Genetically engineered syntheses of tandem repetitive polypeptides consisting of glycine-rich sequence of spider dragline silk.
We described genetically engineered syntheses of tandem repetitive polypeptides consisting of glycine-rich sequence, GlyLeuGlyGlyGlnGlyGlyGlyAlaGlyGlnGlyGlyTyrGly, designated SCAP(1), in spidroin 1 of spider dragline silk from Nephila clavipes and the secondary conformational analyses in the solid state by Fourier transform ir measurements. The polypeptides composed of 4, 5, 6, 7, 11, 12, or 13 repeats of SCAP(1) were expressed in Escherichia coli, purified by nickel chelate affinity chromatography, and then cleaved with cyanogen bromide to release N- and C-terminal extensions. Typical yields were from 1.2 to 5.2 mg of lyophilized uncleaved polypeptides per liter of fermentation medium at an absorbance of 2.0 at 600 nm, and the production levels increased with decreasing the molecular weight of the expressed polypeptides. The lyophilized powder of cleaved SCAP(13) adopted the random coil, whereas the cast film from formic acid formed the beta-sheet structure. The conformational results might indicate that the glycine-rich sequence formed beta-sheet structure in spidroin I. Cleaved SCAP(13) started to decompose under nitrogen at ca. 230 degrees C, which was in agreement with the decomposition temperature of the spider dragline silk from N. clavipes.
Fukushima
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Research & Development Center, Unitika Ltd., Kyoto, Japan.
D000595:Amino Acid Sequence; D001483:Base Sequence; D005998:Glycine / Q000032:analysis*; D019476:Insect Proteins / Q000096:biosynthesis* / Q000737:chemistry; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012091:Repetitive Sequences, Nucleic Acid
D019476:Insect Proteins; D011994:Recombinant Proteins; D005998:Glycine