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  • 1.
    Anfelt, Josefine
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Kaczmarzyk, Danuta
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Shabestary, Kiyan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Renberg, Björn
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Nielsen, Jens
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. Tech Univ Denmark.
    Hudson, Elton P.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Genetic and nutrient modulation of acetyl-CoA levels in Synechocystis for n-butanol production2015Ingår i: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 14, artikel-id 167Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: There is a strong interest in using photosynthetic cyanobacteria as production hosts for biofuels and chemicals. Recent work has shown the benefit of pathway engineering, enzyme tolerance, and co-factor usage for improving yields of fermentation products. Results: An n-butanol pathway was inserted into a Synechocystis mutant deficient in polyhydroxybutyrate synthesis. We found that nitrogen starvation increased specific butanol productivity up to threefold, but cessation of cell growth limited total n-butanol titers. Metabolite profiling showed that acetyl-CoA increased twofold during nitrogen starvation. Introduction of a phosphoketolase increased acetyl-CoA levels sixfold at nitrogen replete conditions and increased butanol titers from 22 to 37 mg/L at day 8. Flux balance analysis of photoautotrophic metabolism showed that a Calvin-Benson-Bassham-Phosphoketolase pathway had higher theoretical butanol productivity than CBB-Embden-Meyerhof-Parnas and a reduced butanol ATP demand. Conclusion: These results demonstrate that phosphoketolase overexpression and modulation of nitrogen levels are two attractive routes toward increased production of acetyl-CoA derived products in cyanobacteria and could be implemented with complementary metabolic engineering strategies.

  • 2.
    Berglund, Lisa
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Björling, Erik
    KTH, Skolan för bioteknologi (BIO).
    Jonasson, Kalle
    KTH, Skolan för bioteknologi (BIO).
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO).
    Fagerberg, Linn
    KTH, Skolan för bioteknologi (BIO).
    Al-Khalili Szigyarto, Cristina
    KTH, Skolan för bioteknologi (BIO).
    Sivertsson, Åsa
    KTH, Skolan för bioteknologi (BIO).
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO).
    A whole-genome bioinformatics approach to selection of antigens for systematic antibody generation2008Ingår i: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 8, nr 14, s. 2832-2839Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Here, we present an antigen selection strategy based on a whole-genome bioinformatics approach, which is facilitated by an interactive visualization tool displaying protein features from both public resources and in-house generated data. The web-based bioinformatics platform has been designed for selection of multiple, non-overlapping recombinant protein epitope signature tags by display of predicted information relevant for antigens, including domain- and epitope sized sequence similarities to other proteins, transmembrane regions and signal peptides. The visualization tool also displays shared and exclusive protein regions for genes with multiple splice variants. A genome-wide analysis demonstrates that antigens for approximately 80% of the human protein-coding genes can be selected with this strategy.

  • 3. Buus, S.
    et al.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Forsström, Björn
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101). KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101). KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Schafer-Nielsen, C.
    High-resolution mapping of linear antibody epitopes using ultrahigh-density peptide microarrays2012Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 11, nr 12, s. 1790-1800Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Antibodies empower numerous important scientific, clinical, diagnostic, and industrial applications. Ideally, the epitope(s) targeted by an antibody should be identified and characterized, thereby establishing antibody reactivity, highlighting possible cross-reactivities, and perhaps even warning against unwanted (e.g. autoimmune) reactivities. Antibodies target proteins as either conformational or linear epitopes. The latter are typically probed with peptides, but the cost of peptide screening programs tends to prohibit comprehensive specificity analysis. To perform high-throughput, high-resolution mapping of linear antibody epitopes, we have used ultrahigh-density peptide microarrays generating several hundred thousand different peptides per array. Using exhaustive length and substitution analysis, we have successfully examined the specificity of a panel of polyclonal antibodies raised against linear epitopes of the human proteome and obtained very detailed descriptions of the involved specificities. The epitopes identified ranged from 4 to 12 amino acids in size. In general, the antibodies were of exquisite specificity, frequently disallowing even single conservative substitutions. In several cases, multiple distinct epitopes could be identified for the same target protein, suggesting an efficient approach to the generation of paired antibodies. Two alternative epitope mapping approaches identified similar, although not necessarily identical, epitopes. These results show that ultrahigh-density peptide microarrays can be used for linear epitope mapping. With an upper theoretical limit of 2,000,000 individual peptides per array, these peptide microarrays may even be used for a systematic validation of antibodies at the proteomic level.

  • 4.
    Forsström, Bjorn
    et al.
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Axnäs, Barbara Bislawska
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Danielsson, Hanna
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Bohlin, Anna
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Dissecting antibodies withregards to linear and conformational epitopesManuskript (preprint) (Övrigt vetenskapligt)
  • 5.
    Forsström, Bjorn
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Axnäs, Barbara Bislawska
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Stengele, Klaus-Peter
    Buehler, Jochen
    Albert, Thomas J.
    Richmond, Todd A.
    Hu, Francis Jingxin
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Hudson, Elton Paul
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Proteome-wide Epitope Mapping of Antibodies Using Ultra-dense Peptide Arrays2014Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 13, nr 6, s. 1585-1597Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Antibodies are of importance for the field of proteomics, both as reagents for imaging cells, tissues, and organs and as capturing agents for affinity enrichment in mass-spectrometry-based techniques. It is important to gain basic insights regarding the binding sites (epitopes) of antibodies and potential cross-reactivity to nontarget proteins. Knowledge about an antibody's linear epitopes is also useful in, for instance, developing assays involving the capture of peptides obtained from trypsin cleavage of samples prior to mass spectrometry analysis. Here, we describe, for the first time, the design and use of peptide arrays covering all human proteins for the analysis of antibody specificity, based on parallel in situ photolithic synthesis of a total of 2.1 million overlapping peptides. This has allowed analysis of on-and off-target binding of both monoclonal and polyclonal antibodies, complemented with precise mapping of epitopes based on full amino acid substitution scans. The analysis suggests that linear epitopes are relatively short, confined to five to seven residues, resulting in apparent off-target binding to peptides corresponding to a large number of unrelated human proteins. However, subsequent analysis using recombinant proteins suggests that these linear epitopes have a strict conformational component, thus giving us new insights regarding how antibodies bind to their antigens.

  • 6.
    Forsström, Björn
    et al.
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Axnäs, Barbara Bislawska
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Danielsson, Hanna
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Bohlin, Anna
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Dissecting Antibodies with Regards to Linear and Conformational Epitopes2015Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, nr 3, artikel-id e0121673Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    An important issue for the performance and specificity of an antibody is the nature of the binding to its protein target, including if the recognition involves linear or conformational epitopes. Here, we dissect polyclonal sera by creating epitope-specific antibody fractions using a combination of epitope mapping and an affinity capture approach involving both synthesized peptides and recombinant protein fragments. This allowed us to study the relative amounts of antibodies to linear and conformational epitopes in the polyclonal sera as well as the ability of each antibody-fraction to detect its target protein in Western blot assays. The majority of the analyzed polyclonal sera were found to have most of the target-specific antibodies directed towards linear epitopes and these were in many cases giving Western blot bands of correct molecular weight. In contrast, many of the antibodies towards conformational epitopes did not bind their target proteins in the Western blot assays. The results from this work have given us insights regarding the nature of the antibody response generated by immunization with recombinant protein fragments and has demonstrated the advantage of using antibodies recognizing linear epitopes for immunoassay involving wholly or partially denatured protein targets.

