title: A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning.
abstract: The extreme strength and elasticity of spider silks originate from the modular nature of their repetitive proteins. To exploit such materials and mimic spider silks, comprehensive strategies to produce and spin recombinant fibrous proteins are necessary. This protocol describes silk gene design and cloning, protein expression in bacteria, recombinant protein purification and fiber formation. With an improved gene construction and cloning scheme, this technique is adaptable for the production of any repetitive fibrous proteins, and ensures the exact reproduction of native repeat sequences, analogs or chimeric versions. The proteins are solubilized in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at 25-30% (wt/vol) for extrusion into fibers. This protocol, routinely used to spin single micrometer-size fibers from several recombinant silk-like proteins from different spider species, is a powerful tool to generate protein libraries with corresponding fibers for structure-function relationship investigations in protein-based biomaterials. This protocol may be completed in 40 d.
authors.affiliation: Department of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071, USA.
authors.forename: Florence
authors.initials: F
authors.lastname: Teulé
authors.forename: Alyssa R
authors.initials: AR
authors.lastname: Cooper
authors.forename: William A
authors.initials: WA
authors.lastname: Furin
authors.forename: Daniela
authors.initials: D
authors.lastname: Bittencourt
authors.forename: Elibio L
authors.initials: EL
authors.lastname: Rech
authors.forename: Amanda
authors.initials: A
authors.lastname: Brooks
authors.forename: Randolph V
authors.initials: RV
authors.lastname: Lewis
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D047011:Silk / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D013112:Spiders / Q000737:chemistry* / Q000235:genetics; D013718:Tensile Strength
chemical_list: D011994:Recombinant Proteins; D047011:Silk