
  • 6.
    Alm, Tove
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Steen, Johanna
    KTH, School of Biotechnology (BIO), Proteomics.
    Ottosson, Jenny
    KTH, School of Biotechnology (BIO), Proteomics.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    High-throughput protein purification under denaturating conditions by the use of cation exchange chromatography2007In: Biotechnology Journal, ISSN 1860-6768, Vol. 2, p. 709-716Article in journal (Refereed)
    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.

  • 7.
    Alm, Tove
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Yderland, Louise
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Nilvebrant, Johan
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Halldin, Anneli
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    A small bispecific protein selected for orthogonal affinity purification2010In: BIOTECHNOL J, ISSN 1860-6768, Vol. 5, no 6, p. 605-617Article in journal (Refereed)
    Abstract [en]

    A novel protein domain with dual affinity has been created by randomization and selection. The small alkali-stabilized albumin-binding domain (ABD(star)), used as scaffold to construct the library, has affinity to human serum albumin (HSA) and is constituted of 46 amino acids of which 11 were randomized. To achieve a dual binder, the binding site of the inherent HSA affinity was untouched and the randomization was made on the opposite side of the molecule. Despite its small size and randomization of almost a quarter of its amino acids, a bifunctional molecule, ABDz1, with ability to bind to both HSA and the Z(2) domain/protein A was successfully selected using phage display. Moreover, the newly selected variant showed improved affinity for HSA compared to the parental molecule. This novel protein domain has been characterized regarding secondary structure and affinity to the two different ligands. The possibility for affinity purification on two different matrices has been investigated using the two ligands, the HSA matrix and the protein A-based, MabSelect SuRe matrix, and the new protein domain was purified to homogeneity. Furthermore, gene fusions between the new domain and three different target proteins with different characteristics were made. To take advantage of both affinities, a purification strategy referred to as orthogonal affinity purification using two different matrices was created. Successful purification of all three versions was efficiently carried out using this strategy.

  • 8. Andersson, K
    et al.
    Gülich, S
    KTH, Superseded Departments (pre-2005), Biochemistry and Biotechnology.
    Hämäläinen, M
    Nygren, P A
    KTH, Superseded Departments (pre-2005), Biochemistry and Biotechnology.
    Hober, Sophia
    KTH, Superseded Departments (pre-2005), Biochemistry and Biotechnology.
    Malmqvist, M
    Kinetic characterization of the interaction of the Z-fragment of protein A with mouse-IgG3 in a volume in chemical space.1999In: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 37, no 3Article in journal (Refereed)
    Abstract [en]

    The kinetic rate parameters for the interaction between a single domain analogue of staphylococcal protein A (Z) and a mouse-IgG3 monoclonal antibody (MAb) were measured in Hepes buffer with different chemical additives. Five buffer ingredients (pH, NaCl, DMSO, EDTA, and KSCN) were varied simultaneously in 16 experiments following a statistical experimental plan. The 16 buffers thus spanned a volume in chemical space. A mathematical model, using data from the buffer composition, was developed and used to predict apparent kinetic parameters in five new buffers within the spanned volume. Association and dissociation parameters were measured in the new buffers, and these agreed with the predicted values, indicating that the model was valid within the spanned volume. The pattern of variation of the kinetic parameters in relation to buffer composition was different for association and dissociation, such that pH influenced both association and dissociation and NaCl influenced only dissociation. This indicated that the recognition mechanism (association) and the stability of the formed complex (dissociation) involve different binding forces, which can be further investigated by kinetic studies in systematically varied buffers.

  • 9. Andersson, Sandra
    et al.
    Konrad, Anna
    KTH, School of Biotechnology (BIO), Protein Technology.
    Ashok, Nikhil
    Pontén, Fredrik
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Protein Technology.
    Asplund, Anna
    Antibodies Biotinylated Using a Synthetic Z-domain from Protein A Provide Stringent In Situ Protein Detection2013In: Journal of Histochemistry and Cytochemistry, ISSN 0022-1554, E-ISSN 1551-5044, Vol. 61, no 11, p. 773-784Article in journal (Refereed)
    Abstract [en]

    Antibody-based protein profiling on a global scale using immunohistochemistry constitutes an emerging strategy for mapping of the human proteome, which is crucial for an increased understanding of biological processes in the cell. Immunohistochemistry is often performed indirectly using secondary antibodies for detection, with the benefit of signal amplification. Direct immunohistochemistry instead brings the advantage of multiplexing; however, it requires labeling of the primary antibody. Many antibody-labeling kits do not specifically target IgG and may therefore cause labeling of stabilizing proteins present in the antibody solution. A new conjugation method has been developed that utilizes a modified Z-domain of protein A (ZBPA) to specifically target the Fc part of antibodies. The aim of the present study was to compare the ZBPA conjugation method and a commercially available labeling kit, Lightning-Link, for in situ protein detection. Fourteen antibodies were biotinylated with each method and stained using immunohistochemistry. For all antibodies tested, ZBPA biotinylation resulted in distinct immunoreactivity without off-target staining, regardless of the presence of stabilizing proteins in the buffer, whereas the majority of the Lightning-Link biotinylated antibodies displayed a characteristic pattern of nonspecific staining. We conclude that biotinylated ZBPA domain provides a stringent method for antibody biotinylation, advantageous for in situ protein detection in tissues.

  • 10. Andersson, Sandra
    et al.
    Konrad, Anna
    KTH, School of Biotechnology (BIO), Proteomics.
    Ashok, Nikhil
    Pontén, Fredrik
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Asplund, Anna
    Antibodies biotinylated using a synthetic Z-domain provide stringent in situ proteindetectionManuscript (preprint) (Other academic)
  • 11.
    Berglund, Lisa
    et al.
    KTH, School of Biotechnology (BIO).
    Björling, Erik
    KTH, School of Biotechnology (BIO).
    Gry, Marcus
    KTH, School of Biotechnology (BIO).
    Asplund, Anna
    Uppsala Univ, Rudbeck laboratory.
    Al-Khalili Szigyarto, Cristina
    KTH, School of Biotechnology (BIO).
    Persson, Anja
    KTH, School of Biotechnology (BIO).
    Ottoson, Jenny
    KTH, School of Biotechnology (BIO).
    Wernérus, Henrik
    KTH, School of Biotechnology (BIO).
    Nilsson, Peter
    KTH, School of Biotechnology (BIO).
    Sivertsson, Åsa
    KTH, School of Biotechnology (BIO).
    Wester, Kenneth
    Uppsala Univ, Rudbeck laboratory.
    Kampf, Caroline
    Uppsala Univ, Rudbeck laboratory.
    Hober, Sophia
    KTH, School of Biotechnology (BIO).
    Pontén, Fredrik
    Uppsala Univ, Rudbeck laboratory.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO).
    Generation of validated antibodies towards the human proteomeArticle in journal (Other academic)
    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.