  • 7. Hansen, Henning Gram
    et al.
    Nilsson, Claes Nymand
    Lund, Anne Mathilde
    Kol, Stefan
    Grav, Lise Marie
    Lundqvist, Magnus
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Lee, Gyun Min
    Andersen, Mikael Rordam
    Kildegaard, Helene Faustrup
    Versatile microscale screening platform for improving recombinant protein productivity in Chinese hamster ovary cells2015Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, artikel-id 18016Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 8.
    Hjelm, Barbara
    et al.
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Fernandez, Carmen Diez
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Johannesson, Henrik
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Uhlen, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Exploring epitopes of antibodies toward the human tryptophanyl-tRNA synthetase2010Ingår i: NEW BIOTECHNOL, ISSN 1871-6784, Vol. 27, nr 2, s. 129-137Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    There is a need to characterize the epitopes of affinity reagents to develop high quality affinity reagents for research, diagnostics and therapy. Here, we describe the analysis of epitopes of antibodies generated toward human tryptophanyl-tRNA synthetase (WARS) using both combinatorial bacterial display and suspension bead array. The bacterial display revealed that the polyclonal antibody binds to three separate epitopes and peptide scanning using 15-mers revealed binding to a 13 amino acid consensus sequence (ELINRIERATGQR). A mouse monoclonal antibody was generated and the mapping approach revealed binding toward a slightly shifted position of the same epitope. Structural analysis showed that the antibodies bind to a-helical regions on the surface of the target protein. An alanine-scanning experiment showed binding to four specific residues. The implications for the systematic analysis of antibody epitopes on the basis of these results are discussed.

  • 9.
    Hjelm, Barbara
    et al.
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Forsström, Björn
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Igel, Ulrika
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Johannesson, Henrik
    Stadler, Charlotte
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Lundberg, Emma
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Ponten, Fredrik
    Sjoberg, Anna
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Schwenk, Jochen M.
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Nilsson, Peter
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Johansson, Christine
    Uhlen, Mathias
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Generation of monospecific antibodies based on affinity capture of polyclonal antibodies2011Ingår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 20, nr 11, s. 1824-1835Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A method is described to generate and validate antibodies based on mapping the linear epitopes of a polyclonal antibody followed by sequential epitope-specific capture using synthetic peptides. Polyclonal antibodies directed towards four proteins RBM3, SATB2, ANLN, and CNDP1, potentially involved in human cancers, were selected and antibodies to several non-overlapping epitopes were generated and subsequently validated by Western blot, immunohistochemistry, and immunofluorescence. For all four proteins, a dramatic difference in functionality could be observed for these monospecific antibodies directed to the different epitopes. In each case, at least one antibody was obtained with full functionality across all applications, while other epitope-specific fractions showed no or little functionality. These results present a path forward to use the mapped binding sites of polyclonal antibodies to generate epitope-specific antibodies, providing an attractive approach for large-scale efforts to characterize the human proteome by antibodies.

  • 10.
    Hjelm, Barbara
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Forsström, Björn
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101). KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Immunizations of inbred rabbits using the same antigen yield antibodies with similar, but not identical, epitopesManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    A problem for the generation of polyclonal antibodies is the potential difficulties to obtain a renewable resource due to batch-to-batch variations when the same antigen is immunized into several separate animals. Here, we have investigated this issue by determining the epitopes of antibodies generated from parallel immunizations of in-bred rabbits with recombinant antigens corresponding to ten human protein targets. The epitopes were mapped by both a suspension bead array approach using overlapping synthetic 15-mer peptides and a bacterial display approach using expression of random fragments of the antigen gene on the surface of bacteria. Both methods determined antibody binding with the aid of fluorescent-based analysis. In addition, one polyclonal antibody was fractionated by peptide-specific affinity capture for in-depth comparison of epitopes. The results show that the same antigen immunized in several in-bred rabbits yields polyclonal antibodies with similar epitopes, but with larger differences in the relative amounts of antibodies to the different epitopes. In some cases, unique epitopes were observed for one of the immunizations. The results suggest that polyclonal antibodies generated by repeated immunizations do not display an identical epitope pattern, although many of the epitopes are similar.

  • 11.
    Hjelm, Barbara
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Forsström, Björn
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101). KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Parallel Immunizations of Rabbits Using the Same Antigen Yield Antibodies with Similar, but Not Identical, Epitopes2012Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, nr 12, s. e45817-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A problem for the generation of polyclonal antibodies is the potential difficulties for obtaining a renewable resource due to batch-to-batch variations when the same antigen is immunized into several separate animals. Here, we have investigated this issue by determining the epitopes of antibodies generated from parallel immunizations of rabbits with recombinant antigens corresponding to ten human protein targets. The epitopes were mapped by both a suspension bead array approach using overlapping synthetic 15-mer peptides and a bacterial display approach using expression of random fragments of the antigen on the surface of bacteria. Both methods determined antibody binding with the aid of fluorescent-based analysis. In addition, one polyclonal antibody was fractionated by peptide-specific affinity capture for in-depth comparison of epitopes. The results show that the same antigen immunized in several rabbits yields polyclonal antibodies with similar epitopes, but with larger differences in the relative amounts of antibodies to the different epitopes. In some cases, unique epitopes were observed for one of the immunizations. The results suggest that polyclonal antibodies generated by repeated immunizations do not display an identical epitope pattern, although many of the epitopes are similar.

  • 12. Hu, Francis Jingxin
    et al.
    Lundqvist, Magnus
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Rockberg, Johan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    SAMURAI (Solid-phase Assisted Mutagenesis by Uracil Restricted Ablation In vitro) for Antibody Affinity Maturation and Paratope MappingManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Mutagenesis libraries are essential for combinatorial protein engineering. Despite improve- ments in gene synthesis and directed mutagenesis, current methodologies still have limitations regarding the synthesis of intact antibody scFv genes and simultaneous diversification of all six CDRs. Here, we de- scribe the generation of mutagenesis libraries for antibody affinity maturation, using a cell-free solid-phase technique for annealing of single-strand mutagenic oligonucleotides. This procedure consists of PCR-based incorporation of uracil into a wild-type template, bead-based capture, and elution of single-strand DNA, and in vitro uracil excision enzyme based degradation of the template DNA. Our approach enabled rapid (8 hours) mutagenesis and automated cloning of 50 position specific alanine mutants for mapping of a scFv antibody paratope. We further exemplify our method by generating affinity maturation libraries with di- versity introduced in critical, nonessential, or all CDR positions randomly. Assessment with Illumina deep sequencing showed >99% functional diversity in two libraries and the ability to diversify all CDR positions simultaneously. Selections of the libraries with bacterial display and deep sequencing evaluation of the selection output showed that diversity introduced in non-essential positions allowed quicker enrichment of improved binders compared to the other two diversification strategies.

