Daniela Bittencourt

A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning.

The extreme strength and elasticity of spider silks originate from the modular nature of their repetitive proteins. To exploit such materials and mimic spider silks, comprehensive strategies to produce and spin recombinant fibrous proteins are necessary. This protocol describes silk gene design and cloning, protein expression in bacteria, recombinant protein purification and fiber formation. With an improved gene construction and cloning scheme, this technique is adaptable for the production of any repetitive fibrous proteins, and ensures the exact reproduction of native repeat sequences, analogs or chimeric versions. The proteins are solubilized in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at 25–30% (wt/vol) for extrusion into fibers. This protocol, routinely used to spin single micrometer-size fibers from several recombinant silk-like proteins from different spider species, is a powerful tool to generate protein libraries with corresponding fibers for structure–function relationship investigations in protein-based biomaterials. This protocol may be completed in 40 d.

Piriform Spider Silk Sequences Reveal Unique Repetitive Elements

Orb-weaving spider silk fibers are assembled from very large, highly repetitive proteins. The repeated segments contain, in turn, short, simple, and repetitive amino acid motifs that account for the physical and mechanical properties of the assembled fiber. Of the six orb-weaver silk fibroins, the piriform silk that makes the attachment discs, which lashes the joints of the web and attaches dragline silk to surfaces, has not been previously characterized. Piriform silk protein cDNAs were isolated from phage libraries of three species: A. trifasciata , N. clavipes , and N. cruentata . The deduced amino acid sequences from these genes revealed two new repetitive motifs: an alternating proline motif, where every other amino acid is proline, and a glutamine-rich motif of 6-8 amino acids. Similar to other spider silk proteins, the repeated segments are large (>200 amino acids) and highly homogenized within a species. There is also substantial sequence similarity across the genes from the three species, with particular conservation of the repetitive motifs. Northern blot analysis revealed that the mRNA is larger than 11 kb and is expressed exclusively in the piriform glands of the spider. Phylogenetic analysis of the C-terminal regions of the new proteins with published spidroins robustly shows that the piriform sequences form an ortholog group.

Spinning Gland Transcriptomics from Two Main Clades of Spiders (Order: Araneae) - Insights on Their Molecular, Anatomical and Behavioral Evolution

Characterized by distinctive evolutionary adaptations, spiders provide a comprehensive system for evolutionary and developmental studies of anatomical organs, including silk and venom production. Here we performed cDNA sequencing using massively parallel sequencers (454 GS-FLX Titanium) to generate ∼80,000 reads from the spinning gland of Actinopus spp. (infraorder: Mygalomorphae) and Gasteracantha cancriformis (infraorder: Araneomorphae, Orbiculariae clade). Actinopus spp. retains primitive characteristics on web usage and presents a single undifferentiated spinning gland while the orbiculariae spiders have seven differentiated spinning glands and complex patterns of web usage. MIRA, Celera Assembler and CAP3 software were used to cluster NGS reads for each spider. CAP3 unigenes passed through a pipeline for automatic annotation, classification by biological function, and comparative transcriptomics. Genes related to spider silks were manually curated and analyzed. Although a single spidroin gene family was found in Actinopus spp., a vast repertoire of specialized spider silk proteins was encountered in orbiculariae. Astacin-like metalloproteases (meprin subfamily) were shown to be some of the most sampled unigenes and duplicated gene families in G. cancriformis since its evolutionary split from mygalomorphs. Our results confirm that the evolution of the molecular repertoire of silk proteins was accompanied by the (i) anatomical differentiation of spinning glands and (ii) behavioral complexification in the web usage. Finally, a phylogenetic tree was constructed to cluster most of the known spidroins in gene clades. This is the first large-scale, multi-organism transcriptome for spider spinning glands and a first step into a broad understanding of spider web systems biology and evolution.

