| 1. |
- Hjelm, Linnea C., et al.
(författare)
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Lysis of Staphylococcal Cells by Modular Lysin Domains Linked via a n-covalent Barnase-Barstar Interaction Bridge
- 2019
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Ingår i: Frontiers in Microbiology. - : Frontiers Media SA. - 1664-302X. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Seijsing, Fredrik, 1988-, et al.
(författare)
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Improved bacteria-phagocyte interaction by means of a fusion protein binding Staphylococcus peptidoglycan and Immunoglobulin G
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The immune system plays an important role in the body´s defense against bacterial infections. Here, we hypothesize that adding an artificial opsonin that binds to antigens on the surface of infecting bacteria as well as to endogenous IgG can increase the interaction between bacteria and immune cells. A chimeric protein was made by fusion of the Src homology domain 3b (SH3b) from the Staphylococcus phage K endolysin (LysK) and the IgG-binding C2 domain from Streptococcus Protein G. SH3b binds to the bacterial cell wall of staphylococci and the C2 domain to the Fab region of the IgG which in turn binds to the Fc receptors of the phagocytes, facilitating interaction between phagocytes and bacteria. Comparative experiments with and without the chimeric protein showed that it both increased the amount of Staphylococcus carnosus cells bound by a humanized monoclonal IgG1 with unrelated specificity and increased the interaction of phagocytes with bacteria. The results justify development of chimeric proteins with the ability to act as artificial opsonins since these possibly can become an addition to future treatments of infections caused by antibiotic resistant bacteria.
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| 3. |
- Seijsing, Fredrik, et al.
(författare)
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Recombinant spider silk coatings functionalized with enzymes targeting bacteria and biofilms
- 2020
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Ingår i: MicrobiologyOpen. - : Wiley. - 2045-8827. ; 9:4
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Tidskriftsartikel (refereegranskat)abstract
- Bacteria forming biofilms on surgical implants is a problem that might be alleviated by the use of antibacterial coatings. In this article, recombinant spider silk was functionalized with the peptidoglycan degrading endolysin SAL-1 from the staphylococcal bacteriophage SAP-1 and the biofilm-matrix-degrading enzyme Dispersin B from Aggregatibacter actinomycetemcomitans using direct genetic fusion and/or covalent protein–protein fusion catalyzed by Sortase A. Spider silk assembly and enzyme immobilization was monitored using quartz crystal microbalance analysis. Enzyme activity was investigated both with a biochemical assay using cleavage of fluorescent substrate analogues and bacterial assays for biofilm degradation and turbidity reduction. Spider silk coatings functionalized with SAL-1 and Disperin B were found to exhibit bacteriolytic effect and inhibit biofilm formation, respectively. The strategy to immobilize antibacterial enzymes to spider silk presented herein show potential to be used as surface coatings of surgical implants and other medical equipment to avoid bacterial colonization.
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| 4. |
- Seijsing, Fredrik, 1988-, et al.
(författare)
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Structure prediction of endolysin SU57e
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Endolysins are used by bacteriophages to break down the cell wall of the bacterial host towards the end of the infection cycle. Endolysins are interesting options for the creation of antibacterial substances and domains from endolysins can be combined with domains from other proteins to create antibacterial constructs. This article aims to find the tertiary structure and the enzymatically active site of the endolysin SU57e. This is done through the use of bioinformatics software, comparative analysis with other endolysins, and attempts at producing and purifying the protein for crystallization. SU57e is encoded by the phage vB_EcoD_SU57 (SU57) previously characterized by Koonjan et al. Bioinformatics analysis indicates that the structure is similar to that of endolysin R21 presented by Sun et al. SU57e also appears to have a signal-anchor-release-domain similar to that of R21.
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| 5. |
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| 6. |
- Liu, Hao, et al.