  • 13.
    Hu, Francis Jingxin
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Lundqvist, Magnus
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Uhlén, Mathias
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap. KTH, Centra, Science for Life Laboratory, SciLifeLab. Tech Univ Denmark, Novo Nord Fdn Ctr Biosustainabil, DK-2970 Horsholm, Denmark..
    Rockberg, Johan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    SAMURAI (Solid-phase Assisted Mutagenesis by Uracil Restriction for Accurate Integration) for antibody affinity maturation and paratope mapping2019Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 47, nr 6, artikel-id e34Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Mutagenesis libraries are essential for combinatorial protein engineering. Despite improvements in gene synthesis and directed mutagenesis, current methodologies still have limitations regarding the synthesis of complete antibody single-chain variable fragment (scFv) genes and simultaneous diversification of all six CDRs. Here, we describe the generation of mutagenesis libraries for antibody affinity maturation using a cell-free solid-phase technique for annealing of single-strand mutagenic oligonucleotides. The procedure consists of PCR-based incorporation of uracil into a wild-type template, bead-based capture, elution of single-strand DNA, and in vitro uracil excision enzyme based degradation of the template DNA. Our approach enabled rapid (8 hours) mutagenesis and automated cloning of 50 position-specific alanine mutants for mapping of a scFv antibody paratope. We further exemplify our method by generating affinity maturation libraries with diversity introduced in critical, nonessential, or all CDR positions randomly. Assessment with Illumina deep sequencing showed less than 1% wild-type in two libraries and the ability to diversify all CDR positions simultaneously. Selections of the libraries with bacterial display and deep sequencing evaluation of the selection output showed that diversity introduced in non-essential positions allowed for a more effective enrichment of improved binders compared to the other two diversification strategies.

  • 14.
    Hu, Francis Jingxin
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Lundqvist, Magnus
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    SPUX - A Solid Phase Uracil Excision Method for Antibody Affinity Maturation and Paratope MappingManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Mutagenesis libraries are the heart of combinatorial protein engineering where proteins such as antibodies are evolved for improved functionality. Despite recent improvements in gene synthesis and selection methodologies, current methods still fail to provide practical means for synthesis of complete antibody scFv and screening of theoretical diversities, hence forcing the user to focused diversity screening and assembly of shorter oligos to avoid synthesis errors and maximize library functionality. Here we demonstrate a way to generate highly functional tailored mutagenesis libraries for efficient antibody affinity maturation using a rapid cell-free solid phase cloning method with single strand diversity oligonucleotides. For this we are utilizing a combination of a high-fidelity polymerase for PCR-based incorporation of Uracil into a wild-type template, bead-based solid-phase technology for elution of single strand DNA, oligonucleotide annealing, extension and automation, and an uracil excision enzyme cocktail for in vitro degradation of template DNA to minimize background. Our method allowed for fast (8 hours) mutagenesis and automated cloning of a complete set of 50 position specific alanine-mutations for mapping of the paratope of a scFv antibody in a single robot run. We further exemplify our method by generating and stratifying a set of antibody scFv affinity maturation libraries with targeted diversity into critical or nonessential paratope positions, as well as by a complete randomization in all positions. The libraries were subjected to bacterial surface display selections and output was followed by Illumina deep sequencing and binding analysis by SPR. The functional quality of our libraries were high, with a yield of >99% functional diversity in the case for two of our libraries. We were further able to target all positions in all loops with diversity, and we could show the ability to target all six loops with diversity at the same time. The comparison of different library focus showed us that scFv libraries with diversity targeted to non-essential enhancing paratope positions more quickly rendered enrichment of improved binders compared to random diversity or paratope-targeted libraries. Surprisingly several of the improved binders from the random library had beneficial mutations in the same positions targeted by the smaller focused non-essential enhancing residue focused library indicating a possible benefit of focusing diversity to these spots. We believe our method for construction of libraries with site directed mutagenesis to be a viable way for generation of functional and diverse genetic libraries, particularly suitable for affinity maturation and paratope mapping of antibodies.

  • 15.
    Hu, Francis Jingxin
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Generation of HER2 monoclonal antibodies using epitopes of a rabbit polyclonal antibody2014Ingår i: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 31, nr 1, s. 35-43Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    One of the issues in using polyclonal antibodies is the limited amount of reagent available from an immunisation, leading to batch-to-batch variation and difficulties in obtaining the same antibody performance when the same antigen is re-immunised into several separate animals. This led to the development of hybridoma technology allowing, at least theoretically, for an unlimited production of a specific binder. Nevertheless, polyclonal antibodies are widely used in research and diagnostics and there exists a need for robust methods to convert a polyclonal antibody with good binding performance into a renewable monoclonal with identical or similar binding specificity. Here we have used precise information regarding the functional recognition sequence (epitope) of a rabbit polyclonal antibody with attractive binding characteristics as the basis for generation of a renewable mouse monoclonal antibody. First, the original protein fragment antigen was used for immunisation and generation of mouse hybridoma, without obtaining binders to the same epitope region. Instead a peptide designed using the functional epitope and structural information was synthesised and used for hybridoma production. Several of the monoclonal antibodies generated were found to have similar binding characteristics to those of the original polyclonal antibody. These monoclonal antibodies detected native HER2 on cell lines and were also able to stain HER2 in immunohistochemistry using xenografted mice, as well as human normal and cancer tissues.

  • 16.
    Hu, Francis Jingxin
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Volk, Anna-Luisa
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Persson, Helena
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab. Lund University, Sweden.
    Säll, Anna
    Borrebaeck, Carl
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Combination of phage and Gram-positive bacterial display of human antibody repertoires enables isolation of functional high affinity binders2017Ingår i: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Surface display couples genotype with a surface exposed phenotype and thereby allows screening of gene-encoded protein libraries for desired characteristics. Of the various display systems available, phage display is by far the most popular, mainly thanks to its ability to harbour large size libraries. Here, we describe the first use of a Gram-positive bacterial host for display of a library of human antibody genes which, when combined with phage display, provides ease of use for screening, sorting and ranking by flow cytometry. We demonstrate the utility of this method by identifying low nanomolar affinity scFv fragments towards human epidermal growth factor receptor 2 (HER2). The ranking and performance of the scFv isolated by flow sorting in surface-immobilised form was retained when expressed as soluble scFv and analysed by biolayer interferometry, as well as after expression as full-length antibodies in mammalian cells. We also demonstrate the possibility of using Gram-positive bacterial display to directly improve the affinity of the identified binders via an affinity maturation step using random mutagenesis and flow sorting. This combined approach has the potential for a more complete scan of the antibody repertoire and for affinity maturation of human antibody formats.