  • 12.
    Berglund, Lisa
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Björling, Erik
    KTH, School of Biotechnology (BIO), Proteomics.
    Oksvold, Per
    KTH, School of Biotechnology (BIO), Proteomics.
    Fagerberg, Linn
    KTH, School of Biotechnology (BIO), Proteomics.
    Al-Khalili Szigyarto, Cristina
    KTH, School of Biotechnology (BIO), Proteomics.
    Persson, Anja
    KTH, School of Biotechnology (BIO), Proteomics.
    Ottosson, Jenny
    KTH, School of Biotechnology (BIO), Proteomics.
    Wernérus, Henrik
    KTH, School of Biotechnology (BIO), Proteomics.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics.
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Proteomics.
    Sivertsson, Åsa
    KTH, School of Biotechnology (BIO), Proteomics.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    et al.,
    A genecentric human protein atlas for expression profiles based on antibodies2008In: Molecular & Cellular Proteomics, ISSN 1535-9476, Vol. 7, no 10, p. 2019-2027Article in journal (Refereed)
    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.

  • 13.
    Björling, Erik
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Lindskog, Cecilia
    Uppsala Univ, Rudbeck Lab.
    Oksvold, Per
    KTH, School of Biotechnology (BIO), Proteomics.
    Linné, Jerker
    Uppsala Univ, Rudbeck Lab.
    Kampf, Caroline
    Uppsala Univ, Rudbeck Lab.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO).
    Pontén, Fredrik
    Uppsala Univ, Rudbeck Lab.
    A web-based tool for in silico biomarker discovery based on tissue-specific protein profiles in normal and cancer tissues2008In: Molecular & Cellular Proteomics, ISSN 1535-9476, Vol. 7, no 5, p. 825-844Article in journal (Refereed)
    Abstract [en]

    Here we report the development of a publicly available Web-based analysis tool for exploring proteins expressed in a tissue- or cancer-specific manner. The search queries are based on the human tissue profiles in normal and cancer cells in the Human Protein Atlas portal and rely on the individual annotation performed by pathologists of images representing immunohistochemically stained tissue sections. Approximately 1.8 million images representing more than 3000 antibodies directed toward human proteins were used in the study. The search tool allows for the systematic exploration of the protein atlas to discover potential protein biomarkers. Such biomarkers include tissue-specific markers, cell type-specific markers, tumor type-specific markers, markers of malignancy, and prognostic or predictive markers of cancers. Here we show examples of database queries to generate sets of candidate biomarker proteins for several of these different categories. Expression profiles of candidate proteins can then subsequently be validated by examination of the underlying high resolution images. The present study shows examples of search strategies revealing several potential protein biomarkers, including proteins specifically expressed in normal cells and in cancer cells from specified tumor types. The lists of candidate proteins can be used as a starting point for further validation in larger patient cohorts using both immunological approaches and technologies utilizing more classical proteomics tools.

  • 14.
    Boström, Maria
    et al.
    KTH, School of Biotechnology (BIO).
    Markland, Katrin
    KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Sandén, Anna Maria
    KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Hedhammar, My
    KTH, School of Biotechnology (BIO), Proteomics. KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics. KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Effect of substrate feed rate on recombinant protein secretion, degradation and invlusion body formation in Escherichia coli2005In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 68, no 1, p. 82-90Article in journal (Refereed)
    Abstract [en]

    The effect of changes in substrate feed rate during fedbatch cultivation was investigated with respect to soluble protein formation and transport of product to the periplasm in Escherichia coli. Production was transcribed from the P-malK promoter; and the cytoplasmic part of the production was compared with production from the P-lacUV5 promoter. The fusion protein product, Zb-MalE, was at all times accumulated in the soluble protein fraction except during high-feed-rate production in the cytoplasm. This was due to a substantial degree of proteolysis in all production systems, as shown by the degradation pattern of the product. The product was also further subjected to inclusion body fori-nation. Production in the periplasm resulted in accumulation of the full-length protein; and this production system led to a cellular physiology where the stringent response could be avoided. Furthermore, the secretion could be used to abort the diauxic growth phase resulting from use of the P-malK promoter. At high feed rate, the accumulation of acetic acid, due to overflow metabolism, could furthermore be completely avoided.

  • 15.
    Boström, Tove
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Danielsson, Frida
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Johansson, Henrik J.
    Karlinska Institute, Cancer Proteomics Mass Spectrometry, Dep. of Oncology-Pathology.
    Tegel, Hanna
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Lehtiö, Janne
    Karolinska Institute, Cancer Proteomics Mass Spectrometry, Dep. of Oncology-Pathology.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Protein Technology.
    Ottosson Takanen, Jenny
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Investigating the correlation of protein and mRNA levels in human cell lines using quantitative proteomics and transcriptomicsManuscript (preprint) (Other academic)
    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.

  • 16.
    Boström, Tove
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Johansson, Henrik J.
    Karolinska Institute.
    Lehtiö, Janne
    Karolinska Institute.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Protein Technology.
    Investigating the Applicability of Antibodies Generated within the Human Protein Atlas as Capture Agents in Immunoenrichment Coupled to Mass Spectrometry2014In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 13, no 10, p. 4424-4435Article in journal (Refereed)
    Abstract [en]

    For identification and characterization of proteins in complex samples, immunoenrichment coupled to mass spectrometry is a good alternative due to the sensitivity of the affinity enrichment and the specificity of mass spectrometry analysis. Antibodies are commonly used affinity agents; however, for high-throughput analysis, antibody availability is usually a bottleneck. Here we present a protocol for immunoenrichment coupled to mass spectrometry in a high-throughput setup, where all steps from bead coupling to mass spectrometry sample preparation are performed in parallel in a 96-well format. Antibodies generated within the Human Protein Atlas project were tested for applicability as capture agents. The antibodies were covalently attached to protein A beads, making it possible to reuse the coupled beads at least three times without destroying the antibody binding efficiency. Target proteins were captured from a U251 MG cell lysate, eluted, digested, and analyzed using mass spectrometry. Of 30 investigated antibodies, around 50% could successfully capture the corresponding native target protein, making the available library of more than 21 000 antibodies a valuable resource for immunoenrichment assays. Due to the diversity of different antibodies regarding affinity and specificity, analyzing antibodies in a high-throughput format is challenging. Even though protocol optimization for individual antibodies can be advantageous for future studies, our method enables a fast screening strategy to determine the usefulness of antibodies in immunoenrichment setups. In addition, we show that the specificity of the antibodies can be investigated by using label-free quantification.

