The review, led by Anton Maraldo and Sean Blamires, traces how scientists construct synthetic genes encoding spidroins, the proteins that make up spider silk. These genes are inserted into expression vectors and transformed into host organisms ranging from Escherichia coli to yeast, plants, silkworms, and even transgenic goats. The hosts then produce the proteins, which are isolated and concentrated into a spinning dope. But generating fibres with qualities that resemble native spider silk remains, according to the authors, one of material science’s grandest challenges. The problem is not just producing the proteins—it is producing them at sufficient quantities, purities, and concentrations high enough to spin into exceptionally robust fibres.

At the heart of the challenge lies the architecture of the spidroin molecule itself. Native spidroins are enormous polypeptides of roughly 200 to 350 kilodaltons, built from a repetitive core flanked by conserved N- and C-terminal regions. The review highlights that the terminal regions are of utmost importance for fibre assembly, while the repetitive domains are responsible for the unique mechanical properties. Across twelve different spider species and multiple fibre types, more than fifty percent of the amino acids in the N- and C-termini were found to be congruent, suggesting these regions are integral to how spider silk proteins assemble. Recombinant constructs have therefore been designed using conserved terminal domains from single or different spider species, creating so-called chimeric structures with varying numbers of conserved glycine- and alanine-rich repeats.

The repetitive domain is where the mechanical magic happens. Most major ampullate spidroin constructs are built from two major subunits: a glycine-rich GGX or GPGXX motif, where X denotes any amino acid and often a sticker residue, followed by a poly-alanine sequence spanning four to seven residues on average. As fibre formation proceeds, these motifs respond to stimuli such as pH shifts, shear forces, and kosmotropic buffer exchange in the spinning duct, forming defined secondary structures. Evidence suggests that poly-alanine sequences form hydrophobic β-sheet crystalline domains that provide silk with high tensile strength, whereas the glycine-rich regions form hydrophilic extensible 3₁₀-helical structures that account for elasticity. The comparatively small size of the most abundant residues enables the formation of highly compact β-sheet content due to minimised interchain distance.

Producing these large, repetitive proteins in heterologous hosts is no small feat. Plasmid stability, difficulties in host-cell translation, protein misfolding, and potential toxicity of certain sequences to host cells all conspire against high yields. Spiders have evolved to overexpress tRNAs for highly abundant residues like glycine and alanine to prevent translational bottlenecking, but common expression hosts lack this specialised machinery. Solutions have taken inspiration from spider physiology: co-expression of recombinant major ampullate spidroins with a plasmid supplying additional glycine and alanine tRNAs showed significant improvement in expression. Combined with incubating bacteria in media with elevated glycine and alanine levels, this strategy allows expression of large, native-like spidroins with comparable yields to smaller repeat proteins. Molecular biology techniques such as recursive directional ligation and, more recently, circular RNA production offer additional avenues for generating native-length templates without duplicating a single motif.

One of the most exciting emerging paradigms in the field is liquid–liquid phase separation, or LLPS. This phenomenon, where proteins separate into a proteinaceous dense phase and a buffer light phase, is increasingly recognised as a critical pre-assembly step in spider silk fibre formation. The sticker-spacer model is often used to describe the residue-residue interactions that drive LLPS: stickers act as molecular glues holding proteins together, while spacers—abundant glycine and alanine residues—provide flexibility and spatially control the interactions between less abundant but more reactive sticker residues like tyrosine, glutamine, and proline, which can enter π-π, π-cation, and charge interactions. Probing of a recombinant MaSp2 showed phosphate ion-induced LLPS at neutral pH resulting in coacervate formation, and when the pH is lowered during spinning—as happens within the spider’s silk gland—the coacervates become fibrous. LLPS is also being explored as a mechanism to control protein solubility during expression in E. coli, and even to generate synthetic organelles capable of performing enzymatically driven reactions within the cell.