  • 17.
    Hu, Francis Jingxin
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Volk, Anna-Luisa
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Persson, Helena
    Department of Immunotechnology, Lund University, Medicon Village (Bldg 406), 223 81 Lund, Sweden..
    Säll, Anna
    Department of Immunotechnology, Lund University, Medicon Village (Bldg 406), 223 81 Lund, Sweden..
    Borrebaeck, Carl
    Department of Immunotechnology, Lund University, Medicon Village (Bldg 406), 223 81 Lund, Sweden..
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Phage and Gram-positive bacterial display of human antibody repertoires enables isolation of functional high affinity bindersIngår i: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347Artikel i tidskrift (Övrigt vetenskapligt)
    Abstract [en]

    Surface display couples genotype with a surface exposed phenotype and thereby allows for screening of gene-encoded protein libraries for desired characteristics. Of the various display systems, phage display is by far the most popular, mainly thanks to its ability to harbor large library sizes. Here, we describe the first use of a grampositive host for display of a library of human antibody genes. The method allows for swift generation of binders by combining phage and gram-positive display, for its ease of use for screening, sorting and ranking by flow cytometry. We demonstrate the utility of this method by identifying specific low nanomolar scFv towards human HER2. The ranking and performance of the scFv isolated by flow sorting in surface immobilized form was retained when expressed as soluble scFv and analyzed by biolayer interferometry as well as after expression as full-length antibodies in mammalian cells. We also show the possibility to use gram-positive display to directly improve the affinity of the identified binders via an affinity maturation step using random mutagenesis and flow sorting. We believe this combined approach has the potential for a more complete scan of the antibody repertoire and for swift affinity maturation of human antibody formats.

  • 18.
    Hudson, Elton P.
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Nikoshkov, Andrej
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101). KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Automated Solid-Phase Subcloning Based on Beads Brought into Proximity by Magnetic Force2012Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, nr 5, s. e37429-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In the fields of proteomics, metabolic engineering and synthetic biology there is a need for high-throughput and reliable cloning methods to facilitate construction of expression vectors and genetic pathways. Here, we describe a new approach for solid-phase cloning in which both the vector and the gene are immobilized to separate paramagnetic beads and brought into proximity by magnetic force. Ligation events were directly evaluated using fluorescent-based microscopy and flow cytometry. The highest ligation efficiencies were obtained when gene- and vector-coated beads were brought into close contact by application of a magnet during the ligation step. An automated procedure was developed using a laboratory workstation to transfer genes into various expression vectors and more than 95% correct clones were obtained in a number of various applications. The method presented here is suitable for efficient subcloning in an automated manner to rapidly generate a large number of gene constructs in various vectors intended for high throughput applications.

  • 19.
    Hudson, Elton P.
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101). KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Multiplex epitope mapping using bacterial surface display reveals both linear and conformational epitopes2012Ingår i: Scientific Reports, ISSN 2045-2322, Vol. 2, s. 706-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    As antibody-based diagnosis and therapy grow at an increased pace, there is a need for methods which rapidly and accurately determine antibody-antigen interactions. Here, we report a method for the multiplex determination of antibody epitopes using bacterial cell-surface display. A protein-fragment library with 107 cell clones, covering 60 clinically-relevant protein targets, was created and characterized with massively parallel sequencing. Using this multi-target fragment library we determined simultaneously epitopes of commercial monoclonal and polyclonal antibodies targeting PSMA, EGFR, and VEGF. Off-target binding was observed for one of the antibodies, which demonstrates the method's ability to reveal cross-reactivity. We exemplify the detection of structural epitopes by mapping the therapeutic antibody Avastin. Based on our findings we suggest this method to be suitable for mapping linear and structural epitopes of monoclonal and polyclonal antibodies in a multiplex fashion and could find applicability in serum profiling as well as other protein-protein interaction studies.

  • 20.
    Häussler, Ragna S.
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinity Proteomics. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Bendes, Annika
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinity Proteomics. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Iglesias, Maria Jesus
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Cellulär och klinisk proteomik. KTH, Centra, Science for Life Laboratory, SciLifeLab. Division of Internal Medicine, University Hospital of North Norway, Tromsø, 9010, Norway.
    Sanchez-Rivera, Laura
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Cellulär och klinisk proteomik. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Dodig-Crnkovic, Tea
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinity Proteomics. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Byström, Sanna
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinity Proteomics. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Fredolini, Claudia
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinity Proteomics. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Birgersson, Elin
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinity Proteomics. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Dale, Matilda
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinity Proteomics. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Edfors, Fredrik
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Fagerberg, Linn
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Rockberg, Johan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Tegel, Hanna
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Uhlèn, Mathias
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi. KTH, Centra, Science for Life Laboratory, SciLifeLab. Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, 2970, Denmark.
    Qundos, Ulrika
    Atlas Antibodies AB, Bromma, 168 69, Sweden.
    Schwenk, Jochen M.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinity Proteomics. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Systematic Development of Sandwich Immunoassays for the Plasma Secretome2019Ingår i: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, artikel-id 1900008Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The plasma proteome offers a clinically useful window into human health. Recent advances from highly multiplexed assays now call for appropriate pipelines to validate individual candidates. Here, a workflow is developed to build dual binder sandwich immunoassays (SIA) and for proteins predicted to be secreted into plasma. Utilizing suspension bead arrays, ≈1800 unique antibody pairs are first screened against 209 proteins with recombinant proteins as well as EDTA plasma. Employing 624 unique antibodies, dilution-dependent curves in plasma and concentration-dependent curves of full-length proteins for 102 (49%) of the targets are obtained. For 22 protein assays, the longitudinal, interindividual, and technical performance is determined in a set of plasma samples collected from 18 healthy subjects every third month over 1 year. Finally, 14 of these assays are compared with with SIAs composed of other binders, proximity extension assays, and affinity-free targeted mass spectrometry. The workflow provides a multiplexed approach to screen for SIA pairs that suggests using at least three antibodies per target. This design is applicable for a wider range of targets of the plasma proteome, and the assays can be applied for discovery but also to validate emerging candidates derived from other platforms.

  • 21. Ko, Bong-Kook
    et al.
    Lee, Sook-Yeon
    Lee, Young-Ha
    Hwang, In-Sik
    Persson, Helena
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Borrebaeck, Carl
    Park, Dongeun
    Kim, Kyu-Tae
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Lee, Jong-Seo
    Combination of novel HER2-targeting antibody 1E11 with trastuzumab shows synergistic antitumor activity in HER2-positive gastric cancer2015Ingår i: Molecular Oncology, ISSN 1878-0261, Vol. 9, nr 2, s. 398-408Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The synergistic interaction of two antibodies targeting the same protein could be developed as an effective anti-cancer therapy. Human epidermal growth factor receptor 2 (HER2) is overexpressed in 20-25% of breast and gastric cancer patients, and HER2-targeted antibody therapy using trastuzumab is effective in many of these patients. Nonetheless, improving therapeutic efficacy and patient survival is important, particularly in patients with HER2-positive gastric cancer. Here, we describe the development of 1E11, a HER2-targeted humanized monoclonal antibody showing increased efficacy in a highly synergistic manner in combination with trastuzumab in the HER2-overexpressing gastric cancer cell lines NCI-N87 and OE-19. The two antibodies bind to sub-domain IV of the receptor, but have non-overlapping epitopes, allowing them to simultaneously bind HER2. Treatment with 1E11 alone induced apoptosis in HER2-positive cancer cells, and this effect was enhanced by combination treatment with trastuzumab. Combination treatment with 1E11 and trastuzumab reduced the levels of total HER2 protein and those of aberrant HER2 signaling molecules including phosphorylated HER3 and EGFR. The synergistic antitumor activity of 1E11 in combination with trastuzumab indicates that it could be a novel potent therapeutic antibody for the treatment of HER2-overexpressing gastric cancers.