  • 17.
    Boström, Tove
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Nilvebrant, Johan
    KTH, School of Biotechnology (BIO), Proteomics.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Purification systems based of bacterial surface proteins2012In: Protein Purification / [ed] Rizwan Ahmad, InTech, 2012, Vol. 1, p. 89-136Chapter in book (Refereed)
  • 18.
    Boström, Tove
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Ottosson Takanen, Jenny
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Protein Technology.
    Antibodies as means for selective mass spectrometry2015In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376XArticle in journal (Refereed)
    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.

  • 19. Colwill, Karen
    et al.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics.
    Sundberg, Mårten
    KTH, School of Biotechnology (BIO), Proteomics.
    Sjöberg, Ronald
    KTH, School of Biotechnology (BIO), Proteomics.
    Sivertsson, Åsa
    KTH, School of Biotechnology (BIO), Proteomics.
    Schwenk, Jochen M
    KTH, School of Biotechnology (BIO), Proteomics.
    Ottosson Takanen, Jenny
    KTH, School of Biotechnology (BIO), Proteomics.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Gräslund, Susanne
    et, al.
    A roadmap to generate renewable protein binders to the human proteome2011In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 8, no 7, p. 551-8Article in journal (Refereed)
    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.

  • 20.
    Edfors, Fredrik
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Boström, Tove
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Forsström, Björn
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Zeiler, Marlis
    Johansson, Henrik J.
    Karlinska Institute.
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Protein Technology.
    Lehtiö, Janne
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Mann, Matthias
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Immunoproteomics using polyclonal antibodies and stable isotope-labeled affinity-purified recombinant proteins2014In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 13, no 6, p. 1611-1624Article in journal (Refereed)
    Abstract [en]

    AThe combination of immuno-based methods and mass spectrometry detection has great potential in the field of quantitative proteomics. Here, we describe a new method (immuno-SILAC) for the absolute quantification of proteins in complex samples based on polyclonal antibodies and stable isotope-labeled recombinant protein fragments to allow affinity enrichment prior to mass spectrometry analysis and accurate quantification. We took advantage of the antibody resources publicly available from the Human Protein Atlas project covering more than 80% of all human protein-coding genes. Epitope mapping revealed that a majority of the polyclonal antibodies recognized multiple linear epitopes, and based on these results, a semi-automated method was developed for peptide enrichment using polyclonal antibodies immobilized on protein A-coated magnetic beads. A protocol based on the simultaneous multiplex capture of more than 40 protein targets showed that approximately half of the antibodies enriched at least one functional peptide detected in the subsequent mass spectrometry analysis. The approach was further developed to also generate quantitative data via the addition of heavy isotope-labeled recombinant protein fragment standards prior to trypsin digestion. Here, we show that we were able to use small amounts of antibodies (50 ng per target) in this manner for efficient multiplex analysis of quantitative levels of proteins in a human HeLa cell lysate. The results suggest that polyclonal antibodies generated via immunization of recombinant protein fragments could be used for the enrichment of target peptides to allow for rapid mass spectrometry analysis taking advantage of a substantial reduction in sample complexity. The possibility of building up a proteome-wide resource for immuno-SILAC assays based on publicly available antibody resources is discussed.

  • 21. Ehn, M.
    et al.
    Ahmadian, Afshin
    KTH, Superseded Departments, Biotechnology.
    Nilsson, Peter
    KTH, Superseded Departments, Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Hober, Sophia
    KTH, Superseded Departments, Biotechnology.
    Escherichia coli single-stranded DNA-binding a molecular tool for improved sequence protein quality in pyrosequencing2002In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 23, no 19, p. 3289-3299Article in journal (Refereed)
    Abstract [en]

    Pyrosequencing is a four-enzyme bioluminometric DNA sequencing technique based on a DNA sequencing by synthesis principle. Currently, the technique is limited to analysis of short DNA sequences exemplified by single-nucleotide polymorphism analysis. In order to expand the field for pyrosequencing, the read length needs to be improved and efforts have been made to purify reaction components as well as add single-stranded DNA-binding protein (SSB) to the pyrosequencing reaction. In this study, we have performed a systematic effort to analyze the effects of SSB by comparing the pyrosequencing result of 103 independent complementary DNA (cDNA) clones. More detailed information about the cause of low quality sequences on templates with different characteristics was achieved by thorough analysis of the pyrograms. Also, real-time biosensor analysis was performed on individual cDNA clones for investigation of primer annealing and SSB binding on these templates. Results from these studies indicate that templates with high performance in pyrosequencing without SSB possess efficient primer annealing and low SSB affinity. Alternative strategies to improve the performance in pyrosequencing by increasing the primer-annealing efficiency have also been evaluated.

  • 22. Ehn, M.
    et al.
    Nilsson, Peter
    KTH, Superseded Departments, Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Hober, Sophia
    KTH, Superseded Departments, Biotechnology.
    Overexpression, rapid isolation, and biochemical characterization of Escherichia coli single-stranded DNA-binding protein2001In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 22, no 1, p. 120-127Article in journal (Refereed)
    Abstract [en]

    Escherichia coli (E. coli) single-stranded binding protein (SSB) is a valuable protein for various biotechnical applications, such as PCR and DNA sequencing, Here we describe an efficient expression and purification scheme where the tendency of SSB to aggregate at low salt concentration and high protein concentration is avoided. The method contains fewer steps of purification and results in high protein yield, compared to previous published protocols. In our protocol, cells are harvested after cultivation overnight and SSB is isolated by ammonium sulfate precipitation followed by anion-exchange chromatography. The yield from a 2-liter fed-batch fermenter is 2 g protein, which is higher than all production methods for SSB earlier reported, Moreover, the two classical isolation steps combined in the purification scheme are robust, cost-efficient, and suitable for scaling up. The resulting SSB is pure and a correctly folded tetramer with an apparent binding to single-stranded DNA with a K-D of 10(-8) M, as determined by surface plasmon resonance.

  • 23.
    Ehn, Maria
    et al.
    KTH, Superseded Departments, Biotechnology.
    Nourizad, Nader
    KTH, Superseded Departments, Biotechnology.
    Bergstrom, Kristina
    KTH, Superseded Departments, Biotechnology.
    Ahmadian, Afshin
    KTH, Superseded Departments, Biotechnology.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Hober, Sophia
    KTH, Superseded Departments, Biotechnology.
    Toward pyrosequencing on surface-attached genetic material by use of DNA-binding luciferase fusion proteins2004In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 329, no 1, p. 11-20Article in journal (Refereed)
    Abstract [en]

    Mutation detection and single-nucleotide polymorphisin genotyping require screening of large samples of materials and therefore the importance of high-throughput DNA analysis techniques is significant. Pyrosequencing is a four-enzyme bioluminometric DNA sequencing technology based on the sequencing-by-synthesis principle. Currently, the technique is limited to simultaneous analysis of 96 or 384 samples. Earlier, attempts to increase the sample capacity were made using micromachined filter chamber arrays where parallel analyses of nanoliter samples could be monitored in real time. We have developed a strategy for specific immobilization of the light-producing enzyme luciferase to the DNA template within a reaction chamber. By this approach, luciferase is genetically fused to a DNA-binding protein (Klenow polymerase or Escherichia coli single-stranded DNA-binding (SSB) protein) and to a purification handle (Z(basic)). The proteins are produced in E. coli and purified using cation and anion exchange chromatography with removal of Z(basic). The produced proteins have been analyzed using an assay for complete primer extension of DNA templates immobilized on magnetic beads detected by pyrosequencing chemistry. Results from these experiments show that the proteins bind selectively to the immobilized DNA and that their enzymatic domains were active. Z(basic)-SSB-luciferase produced the highest signal in this assay and was further exploited as enzymatic reagent for DNA sequencing.