(författare)
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Target-specific cytotoxic effects on HER2-expressing cells by the tripartite fusion toxin Z(HER2:2891)-ABD-PE38X8, including a targeting affibody molecule and a half-life extension domain
- 2015
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Ingår i: International Journal of Oncology. - : Spandidos Publications. - 1019-6439 .- 1791-2423. ; 47:2, s. 601-609
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Tidskriftsartikel (refereegranskat)abstract
- Development of cancer treatment regimens including immunotoxins is partly hampered by their immunogenicity. Recently, deimmunized versions of toxins have been described, potentially being better suited for translation to the clinic. In this study, a recombinant tripartite fusion toxin consisting of a deimmunized version of exotoxin A from Pseudomonas aeruginosa (PE38) genetically fused to an affibody molecule specifically interacting with the human epidermal growth factor receptor 2 (HER2), and also an albumin binding domain (ABD) for half-life extension, has been produced and characterized in terms of functionality of the three moieties. Biosensor based assays showed that the fusion toxin was able to interact with human and mouse serum albumin, but not with bovine serum albumin and that it interacted with HER2 (K-D=5 nM). Interestingly, a complex of the fusion toxin and human serum albumin also interacted with HER2 but with a somewhat weaker affinity (K-D=12 nM). The IC50-values of the fusion toxin ranged from 6 to 300 pM on SKOV-3, SKBR-3 and A549 cells and was lower for cells with higher surface densities of HER2. The fusion toxin was found specific for HER2 as shown by blocking available HER2 receptors with free affibody molecule before subjecting the cells to the toxin. Analysis of contact time showed that 10 min was sufficient to kill 50% of the cells. In conclusion, all three regions of the fusion toxin were found to be functional.
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| 7. |
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| 8. |
- Nilebäck, Linnea, et al.
(författare)
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Bioactive Silk Coatings Reduce the Adhesion of Staphylococcus aureus while Supporting Growth of Osteoblast-like Cells
- 2019
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Ingår i: ACS Applied Materials and Interfaces. - : American Chemical Society (ACS). - 1944-8244 .- 1944-8252. ; 11:28, s. 24999-25007
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Tidskriftsartikel (refereegranskat)abstract
- Orthopedic and dental implants are associated with a substantial risk of failure due to biomaterial-associated infections and poor osseointegration. To prevent such outcomes, a coating can be applied on the implant to ideally both reduce the risk of bacterial adhesion and support establishment of osteoblasts. We present a strategy to construct dual-functional silk coatings with such properties. Silk coatings were made from a recombinant partial spider silk protein either alone (silk(wt)) or fused with a cell-binding motif derived from fibronectin (FN-silk). The biofilm-dispersal enzyme Dispersin B (DspB) and two peptidoglycan degrading endolysins, PlySs2 and SAL-1, were produced recombinantly. A sortase recognition tag (SrtTag) was included to allow site-specific conjugation of each enzyme onto silk(wt) and FN-silk coatings using an engineered variant of the transpeptidase Sortase A (SrtA*). To evaluate bacterial adhesion on the samples, Staphylococcus aureus was incubated on the coatings and subsequently subjected to live/dead staining. Fluorescence microscopy revealed a reduced number of bacteria on all silk coatings containing enzymes. Moreover, the bacteria were mobile to a higher degree, indicating a negative influence on the bacterial adhesion. The capability to support mammalian cell interactions was assessed by cultivation of the osteosarcoma cell line U-2 OS on dual-functional surfaces, prepared by conjugating the enzymes onto FN-silk coatings. U-2 OS cells could adhere to silk coatings with enzymes and showed high spreading and viability, demonstrating good cell compatibility.
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| 9. |
- Seijsing, Johan, et al.
(författare)
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An engineered affibody molecule with pH-dependent binding to FcRn mediates extended circulatory half-life of a fusion protein
- 2014
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 111:48, s. 17110-17115
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Tidskriftsartikel (refereegranskat)abstract
- Proteins endocytosed from serum are degraded in the lysosomes. However, serum albumin (SA) and IgG, through its Fc part, bind to the neonatal Fc receptor (FcRn) at low pH in the endosome after endocytosis, and are transported back to the cellular surface, where they are released into the bloodstream, resulting in an extended serum circulation time. Association with Fc or SA has been used to prolong the in vivo half-life of biopharmaceuticals, using the interaction with FcRn to improve treatment regimens. This has been achieved either directly, by fusion or conjugation to Fc or SA, or indirectly, using SA-binding proteins. The present work takes this principle one step further, presenting small affinity proteins that bind directly to FcRn, mediating extension of the serum half-life of fused biomolecules. Phage display technology was used to select affibody molecules that can bind to FcRn in the pH-dependent manner required for rescue by FcRn. The biophysical and binding properties were characterized in vitro, and the affibody molecules were found to bind to FcRn more strongly at low pH than at neutral pH. Attachment of the affibody molecules to a recombinant protein, already engineered for increased halflife, resulted in a nearly threefold longer half-life in mice. These tags should have general use as fusion partners to biopharmaceuticals to extend their half-lives in vivo.
