title: Antimicrobial functionalized genetically engineered spider silk.
abstract: Genetically engineered fusion proteins offer potential as multifunctional biomaterials for medical use. Fusion or chimeric proteins can be formed using recombinant DNA technology by combining nucleotide sequences encoding different peptides or proteins that are otherwise not found together in nature. In the present study, three new fusion proteins were designed, cloned and expressed and assessed for function, by combining the consensus sequence of dragline spider silk with three different antimicrobial peptides. The human antimicrobial peptides human neutrophil defensin 2 (HNP-2), human neutrophil defensins 4 (HNP-4) and hepcidin were fused to spider silk through bioengineering. The spider silk domain maintained its self-assembly features, a key aspect of these new polymeric protein biomaterials, allowing the formation of β-sheets to lock in structures via physical interactions without the need for chemical cross-linking. These new functional silk proteins were assessed for antimicrobial activity against Gram - Escherichia coli and Gram + Staphylococcus aureus and microbicidal activity was demonstrated. Dynamic light scattering was used to assess protein aggregation to clarify the antimicrobial patterns observed. Attenuated-total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and circular dichroism (CD) were used to assess the secondary structure of the new recombinant proteins. In vitro cell studies with a human osteosarcoma cell line (SaOs-2) demonstrated the compatibility of these new proteins with mammalian cells.
authors: 3B's Research Group - Biomaterials, Biodegradables and Biomimetics, Department of Polymer Engineering, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Indústrial da Gandra, 4806-909 Caldas das Taipas, Guimarães, Portugal.
authors: Sílvia C
authors: SC
authors: Gomes
authors: Isabel B
authors: IB
authors: Leonor
authors: João F
authors: JF
authors: Mano
authors: Rui L
authors: RL
authors: Reis
authors: David L
authors: DL
authors: Kaplan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry* / Q000235:genetics* / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects; D064451:Hepcidins; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D047011:Silk / Q000737:chemistry / Q000235:genetics; D017550:Spectroscopy, Fourier Transform Infrared; D013112:Spiders / Q000737:chemistry / Q000235:genetics; D013211:Staphylococcus aureus / Q000187:drug effects; D023084:alpha-Defensins / Q000737:chemistry* / Q000235:genetics* / Q000494:pharmacology
chemical_list: D023181:Antimicrobial Cationic Peptides; C578702:HAMP protein, human; D064451:Hepcidins; D011993:Recombinant Fusion Proteins; D047011:Silk; D023084:alpha-Defensins; C406416:human neutrophil peptide 2; C406428:human neutrophil peptide 4