  • 24. Ek, S.
    et al.
    Andreasson, U.
    Hober, Sophia
    KTH, School of Biotechnology (BIO).
    Kampf, Caroline
    KTH, School of Biotechnology (BIO).
    Ponten, Fredrik K.
    KTH, School of Biotechnology (BIO).
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO).
    Merz, H.
    Borrebaeck, C. A. K.
    From gene expression analysis to tissue microarrays - A rational approach to identify therapeutic and diagnostic targets in lymphoid malignancies2006In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 5, no 6, p. 1072-1081Article in journal (Refereed)
    Abstract [en]

    Mantle cell lymphoma (MCL) is an aggressive lymphoid malignancy for which better treatment strategies are needed. To identify potential diagnostic and therapeutic targets, a signature consisting of MCL-associated genes was selected based on a comprehensive gene expression analysis of malignant and normal B cells. The corresponding protein epitope signature tags were identified and used to raise monospecific, polyclonal antibodies, which were subsequently analyzed on paraffin-embedded sections of malignant and normal tissue. In this study, we demonstrate that the initial selection strategy of MCL-associated genes successfully allows identification of protein antigens either uniquely expressed or overexpressed in MCL compared with normal lymphoid tissues. We propose that genome-based, affinity proteomics, using protein epitope signature tag-induced antibodies, is an efficient way to rapidly identify a number of disease-associated protein candidates of both previously known and unknown identities.

  • 25.
    Eriksson, Cecilia
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Agaton, Charlotta
    KTH, School of Biotechnology (BIO).
    Kånge, Rikard
    Sundberg, Marten
    KTH, School of Biotechnology (BIO).
    Nilsson, Peter
    KTH, School of Biotechnology (BIO).
    Ek, Bo
    Uhlen, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Gustafsson, Magnus
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Microfluidic analysis of antibody specificity in a compact disk format2006In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 5, no 7, p. 1568-1574Article in journal (Refereed)
    Abstract [en]

    A new and flexible technology for high throughput analysis of antibody specificity and affinity is presented. The method is based on microfluidics and takes advantage of compact disks (CDs) in which the centrifugal force moves fluids through microstructures containing immobilized metal affinity chromatography columns. Analyses are performed as a sandwich assay, where antigen is captured to the column via a genetically attached His(6)-tag. The antibodies to be analyzed are applied onto the columns. Thereafter, fluorescently labeled secondary antibodies recognize the bound primary antibodies, and detection is carried out by laser-induced fluorescence. The CDs contain 104 microstructures enabling analysis of antibodies against more than 100 different proteins using a single CD. Importantly, through the three- dimensional visualization of the binding patterns in a column it is possible to separate high affinity from low affinity binding. The method presented here is shown to be very sensitive, flexible and reproducible.

  • 26.
    Eriksson, Cecilia
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Pereira, R. A.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Towards an immunobased tool for the qualification of bookmarker candidates-compact disk enclosed nanocolumns with orientation-immobilized monospecific antibodies.Article in journal (Other academic)
  • 27.
    Eriksson, Cecilia
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Schwenk, Jochen M.
    KTH, School of Biotechnology (BIO), Proteomics.
    Sjöberg, A.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Affibody molecule mediated depletion of HSA and IgG performed in singlet or in a rapid high throughput format2009Article in journal (Other academic)
  • 28.
    Eriksson, Cecilia
    et al.
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Schwenk, Jochen M.
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Sjöberg, Anna
    KTH, School of Biotechnology (BIO).
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Affibody molecule-mediated depletion of HSA and IgG using different buffer compositions: a 15 min protocol for parallel processing of 1-48 samples2010In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 56, p. 49-57Article in journal (Refereed)
    Abstract [en]

    High-abundant plasma proteins pose a challenge in a large number of proteomics-based technologies. Depletion of these high-abundant proteins has proven to be a fruitful strategy to circumvent masking of lower-abundant proteins that could serve as valuable biomarker candidates. However, current strategies often do not meet the throughput requirements of large-scale proteomic studies. In the present paper, a flexible and parallelized method for the depletion of high-abundant proteins is described, allowing the removal of the two most abundant proteins from 48 blood-derived samples in less than 15 min using Affibody molecules as affinity ligands. A sample-processing platform like this should be suitable for a number of proteomics technologies; its flexibility in buffer composition allows for different types of downstream applications.

  • 29.
    Fagerberg, Linn
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Oksvold, Per
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Skogs, Marie
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Älgenäs, C.
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Pontén, F.
    Sivertsson, Åsa
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Odeberg, Jacob
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Klevebring, Daniel
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Kampf, C.
    Asplund, A.
    Sjöstedt, E.
    Al-Khalili Szigyarto, C.
    Edqvist, P. -H
    Olsson, I.
    Rydberg, U.
    Hudson, P.
    Ottosson Takanen, J.
    Berling, H.
    Björling, Lisa
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Tegel, Hanna
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Rockberg, J.
    Nilsson, Peter
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Navani, S.
    Jirström, K.
    Mulder, J.
    Schwenk, Jochen M.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Zwahlen, Martin
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hober, Sophia
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Forsberg, Mattias
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Von Feilitzen, Kalle
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Contribution of antibody-based protein profiling to the human chromosome-centric proteome project (C-HPP)2013In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 12, no 6, p. 2439-2448Article in journal (Refereed)
    Abstract [en]

    A gene-centric Human Proteome Project has been proposed to characterize the human protein-coding genes in a chromosome-centered manner to understand human biology and disease. Here, we report on the protein evidence for all genes predicted from the genome sequence based on manual annotation from literature (UniProt), antibody-based profiling in cells, tissues and organs and analysis of the transcript profiles using next generation sequencing in human cell lines of different origins. We estimate that there is good evidence for protein existence for 69% (n = 13985) of the human protein-coding genes, while 23% have only evidence on the RNA level and 7% still lack experimental evidence. Analysis of the expression patterns shows few tissue-specific proteins and approximately half of the genes expressed in all the analyzed cells. The status for each gene with regards to protein evidence is visualized in a chromosome-centric manner as part of a new version of the Human Protein Atlas (www.proteinatlas.org).

