title: Purification and cytotoxicity of tag-free bioengineered spider silk proteins.
abstract: Bioengineered spider silk-like proteins can serve as biomaterials for various biomedical applications. These proteins can be assembled in several morphological forms such as films, microcapsules, spheres, fibers, gels, and scaffolds. However, crucial points for recombinant spider silks for human use are toxicity and immunogenicity. To assess this issue, two bioengineered spider silk proteins composed of different numbers of repetitive motifs of the consensus repeats from spidroin-1 from Nephila clavipes (15X and 6X) were cloned and expressed in Escherichia coli. The proteins were free of tag sequence and were purified using two methods based on (1) thermal and (2) organic acid resistance of the spider silks. The soluble spider silk proteins were not cytotoxic and did not activate macrophages over a wide range of concentrations, except when present at the highest concentration. Films made of the different silk variants supported the growth of the cells. Based on these data, and as the biodegradation rate of silk is very slow, the bioengineered spider silks are presumed safe biomaterials for biomedical applications.
authors.affiliation: Department of Cancer Diagnostics and Immunology, Greater Poland Cancer Centre, Poznan 61-866, Poland. hanna.dams-kozlowska@wco.pl
authors.forename: Hanna
authors.initials: H
authors.lastname: Dams-Kozlowska
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Majer
authors.forename: Paulina
authors.initials: P
authors.lastname: Tomasiewicz
authors.forename: Jolanta
authors.initials: J
authors.lastname: Lozinska
authors.forename: David L
authors.initials: DL
authors.lastname: Kaplan
authors.forename: Andrzej
authors.initials: A
authors.lastname: Mackiewicz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D057005:Bioengineering / Q000379:methods*; D053148:Caspase 3 / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects* / Q000201:enzymology; D005349:Fibroins / Q000737:chemistry / Q000302:isolation & purification* / Q000633:toxicity*; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008262:Macrophage Activation / Q000187:drug effects; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D011994:Recombinant Proteins / Q000302:isolation & purification; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; C086087:spidroin 1; D005349:Fibroins; D007770:L-Lactate Dehydrogenase; D053148:Caspase 3