RRC ID 62225
Author Asakura T, Suzuki Y, Nagano A, Knight D, Kamiya M, Demura M.
Title Synthesis and characterization of water-soluble silk peptides and recombinant silk protein containing polyalanine, the integrin binding site, and two glutamic acids at each terminal site as a possible candidate for use in bone repair materials.
Journal Biomacromolecules
Abstract The recombinant proteins [EE(A)12EETGRGDSPAAS]n (n = 5,10) were prepared as a potential scaffold material for bone repair. The construct was based on Antheraea perni silk fibroin to which cells adhere well and combined poly(alanine), the integrin binding site TGRGDSPA, and a pair of glutamic acids (E2) at both the N- and C-terminal sites to render the construct water-soluble and with the hope that it might enhance mineralization with hydroxyapatite. Initially, two peptides E2(A)nE2TGRGDSPAE2(A)nE2 (n = 6, 12) were prepared by solid state synthesis to examine the effect of size on conformation and on cell binding. The larger peptide bound osteoblasts more readily and had a higher helix content than the smaller one. Titration of the side chain COO(-) to COOH of the E2 and D units in the peptide was monitored by solution NMR. On the basis of these results, we produced the related recombinant His tagged protein [EE(A)12EETGRGDSPAAS]n (n = 5,10) by expression in Escherichia coli . The solution NMR spectra of the recombinant protein indicated that the poly(alanine) regions are helical, and one E2 unit is helical and the other is a random coil. A molecular dynamics simulation of the protein supports these conclusions from NMR. We showed that the recombinant protein, especially, [EE(A)12EETGRGDSPAAS]10 has some of the properties required for bone tissue engineering scaffold including insolubility, and evidence of enhanced cell binding through focal adhesions, and enhanced osteogenic expression of osteoblast-like cells bound to it, and has potential for use as a bone repair material.
Volume 14(10)
Pages 3731-41
Published 2013-10-14
DOI 10.1021/bm401118m
PMID 24004453
MeSH Animals Binding Sites Bone Substitutes / chemistry* Cell Adhesion Cell Line Fibroins / chemical synthesis Fibroins / chemistry* Fibroins / metabolism* Glutamates / analysis* Hydrogen-Ion Concentration Integrins / metabolism* Mice Molecular Dynamics Simulation Moths Nuclear Magnetic Resonance, Biomolecular Osteoclasts / chemistry Osteoclasts / cytology Peptides / analysis* Protein Conformation Recombinant Proteins / chemical synthesis Recombinant Proteins / chemistry Recombinant Proteins / metabolism Solubility Surface Properties Water / chemistry
IF 6.092
Human and Animal Cells MC3T3-E1(RCB1126)