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.
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.
authors.affiliation: Department of Biotechnology, Tokyo University of Agriculture and Technology , Koganei, Tokyo 184-8588, Japan.
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Asakura
authors.forename: Yu
authors.initials: Y
authors.lastname: Suzuki
authors.forename: Aya
authors.initials: A
authors.lastname: Nagano
authors.forename: David
authors.initials: D
authors.lastname: Knight
authors.forename: Masakatsu
authors.initials: M
authors.lastname: Kamiya
authors.forename: Makoto
authors.initials: M
authors.lastname: Demura
mesh_terms: D000818:Animals; D001665:Binding Sites; D018786:Bone Substitutes / Q000737:chemistry*; D002448:Cell Adhesion; D002460:Cell Line; D005349:Fibroins / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D005971:Glutamates / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D016023:Integrins / Q000378:metabolism*; D051379:Mice; D056004:Molecular Dynamics Simulation; D009036:Moths; D019906:Nuclear Magnetic Resonance, Biomolecular; D010010:Osteoclasts / Q000737:chemistry / Q000166:cytology; D010455:Peptides / Q000032:analysis*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D013499:Surface Properties; D014867:Water / Q000737:chemistry
chemical_list: D018786:Bone Substitutes; D005971:Glutamates; D016023:Integrins; D010455:Peptides; D011994:Recombinant Proteins; D014867:Water; C019529:polyalanine; D005349:Fibroins