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      High-yield Production of Amyloid-β Peptide Enabled by a Customized Spider Silk Domain

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          Abstract

          During storage in the silk gland, the N-terminal domain (NT) of spider silk proteins (spidroins) keeps the aggregation-prone repetitive region in solution at extreme concentrations. We observe that NTs from different spidroins have co-evolved with their respective repeat region, and now use an NT that is distantly related to previously used NTs, for efficient recombinant production of the amyloid-β peptide (Aβ) implicated in Alzheimer’s disease. A designed variant of NT from Nephila clavipes flagelliform spidroin, which in nature allows production and storage of β-hairpin repeat segments, gives exceptionally high yields of different human Aβ variants as a solubility tag. This tool enables efficient production of target peptides also in minimal medium and gives up to 10 times more isotope-labeled monomeric Aβ peptides per liter bacterial culture than previously reported.

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          Most cited references36

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          An analytical solution to the kinetics of breakable filament assembly.

          We present an analytical treatment of a set of coupled kinetic equations that governs the self-assembly of filamentous molecular structures. Application to the case of protein aggregation demonstrates that the kinetics of amyloid growth can often be dominated by secondary rather than by primary nucleation events. Our results further reveal a range of general features of the growth kinetics of fragmenting filamentous structures, including the existence of generic scaling laws that provide mechanistic information in contexts ranging from in vitro amyloid growth to the in vivo development of mammalian prion diseases.
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            A facile method for expression and purification of the Alzheimer’s disease-associated amyloid β-peptide

            We report the development of a high-level bacterial expression system for the Alzheimer’s disease-associated amyloid β-peptide (Aβ), together with a scaleable and inexpensive purification procedure. Aβ(1–40) and Aβ(1–42) coding sequences together with added ATG codons were cloned directly into a Pet vector to facilitate production of Met-Aβ(1–40) and Met-Aβ(1–42), referred to as Aβ(Μ1–40) and Aβ(Μ1–42), respectively. The expression sequences were designed using codons preferred by Escherichia coli, and the two peptides were expressed in this host in inclusion bodies. Peptides were purified from inclusion bodies using a combination of anion-exchange chromatography and centrifugal filtration. The method described requires little specialized equipment and provides a facile and inexpensive procedure for production of large amounts of very pure Aβ peptides. Recombinant peptides generated using this protocol produced amyloid fibrils that were indistinguishable from those formed by chemically synthesized Aβ1–40 and Aβ1–42. Formation of fibrils by all peptides was concentration-dependent, and exhibited kinetics typical of a nucleation-dependent polymerization reaction. Recombinant and synthetic peptides exhibited a similar toxic effect on hippocampal neurons, with acute treatment causing inhibition of MTT reduction, and chronic treatment resulting in neuritic degeneration and cell loss.
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              The Nephila clavipes genome highlights the diversity of spider silk genes and their complex expression

              Benjamin Voight and colleagues report the annotated genome of the golden orb-weaver spider. They describe 28 spider silk genes (spidroins), characterize their expression in distinct silk gland types and identify non-spidroin genes with expression patterns suggesting potential roles in silk production.
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                Author and article information

                Contributors
                henrik.biverstal@ki.se
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                14 January 2020
                14 January 2020
                2020
                : 10
                : 235
                Affiliations
                [1 ]ISNI 0000 0004 1937 0626, GRID grid.4714.6, Department of Neurobiology, Care Sciences and Society, Center for Alzheimer Research, Division of Neurogeriatrics, Karolinska Institutet, ; 141 52 Huddinge, Sweden
                [2 ]ISNI 0000 0004 0395 6526, GRID grid.419212.d, Department of Physical Organic Chemistry, Latvian Institute of Organic Synthesis, ; Riga, LV-1006 Latvia
                [3 ]ISNI 0000 0004 1937 0626, GRID grid.4714.6, Department of Molecular Tumor and Cell Biology, Karolinska Institutet, ; 171 65 Solna, Sweden
                [4 ]ISNI 0000 0004 0389 8485, GRID grid.55325.34, Department of Pathology, University of Oslo/Oslo University Hospital, ; N-0424 Oslo, Norway
                [5 ]ISNI 0000 0001 0057 2672, GRID grid.4562.5, LIED, University of Lübeck, ; D-23538 Lübeck, Germany
                [6 ]ISNI 0000 0001 0775 3222, GRID grid.9845.0, Department of Pharmacology, Medical Faculty, University of Latvia, ; Riga, LV-1004 Latvia
                [7 ]ISNI 0000 0000 8578 2742, GRID grid.6341.0, Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, ; 750 07 Uppsala, Sweden
                Article
                57143
                10.1038/s41598-019-57143-x
                6959368
                31937841
                0967fcb3-f16c-4e73-bf6a-ba103503b16c
                © The Author(s) 2020

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 12 October 2019
                : 18 December 2019
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100006285, Magnus Bergvalls Stiftelse;
                Funded by: Åhlén-stiftelsen
                Funded by: FundRef http://dx.doi.org/10.13039/100010815, Stiftelsen för Gamla Tjänarinnor;
                Funded by: FundRef http://dx.doi.org/10.13039/100010771, Loo och Hans Ostermans Stiftelse för Medicinsk Forskning;
                Funded by: Geriatric Diseases Foundation at Karolinska Institutet
                Funded by: INSTRUCT
                Award ID: APPID 272
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100004200, Stiftelsen Olle Engkvist Byggmästare;
                Funded by: FundRef http://dx.doi.org/10.13039/501100008599, Alzheimerfonden;
                Funded by: FundRef http://dx.doi.org/10.13039/100010769, Petrus och Augusta Hedlunds Stiftelse;
                Funded by: FundRef http://dx.doi.org/10.13039/501100005375, Latvijas Zinatnes Padome;
                Award ID: 2018/1-0275
                Award ID: 2018/1-0275
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100005416, Norges Forskningsråd;
                Funded by: FundRef http://dx.doi.org/10.13039/100013278, EU Joint Programme - Neurodegenerative Disease Research;
                Award ID: PROP-AD 643417
                Award ID: PROP-AD 643417
                Award Recipient :
                Funded by: Center for innovative medicine (CIMED), Karolinska Insitutet
                Funded by: FundRef http://dx.doi.org/10.13039/501100004359, Vetenskapsrådet;
                Funded by: FundRef http://dx.doi.org/10.13039/501100001862, Svenska Forskningsrådet Formas;
                Funded by: FundRef http://dx.doi.org/10.13039/501100001858, VINNOVA;
                Categories
                Article
                Custom metadata
                © The Author(s) 2020

                Uncategorized
                biochemistry,biophysics,biosynthesis,peptides,proteins,neurodegeneration,neurodegenerative diseases

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