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| 10. |
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| 11. |
- Seijsing, Johan, et al.
(författare)
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In vivo depletion of serum IgG by an affibody molecule binding the neonatal Fc receptor
- 2018
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Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Lowering the total level of Immunoglobulin G (IgG) in circulation is a promising general treatment option for many autoimmune diseases driven by pathogenic autoantibodies. The half-life of IgG in circulation is unusually long as a consequence of its interaction with the neonatal Fc receptor (FcRn), which protects it from lysosomal degradation by cells in contact with blood. Blocking the IgG/FcRn interaction prevents FcRn-mediated rescue, which may lead to increased catabolism and a lowering of the total IgG level. Here, we find that an engineered alternative scaffold protein, an affibody molecule, interacting specifically with FcRn, is able to block the IgG/FcRn interaction in vitro. The affibody molecule (Z(FcRn)) was expressed alone or as a fusion to an albumin binding domain (ABD), to extend its half-life in circulation, in both cases with retained affinity and blocking potential. Repeated i.v. injections in mice of Z(FcRn) and Z(FcRn)-ABD were found to result in an up to 40% reduction of the IgG serum-level after 5 days. Potential applications of Z(FcRn) as a general treatment modality for autoimmune diseases are discussed.
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| 12. |
- Seijsing, Johan, 1986-
(författare)
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Rational and Combinatorial Engineering of Affinity Proteins Towards Therapeutical Applications
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Protein engineering has had an immense impact on the development of biological drugs, including replacement therapies with engineered versions of insulin or factor VIII to treat diabetes or bleeding disorders, and monoclonal antibodies to treat cancer and various other malignancies. Now, with the next generation of treatment modalities coming up, including monoclonal reagents based on alternative scaffolds, gene and cell therapies, the importance of protein engineering to tailor-make these treatments is likely to increase further.The neonatal Fc receptor (FcRn) is widely expressed in the body. One of the receptor's interesting functions is to rescue immunoglobulin G (IgG) and serum albumin (SA) from degradation by cells in contact with blood. When serum proteins are endocytosed by a cell, they are transported via the endosome to the lysosome for degradation. However, IgG and SA associate with the FcRn already at the slightly acidic pH of the endosome followed by transport back to the cell surface. There the complex encounters the neutral pH of the blood, at which the binding affinity to FcRn is lost, and IgG and SA are released back into circulation. In the main part of this thesis, efforts are described aiming to take use of, or block, the FcRn recycling mechanism to control the serum circulation half-life of proteins.In the first study (Study I), a robust expression strategy for human FcRn was designed and evaluated. The extracellular domain was produced in the human SKOV3 cell-line after facile lentiviral delivery of the expression cassettes. This lead to continuous expression of secreted protein that could be purified to homogeneity by a single affinity chromatographic step, using the intrinsic pH-dependent interaction between FcRn and IgG, where the latter was immobilized in a column. The amount of purified protein was 1.4 mg per liter medium. The protein was characterized by SDS-PAGE, western blotting, circular dichroism spectroscopy, ELISA, surface plasmon resonance and a temperature stability assay. The results suggested a fully functional and stable protein of high purity. In addition, the gene encoding full-length FcRn as a fusion to eGFP was delivered to HeLa cells utilizing the same lentiviral system. Subsequent analysis by flow cytometry and confocal fluorescence microscopy indicated a wide distribution of eGFP/FcRn expression among the cells. Binding of IgG and HSA was found to correlate well with the amount of eGFP/FcRn expressed by the cells.In a following study (Study II), the goal was to generate affinity proteins interacting with human FcRn in a pH-dependent manner similar to that of FcRn's natural ligands. The affinity proteins used are denoted affibody molecules, a class of small alternative scaffold proteins with a three-helical structure. Affibody molecules were selected from