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

摘要

The recombinant proteins [EE(A)12EETGRGDSPAAS]" (n = 5,10) were prepared as a potential scaffold material for bone repair. The construct was based on Antheraea pemi 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)_(12)EETGRGDSPAAS]_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)_(12)EETGRGDSPAAS]_(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.