  • 30.
    Falk, Ronny
    et al.
    KTH, Superseded Departments, Biotechnology.
    Agaton, Charlotta
    KTH, Superseded Departments, Biotechnology.
    Kiesler, E.
    Jin, S.
    Wieslander, L.
    Visa, N.
    Hober, Sophia
    KTH, Superseded Departments, Biotechnology.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    An improved dual-expression concept, generating high-quality antibodies for proteomics research2003In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 38, p. 231-239Article in journal (Refereed)
    Abstract [en]

    A novel, improved dual bacterial-expression system, designed for large-scale generation of high-quality polyclonal antibody preparations intended for proteomics research, is presented. The concept involves parallel expression of cDNA-encoded proteins, as a fusion with two different tags in two separate vector systems. Both systems enable convenient blotting procedures for expression screening on crude bacterial cell cultures and single-step affinity purification under denaturing conditions. One of the fusion proteins is used to elicit antibodies, and the second fusion protein is used in an immobilized form as an affinity ligand to enrich antibodies with selective reactivity to the cDNA-encoded part, common for the two fusion proteins. To evaluate the system, four cDNA clones from putative nuclear proteins from the non-biting midge Chironomus tentans were expressed. Antibodies to these cDNA-encoded proteins were generated, enriched and used in blotting and immunofluorescence procedures to determine expression patterns for the native proteins corresponding to the cDNAs. The four antibody preparations showed specific reactivity to the corresponding recombinant cDNA-encoded proteins, and three of the four antibodies gave specific staining in Western-blot analysis of nuclear cell extracts. Furthermore, two of the antibody preparations gave specific staining in immunofluorescence analysis of C. tentans cells. We conclude that the dual-vector concept presented offers a highly stringent strategy for the generation of monospecific polyclonal antibodies, which are useful in proteomics research.

  • 31. Falk, Ronny
    et al.
    Ramstrom, Margareta
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Approaches for systematic proteome exploration2007In: Biomolecular Engineering, ISSN 1389-0344, E-ISSN 1878-559X, Vol. 24, no 2, p. 155-168Article, review/survey (Refereed)
    Abstract [en]

    With the completion of the human genome project (HUGO) during recent years, gene function, protein abundance and expression patterns in tissues and cell types have emerged as central areas for the scientific community. A mapped human proteome will extend the value of the genome sequence and large-scale efforts aiming at elucidating protein localization, abundance and function are invaluable for biomarker and drug discovery. This research area, termed proteomics, is more demanding than any genome sequencing effort and to perform this on a wide scale is a highly diverse task. Therefore, the proteornics field employs a range of methods to examine different aspects of proteomics including protein localization, protein-protein interactions, posttranslational modifications and alteration of protein composition (e.g. differential expression) in tissues and body fluids. Here, some of the most commonly used methods, including chromatographic separations together with mass spectrometry and a number of affinity proteomics concepts are discussed and exemplified.

  • 32.
    Falk, Ronny
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Ramström, Margareta
    KTH, School of Biotechnology (BIO), Proteomics.
    Eriksson, Cecilia
    KTH, School of Biotechnology (BIO), Proteomics.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Wernérus, Henrik
    KTH, School of Biotechnology (BIO), Proteomics.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Targeted protein pullout from human tissue samples using competitive elution2011In: Biotechnology Journal, ISSN 1860-6768, Vol. 6, no 1, p. 28-37Article in journal (Refereed)
    Abstract [en]

    One commonly used strategy to gain information on the proteins in a cell is to isolate the proteins of interest by specific binders, often antibodies. Not only the specificity of the capturing antibodies but also the washing and elution conditions are crucial to avoid false-positive protein identifications. Eluting the target protein from the matrix, while avoiding the release of unrelated background proteins, should both provide more correct information on the target protein and its interaction partners, and minimize the effort to perform downstream analyses through the reduced number of eluted proteins. In this study, a novel approach for selective protein pullout is presented. Monospecific antibodies were used to selectively pullout target proteins from a complex biosample. Subsequently, the target proteins were competitively eluted from the affinity media with the recombinant antigen. To deplete the antigen from the eluted sample, I MAC spin columns were utilized to bind the N-terminal His-tag of the antigens. The competitive elution method was applied both to a model system, and for the extraction of a native human target protein. In the model system the recombinant target protein BBC7 was spiked into a protein extract of human liver, whereas an endogenously expressed target protein, cTAGE5, was extracted from the liver extract directly. SDS-PAGE analysis and mass spectrometry confirmed affinity isolation of expected target proteins. More selective elution was obtained using the competitive procedure as compared to elution at low pH. Competitive elution has thus been shown to offer an effective approach for wide-scale pullout experiments where proteins and their interaction partners are to be studied.

  • 33. Garousi, J.
    et al.
    Lindbo, S.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Honarvar, H.
    Velletta, J.
    Mitran, B.
    Altai, M.
    Orlova, A.
    Tolmachev, V.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Protein Technology.
    Influence of the N-terminal amino acid sequence on imaging properties of In-111-labeled anti-HER2 scaffold protein ADAPT62016In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 43, p. S55-S55Article in journal (Refereed)
  • 34. Garousi, J.
    et al.
    Lindbo, Sarah
    KTH, School of Biotechnology (BIO), Protein Technology. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Orlova, A.
    Åstrand, Mikael
    KTH, School of Biotechnology (BIO), Protein Technology. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Nilvebrant, Johan
    KTH, School of Biotechnology (BIO), Protein Technology. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Buijs, J.
    Sandstrom, M.
    Honarvar, H.
    Tolmachev, V.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Protein Technology. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Development of ADAPT6 as a new scaffold protein for radionuclide molecular imaging2014In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 41, p. S309-S309Article in journal (Refereed)
  • 35. Garousi, Javad
    et al.
    Lindbo, Sarah
    KTH, School of Biotechnology (BIO), Protein Technology.
    Honarvar, Hadis
    Velletta, Justin
    Mitran, Bogdan
    Altai, Mohamed
    Orlova, Anna
    Tolmachev, Vladimir
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Protein Technology.
    Influence of the N -Terminal Composition on Targeting Properties of Radiometal-Labeled Anti-HER2 Scaffold Protein ADAPT62016In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 27, no 11, p. 2678-2688Article in journal (Refereed)
    Abstract [en]