a combinatorial library displayed on phage where binding took place at pH 5.5 and elution was performed at pH 2.2 or 8.0. Selected variants were characterized by developed in vitro and cell based assays, and some were found to have the desired pH-dependent binding to FcRn. In vivo studies in mice showed that the serum half-life of a model protein, genetically fused to the FcRn binding affibody molecules, was extended up to almost three-fold compared to a control protein (from 33 to 91 hours).In a subsequent study (Study III), the use of a FcRn binding affibody molecule to reduce, rather than prolong, the serum half-life of proteins was explored. Here, the rationale was to investigate if injection of a FcRn binding affibody could hinder endogenous IgG from being rescued by FcRn, which could lead to depletion of IgGs by lysosomal degradation. In autoimmune diseases, such depletion of IgG would include also pathogenic IgG and could thus mitigate the symptoms of the disease. Using cell based assays, it was found that one affibody molecule, selected in Study II, could readily block IgG from binding both human and murine FcRn. A following in vivo study in mice showed that systemic injection of the molecule reduced the amount of endogenous IgG by 39%.In a fourth study (Study IV), the goal was to use a different class of affinity proteins to regulate the level of an enzyme in the brain. More specifically, artificial zinc finger-based transcription factors regulating the level of GAD67, which is the rate-limiting enzyme in synthesis of gamma-aminobutyric acid (GABA), were designed. Imbalances in GABA-signaling is involved in different diseases, including Parkinson's disease and epilepsy, and regulation of GAD67 at particular sites in the brain might be a route to ameliorate symptoms associated with such diseases. ELISA-based investigation showed that one of the designed zinc fingers, denoted G3, bound selectively to its intended target DNA sequence. A construct encoding G3 fused to a general transcriptional activator (VP64) was delivered to PC12-cells, using a lentivirus-based gene delivery system, resulting in a significant up-regulation of endogenous GAD67 expression. The same construct was subsequently delivered to the striatum of rats, with an induced disease model of Parkinson's disease. Western blot of striatal samples showed a significant up-regulation of GAD67 expression in lesioned striatum compared to intact striatum, and a tendency towards up-regulation compared to lesioned striatum.Taken together, the protein engineering efforts described in this thesis concerning affinity proteins binding other proteins or DNA, has the potential to find use in drug development and may benefit patients in the future.
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| 13. |
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| 14. |
- Seijsing, Johan, et al.
(författare)
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Robust Expression of the Human Neonatal Fc Receptor in a Truncated Soluble Form and as a Full-Length Membrane-Bound Protein in Fusion with eGFP
- 2013
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:11, s. e81350-
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Tidskriftsartikel (refereegranskat)abstract
- Studies on the neonatal Fc receptor (FcRn) have revealed a multitude of important functions in mammals, including protection of IgG and serum albumin (SA) from lysosomal degradation. The pharmacokinetic behavior of therapeutic antibodies, IgG-Fc- and SA-containing drugs is therefore influenced by their interaction with FcRn. Pre-clinical development of such drugs is facilitated if their interaction with FcRn can be studied in vitro. For this reason we have developed a robust system for production of the soluble extracellular domain of human FcRn as well as the full-length receptor as fusion to green fluorescent protein, taking advantage of a lentivirus-based gene delivery system where stable over-expressing cells are easily and rapidly generated. Production of the extracellular domain in multiple-layered culture flasks, followed by affinity purification using immobilized IgG, resulted in capture of milligram amounts of soluble receptor per liter cell culture with retained IgG binding. The receptor was further characterized by SDS-PAGE, western blotting, circular dichroism spectroscopy, ELISA, surface plasmon resonance and a temperature stability assay showing a functional and stable protein of high purity. The full-length receptor was found to be successfully over-expressed in a membrane-bound form with retained pH-dependent IgG- and SA-binding.
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