The high tensile strength and biocompatibility of spider dragline silk makes it a desirable material in many engineering and tissue regeneration applications. Here, we present the feasibility to produce recombinant proteins in transgenic tobacco Nicotiana tabacum with sequences representing spider silk protein building blocks . Recombinant mini-spidroins contain native N- and C-terminal domains of major ampullate spidroin 1 (rMaSp1) or rMaSp2 flanking an abbreviated number (8, 16 or 32) of consensus repeat domains. Two different expression plasmid vectors were tested and a downstream chitin binding domain and self-cleavable intein were included to facilitate protein purification. We confirmed gene insertion and RNA transcription by PCR and reverse-transcriptase PCR, respectively. Mini-spidroin production was detected by N-terminus specific antibodies. Purification of mini-spidroins was performed through chitin affinity chromatography and subsequent intein activation with reducing reagent. Mini-spidroins, when dialyzed and freeze-dried, formed viscous gelatin-like fluids.

Spider dragline silk is a proteinaceous material that combines superior toughness and biocompatibility, which makes it a promising biomaterial. The distinct protein structure and the fiber formation process contribute to the superior toughness of dragline silk. Previously, we have produced recombinant spider silk-like proteins in transgenic tobacco that are readily purified from plant extracts. The plant-derived spidroin-like proteins consisted of native major ampullate spidroin 1 or spidroin 2 N- and C-termini flanking 8, 16, or 32 copies of their respective consensus block repeats (mini-spidroins). Here, we present the generation of fibers from mini-spidroins (rMaSp1R8 and rMaSp2R8) by polyelectrolyte complex formation using an anionic polyelectrolyte, gellan gum. Mini-spidroins, when treated with acetic acid and cross-linked by glutaraldehyde, formed a thin film at the interface when overlaid with a gellan gum solution. Immediate pulling of the film resulted in autofluorescent fibrous materials from either mini-spidroin alone or a combination of rMaSp1R8 and rMaSp2R8 (70:30). Addition of chitosan to the mini-spidroin solutions permitted continuous fiber production until the spinning dope supply was exhausted. When air-dried as-spun fibers were rehydrated and stretched in water, the fiber diameter decreased and the overall toughness improved. This study showed that spider silk-like fibers can be produced in large quantities through charge attraction that assembles chitosan, mini-spidroins, and gellan gum into fibrous complexes. We speculate that the spider silk self-assembly process in the duct may involve attraction of variously charged chitinous polymers, spidroins, and glycoproteins.

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Background and ObjectivesTo expand the clinical knowledge of GPAA1-related glycosylphosphatidylinositol (GPI) deficiency.MethodsAn international case series of 7 patients with biallelic GPAA1 variants were identified. Clinical, biochemical, and neuroimaging data were collected for comparison. Where possible, GPI-anchored proteins were assessed using flow cytometry.ResultsTen novel variants were identified in 7 patients. Flow cytometry samples of 3 available patients confirmed deficiency of several GPI-anchored proteins on leukocytes. Extensive phenotypic information was available for each patient. The majority experienced developmental delay, seizures, and hypotonia. Neuroimaging revealed cerebellar anomalies in the majority of the patients. Alkaline phosphatase was within the normal range in 5 individuals and low in 1 individual, as has been noted in other transamidase defects. We notably describe individuals either less affected or older than the ones published previously.DiscussionClinical features of the cases reported broaden the spectrum of the known phenotype of GPAA1-related GPI deficiency, while outlining the importance of using functional studies such as flow cytometry to aid in variant classification.