  • 22.
    Kronqvist, Nina
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Malm, Magdalena
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hjelm, Barbara
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Staphylococcal surface display in combinatorial protein engineering and epitope mapping of antibodies2010Ingår i: Recent Patents on Biotechnology, ISSN 1872-2083, Vol. 4, nr 3, s. 171-182Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The field of combinatorial protein engineering for generation of new affinity proteins started in the mid 80s by the development of phage display. Although phage display is a prime example of a simple yet highly efficient method, manifested by still being the standard technique 25 years later, new alternative technologies are available today. One of the more successful new display technologies is cell display. Here we review the field of cell display for directed evolution purposes, with focus on a recently developed method employing Gram-positive staphylococci as display host. Patents on the most commonly used cell display systems and on different modifications as well as specific applications of these systems are also included. General strategies for selection of new affinity proteins from cell-displayed libraries are discussed, with detailed examples mainly from studies on the staphylococcal display system. In addition, strategies for characterization of recombinant proteins on the staphylococcal cell surface, with an emphasis on an approach for epitope mapping of antibodies, are included.

  • 23.
    Kronqvist, Nina
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hjelm, Barbara
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    New Ways for Discovery of Biopharmaceuticals: Emerging Techniques using Surface Display on Gram-positive Bacteria for Combinatorial Protein Engineering and Characterization2009Ingår i: Bioforum Europe, ISSN 1611-597X, Vol. 13, nr 6-7, s. 022-Artikel i tidskrift (Refereegranskat)
  • 24.
    Lindskog, Mats
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO).
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO).
    Sterky, Fredrik
    KTH, Skolan för bioteknologi (BIO).
    Selection of protein epitopes for antibody production2005Ingår i: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 38, nr 5, s. 723-727Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Protein functional analysis in the post-genomic era is a huge task that has to be approached by different methods in parallel. The use of protein-specific antibodies in conjunction with tissue microarrays has proven to be one important technology. In this study, we present a strategy for the optimized design of protein subfragments for subsequent antibody production. The fragments are selected based on a principle of lowest sequence similarity to other human proteins, optimally to generate antibodies with high selectivity. Furthermore, the fragments should have properties optimized for efficient protein production in Escherichia coli. The strategy has been implemented in Bishop, which is a Java-based software enabling the high-throughput production of protein fragments. Bishop allows for the avoidance of certain restriction enzyme sites, transmembrane regions, and signal peptides. A Basic Local Alignment Search Tool (BLAST) scanning procedure permits the selection of fragments of a selected size with a minimal sequence similarity to other proteins. The software and the strategy were evaluated on a human test data set and verified to fulfill the requested criteria.

  • 25.
    Lundqvist, Magnus
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Edfors, Fredrik
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Sivertsson, Åsa
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Hallström, Björn M
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Hudson, Elton P.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Tegel, Hanna
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Holmberg, Anders
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Solid-phase cloning for high-throughput assembly of single and multiple DNA parts2015Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 43, nr 7, artikel-id e49Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We describe solid-phase cloning (SPC) for high-throughput assembly of expression plasmids. Our method allows PCR products to be put directly into a liquid handler for capture and purification using paramagnetic streptavidin beads and conversion into constructs by subsequent cloning reactions. We present a robust automated protocol for restriction enzyme based SPC and its performance for the cloning of >60 000 unique human gene fragments into expression vectors. In addition, we report on SPC-based single-strand assembly for applications where exact control of the sequence between fragments is needed or where multiple inserts are to be assembled. In this approach, the solid support allows for head-to-tail assembly of DNA fragments based on hybridization and polymerase fill-in. The usefulness of head-to-tail SPC was demonstrated by assembly of >150 constructs with up to four DNA parts at an average success rate above 80%. We report on several applications for SPC and we suggest it to be particularly suitable for high-throughput efforts using laboratory workstations.

  • 26.
    Lundqvist, Magnus
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Thalén, Niklas
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Volk, Anna-Luisa
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Hansen, Henning Gram
    Tech Univ Denmark, Novo Nordisk Fdn Ctr Biosustainabil, Lyngby, Denmark..
    von Otter, Eric
    Nanyang Technol Univ, Sch Biol Sci, Singapore 637551, Singapore..
    Nygren, Per-Åke
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap. The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby, Denmark.
    Rockberg, Johan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Chromophore pre-maturation for improved speed and sensitivity of split-GFP monitoring of protein secretion2019Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, artikel-id 310Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Complementation-dependent fluorescence is a powerful way to study co-localization or interactions between biomolecules. A split-GFP variant, involving the self-associating GFP 1-10 and GFP 11, has previously provided a convenient approach to measure recombinant protein titers in cell supernatants. A limitation of this approach is the slow chromophore formation after complementation. Here, we alleviate this lag in signal generation by allowing the GFP 1-10 chromophore to mature on a solid support containing GFP 11 before applying GFP 1-10 in analyses. The pre-maturated GFP 1-10 provided up to 150-fold faster signal generation compared to the non-maturated version. Moreover, pre-maturated GFP 1-10 significantly improved the ability of discriminating between Chinese hamster ovary (CHO) cell lines secreting GFP 11-tagged erythropoietin protein at varying rates. Its improved kinetics make the pre-maturated GFP 1-10 a suitable reporter molecule for cell biology research in general, especially for ranking individual cell lines based on secretion rates of recombinant proteins.

  • 27.
    Lundqvist, Magnus
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Thalén, Niklas
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Volk, Anna-Luisa
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    von Otter, Eric
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Nygren, Per-Åke
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab. Technical University of Denmark, Denmark.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Chromophore prematuration for improved speed and sensitivity of split-GFP in vitro applicationsManuskript (preprint) (Övrigt vetenskapligt)
  • 28.
    Nilvebrant, Johan
    et al.
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Rockberg, Johan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    An introduction to epitope mapping2018Ingår i: Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029, Vol. 1785, s. 1-10Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Antibodies are protein molecules used routinely for therapeutic, diagnostic, and research purposes due to their exquisite ability to selectively recognize and bind a given antigen. The particular area of the antigen recognized by the antibody is called the epitope, and for proteinaceous antigens the epitope can be of complex nature. Information about the binding epitope of an antibody can provide important mechanistic insights and indicate for what applications an antibody might be useful. Therefore, a variety of epitope mapping techniques have been developed to localize such regions. Although the real picture is even more complex, epitopes in protein antigens are broadly grouped into linear or discontinuous epitopes depending on the positioning of the epitope residues in the antigen sequence and the requirement of structure. Specialized methods for mapping of the two different classes of epitopes, using high-throughput or high-resolution methods, have been developed. While different in their detail, all of the experimental methods rely on assessing the binding of the antibody to the antigen or a set of antigen mimics. Early approaches utilizing sets of truncated proteins, small numbers of synthesized peptides, and structural analyses of antibody-antigen complexes have been significantly refined. Current state-of-the-art methods involve combinations of mutational scanning, protein display, and high-throughput screening in conjunction with bioinformatic analyses of large datasets.

