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      Recombinant spider silk from aqueous solutions via a bio-inspired microfluidic chip

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          Abstract

          Spiders achieve superior silk fibres by controlling the molecular assembly of silk proteins and the hierarchical structure of fibres. However, current wet-spinning process for recombinant spidroins oversimplifies the natural spinning process. Here, water-soluble recombinant spider dragline silk protein (with a low molecular weight of 47 kDa) was adopted to prepare aqueous spinning dope. Artificial spider silks were spun via microfluidic wet-spinning, using a continuous post-spin drawing process (WS-PSD). By mimicking the natural spinning apparatus, shearing and elongational sections were integrated in the microfluidic spinning chip to induce assembly, orientation of spidroins, and fibril structure formation. The additional post-spin drawing process following the wet-spinning section partially mimics the spinning process of natural spider silk and substantially contributes to the compact aggregation of microfibrils. Subsequent post-stretching further improves the hierarchical structure of the fibres, including the crystalline structure, orientation, and fibril melting. The tensile strength and elongation of post-treated fibres reached up to 510 MPa and 15%, respectively.

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          Spider silk fibers spun from soluble recombinant silk produced in mammalian cells.

          Spider silks are protein-based "biopolymer" filaments or threads secreted by specialized epithelial cells as concentrated soluble precursors of highly repetitive primary sequences. Spider dragline silk is a flexible, lightweight fiber of extraordinary strength and toughness comparable to that of synthetic high-performance fibers. We sought to "biomimic" the process of spider silk production by expressing in mammalian cells the dragline silk genes (ADF-3/MaSpII and MaSpI) of two spider species. We produced soluble recombinant (rc)-dragline silk proteins with molecular masses of 60 to 140 kilodaltons. We demonstrated the wet spinning of silk monofilaments spun from a concentrated aqueous solution of soluble rc-spider silk protein (ADF-3; 60 kilodaltons) under modest shear and coagulation conditions. The spun fibers were water insoluble with a fine diameter (10 to 40 micrometers) and exhibited toughness and modulus values comparable to those of native dragline silks but with lower tenacity. Dope solutions with rc-silk protein concentrations >20% and postspinning draw were necessary to achieve improved mechanical properties of the spun fibers. Fiber properties correlated with finer fiber diameter and increased birefringence.
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            Self-assembly of spider silk proteins is controlled by a pH-sensitive relay.

            Nature's high-performance polymer, spider silk, consists of specific proteins, spidroins, with repetitive segments flanked by conserved non-repetitive domains. Spidroins are stored as a highly concentrated fluid dope. On silk formation, intermolecular interactions between repeat regions are established that provide strength and elasticity. How spiders manage to avoid premature spidroin aggregation before self-assembly is not yet established. A pH drop to 6.3 along the spider's spinning apparatus, altered salt composition and shear forces are believed to trigger the conversion to solid silk, but no molecular details are known. Miniature spidroins consisting of a few repetitive spidroin segments capped by the carboxy-terminal domain form metre-long silk-like fibres irrespective of pH. We discovered that incorporation of the amino-terminal domain of major ampullate spidroin 1 from the dragline of the nursery web spider Euprosthenops australis (NT) into mini-spidroins enables immediate, charge-dependent self-assembly at pH values around 6.3, but delays aggregation above pH 7. The X-ray structure of NT, determined to 1.7 A resolution, shows a homodimer of dipolar, antiparallel five-helix bundle subunits that lack homologues. The overall dimeric structure and observed charge distribution of NT is expected to be conserved through spider evolution and in all types of spidroins. Our results indicate a relay-like mechanism through which the N-terminal domain regulates spidroin assembly by inhibiting precocious aggregation during storage, and accelerating and directing self-assembly as the pH is lowered along the spider's silk extrusion duct.
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              Design of superior spider silk: from nanostructure to mechanical properties.

              Spider dragline silk is of practical interest because of its excellent mechanical properties. However, the structure of this material is still largely unknown. In this article, we report what we believe is a new model of the hierarchical structure of silk based on scanning electron microscope and atomic force microscope images. This hierarchical structure includes beta-sheet, polypeptide chain network, and silk fibril. It turns out that an exceptionally high strength of the spider dragline silk can be obtained by decreasing the size of the crystalline nodes in the polypeptide chain network while increasing the degree of orientation of the crystalline nodes. Based on this understanding, how the reeling speed affects mechanical properties of spider dragline silk can be understood properly. Hopefully, the understanding obtained in this study will shed light on the formation of spider silk, and consequently, on the principles for the design of ultrastrong silk.
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                Author and article information

                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group
                2045-2322
                07 November 2016
                2016
                : 6
                : 36473
                Affiliations
                [1 ]State Key Laboratory for Modification of Chemical Fibres and Polymer Materials, College of Materials Science and Engineering, Donghua University , Shanghai, 201620, China
                [2 ]School of Life Sciences and Biotechnology, Shanghai Jiao Tong University , Shanghai 200240, China
                Author notes
                [*]

                These authors contributed equally to this work.

                Article
                srep36473
                10.1038/srep36473
                5098227
                27819339
                2dc16a87-7a3a-4303-97a7-7ceacc507129
                Copyright © 2016, The Author(s)

                This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

                History
                : 21 July 2016
                : 14 October 2016
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