title: Accelerated Nucleation of Hydroxyapatite Using an Engineered Hydrophobin Fusion Protein.
abstract: Calcium phosphate mineralization is of particular interest in dental repair. A biomimetic approach using proteins or peptides is a highly promising way to reconstruct eroded teeth. In this study, the screening of several proteins is described for their binding and nucleating activities toward hydroxyapatite. Out of 27 tested candidates, only two hydrophobin fusion proteins showed binding abilities to hydroxyapatite in a mouthwash formulation and an increased nucleation in artificial saliva. Using a semirational approach, one of the two candidates (DEWA_5), a fusion protein consisting of a truncated section of the Bacillus subtilis synthase YaaD, the Aspergillus nidulans hydrophobin DEWA, and the rationally designed peptide P11-4 described in the literature, could be further engineered toward a faster mineral formation. The variants DEWA_5a (40aaYaaD-SDSDSD-DEWA) and DEWA_5b (40aaYaaD-RDRDRD-DEWA) were able to enhance the nucleation activity without losing the ability to form hydroxyapatite. In the case of variant DEWA_5b, an additional increase in the binding toward hydroxyapatite could be achieved. Especially with the variant DEWA_5a, the protein engineering of the rationally designed peptide sequence resulted in a resemblance of an amino acid motif that is found in nature. The engineered peptide resembles the amino acid motif in dentin phosphoprotein, one of the major proteins involved in dentinogenesis.
authors.affiliation: Institute of Technical Biochemistry, University of Stuttgart , Allmandring 31, 70569 Stuttgart, Germany.
authors.forename: Melanie
authors.initials: M
authors.lastname: Melcher
authors.affiliation: Institute of Technical Biochemistry, University of Stuttgart , Allmandring 31, 70569 Stuttgart, Germany.
authors.forename: Sandra J
authors.initials: SJ
authors.lastname: Facey
authors.affiliation: Institute of Technical Biochemistry, University of Stuttgart , Allmandring 31, 70569 Stuttgart, Germany.
authors.forename: Thorsten M
authors.initials: TM
authors.lastname: Henkes
authors.affiliation: Fine Chemicals Research, BASF SE, 67056 Ludwigshafen, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Subkowski
authors.affiliation: Institute of Technical Biochemistry, University of Stuttgart , Allmandring 31, 70569 Stuttgart, Germany.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Hauer
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017886:Durapatite / Q000737:chemistry / Q000378:metabolism*; D016326:Extracellular Matrix Proteins; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012464:Saliva, Artificial / Q000737:chemistry*; D012795:Sialoglycoproteins; D014070:Tooth / Q000378:metabolism; D000099311:Dentin Sialophosphoprotein
chemical_list: D001426:Bacterial Proteins; D017886:Durapatite; D016326:Extracellular Matrix Proteins; D005656:Fungal Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D012464:Saliva, Artificial; D012795:Sialoglycoproteins; D000099311:Dentin Sialophosphoprotein; C095254:DEWA protein, Asperigillus nidulans