    Radionuclide-imaging-based stratification of patients to targeted therapies makes cancer treatment more personalized and therefore more efficient. Albumin-binding domain derived affinity proteins (ADAPTs) constitute a novel group of imaging probes based on the scaffold of an albumin binding domain (ABD). To evaluate how different compositions of the N-terminal sequence of ADAPTS influence their biodistribution, a series of human epidermal growth factor receptor type 2 (HER2)-binding ADAPT6 derivatives with different N-terminal sequences were created: GCH(6)DANS (2), GC(HE)(3)DANS (3), GCDEAVDANS (4), and GCVD.ANS(5). These were compared with the parental variant: GCSS(HE)(3)DEAVDANS (1). All variants were site-specifically conjugated with a maleimido-derivative of a DOTA chelator and labeled with In-III. Binding to HER2-expressing cells in vitro, in vivo biodistribution as well as targeting properties of the new variants were compared with properties of the In-III-labeled parental ADAPT variant 1 (In-III-DOTA-1). The composition of the N-terminal sequence had an apparent influence on biodistribution of ADAPT6 in mice. The use of a hexahistidine tag in (InD)-In-III-OTA-2 was associated with elevated hepatic uptake compared to the (HE)(3)-containing counterpart, In-III-DOTA-3. All new variants without a hexahistidine tag demonstrated lower uptake in blood, lung, spleen, and muscle compared to uptake in the parental variant. The best new variants, In-III-DOTA-3 and In-III-DOTA-5, provided tumor uptakes of 14.6 +/- 2.4 and 12.5 +/- 1.3% ID/g at 4 h after injection, respectively. The tumor uptake of In-III-DOTA-3 was significantly higher than the uptake of the parental In-III-DOTA-1 (9.1 +/- 2.0% ID/g). The tumor-to-blood ratios of 395 +/- 75 and 419 +/- 91 at 4 h after injection were obtained for In-III-DOTA-5 and (IIII)n-DOTA-3, respectively. In conclusion, the N-terminal sequence composition affects the biodistribution and targeting properties of ADAPT-based imaging probes, and its optimization may improve imaging contrast.

  • 36. Garousi, Javad
    et al.
    Lindbo, Sarah
    KTH, School of Biotechnology (BIO), Protein Technology.
    Mitran, Bogdan
    Buijs, Jos
    Vorobyeva, Anzhelika
    Orlova, Anna
    Tolmachev, Vladimir
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Protein Technology.
    Comparative evaluation of tumor targeting using the anti-HER2 ADAPT scaffold protein labeled at the C-terminus with indium-111 or technetium-99m2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 14780Article in journal (Refereed)
    Abstract [en]

    ABD-Derived Affinity Proteins (ADAPTs) is a novel class of engineered scaffold proteins derived from an albumin-binding domain of protein G. The use of ADAPT6 derivatives as targeting moiety have provided excellent preclinical radionuclide imaging of human epidermal growth factor 2 (HER2) tumor xenografts. Previous studies have demonstrated that selection of nuclide and chelator for its conjugation has an appreciable effect on imaging properties of scaffold proteins. In this study we performed a comparative evaluation of the anti-HER2 ADAPT having an aspartate-glutamate-alanine-valine-aspartate-alanine-asparagine-serine (DEAVDANS) N-terminal sequence and labeled at C-terminus with (99)mTc using a cysteine-containing peptide based chelator, glycine-serine-serine-cysteine (GSSC), and a similar variant labeled with In-111 using a maleimido derivative of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator. Both (99)mTc-DEAVDANS-ADAPT6-GSSC and In-111-DEAVDANS-ADAPT6-GSSC-DOTA accumulated specifically in HER2-expressing SKOV3 xenografts. The tumor uptake of both variants did not differ significantly and average values were in the range of 19-21% ID/g. However, there was an appreciable variation in uptake of conjugates in normal tissues that resulted in a notable difference in the tumor-to-organ ratios. The In-111-DOTA label provided 2-6 fold higher tumor-to-organ ratios than (99)mTc-GSSC and is therefore the preferable label for ADAPTs.

  • 37. Garousi, Javad
    et al.
    Lindbo, Sarah
    KTH, School of Biotechnology (BIO), Protein Technology.
    Nilvebrant, Johan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Åstrand, Mikael
    KTH, School of Biotechnology (BIO), Protein Technology.
    Buijs, Jos
    Sandstrom, Mattias
    Honarvar, Hadis
    Orlova, Anna
    Tolmachev, Vladimir
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Protein Technology.
    ADAPT, a Novel Scaffold Protein-Based Probe for Radionuclide Imaging of Molecular Targets That Are Expressed in Disseminated Cancers2015In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 75, no 20, p. 4364-4371Article in journal (Refereed)
    Abstract [en]

    Small engineered scaffold proteins have attracted attention as probes for radionuclide-based molecular imaging. One class of these imaging probes, termed ABD-Derived Affinity Proteins (ADAPT), has been created using the albumin-binding domain (ABD) of streptococcal protein G as a stable protein scaffold. In this study, we report the development of a clinical lead probe termed ADAPT6 that binds HER2, an oncoprotein overexpressed in many breast cancers that serves as a theranostic biomarker for several approved targeting therapies. Surface-exposed amino acids of ABD were randomized to create a combinatorial library enabling selection of high-affinity binders to various proteins. Furthermore, ABD was engineered to enable rapid purification, to eradicate its binding to albumin, and to enable rapid blood clearance. Incorporation of a unique cysteine allowed site-specific conjugation to a maleimido derivative of a DOTA chelator, enabling radionuclide labeling, In-111 for SPECT imaging and Ga-68 for PET imaging. Pharmacologic studies in mice demonstrated that the fully engineered molecule In-111/Ga-68-DOTA(HE) 3-ADAPT6 was specifically bound and taken up by HER2-expressing tumors, with a high tumor-to-normal tissue ratio in xenograft models of human cancer. Unbound tracer underwent rapid renal clearance followed by high renal reabsorption. HER2-expressing xenografts were visualized by gamma-camera or PET at 1 hour after infusion. PET experiments demonstrated feasibility for discrimination of xenografts with high or low HER2 expression. Our results offer a preclinical proof of concept for the use of ADAPT probes for noninvasive in vivo imaging.

  • 38.
    Gräslund, Torbjörn
    et al.
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Ehn, Maria
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Gunnel, Lundin
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Hedhammar, My
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Strategy for highly selective ion-exchange capture using a charge-polarizing fusion partner2002In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 942, no 1-2, p. 157-166Article in journal (Refereed)
    Abstract [en]

    To achieve efficient recovery of recombinantly produced target proteins using cation-exchange chromatography, a novel basic protein domain is used as a purification handle. The proteolytic instability usually encountered for basic peptide tags is avoided by the use of a highly constrained α-helical domain based on staphylococcal protein A into which positively charged amino acids have been introduced. Here we show that this domain, consisting of 58 amino acids with a calculated isoelectric point (pI) of 10.5, can be used to efficiently capture different fused target proteins, such as a bacterial DNA polymerase (Klenow fragment), a viral protease (3C) and a fungal lipase (Cutinase). In contrast to standard cation-exchange chromatography, efficient capture can be achieved also at a pH value higher than the pI of the fusion protein, demonstrated here by Zbasic-Klenow polymerase (pI≈5.8) and ZZ-Cutinase-Zbasic (pI≈7.2) both purified at a pH of 7.5. These results show that the Zbasic domain is able to confer a regional concentration of positive charge on the fusion protein even at a relatively high pH. Hence, the data suggest that this domain could be used for highly efficient and selective capture of target proteins at conditions where most host-cell proteins do not bind to the chromatographic resin. The obtained purity after this one-step procedure suggests that the strategy could be an alternative to standard affinity chromatography. Methods for site-specific proteolysis of the fusion proteins to release native target proteins are also discussed.