  • 29.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO).
    Methods for Generation and Characterization of Monospecific Antibodies2008Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Recent advances in biotechnology have generated possibilities to investigate and measure parts of life previously left for believers to explain. Utilizing the same book of recipes, the genome, our cells produce selections of proteins at a time and thereby niche into a multitude of specialized cell types, tissues and organs comprising our body. Knowledge of the precise protein composition in a given organ at normal and disease condition would be of invaluable importance, both for identification of disease causes and the design of new pharmaceuticals, as well as for a deeper understanding of the processes of life. This doctoral thesis describes the start and progress of a visionary project (HPR) to localize all human proteins in our body, with emphasis on the generation and characterization of antibodies used as protein targeting missiles. To facilitate the identification of one human protein in a complex environment like our body, it is of significant importance to have precise and specific means of detection. The first two papers (I-II), describe software developed for generation of monospecific antibodies satisfying such needs, using a set of rules for antigen optimization. Five years after project start a large amount of antibodies with documented characteristics have been generated. The third paper (III), illustrates an attempt to sieve these antibody characteristics to develop a tool, for further improvement of antigen selection, based on the correlation between antigen sequence and amount of specific antibody generated.Having a panel of protein-specific antibodies is a possession of a great value, not only for localization studies, but also as possible target-directed pharmaceuticals. In such cases, knowledge of the precise epitope recognized by the antibody on its target protein, is an important aid, both for understanding its effect as well as unwanted cross-reactivity. Paper (IV) describes the development of a high-resolution method for epitope mapping of antibodies using staphylococcal display. An application of the method is described in the last paper (V) where it is used to map an anti-HER2 monospecific antibody with growth-inhibiting effects on breast cancer cells. The monospecific antibody was fractionated into separate populations and five novel epitopes related to cancer cell growth-inhibition was determined.Altogether these methods are valuable tools for generation and characterization of monospecific antibodies.

  • 30.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Split-GFP and droplet microfluidics allows high-throughput screening of mammalian cell factories2016Ingår i: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 33, s. S51-S51Artikel i tidskrift (Refereegranskat)
  • 31.
    Rockberg, Johan
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Hjelm, Barbara
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Epitope mapping of antibodies using bacterial surface display2008Ingår i: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 5, nr 12, s. 1039-1045Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We describe a method for mapping the epitopes recognized by antibodies, based on bacterial surface expression of antigen protein fragments followed by antibody-based flow-cytometric sorting. We analyzed the binding sites of both monoclonal and polyclonal antibodies directed to three human protein targets: (i) the human epidermal growth factor receptor 2 (HER2), (ii) ephrin-B3 and (iii) the transcription factor SATB2. All monoclonal antibodies bound a single epitope, whereas the polyclonal antibodies showed, in each case, a binding pattern with one to five separate epitopes. A comparison of polyclonal and monoclonal antibodies raised to the same antigen showed overlapping binding epitopes. We also demonstrated that bacterial cells with displayed protein fragments can be used as affinity ligands to generate epitope-specific antibodies. Our approach shows a path forward for systematic validation of antibodies for epitope specificity and cross-reactivity on a whole-proteome level.

  • 32.
    Rockberg, Johan
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Hjelm, Barbara
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Epitope mapping using gram-positive surface display2010Ingår i: Current Protocols in Immunology, ISSN 1934-3671, nr SUPPL. 90, s. 9.9.1-9.9.17Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Antibodies have proven to be invaluable tools for a vast number of applications during the last decades, including protein purification and characterization, medical diagnosis and imaging, and treatment using therapeutic antibiotics. No matter what the aims of the application are, the antibodys binding characteristics will still be the main features determining the assays reliability. Here, we describe a protocol for determination of antibody-binding epitopes using an antigen-focused, library-based approach where library members are generated by fragmentation of antigen DNA and presented as cloned peptides on the cell surface of the Gram-positive bacterium Staphylococcus carnosus. The rigid cell structure of this organism allows for multivalent expression and permits rapid library analysis and sorting of antibody-binding cells using flow-sorting devices. Epitopes are determined by DNA sequencing of the sorted cells and alignment back to the antigen sequence. The protocol described here has been shown useful for mapping of both monoclonal and polyclonal binders with varying epitope lengths.

  • 33.
    Rockberg, Johan
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Schwenk, Jochen M.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Discovery of epitopes for targeting the human epidermal growth factor receptor 2 (HER2) with antibodies2009Ingår i: Molecular Oncology, ISSN 1574-7891, Vol. 3, nr 3, s. 238-247Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Antibodies have become valuable therapeutic agents for targeting of extracellular proteins in various diseases, including cancer, autoimmunity and cardiovascular disorders. For breast cancer, antibodies targeting the human HER2 have been shown to result in cell growth inhibition both in vitro and in patients with breast tumors. There is evidence to suggest that targeting multiple HER2 epitopes may result in increased growth inhibition making it interesting to find antibodies targeting new epitopes. Here, we report on a new scheme to discover antibodies directed to new epitopes using the extracellular domain of the HER2 as a model. Polyclonal antibodies were generated using recombinant protein fragments and affinity purified fractions of the antibodies were functionally characterized and precisely epitope mapped using bacterial surface display. Polyclonal antibodies towards a 127 amino acid recombinant protein fragment spanning between domains II and III of the HER2 were shown to bind to human ductal carcinoma cell line BT474 resulting in growth inhibition. Affinity purification demonstrated that antibodies to two separate regions from the N- and C-terminal end of the fragment exhibited the growth inhibition. Epitope mapping of the C-terminal antibodies revealed a 25 amino acid region (LPESFDGDPASNTAPLQPEQLQVF) with two distinct epitopes mediating efficient growth inhibition. The results suggest that antibodies directed towards this region of domain III of the HER2, distinct from the well-known monoclonal antibodies trastuzumab and pertuzumab, bind to the HER2 on living cells and exhibit growth inhibition. The work describes a new strategy to develop antibodies directed to non-overlapping epitopes and shows a path of pursuit to explore the epitope space of a target protein.