A MaSp2-like gene found in the Amazon mygalomorph spider Avicularia juruensis

Two unique spidroins are present in the silk of the Amazon mygalomorph spider - Avicularia juruensis (Theraphosidae), and for the first time the presence and expression of a major ampullate spidroin 2-like in Mygalomorphae are demonstrated. Molecular analysis showed the presence of (GA)(n,) poly-A and GPGXX motifs in the amino acid sequence of Spidroin 2, the last being a motif described so far only in MaSp2 and Flag spidroins. Phylogenetic analysis confirmed the previously known orthologous silk gene clusters, and placed this gene firmly within the orbicularian MaSp2 clade. Gene tree-species tree reconciliations show a pattern of multiple gene duplication throughout spider silk evolution, and pinpoint the oldest speciation in which MaSps must have been present in spiders on the mygalomorph-araneomorph split, 240 MYA. Therefore, while not refuting orb weaver monophyly, MaSp2s, and major ampullate silks in general cannot be classified as orbicularian synapomorphies, but have to be considered plesiomorphic for Opisthothelae. The evidence presented here challenges the simplified notion that mygalomorphs spin only one kind of silk, and adds to the suite of information suggesting a pattern of early niche diversification between Araneomorphae and Mygalomorphae. Additionally, mygalomorph MaSp2-like might accommodate mechanical demands arising from the arboreal habitat preference of Avicularia.

Characterization of Aspergillus species on Brazil nut from the Brazilian Amazonian region and development of a PCR assay for identification at the genus level.

BackgroundBrazil nut is a protein-rich extractivist tree crop in the Amazon region. Fungal contamination of shells and kernel material frequently includes the presence of aflatoxigenic Aspergillus species from the section Flavi. Aflatoxins are polyketide secondary metabolites, which are hepatotoxic carcinogens in mammals. The objectives of this study were to identify Aspergillus species occurring on Brazil nut grown in different states in the Brazilian Amazon region and develop a specific PCR method for collective identification of member species of the genus Aspergillus.ResultsPolyphasic identification of 137 Aspergillus strains isolated from Brazil nut shell material from cooperatives across the Brazilian Amazon states of Acre, Amapá and Amazonas revealed five species, with Aspergillus section Flavi species A. nomius and A. flavus the most abundant. PCR primers ASP_GEN_MTSSU_F1 and ASP_GEN_MTSSU_R1 were designed for the genus Aspergillus, targeting a portion of the mitochondrial small subunit ribosomal RNA gene. Primer specificity was validated through both electronic PCR against target gene sequences at Genbank and in PCR reactions against DNA from Aspergillus species and other fungal genera common on Brazil nut. Collective differentiation of the observed section Flavi species A. flavus, A. nomius and A. tamarii from other Aspergillus species was possible on the basis of RFLP polymorphism.ConclusionsGiven the abundance of Aspergillus section Flavi species A. nomius and A. flavus observed on Brazil nut, and associated risk of mycotoxin accumulation, simple identification methods for such mycotoxigenic species are of importance for Hazard Analysis Critical Control Point system implementation. The assay for the genus Aspergillus represents progress towards specific PCR identification and detection of mycotoxigenic species.

Spider Silks and Their Biotechnological Applications

Spiders have developed specialized silks with outstanding biophysical properties over millions of years of evolution. As biopolymers composed by highly repetitive amino acid motifs, spider silks have been the focus of research for years. Due to recent advances in genetic engineering, recombinant spider silks have been produced, revealing the relationships between their protein structure and their mechanical properties. Each amino acid motif present in the silk adopts a particular secondary structure responsible for conferring a specific mechanical property to it. This feature has opened up the possibility to produce recombinant silks with controlled properties for various biotechnological applications. Moreover, spider silks are biocompatible and biodegradable biomaterials, which also allow their application in medicine. Accordingly, the relationship between molecular composition, secondary structure, and mechanical properties of spider silks is described in this chapter, along with a discussion of the current strategies for the production of recombinant spider silks, their importance in new biotechnological applications, and the current status of the field.