Conferring biological activity to native spider silk: A biofunctionalized protein‐based microfiber

Spider silk is an extraordinary material with physical properties comparable to the best scaffolding/structural materials, and as a fiber it can be manipulated with ease into a variety of configurations. Our work here demonstrates that natural spider silk fibers can also be used to organize biological components on and in devices through rapid and simple means. Micron scale spider silk fibers (5–10 μm in diameter) were surface modified with a variety of biological entities engineered with pentaglutamine tags via microbial transglutaminase (mTG). Enzymes, enzyme pathways, antibodies, and fluorescent proteins were all assembled onto spider silk fibers using this biomolecular engineering/biofabrication process. Additionally, arrangement of biofunctionalized fiber should in of itself generate a secondary level of biomolecular organization. Toward this end, as proofs of principle, spatially defined arrangement of biofunctionalized spider silk fiber was shown to generate effects specific to silk position in two cases. In one instance, arrangement perpendicular to a flow produced selective head and neck carcinoma cell capture on silk with antibodies complexed to conjugated protein G. In a second scenario, asymmetric bacterial chemotaxis arose from asymmetric conjugation of enzymes to arranged silk. Overall, the biofabrication processes used here were rapid, required no complex chemistries, were biologically benign, and also the resulting engineered silk microfibers were flexible, readily manipulated and functionally active. Deployed here in microfluidic environments, biofunctional spider silk fiber provides a means to convey complex biological functions over a range of scales, further extending its potential as a biomaterial in biotechnological settings. Biotechnol. Bioeng. 2017;114: 83–95. © 2016 Wiley Periodicals, Inc.

[1]  D. Kaplan,et al.  Multifunctional spider silk polymers for gene delivery to human mesenchymal stem cells. , 2015, Journal of biomedical materials research. Part B, Applied biomaterials.

[2]  T. Scheibel,et al.  To spin or not to spin: spider silk fibers and more , 2015, Applied Microbiology and Biotechnology.

[3]  Fritz Vollrath,et al.  Silk Reconstitution Disrupts Fibroin Self-Assembly. , 2015, Biomacromolecules.

[4]  J. Kong,et al.  Obtaining information about protein secondary structures in aqueous solution using Fourier transform IR spectroscopy , 2015, Nature Protocols.

[5]  Ovijit Chaudhuri,et al.  Biological materials and molecular biomimetics - filling up the empty soft materials space for tissue engineering applications. , 2015, Journal of materials chemistry. B.

[6]  F. Gräter,et al.  Viscous Friction between Crystalline and Amorphous Phase of Dragline Silk , 2014, PloS one.

[7]  Eileen Fong,et al.  Recombinant elastomeric protein biopolymers: Progress and prospects , 2014 .

[8]  M. Hedhammar,et al.  Recombinant spider silk genetically functionalized with affinity domains. , 2014, Biomacromolecules.

[9]  P. Vogt,et al.  Biomechanics and Biocompatibility of Woven Spider Silk Meshes During Remodeling in a Rodent Fascia Replacement Model , 2014, Annals of surgery.

[10]  E. Goormaghtigh,et al.  ATR-FTIR: a "rejuvenated" tool to investigate amyloid proteins. , 2013, Biochimica et biophysica acta.

[11]  François Paquet-Mercier,et al.  Native spider silk as a biological optical fiber , 2013 .

[12]  H. Tseng,et al.  Capture and Stimulated Release of Circulating Tumor Cells on Polymer‐Grafted Silicon Nanostructures , 2013, Advanced materials.

[13]  Lingling Xu,et al.  Recombinant Minimalist Spider Wrapping Silk Proteins Capable of Native-Like Fiber Formation , 2012, PloS one.

[14]  M. Richter,et al.  Enzyme-catalyzed protein crosslinking , 2012, Applied Microbiology and Biotechnology.

[15]  A. Jayaraman,et al.  Chemotaxis to the Quorum-Sensing Signal AI-2 Requires the Tsr Chemoreceptor and the Periplasmic LsrB AI-2-Binding Protein , 2010, Journal of bacteriology.

[16]  Young Hwan Park,et al.  Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber , 2010, Proceedings of the National Academy of Sciences.

[17]  W. Bentley,et al.  Engineered biological nanofactories trigger quorum sensing response in targeted bacteria. , 2010, Nature nanotechnology.

[18]  Q. Wang,et al.  Transglutaminase‐mediated crosslinking of gelatin onto wool surfaces to improve the fabric properties , 2009 .

[19]  W. Bentley,et al.  Biofabrication of antibodies and antigens via IgG‐binding domain engineered with activatable pentatyrosine pro‐tag , 2009, Biotechnology and bioengineering.

[20]  S. Rammensee,et al.  Assembly mechanism of recombinant spider silk proteins , 2008, Proceedings of the National Academy of Sciences.