  • 34.
    Rockberg, Johan
    et al.
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Szigyarto, Cristina AI-Khalili
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Uhlén, Mathias
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Antigen selection for design of protein epitope signature tags2004Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 3, nr 10, s. S5-S5Artikel i tidskrift (Övrigt vetenskapligt)
  • 35.
    Rockberg, Johan
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Prediction of antibody response using recombinant human protein fragments as antigen2009Ingår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 18, nr 11, s. 2346-2355Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A great need exists for prediction of antibody response for the generation of antibodies toward protein targets. Earlier studies have suggested that prediction methods based on hydrophilicity propensity scale, in which the degree of exposure of the amino acid in an aqueous solvent is calculated, has limited value. Here, we show a comparative analysis based on 12,634 affinity-purified antibodies generated in a standardized manner against human recombinant protein fragments. The antibody response (yield) was measured and compared to theoretical predictions based on a large number (544) of published propensity scales. The results show that some of the scales have predictive power, although the overall Pearson correlation coefficient is relatively low (0.2) even for the best performing amino acid indices. Based on the current data set, a new propensity scale was calculated with a Pearson correlation coefficient of 0.25. The values correlated in some extent to earlier scales, including large penalty for hydrophobic and cysteine residues and high positive contribution from acidic residues, but with relatively low positive contribution from basic residues. The fraction of immunogens generating low antibody responses was reduced from 30% to around 10% if immunogens with a high propensity score (>0.48) were selected as compared to immunogens with lower scores (<0.29). The study demonstrates that a propensity scale might be useful for prediction of antibody response generated by immunization of recombinant protein fragments. The data set presented here can be used for further studies to design new prediction tools for the generation of antibodies to specific protein targets.

  • 36. Sastry, Anand
    et al.
    Monk, Jonathan
    Tegel, Hanna
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. Technical University of Denmark - DTU.
    Pålsson, Bernhard O.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Brunk, Elizabeth
    Machine learning in computational biology to accelerate high-throughput protein expression2017Ingår i: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 33, nr 16, s. 2487-2495Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Motivation: The Human Protein Atlas (HPA) enables the simultaneous characterization of thousands of proteins across various tissues to pinpoint their spatial location in the human body. This has been achieved through transcriptomics and high-throughput immunohistochemistry-based approaches, where over 40 000 unique human protein fragments have been expressed in E. coli. These datasets enable quantitative tracking of entire cellular proteomes and present new avenues for understanding molecular-level properties influencing expression and solubility. Results: Combining computational biology and machine learning identifies protein properties that hinder the HPA high-throughput antibody production pipeline. We predict protein expression and solubility with accuracies of 70% and 80%, respectively, based on a subset of key properties (aromaticity, hydropathy and isoelectric point). We guide the selection of protein fragments based on these characteristics to optimize high-throughput experimentation.

  • 37.
    Schwarz, Hubert
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Zhang, Ye
    KTH, Skolan för bioteknologi (BIO), Centra, Centrum för Bioprocessteknik, CBioPT.
    Zhan, Caijuan
    KTH, Skolan för teknikvetenskap (SCI). KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Malm, Magdalena
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Field, Raymond
    Biopharmaceutical Development, MedImmune, Cambridge, UK.
    Turner, Richard
    Biopharmaceutical Development, MedImmune, Cambridge, UK.
    Sellick, Christopher
    Biopharmaceutical Development, MedImmune, Cambridge, UK.
    Varley, Paul
    Biopharmaceutical Development, MedImmune, Cambridge, UK.
    Rockberg, Johan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Chotteau, Véronique
    KTH, Skolan för teknikvetenskap (SCI).
    Small-scale bioreactor supports high density HEK293 cell perfusion culture for the production of recombinant ErythropoietinManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Process intensification in mammalian cell culture-based recombinant protein production has been achieved by high cell density perfusion exceeding 108 cells/mL in the recent years. As the majority of therapeutic proteins are produced in Chinese Hamster Ovary (CHO) cells, intensified perfusion processes have been mainly developed for this type of host cell line. However, the use of CHO cells can result in non-human posttranslational modifications of the protein of interest, which may be disadvantageous compared with human cell lines.

    In this study, we developed a high cell density perfusion process of Human Embryonic Kidney (HEK293) cells producing recombinant human Erythropoietin (rhEPO). Firstly, a small-scale perfusion system from commercial bench-top screening bioreactors was developed for <250 mL working volume. Then, after the first trial runs with CHO cells, the system was modified for HEK293 cells (more sensitive than CHO cells) to achieve a higher oxygen transfer under mild aeration and agitation conditions. Steady states for medium (20 x 106 cells/mL) and high cell densities (80 x 106 cells/mL), normal process temperature (37 °C) and mild hypothermia (33 °C) as well as different cell specific perfusion rates (CSPR) from 10 to 60 pL/cell/day were applied to study the performance of the culture. The volumetric productivity was maximized for the high cell density steady state but decreased when an extremely low CSPR of 10 pL/cell/day was applied. The shift from high to low CSPR strongly reduced the nutrient uptake rates. The results from our study show that human cell lines, such as HEK293 can be used for intensified perfusion processes. 

  • 38.
    Sterky, Fredrik
    et al.
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Berglund, L.
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Lindskog, M.
    KTH, Skolan för bioteknologi (BIO).
    Rockberg, Johan
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Al-Khalili Szigyarto, Cristina
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Uhlén, Mathias
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Strategies and software for design of protein Epitopes within HPR2004Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 3, nr 10, s. S4-S4Artikel i tidskrift (Övrigt vetenskapligt)
  • 39.
    Szigyarto, C. Al-Khalili
    et al.
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Berglund, L.
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Sivertsson, Åsa
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Lindskog, M.
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Rockberg, J.
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Westberg, J.
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Agaton, L.
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Persson, A.
    KTH, Tidigare Institutioner (före 2005), Bioteknologi. Royal Inst Technol, Dept Mol Biotechnol, Stockholm, Sweden..
    Uhlén, Mathias
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Statistics of protein epitope signature tag design within the Swedish human proteom resource2004Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 3, nr 10, s. S91-S91Artikel i tidskrift (Övrigt vetenskapligt)
  • 40.
    Thul, Peter J.
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Åkesson, Lovisa
    KTH, Skolan för bioteknologi (BIO). KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Wiking, Mikaela
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Mahdessian, Diana
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Geladaki, A.
    Ait Blal, Hammou
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Alm, Tove L.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Asplund, A.
    Björk, Lars
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Breckels, L. M.
    Bäckström, Anna
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Danielsson, Frida
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Fagerberg, Linn
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Fall, Jenny
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Gatto, L.
    Gnann, Christian
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Hjelmare, Martin
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Johansson, Fredric
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Lee, Sunjae
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Lindskog, C.
    Mulder, J.
    Mulvey, C. M.
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Oksvold, Per
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Schutten, Rutger
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Sivertsson, Åsa
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Sjöstedt, E.
    Skogs, Marie
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Stadler, Charlotte
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Sullivan, Devin P.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Tegel, Hanna
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Winsnes, Casper F.
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Zhang, Cheng
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Zwahlen, Martin
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Mardinoglu, Adil
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Pontén, F.
    von Feilitzen, Kalle
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Lilley, K. S.
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Lundberg, Emma
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    A subcellular map of the human proteome2017Ingår i: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 356, nr 6340, artikel-id 820Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Resolving the spatial distribution of the human proteome at a subcellular level can greatly increase our understanding of human biology and disease. Here we present a comprehensive image-based map of subcellular protein distribution, the Cell Atlas, built by integrating transcriptomics and antibody-based immunofluorescence microscopy with validation by mass spectrometry. Mapping the in situ localization of 12,003 human proteins at a single-cell level to 30 subcellular structures enabled the definition of the proteomes of 13 major organelles. Exploration of the proteomes revealed single-cell variations in abundance or spatial distribution and localization of about half of the proteins to multiple compartments. This subcellular map can be used to refine existing protein-protein interaction networks and provides an important resource to deconvolute the highly complex architecture of the human cell.