  • 39.
    Gräslund, Torbjörn
    et al.
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Hedhammar, My
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Hober, Sophia
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Integrated strategy for selective expanded bed ion-exchange adsorption and site-specific protein processing using gene fusion technology2002In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 96, no 1, p. 93-102Article in journal (Refereed)
    Abstract [en]

    The highly charged domain Z(basic) can be used as a fusion partner to enhance adsorption of target proteins to cation exchanging resins at high pH-values. In this paper, we describe a strategy for purification of target proteins fused to Z(basic) at a constant physiological pH using cation exchange chromatography in an expanded bed mode. We show that two proteins, Klenow DNA polymerase and the viral protease 3C, can be efficiently purified from unclarified Escherichia coli homogenates in a single step with a selectivity analogous to what is normally achieved by affinity chromatography. The strategy also includes an integrated site-specific removal of the Z(basic) purification handle to yield a free target protein.

  • 40.
    Gräslund, Torbjörn
    et al.
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Lundin, Gunnel
    Uhlén, Mathias
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Hober, Sophia
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Charge engineering of a protein domain to allow efficient ion-exchange recovery2000In: Protein Engineering, ISSN 0269-2139, E-ISSN 1460-213X, Vol. 13, no 10, p. 703-709Article in journal (Refereed)
    Abstract [en]

    We have created protein domains with extreme surface charge. These mutated domains allow for ion-exchange chromatography under conditions favourable for selective and efficient capture, using Escherichia coli as a host organism. The staphylococcal protein A-derived domain Z (Z(wt)) was used asa scaffold when constructing two mutants, Z(basic1) and Z(basic2), with high positive surface charge. Far-ultraviolet circular dichroism measurements showed that they have a secondary structure content comparable to the parental molecule Z(wt). Although melting temperatures (T-m) of the engineered domains were lower than that of the wild-type Z domain, both mutants could be produced successfully as intracellular full-length products in E. coli and purified to homogeneity by ion-exchange chromatography. Further studies performed on Z(basic1) and Z(basic2) showed that they were able to bind to a cation exchanger even at pH values in the 9 to 11 range. A gene fusion between Z(basic2) and the acidic human serum albumin binding domain (ABD), derived from streptococcal protein G, was also constructed. The gene product Z(basic2)-ABD could be purified using cation-exchange chromatography from a whole cell lysate to more than 90% purity.

  • 41.
    Grönwall, Caroline
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology. KTH, School of Biotechnology (BIO), Proteomics.
    Sjöberg, Anna
    Affibody AB, Bromma.
    Ramström, Margareta
    KTH, School of Biotechnology (BIO), Molecular Biotechnology. KTH, School of Biotechnology (BIO), Proteomics.
    Höidén-Guthenberg, Ingmarie
    Affibody AB, Bromma.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Molecular Biotechnology. KTH, School of Biotechnology (BIO), Proteomics.
    Jonasson, Per
    Affibody AB, Bromma.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology. KTH, School of Biotechnology (BIO), Proteomics.
    Affibody-mediated transferrin depletion for proteomics applications2007In: Biotechnology Journal, ISSN 1860-6768, Vol. 2, no 11, p. 1389-1398Article in journal (Refereed)
    Abstract [en]

    An Affibody® (Affibody) ligand with specific binding to human transferrin was selected by phage display technology from a combinatorial protein library based on the staphylococcal protein A (SpA)-derived Z domain. Strong and selective binding of the selected Affibody ligand to transferrin was demonstrated using biosensor technology and dot blot analysis. Impressive specificity was demonstrated as transferrin was the only protein recovered by affinity chromatography from human plasma. Efficient Affibody-mediated capture of transferrin, combined with IgG- and HSA-depletion, was demonstrated for human plasma and cerebrospinal fluid (CSF). For plasma, 85% of the total transferrin content in the samples was depleted after only two cycles of transferrin removal, and for CSF, 78% efficiency was obtained in single-step depletion. These results clearly suggest a potential for the development of Affibody-based resins for the removal of abundant proteins in proteomics analyses.

  • 42. Gulich, S.
    et al.
    Linhult, M.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Hober, Sophia
    KTH, Superseded Departments, Biotechnology.
    Stability towards alkaline conditions can be engineered into a protein ligand2000In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 80, no 2, p. 169-178Article in journal (Refereed)
    Abstract [en]

    One of the problems with a proteinaceous affinity ligand is their sensitivity to alkaline conditions. Here, we show that a simple and straightforward strategy consisting of replacing all asparagine residues with other amino acids can dramatically improve the chemical stability of a protein towards alkaline conditions. As a model, a Streptococcal albumin-binding domain (ABD) was used. The engineered variant showed higher stability towards 0.5 M NaOH, as well as higher thermal stability compared to its native counterpart. This protein engineering approach could potentially also be used for other protein ligands to eliminate the sensitivity to alkaline cleaning-in-place (CIP) conditions.

  • 43. Gulich, S.
    et al.
    Linhult, M.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Hober, Sophia
    KTH, Superseded Departments, Biotechnology.
    Engineering streptococcal protein G for increased alkaline stability2002In: Protein Engineering, ISSN 0269-2139, E-ISSN 1460-213X, Vol. 15, no 10, p. 835-842Article in journal (Refereed)
    Abstract [en]

    Most protein-based affinity chromatography media are very sensitive towards alkaline treatment, which is a preferred method for regeneration and removal of contaminants from the purification devices in industrial applications. In a previous study, we concluded that a simple and straightforward strategy consisting of replacing asparagine residues could improve the stability towards alkaline conditions. In this study, we have shown the potential of this rationale by stabilizing an IgG-binding domain of streptococcal protein G, i.e. the C2 domain. In order to analyze the contribution of the different amino acids to the alkaline sensitivity of the domain we used a single point mutation strategy. Amino acids known to be susceptible towards high pH, asparagine and glutamine, were substituted for less-alkali-susceptible residues. In addition, aspartic acid residues were mutated to evaluate if the stability could be further increased. The stability of the different C2 variants was subsequently analyzed by exposing them to NaOH. The obtained results reveal that the most sensitive amino acid towards alkaline conditions in the structure of C2 is Asn36. The double mutant, C2(N7,36A), was found to be the most stable mutant constructed. In addition to the increased alkaline stability and also very important for potential use as an affinity ligand, this mutated variant also retains the secondary structure, as well as the affinity to the Fc fragment of IgG.