[21]  David L Kaplan,et al.  Spider silks and their applications. , 2008, Trends in biotechnology.

[22]  I. Tso,et al.  Signaling by decorating webs: luring prey or deterring predators? , 2007 .

[23]  D. Beebe,et al.  Protocol for the fabrication of enzymatically crosslinked gelatin microchannels for microfluidic cell culture , 2007, Nature Protocols.

[24]  M. Ishii,et al.  Evaluation of the pH- and thermal stability of the recombinant green fluorescent protein (GFP) in the presence of sodium chloride , 2007, Applied biochemistry and biotechnology.

[25]  David L Kaplan,et al.  RGD-functionalized bioengineered spider dragline silk biomaterial. , 2006, Biomacromolecules.

[26]  Peter M Vogt,et al.  Use of spider silk fibres as an innovative material in a biocompatible artificial nerve conduit , 2006, Journal of cellular and molecular medicine.

[27]  Todd A. Blackledge,et al.  Variation in the material properties of spider dragline silk across species , 2006 .

[28]  Nathan C Shaner,et al.  A guide to choosing fluorescent proteins , 2005, Nature Methods.

[29]  M. Herberstein,et al.  Spider signals: are web decorations visible to birds and bees? , 2005, Biology Letters.

[30]  Hsuan-Chen Wu,et al.  Giant wood spider Nephila pilipes alters silk protein in response to prey variation , 2005, Journal of Experimental Biology.

[31]  Fritz Vollrath,et al.  Characterization of the protein components of Nephila clavipes dragline silk. , 2005, Biochemistry.

[32]  P. Zhou,et al.  Toughness of Spider Silk at High and Low Temperatures , 2005 .

[33]  R. Tsien,et al.  Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein , 2004, Nature Biotechnology.

[34]  Thomas Scheibel,et al.  Spider silks: recombinant synthesis, assembly, spinning, and engineering of synthetic proteins , 2004, Microbial cell factories.

[35]  I. Pastan,et al.  Abrogation of Head and Neck Squamous Cell Carcinoma Growth by Epidermal Growth Factor Receptor Ligand Fused to Pseudomonas Exotoxin Transforming Growth Factor α-PE38 , 2004, Clinical Cancer Research.

[36]  K. Yokoyama,et al.  Properties and applications of microbial transglutaminase , 2004, Applied Microbiology and Biotechnology.

[37]  Kim R Hardie,et al.  LuxS: its role in central metabolism and the in vitro synthesis of 4-hydroxy-5-methyl-3(2H)-furanone. , 2002, Microbiology.

[38]  M. Surette,et al.  The LuxS family of bacterial autoinducers: biosynthesis of a novel quorum‐sensing signal molecule , 2001, Molecular microbiology.

[39]  Takeshi Watanabe Effects of Web Design on the Prey Capture Efficiency of the Uloborid Spider Octonoba sybotides under Abundant and Limited Prey Conditions , 2001 .

[40]  R. Lewis,et al.  Hypotheses that correlate the sequence, structure, and mechanical properties of spider silk proteins. , 1999, International journal of biological macromolecules.

[41]  G. Phillips,et al.  The molecular structure of green fluorescent protein , 1996, Nature Biotechnology.

[42]  W. Stemmer,et al.  Improved Green Fluorescent Protein by Molecular Evolution Using DNA Shuffling , 1996, Nature Biotechnology.

[43]  R. Lewis,et al.  Structure of a protein superfiber: spider dragline silk. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[44]  A. Matsuura,et al.  Polymerization of Several Proteins by Ca2+-Independent Transglutaminase Derived from Microorganisms , 1989 .

[45]  H. Susi,et al.  Examination of the secondary structure of proteins by deconvolved FTIR spectra , 1986, Biopolymers.

[46]  Friedrich G. Barth,et al.  Vibratory signals and prey capture in orb-weaving spiders (Zygiella x-notata, Nephila clavipes; Araneidae) , 1982, Journal of comparative physiology.

[47]  G L Hazelbauer,et al.  Chemotaxis Toward Sugars in Escherichia coli , 1973, Journal of bacteriology.

[48]  John Chamberlain,et al.  The determination of refractive index spectra by fourier spectrometry , 1969 .

[49]  Fritz Vollrath,et al.  Structural disorder in silk proteins reveals the emergence of elastomericity. , 2008, Biomacromolecules.

[50]  T. Penna,et al.  Stability of recombinant green fluorescent protein (GFPuv) in glucose solutions at different concentrations and pH values , 2005, Applied biochemistry and biotechnology.

[51]  D. Kaplan,et al.  The amino acid composition of major ampullate gland silk (dragline) of Nephila clavipes (Araneae, Tetragnathidae). , 1990 .