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Here, we review the processes involved in producing and assessing the quality of recombinant spider silk proteins (spidroins) and the challenges associated with their synthesis and spinning into robust fibres. We provide an overview of the techniques used to produce the proteins, from gene synthesis to expression in various host organisms. Evidence suggests that the N- and C-terminal regions of spidroins are of utmost importance for fibre assembly and the repetitive domains are responsible for the unique mechanical properties in both native and recombinant versions of spider silks. We describe the role of liquid–liquid phase separation (LLPS) in spidroin assembly and its importance in subsequent fibre formation. Recent developments in recombinant spidroin production and co-expression strategies for improving yield and scalability are highlighted. Techniques such as mass photometry and size exclusion chromatography (SEC) for analysing protein purity and assembly behaviour are thereupon detailed. Finally, we address the role that predictive computational methods play in the future of designing novel and high-performing materials inspired by spidroins. 

Natural glues offer great potential as bio-inspired solutions to problems associated with the performance of synthetic adhesives. Spider viscous glues are elastic pressure sensitive adhesives (PSAs) that physically adhere to surfaces on contact across a range of environmental conditions. Extracting useful components from these secretions remains a challenge that can be met by the comparative analyses of functional analogues. Here we used 1 H NMR spectroscopy and mass spectrometry to ascertain the organic salt compositions of the PSAs of four different species of Australian spiders belonging to two lineages that independently acquired aqueous gluey secretions: the St Andrew’s cross ( Argiope keyserlingi ), the redback ( Latrodectus hasselti ), the false widow ( Steatoda grossa ), and the daddy long-legs spider ( Pholcus phalangiodes ). The PSAs from each of these spiders contained similar organic salts, albeit in variable concentrations. The adhesives of the false widow and daddy long-legs spider had mixtures of only a few components, of which betaine predominated, while the PSAs of the other spiders predominantly contained small organic acids such as GABA/GABA-amide, isethionate, and choline salts. Our results suggest that the PSA composition of spiders is likely to be influenced more by environmental factors than evolutionary history and are guided by common principles. Our findings could be valuable for facilitating the design of more sustainable synthetic glues. 

To enhance the solubility of orally administered pharmaceuticals, liquid capsules or amorphous tablets are often preferred over crystalline drug products. However, little is known regarding the variation in bonding mechanisms between pharmaceutical molecules in their different disordered forms. In this study, liquid and melt-quenched glassy carbamazepine have been studied using high energy X-ray diffraction and modeled using Empirical Potential Structure Refinement. The results show significant structural differences between the liquid and glassy states. The liquid shows a wide range of structures; from isolated molecules, to aromatic ring correlations and NH-O hydrogen bonding. Upon quenching from the liquid to the glass the number of hydrogen bonds per molecule increases by ~50% at the expense of a ~30% decrease in the close contact (non-bonded) carbon-carbon interactions between aromatic rings. During the cooling process, there is an increase in both singly and doubly hydrogen-bonded adjacent molecules. Although hydrogen-bonded dimers found in the crystalline states persist in the glassy state, the absence of a crystalline lattice also allows small, hydrogen-bonded NH-O trimers and tetramers to form. This proposed model for the structure of glassy carbamazepine is consistent with the results from vibrational spectroscopy and nuclear magnetic resonance. 

Many natural silks produced by spiders and insects are unique materials in their exceptional toughness and tensile strength, while being lightweight and biodegradable–properties that are currently unparalleled in synthetic materials. Myriad approaches have been attempted to prepare artificial silks from recombinant spider silk spidroins but have each failed to achieve the advantageous properties of the natural material. This is because of an incomplete understanding of the in vivo spidroin-to-fiber spinning process and, particularly, because of a lack of knowledge of the true morphological nature of spidroin nanostructures in the precursor dope solution and the mechanisms by which these nanostructures transform into micrometer-scale silk fibers. Herein we determine the physical form of the natural spidroin precursor nanostructures stored within spider glands that seed the formation of their silks and reveal the fundamental structural transformations that occur during the initial stages of extrusion en route to fiber formation. Using a combination of solution phase diffusion NMR and cryogenic transmission electron microscopy (cryo-TEM), we reveal direct evidence that the concentrated spidroin proteins are stored in the silk glands of black widow spiders as complex, hierarchical nanoassemblies (∼300 nm diameter) that are composed of micellar subdomains, substructures that themselves are engaged in the initial nanoscale transformations that occur in response to shear. We find that the established micelle theory of silk fiber precursor storage is incomplete and that the first steps toward liquid crystalline organization during silk spinning involve the fibrillization of nanoscale hierarchical micelle subdomains.