  • 44. Gulich, S.
    et al.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Hober, Sophia
    KTH, Superseded Departments, Biotechnology.
    Protein engineering of an IgG-binding domain allows milder elution conditions during affinity chromatography2000In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 76, no 03-feb, p. 233-244Article in journal (Refereed)
    Abstract [en]

    One of the problems in the recovery of antibodies by affinity chromatography is the low pH, which is normally essential to elute the bound material from the column. Here, we have addressed this problem by constructing destabilized mutants of a domain analogue (domain Z) from an IgG-binding bacterial receptor, protein A. In ol-der to destabilize the IgG-binding domain, two protein engineered variants were constructed using site-directed mutagenesis of the second loop of this antiparallel three-helix bundle domain. In the first mutant (Z6C), the second loop was extended with six glycines in order to evaluate the significance of the loop length. In the second mutant (ZL4G), the original loop sequence was exchanged for glycines in order to evaluate the importance of the loop forming residues. Both mutated variants have a lower a-helical content, as well as a lower thermal and chemical stability compared to the parent 2-molecule. The affinity to IgG was slightly lowered in both cases, mainly due to higher dissociation rates. Interestingly, the elution studies showed that most of the bound IgG-molecules could be eluted at a pH as high as 4.5 from columns with the engineered ligands, while only 70% of the bound IgG could be eluted from the matrix with the parent Z as ligand.

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

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

  • 46. Hedhammar, M.
    et al.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Protein engineering strategies for selective protein purification2005In: Chemical Engineering & Technology, ISSN 0930-7516, E-ISSN 1521-4125, Vol. 28, no 11, p. 1315-1325Article, review/survey (Refereed)
    Abstract [en]

    When producing and purifying recombinant proteins it is of importance to minimize the number of unit operations during the purification procedure. This is accomplished by increasing the selectivity in each step. Due to the high selectivity of affinity chromatography it has a widespread use in protein purification. However, most target proteins lack a suitable affinity ligand usable for capture oil a solid matrix. A way to circumvent this obstacle is to genetically fuse the gene encoding the target protein with a gene encoding a purification tag. When the chimeric protein is expressed, the tag allows for specific capture of the fusion protein. In industrial-scale production, extension of the target protein often is unwanted since it might interfere with the function of the target protein. Hence, a purification scheme developed for the native protein is desired. In this review, different fusion strategies used for protein purification are discussed. Also, the development of ligands for selective affinity purification of native target proteins is surveyed.

  • 47.
    Hedhammar, My
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Alm, Tove
    KTH, School of Biotechnology (BIO), Proteomics.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Proteomics.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Single-step recovery and solid-phase refolding of inclusion body proteins using a polycationic purification tag2006In: Biotechnology Journal, ISSN 1860-6768, Vol. 1, p. 187-196Article in journal (Refereed)
    Abstract [en]

    A strategy for purification of inclusion body-forming proteins is described, in which the positively charged domain Z(basic) is used as a fusion partner for capture of denatured proteins on a cation exchange column. It is shown that the purification tag is selective under denaturing conditions. Furthermore, the new strategy for purification of proteins from inclusion bodies is compared with the commonly used method for purification of His(6)-tagged inclusion body proteins. Finally, the simple and effective means of target protein capture provided by the Z(basic) tag is further successfully explored for solid-phase refolding. This procedure has the inherited advantage of combining purification and refolding in one step and offers the advantage of eluting the concentrated product in a suitable buffer.

  • 48.
    Hedhammar, My
    et al.
    KTH, Superseded Departments, Biotechnology.
    Gräslund, Torbjörn
    KTH, Superseded Departments, Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Hober, Sophia
    KTH, Superseded Departments, Biotechnology.
    Negatively charged purification tags for selective anion-exchange recovery2004In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 17, no 11, p. 779-786Article in journal (Refereed)
    Abstract [en]

     A novel strategy for the highly selective purification of recombinant fusion proteins using negatively charged protein domains, which were constructed by protein design, is described. A triple alpha-helical domain of 58 amino acids was used as scaffold. Far-ultraviolet circular dichroism measurements showed that the designed domains had very low alpha-helicity in a low-conductivity environment in contrast to the scaffold. The secondary structure could be induced by adding salt, giving a structure comparable to the parental molecule. Further studies showed that the new domains were able to bind to an anion exchanger even at pH values down to 5 and 6. Gene fusions between one of the designed domains and different target proteins, such as green fluorescent protein (GFP), maltose binding protein (MBP) and firefly luciferase, were also constructed. These gene products could be efficiently purified from whole cell lysates at pH 6 using anion-exchange chromatography.

  • 49.
    Hedhammar, My
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Z(basic) - A novel purification tag for efficient protein recovery2007In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1161, no 1-2, p. 22-28Article in journal (Refereed)
    Abstract [en]

    A positively charged protein domain, Z(basic) can be used as a general purification tag to achieve efficient recovery of recombinantly produced target proteins using cation-exchange chromatography. To construct a protein domain usable for ion-exchange chromatography, the surface of protein Z was engineered to be highly charged, which allowed for selective capture of target proteins on a cation-exchanger at physiological pH values. Interestingly, the novel domain, denoted Z(basic) was shown to be selective also under denaturing conditions and could preferably be used for purification of proteins solubilised from inclusion bodies. Moreover, a flexible process for solid-phase refolding was developed, using Z(basic) as a reversible linker to the cation-exchanger resin. This procedure has the inherited advantage of combining purification and refolding into a single step and still enabling elution of a concentrated product in a suitable buffer. This article summarizes development and use of the Z(basic), tag in small and pilot-plant-scale downstream processing.

  • 50.
    Hedhammar, My
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Jung, H. R.
    University of Southern Denmark, Department for Biochemistry and Molecular Biology.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Enzymatic cleavage of fusion proteins using immobilised protease 3C2006In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 47, no 2, p. 422-426Article in journal (Refereed)
    Abstract [en]

    A strategy for efficient cleavage of fusion proteins using an immobilised protease has been developed. Protease 3C from coxsackie virus was recombinantly produced in Escherichia coli and covalently immobilised onto a solid support. Thereafter, Z(basic) tagged fusion proteins, with a specific cleavage sequence between the domains, were flown through the proteolytic column and circulated until complete cleavage. Subsequently, the processed protein solution was applied on a cation exchanger. Thereby, removal of the released, positively charged fusion tag, Z(basic), was done by adsorption to the matrix while the target proteins were recovered in the flow through. Interestingly, the columns were shown to be reusable without any measurable decrease in activity. Moreover, after storage in 4 degrees C for two months the activity was almost unaffected.

123 1 - 50 of 131
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf