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Lundqvist, M., Thalén, N., Volk, A.-L., Hansen, H. G., von Otter, E., Nygren, P.-Å., . . . Rockberg, J. (2019). Chromophore pre-maturation for improved speed and sensitivity of split-GFP monitoring of protein secretion. Scientific Reports, 9, Article ID 310.
Open this publication in new window or tab >>Chromophore pre-maturation for improved speed and sensitivity of split-GFP monitoring of protein secretion
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2019 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 310Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2019
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-243949 (URN)10.1038/s41598-018-36559-x (DOI)000456282100065 ()30670736 (PubMedID)2-s2.0-85060382656 (Scopus ID)
Note

QC 20190305

Available from: 2019-03-05 Created: 2019-03-05 Last updated: 2019-03-05Bibliographically approved
Hjelm, L. C., Ninebrant, J., Nygren, P.-Å., Nilsson, A. S. & Seijsing, J. (2019). Lysis of Staphylococcal Cells by Modular Lysin Domains Linked via a n-covalent Barnase-Barstar Interaction Bridge. Frontiers in Microbiology, 10, Article ID 558.
Open this publication in new window or tab >>Lysis of Staphylococcal Cells by Modular Lysin Domains Linked via a n-covalent Barnase-Barstar Interaction Bridge
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2019 (English)In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 10, article id 558Article in journal (Refereed) Published
Abstract [en]

Bacteriophage endolysins and bacterial exolysins are capable of enzymatic degradation of the cell wall peptidoglycan layer and thus show promise as a new class of antimicrobials. Both exolysins and endolysins often consist of different modules, which are responsible for enzymatic functions and cell wall binding, respectively. Individual modules from different endo- or exolysins with different binding and enzymatic activities, can via gene fusion technology be re-combined into novel variants for investigations of arrangements of potential clinical interest. The aim of this study was to investigate if separately produced cell wall binding and enzyme modules could be assembled into a functional lysin via a non-covalent affinity interaction bridge composed of the barnase ribonuclease from Bacillus amyloliquefaciens and its cognate inhibitor barstar, known to form a stable heterodimeric complex. In a proof-of-principle study, using surface plasmon resonance, flow cytometry and turbidity reduction assays, we show that separately produced modules of a lysin cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) from Staphylococcus aureus bacteriophage K endolysin (LysK) fused to barnase and a cell wall binding Src homology 3 domain (SH3b) from the S. simulans exolysin lysostaphin fused to barstar can be non-covalently assembled into a functional lysin showing both cell wall binding and staphylolytic activity. We hypothesize that the described principle for assembly of functional lysins from separate modules through appended hetero-dimerization domains has a potential for investigations of also other combinations of enzymatically active and cell wall binding domains for desired applications.

Place, publisher, year, edition, pages
Frontiers Media SA, 2019
Keywords
endolysin, exolysin, barnase, barstar, fusion protein, non-covalent interaction, Staphylococcus, antibiotic alternative
National Category
Microbiology
Identifiers
urn:nbn:se:kth:diva-248331 (URN)10.3389/fmicb.2019.00558 (DOI)000461983900001 ()
Note

QC 20190409

Available from: 2019-04-09 Created: 2019-04-09 Last updated: 2019-04-09Bibliographically approved
Subramanian, K., Neill, D. R., Malak, H. A., Spelmink, L., Khandaker, S., Marchiori, G. D., . . . Henriques-Normark, B. (2019). Pneumolysin binds to the mannose receptor C type 1 (MRC-1) leading to anti-inflammatory responses and enhanced pneumococcal survival. Nature Microbiology, 4(1), 62-70
Open this publication in new window or tab >>Pneumolysin binds to the mannose receptor C type 1 (MRC-1) leading to anti-inflammatory responses and enhanced pneumococcal survival
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2019 (English)In: Nature Microbiology, E-ISSN 2058-5276, Vol. 4, no 1, p. 62-70Article in journal (Refereed) Published
Abstract [en]

Streptococcus pneumoniae (the pneumococcus) is a major cause of mortality and morbidity globally, and the leading cause of death in children under 5 years old. The pneumococcal cytolysin pneumolysin (PLY) is a major virulence determinant known to induce pore-dependent pro-inflammatory responses. These inflammatory responses are driven by PLY-host cell membrane cholesterol interactions, but binding to a host cell receptor has not been previously demonstrated. Here, we discovered a receptor for PLY, whereby pro-inflammatory cytokine responses and Toll-like receptor signalling are inhibited following PLY binding to the mannose receptor C type 1 (MRC-1) in human dendritic cells and mouse alveolar macrophages. The cytokine suppressor SOCS1 is also upregulated. Moreover, PLY-MRC-1 interactions mediate pneumococcal internalization into non-lysosomal compartments and polarize naive T cells into an interferon-gamma(low), interleukin-4(high) and FoxP3(+) immunoregulatory phenotype. In mice, PLY-expressing pneumococci colocalize with MRC-1 in alveolar macrophages, induce lower pro-inflammatory cytokine responses and reduce neutrophil infiltration compared with a PLY mutant. In vivo, reduced bacterial loads occur in the airways of MRC-1-deficient mice and in mice in which MRC-1 is inhibited using blocking antibodies. In conclusion, we show that pneumococci use PLY-MRC-1 interactions to downregulate inflammation and enhance bacterial survival in the airways. These findings have important implications for future vaccine design.

Place, publisher, year, edition, pages
Nature Publishing Group, 2019
National Category
Microbiology
Identifiers
urn:nbn:se:kth:diva-240700 (URN)10.1038/s41564-018-0280-x (DOI)000453056100011 ()30420782 (PubMedID)2-s2.0-85056488264 (Scopus ID)
Funder
Swedish Research CouncilSwedish Foundation for Strategic Research Knut and Alice Wallenberg Foundation
Note

QC 20190110

Available from: 2019-01-10 Created: 2019-01-10 Last updated: 2019-01-10Bibliographically approved
Hafstrand, I., Sayitoglu, E. C., Apavaloaei, A., Josey, B. J., Sun, R., Han, X., . . . Achour, A. (2019). Successive crystal structure snapshots suggest the basis for MHC class I peptide loading and editing by tapasin. Proceedings of the National Academy of Sciences of the United States of America, 116(11), 5055-5060
Open this publication in new window or tab >>Successive crystal structure snapshots suggest the basis for MHC class I peptide loading and editing by tapasin
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2019 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 116, no 11, p. 5055-5060Article in journal (Refereed) Published
Abstract [en]

MHC-I epitope presentation to CD8(+) T cells is directly dependent on peptide loading and selection during antigen processing. However, the exact molecular bases underlying peptide selection and binding by MHC-I remain largely unknown. Within the peptide-loading complex, the peptide editor tapasin is key to the selection of MHC-I-bound peptides. Here, we have determined an ensemble of crystal structures of MHC-I in complex with the peptide exchange-associated dipeptide GL, as well as the tapasin-associated scoop loop, alone or in combination with candidate epitopes. These results combined with mutation analyses allow us to propose a molecular model underlying MHC-I peptide selection by tapasin. The N termini of bound peptides most probably bind first in the N-terminal and middle region of the MHC-I peptide binding cleft, upon which the peptide C termini are tested for their capacity to dislodge the tapasin scoop loop from the F pocket of the MHC-I cleft. Our results also indicate important differences in peptide selection between different MHC-I alleles.

Place, publisher, year, edition, pages
NATL ACAD SCIENCES, 2019
Keywords
MHC-I, tapasin, peptide editing, TAPBPR
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-247808 (URN)10.1073/pnas.1807656116 (DOI)000460911500051 ()30808808 (PubMedID)2-s2.0-85062827860 (Scopus ID)
Note

QC 20190401

Available from: 2019-04-01 Created: 2019-04-01 Last updated: 2019-04-01Bibliographically approved
Yu, F., Alesand, V. & Nygren, P.-Å. (2018). Site-Specific Photoconjugation of Beta-Lactamase Fragments to Monoclonal Antibodies Enables Sensitive Analyte Detection via Split-Enzyme Complementation. Biotechnology Journal, 13(7), Article ID 1700688.
Open this publication in new window or tab >>Site-Specific Photoconjugation of Beta-Lactamase Fragments to Monoclonal Antibodies Enables Sensitive Analyte Detection via Split-Enzyme Complementation
2018 (English)In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 13, no 7, article id 1700688Article in journal (Refereed) Published
Abstract [en]

Protein fragment complementation assays (PCA) rely on a proximity-driven reconstitution of a split reporter protein activity, typically via interaction between bait and prey units separately fused to the reporter protein halves. The PCA principle can also be formatted for use in immunossays for analyte detection, e.g., via the use of small immunoglobulin binding proteins (IgBp) as fusion partners to split-reporter protein fragments for conversion of pairs of antibodies into split-protein half-probes. However, the non-covalent binding between IgBp and antibodies is not ideal for development of robust assays. Here, the authors describe how split-enzyme reporter halves can be both site-specifically and covalently photoconjugated at antibody Fc-parts for use in homogeneous dual-antibody in vitro immunoassays based on analyte-dependent split-enzyme fragment complementation. The half-probes consist of parts of a beta-lactamase split-protein reporter fused to an immunoglobulin Fc binding domain equipped with a unique cysteine residue at which a photoactivable maleimide benzophenone group (MBP) is attached. Using such antibody conjugates the authors obtain an analyte-driven complementation of the reporter enzyme fragments monitored via conversion of a chromogenic substrate. Results from detection of human interferon-gamma and the extracellular domain of HER2 is shown. The described principles for site-specific conjugation of proteins to antibodies should be broadly applicable.

Place, publisher, year, edition, pages
WILEY-V C H VERLAG GMBH, 2018
Keywords
affibody, antibody, complementation, homogeneous assay, nitrocefin, photoconjugation, split-beta-lactamase
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-232401 (URN)10.1002/biot.201700688 (DOI)000437281600007 ()29485240 (PubMedID)2-s2.0-85043698701 (Scopus ID)
Funder
VINNOVA
Note

QC 20180726

Available from: 2018-07-26 Created: 2018-07-26 Last updated: 2018-07-26Bibliographically approved
Ståhl, S., Gräslund, T., Eriksson Karlström, A., Frejd, F. Y., Nygren, P.-Å. & Löfblom, J. (2017). Affibody Molecules in Biotechnological and Medical Applications. Trends in Biotechnology, 35(8), 691-712
Open this publication in new window or tab >>Affibody Molecules in Biotechnological and Medical Applications
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2017 (English)In: Trends in Biotechnology, ISSN 0167-7799, E-ISSN 1879-3096, Vol. 35, no 8, p. 691-712Article, review/survey (Refereed) Published
Abstract [en]

Affibody molecules are small (6.5-kDa) affinity proteins based on a three-helix bundle domain framework. Since their introduction 20 years ago as an alternative to antibodies for biotechnological applications, the first therapeutic affibody molecules have now entered clinical development and more than 400 studies have been published in which affibody molecules have been developed and used in a variety of contexts. In this review, we focus primarily on efforts over the past 5 years to explore the potential of affibody molecules for medical applications in oncology, neurodegenerative, and inflammation disorders, including molecular imaging, receptor signal blocking, and delivery of toxic payloads. In addition, we describe recent examples of biotechnological applications, in which affibody molecules have been exploited as modular affinity fusion partners.

Place, publisher, year, edition, pages
Elsevier, 2017
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-211597 (URN)10.1016/j.tibtech.2017.04.007 (DOI)000405847200005 ()
Note

QC 20170815

Available from: 2017-08-15 Created: 2017-08-15 Last updated: 2017-08-15Bibliographically approved
Allerbring, E. B., Duru, A. D., Chadderton, J., Markov, N., Uchtenhagen, H., Popov, A., . . . Achour, A. (2015). Structural and thermodynamic basis underlying in vivo reestablishment of T-cell recognition of a viral escape mutant. Paper presented at 8th International Workshop on Antigen Processing and Presentation, JUN 10-13, 2014, Philadelphia, PA. Molecular Immunology, 68(2), 151-151
Open this publication in new window or tab >>Structural and thermodynamic basis underlying in vivo reestablishment of T-cell recognition of a viral escape mutant
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2015 (English)In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 68, no 2, p. 151-151Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
Elsevier, 2015
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-180623 (URN)000366767700073 ()
Conference
8th International Workshop on Antigen Processing and Presentation, JUN 10-13, 2014, Philadelphia, PA
Note

QC 20160120

Available from: 2016-01-20 Created: 2016-01-19 Last updated: 2017-11-30Bibliographically approved
Sekiguchi, M., Iwasaki, T., Shibasaki, S., Nygren, P.-Å., Gräslund, T. & Sano, H. (2014). Affibody Molecules Inhibiting The Interaction Between Ras And Raf Suppress The Proliferation And The Production Of Inflammatory Mediators By Synovial Cells. Paper presented at 15th Annual European Congress of Rheumatology (EULAR), JUN 11-14, 2014, Paris, France. Annals of the Rheumatic Diseases, 73, 848-848
Open this publication in new window or tab >>Affibody Molecules Inhibiting The Interaction Between Ras And Raf Suppress The Proliferation And The Production Of Inflammatory Mediators By Synovial Cells
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2014 (English)In: Annals of the Rheumatic Diseases, ISSN 0003-4967, E-ISSN 1468-2060, Vol. 73, p. 848-848Article in journal, Meeting abstract (Other academic) Published
National Category
Rheumatology and Autoimmunity
Identifiers
urn:nbn:se:kth:diva-169164 (URN)10.1136/annrheumdis-2014-eular.2553 (DOI)000346919804447 ()
Conference
15th Annual European Congress of Rheumatology (EULAR), JUN 11-14, 2014, Paris, France
Note

QC 20150612

Available from: 2015-06-12 Created: 2015-06-11 Last updated: 2017-12-04Bibliographically approved
Yu, F., Gudmundsdotter, L., Akal, A., Gunneriusson, E., Frejd, F. & Nygren, P.-Å. (2014). An affibody-adalimumab hybrid blocks combined IL-6 and TNF-triggered serum amyloid A secretion in vivo. mAbs, 6(6), 1598-1607
Open this publication in new window or tab >>An affibody-adalimumab hybrid blocks combined IL-6 and TNF-triggered serum amyloid A secretion in vivo
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2014 (English)In: mAbs, ISSN 1942-0862, Vol. 6, no 6, p. 1598-1607Article in journal (Refereed) Published
Abstract [en]

In inflammatory disease conditions, the regulation of the cytokine system is impaired, leading to tissue damages. Here, we used protein engineering to develop biologicals suitable for blocking a combination of inflammation driving cytokines by a single construct. From a set of interleukin (IL)-6-binding affibody molecules selected by phage display, five variants with a capability of blocking the interaction between complexes of soluble IL-6 receptor a (sIL-6R alpha) and IL6 and the co-receptor gp130 were identified. In cell assays designed to analyze any blocking capacity of the classical or the alternative (trans) signaling IL-6 pathways, one variant, Z(IL-6_13) with an affinity (K-D) for IL-6 of similar to 500 pM, showed the best performance. To construct fusion proteins ("AffiMabs") with dual cytokine specificities, Z(IL-6_13) was fused to either the N-or C-terminus of both the heavy and light chains of the anti-tumor necrosis factor (TNF) monoclonal antibody adalimumab (Humira (R)). One AffiMab construct with Z(IL-6_13) positioned at the N-terminus of the heavy chain, denoted Z(IL-6_13)-HCAda, was determined to be the most optimal, and it was subsequently evaluated in an acute Serum Amyloid A (SAA) model in mice. Administration of the AffiMab or adalimumab prior to challenge with a mix of IL-6 and TNF reduced the levels of serum SAA in a dose-dependent manner. Interestingly, the highest dose (70 mg/kg body weight) of adalimumab only resulted in a 50% reduction of SAA-levels, whereas the corresponding dose of the Z(IL-6_13)-HCAda AffiMab with combined IL-6/TNF specificity, resulted in SAA levels below the detection limit.

Keywords
AffiMab, Antibody, IL-6, TNF, adalimumab, affibody, affinity, fusion, inflammation
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-159385 (URN)10.4161/mabs.36089 (DOI)000346878600024 ()2-s2.0-84920827599 (Scopus ID)
Funder
Swedish Research Council
Note

QC 20150203

Available from: 2015-02-03 Created: 2015-01-29 Last updated: 2015-04-16Bibliographically approved
Shibasaki, S., Karasaki, M., Gräslund, T., Nygren, P.-Å., Sano, H. & Iwasaki, T. (2014). Inhibitory effects of H-Ras/Raf-1–binding affibody molecules on synovial cell function. AMB Express, 4(82)
Open this publication in new window or tab >>Inhibitory effects of H-Ras/Raf-1–binding affibody molecules on synovial cell function
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2014 (English)In: AMB Express, ISSN 2191-0855, E-ISSN 2191-0855, Vol. 4, no 82Article in journal (Refereed) Published
Abstract [en]

Affibody molecules specific for H-Ras and Raf-1 were evaluated for their ability to inhibit synovial cell function. Affibody molecules targeting H-Ras (Zras122, Zras220, and Zras521) or Raf-1 (Zraf322) were introduced into the MH7A synovial cell line using two delivery methods: transfection with plasmids encoding the affibody molecules or direct introduction of affibody protein using a cell-penetrating peptide reagent. Interleukin-6 (IL-6) and prostaglandin E2 (PGE2) production by MH7A cells were analyzed by enzyme-linked immunosorbent assay after stimulation with tumor necrosis factor-alpha (TNF-α). Cell proliferation was also analyzed. Phosphorylation of extracellular signal-regulated kinase (ERK) was analyzed by western blot. All affibody molecules could inhibit IL-6 and PGE2 production in TNF-α-stimulated MH7A cells. The inhibitory effect was stronger when affibody molecules were delivered as proteins via a cell-penetrating peptide reagent than when plasmid-DNA encoding the affibody moelcules was transfected into the cells. Plasmid-expressed Zras220 inhibited phosphorylation of ERK in TNF-α-stimulated MH7A cells. Protein-introduced Zraf322 inhibited the production of IL-6 and PGE2 and inhibited cell proliferation in MH7A cells. These findings suggest that affibody molecules specific for H-Ras and Raf-1 can affect intracellular signal transduction through the MAP kinase pathway to inhibit cell proliferation and production of inflammatory mediators by synovial cells.

Place, publisher, year, edition, pages
Springer Berlin/Heidelberg, 2014
Keywords
affinity cancer
National Category
Biological Sciences
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-164186 (URN)10.1186/s13568-014-0082-3 (DOI)000358068500001 ()2-s2.0-84957590363 (Scopus ID)
Note

QC 20150420

Available from: 2015-04-14 Created: 2015-04-14 Last updated: 2017-12-04Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-4214-6991

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