Once proteins are produced, purification becomes the next bottleneck. Contaminants from E. coli or other expression systems could degrade the protein of interest or obstruct fibre assembly. Cell lysis methods—ultrasonication, pressure homogenisation, freeze-thaw cycles, or chemical lysing agents—must be tailored to the specific case. The overexpressed proteins may be soluble in the cytosol, or may form insoluble inclusion bodies requiring solubilisation and refolding. A multistep purification protocol is generally recommended, beginning with an affinity step using tags such as the traditional 6-His tag, streptavidin-based tags eluted with biotin, maltose binding protein, or glutathione-S-transferase. Recently, a new generation of tags has been released in which the desired protein self-cleaves from its tag during purification, leaving no residual amino acids. Ion-exchange and hydrophobic interaction chromatography offer alternative strategies that exploit the natural polarity of the protein construct.

Characterising the purified proteins requires sophisticated analytical techniques. Size exclusion chromatography remains an established method for fractionating polydisperse spidroin solutions and isolating the monomeric fraction, though its efficiency in capturing real-time assembly is limited. A newly developed light-based method called mass photometry can accurately determine the molecular weights of single molecules and protein assemblies with extremely low volumes and concentrations, making it a powerful tool for quantifying assembly properties, polydispersity, and protein behaviour in various buffers. Spectrofluorimetric analysis using Thioflavin T—originally adopted from neurodegenerative research into amyloid-forming peptides—reports β-sheet formation through fluorescence that only occurs once the dye has intercalated into the β-sheet structure. Bright-field and confocal microscopy characterise spidroin phase separation, while Fluorescence Recovery after Photobleaching (FRAP) determines the diffusion properties of proteins within dense droplets. Recent work utilised FRAP analysis of a recombinantly expressed MaSp2 to determine physical property shifts when buffered into environments with progressively decreasing pH, with lower pH samples showing a significant decrease in fluorescence recovery—characterising the transition from pre-assembly droplets into more rigid nanofibril structures.

Machine learning and artificial intelligence are now transforming how researchers design and evaluate spidroin constructs. The release of AlphaFold2 in 2021 revolutionised protein structure prediction, and ColabFold further enhanced accessibility by making fast, accurate predictions available to the wider scientific community—running five times faster than AlphaFold2 for single predictions. AlphaFold3, released during the preparation of the review, offers even greater speed and ease of use, though it remains limited to 5000 tokens per run, equating to a maximum of about fourteen MaSp1 units. Fold prediction tools can estimate terminal domain structures with high confidence, but their ability to predict the repetitive domain remains limited to low-confidence outputs. Beyond AlphaFold, applying deep learning to proteome datasets has enabled accurate predictions of radius of gyration, end-to-end distance, polymer-scaling exponents, asphericity, and phase separation propensity. Specialised spidroin tools have also been developed, ranging from coarse-grained modelling of silk synthesis throughout the gland to generative design of novel spidroin sequences trained on a dataset of over one thousand silk sequences annotated with physical properties such as elasticity, tensile strength, and water content.

The review concludes that the future of recombinant spider silk production lies in optimising both expression systems and spinning methodologies to develop economically viable methods for creating fibres with properties approaching those of their natural counterparts. Further investigation into liquid–liquid phase separation and other pre-assembly processes offers exciting avenues for enhancing fibre quality and consistency. The authors argue that future research should prioritise understanding the unique silk gland environments across spider species, as factors such as pH shifts, shear forces, ion concentrations, and duct physiology influence mechanical properties in ways that require closer examination. Comparative studies across species could reveal crucial differences in gland morphology and function, offering clues to refining recombinant processes. With advances in bioengineering approaches such as gene editing and expression systems, alongside improvements in synthetic spinning methodologies, the field seems poised to deliver on the promise of a material that has captivated scientists for generations—one that could transform medicine, textiles, wearables, and materials engineering alike.

Subject of Research: Biochemical methods for producing and characterising recombinant spider silks

Article Title: Biochemical methods for producing and characterising recombinant spider silks

Article References: Biochemical methods for producing and characterising recombinant spider silks. (n.d.). https://doi.org/10.3389/frchs.2024.1488680

Image Credits: AI Generated

DOI: 10.3389/frchs.2024.1488680

Keywords: Biochemical, methods, producing, characterising, recombinant, spider, silks, scientific research