| Bombyx mori sericin, Nephila clavipes dragline silk, human type I collagen are fibrous proteins that play important structural roles in forming materials that have superb mechanical properties for performing various functions. Genetic engineering provides a useful tool to prepare monodisperse biopolymers for more accurate study of the assembly and structure of these fibrous proteins.; Recombinant sericin proteins of different molecular weights (17.4, 31.9, 46.5 kDa), based on the 38 amino acid repetitive motif of native sericin, were cloned, expressed and purified. The recombinant sericin self-assembled during dialysis (starting concentration of 2.5 mg/ml) or incubation on mica surface, forming twisted fibers. Circular dichroism and Fourier transform infrared spectroscopy studies demonstrated protein conformational transitions occurred from random coil to beta-sheets. Congo red stained recombinant sericin fibrils exhibited applegreen birefringence, indicating long-range order in the array of beta-sheets. Biosynthetic sericin has a high content of polar amino acids (e.g. > 40 mole % serine), leading to a beta-sheet conformation formed by hydrogen bonding via polar zipper interactions. Analysis of recombinant sericin sequence using Mandel-Gutfreund's (1)definition of polar and nonpolar amino acids showed that the hydrophobicity pattern resembles the most frequent pattern of amyloidogenic proteins, polar amino acid aggregates (PPPPP). Many beta-proteins and peptides are designed to study amyloidogenesis using a polar/nonpolar alternating pattern (PNPNPN). Sericin-like proteins or peptides provide an alternative model in terms of hydrophobicity pattern with which to explore questions related to beta-sheet formation and amyloidogenesis. The glue-like property of sericin is attributed to the hydrogen bonding between serine residues of sericin with serine residues in the fibroin structural components of silk fibers.; Spider silks exhibit remarkable mechanical properties while dentin matrix protein 1 from rat provides controlled nucleation and hydroxyapatite growth. In the present work, these two attributes were combined via genetic engineering to form a chimera, a clone encoding consensus repeats from the major protein in the spider dragline silk of N. clavipes fused to the carboxyl terminal domain of dentin matrix protein 1 (CDMP1). The objective was to exploit the self-assembly and material properties of silk proteins with controlled hydroxyapatite formation from CDMP1, for novel biomaterial composites. (Abstract shortened by UMI.)... |