  • 41.
    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 proteomics2005Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 4, nr 12, s. 1920-1932Artikel i tidskrift (Refereegranskat)
    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.

  • 42.
    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 proteome2015Ingår i: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 347, nr 6220, s. 1260419-Artikel i tidskrift (Refereegranskat)
    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.

  • 43.
    Volk, Anna Luisa
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Hu, Francis jingxin
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Epitope mapping of monoclonal and polyclonal antibodies using bacterial cell surface display2014Ingår i: Monoclonal antibodies, Humana Press, 2014, s. 485-500Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

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

  • 44.
    Volk, Anna-Luisa
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Hansen, Henning G.
    Lundqvist, Magnus
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Hammar, Petter
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Bai, Yunpeng
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Kol, Stefan
    Kildegaard, Helene F.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab. Technical University of Denmark, Denmark.
    Joensson, Haakan N.
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Droplet microfluidics and split-GFP complementation enable selection of Chinese hamster ovary cells with high specific productivity of therapeutic glycoproteinsManuskript (preprint) (Övrigt vetenskapligt)
  • 45.
    Volk, Anna-Luisa
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. Skolan för bioteknologi (BIO), KTH, Centra, Science for Life Laboratory, SciLifeLab, KTH Centrum för tillämpad proteomik (KCAP).
    Hu, Francis Jingxin
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. Skolan för bioteknologi (BIO), KTH, Centra, Science for Life Laboratory, SciLifeLab, KTH Centrum för tillämpad proteomik (KCAP).
    Berglund, Magnus M.
    Nordling, Erik
    Strömberg, Patrik
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH - Center for Applied Proteomics; Technical University of Denmark, Denmark.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH - Center for Applied Proteomics.
    Stratification of responders towards eculizumab using a structural epitope mapping strategy2016Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, artikel-id 31365Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The complement component 5 (C5)-binding antibody eculizumab is used to treat patients with paroxysmal nocturnal hemoglobinuria (PNH) and atypical haemolytic uremic syndrome (aHUS). As recently reported there is a need for a precise classification of eculizumab responsive patients to allow for a safe and cost-effective treatment. To allow for such stratification, knowledge of the precise binding site of the drug on its target is crucial. Using a structural epitope mapping strategy based on bacterial surface display, flow cytometric sorting and validation via haemolytic activity testing, we identified six residues essential for binding of eculizumab to C5. This epitope co-localizes with the contact area recently identified by crystallography and includes positions in C5 mutated in non-responders. The identified epitope also includes residue W917, which is unique for human C5 and explains the observed lack of cross-reactivity for eculizumab with other primates. We could demonstrate that Ornithodorus moubata complement inhibitor (OmCI), in contrast to eculizumab, maintained anti-haemolytic function for mutations in any of the six epitope residues, thus representing a possible alternative treatment for patients non-responsive to eculizumab. The method for stratification of patients described here allows for precision medicine and should be applicable to several other diseases and therapeutics.

  • 46.
    Volk, Anna-Luisa
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Hu, Francis Jingxin
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH).
    Rockberg, Johan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH).
    Epitope mapping of antibodies using bacterial cell surface display of gene fragment libraries2018Ingår i: Epitope Mapping Protocols, Humana Press, 2018, s. 141-157Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

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

  • 47.
    Volk, Anna-Luisa
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Ko, Bong-Kook
    Lundqvist, Magnus
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Lee, Hyun-Jong
    Frejd, Fredrik Y.
    Kim, Kyu-Tae
    Lee, Jong-Seo
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Bi-specific antibody molecule inhibits tumor cell proliferation more efficiently than the two-molecule combinationManuskript (preprint) (Övrigt vetenskapligt)
  • 48.
    Volk, Anna-Luisa
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Rockberg, Johan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Discontinuous epitope mapping of antibodies using bacterial cell surface display of folded domains2018Ingår i: Epitope Mapping Protocols, Humana Press, 2018, s. 159-183Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

    Knowledge of the exquisite-binding surface of an antibody on its target protein is of great value, in particular for therapeutic antibodies for understanding method of action and for stratification of patients carrying the necessary epitope for desired drug efficacy, but also for capture assays under native conditions. Several epitope mapping methodologies have been described for this purpose, with the laborious X-ray crystallography method being the ideal method for mapping of discontinuous epitopes in antibody-antigen crystal complexes and high-throughput peptide-based methods for mapping of linear epitopes. We here report on the usage of a bacterial surface display-based method for mapping of structural epitopes by display of folded domains on the surface of Gram positive bacteria, followed by domain-targeted mutagenesis and library analysis for the identification of key-residues by flow sorting and sequencing. Identified clones with reduced affinity are validated by single clone FACS and subsequent full-length expression in mammalian cells for validation.

  • 49.
    Zhan, Caijuan
    et al.
    KTH, Skolan för teknikvetenskap (SCI). KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Hubert, Schwarz
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Malm, Magdalena
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Sellick, Christopher
    Rockberg, Johan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Chotteau, Véronique
    KTH, Skolan för teknikvetenskap (SCI). KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Hydrodynamic shear stress in hollow filter for perfusion culture of human cellsManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    High cell density perfusion process is an economical way to produce biopharmaceuticals at high yield. To achieve high density of healthy cells, the cell culture conditions should be free from mechanically detriment. Human embryonic kidney (HEK) K293 cells, interesting for the production of therapeutic glycoproteins, are known as shear sensitive. In order to obtain the optimal hydrodynamics conditions with reduced mechanical damage, we investigated the fact of the shear stress compatible with HEK293 cells. We reviewed hollow filter based tangential flow filtration strategies, tangential flow filtration (TFF) and alternating tangential flow filtration (ATF). We studied shear stress introduced by these two flow filtration methods. By theoretical study, we obtained that lower shear stress introduced by alternating tangential flow filtration result in lower average shear stress comparing to tangential flow filtration with same flow rate.  In our experimental runs, we achieved different shear stress levels by applying different flow rates. 5-Days batch cultivations were performed to examine the influence of shear stress on cell growing and metabolic behaviour. We identified that the shear stress potentially reduce the growth rate and productivity of HEK293 cells and found the cell metabolism associated with shear stress levels. By documenting these cell responses to shear stress, we confirmed our theoretical results and could further optimize the hydrodynamic conditions for perfusion process of HEK 293 cells. 

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