title: Characterization and assembly of a GFP-tagged cylindriform silk into hexameric complexes.
abstract: Spider silk has been studied extensively for its attractive mechanical properties and potential applications in medicine and industry. The production of spider silk, however, has been lagging behind for lack of suitable systems. Our approach focuses on solving the production of spider silk by designing, expressing, purifying and characterizing the silk from cylindriform glands. We show that the cylindriform silk protein, in contrast to the commonly used dragline silk protein, is fully folded and stable in solution. With the help of GFP as a fusion tag we enhanced the expression of the silk protein in Escherichia coli and could optimize the downstream processing. Secondary structures analysis by circular dichroism and FTIR shows that the GFP-silk fusion protein is predominantly α-helical, and that pH can trigger a α- to β-transition resulting in aggregation. Structural analysis by small angle X-ray scattering suggests that the GFP-Silk exists in the form of a hexamer in solution.
authors: Division of Pure and Applied Biochemistry, Chemical Center, Lund University, Getingevägen 60, 222 41, Lund, Sweden.
authors: Carl
authors: C
authors: Öster
authors: Johan
authors: J
authors: Svensson Bonde
authors: Leif
authors: L
authors: Bülow
authors: Cedric
authors: C
authors: Dicko
mesh_terms: D000818:Animals; D002942:Circular Dichroism; D000067493:Dynamic Light Scattering; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D055503:Protein Multimerization; D058849:Protein Refolding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D053838:Scattering, Small Angle; D047011:Silk / Q000737:chemistry*; D012995:Solubility; D017550:Spectroscopy, Fourier Transform Infrared; D013112:Spiders; D014961:X-Ray Diffraction
chemical_list: D011993:Recombinant Fusion Proteins; D047011:Silk; D049452:Green Fluorescent Proteins
keywords: biophysical properties; cylindriform silk; recombinant silk; self assembly