Designing recombinant spider silk proteins to control assembly.
The consensus repeat sequence found in the dragline silk from the spider, Nephila clavipes, was redesigned to incorporate a redox trigger flanking the beta-sheet forming polyalanine sequences. The methionine redox trigger, in the oxidized state, was incorporated to prevent the formation of the beta sheets, while in the reduced state would not result in sterical limitations to beta sheet formation. A synthetic gene incorporating the trigger was constructed, cloned and then expressed in Escherichia coli. The purified protein, about 25 kDa, contained the expected amino acid composition and migration behavior on SDS-PAGE. The recombinant protein was analyzed by X-ray diffraction, TEM, electron diffraction and circular dichroism in both oxidized and reduced states. Based on the results, the incorporation of a redox trigger appears to be a powerful experimental strategy to explore the self-assembly of fibrous proteins such as silks.
Winkler
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Tufts University, Biotechnology Center, Department of Chemical Engineering, Medford, MA 02155, USA.
Szela
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Avtges
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Valluzzi
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Kirschner
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Kaplan
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D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D019476:Insect Proteins / Q000737:chemistry* / Q000235:genetics* / Q000648:ultrastructure; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D047011:Silk; D013112:Spiders / Q000737:chemistry*; D014961:X-Ray Diffraction
D019476:Insect Proteins; D011993:Recombinant Fusion Proteins; D047011:Silk