Abstract The development of bioactive protein-based materials hinges on scalable expression systems and robust and mild processing methods. Here we report the successful bacterial overexpression and purification of a fusion protein comprising the mini-spidroin (A3I)3-A14 and the Immunoglobulin G (IgG)-degrading enzyme IdeS. High-density fed-batch bioreactor cultivation resulted in expression levels of 7.2 g/L, demonstrating the scalability of the system. Structural and functional characterization confirmed that the IdeS moeity was folded and retained enzymatic activity. Importantly, the high solubility of the fusion protein ( > 200 mg/mL), combined with the mini-spidroin’s ability to assemble into gels and fibers, allowed us to develop self-assembled hydrogels and wet-spun fibers that maintained enzymatic function. Notably, enzymatic activity was preserved in fibers stored for prolonged times under both wet and dry conditions. These findings illustrate the potential of spidroin fusion proteins as a modular platform for producing robust bioactive materials in a scalable process, offering new avenues for biomedical applications.

Large-scale production of artificial spider silk fibers requires heterologous expression of spider silk proteins (spidroins), yet current methods remain limited by low yields and costly purification processes. To overcome these challenges, we engineered mini-spidroins in which the poly-alanine motifs of the repetitive region were replaced with the non-natural amyloidogenic β16 peptide, significantly enhancing expression yields and solubility. Furthermore, we developed a simple, chromatography-free purification method for these constructs based on NaCl-induced liquid-liquid phase separation (LLPS). This one-step purification strategy reduced processing costs by up to 99% compared to conventional affinity chromatography while achieving yields of ~300 mg of purified protein per liter of shake flask culture and ~25 g L-1 from bioreactor cultivations. The purified engineered mini-spidroins could be spun into continuous fibers using an all-aqueous, biomimetic spinning process triggered by a pH drop. The resulting fibers exhibited mechanical properties comparable to those produced from the mini-spidroin NT2RepCT, which requires conventional chromatographic purification. Together, our protein-engineering approach and LLPS-based purification method provide a potentially scalable, sustainable, and cost-effective platform for artificial spider silk, representing a major step toward the commercial viability of recombinant silk-based materials.

Every year, our clothes release huge amounts of plastics into the environment, and some of those plastics end up in our bodies. To make cleaner and safer fabrics, we can look at one of nature’s best fiber-makers: spiders! Spider silk is strong like steel, but seven times lighter, much stretchier, and biodegradable. Even better, spiders make it at room temperature using only water. Unfortunately, we cannot farm spiders to make silk, so we have learned to copy their tricks. We put spider silk genes into bacteria, so the bacteria made lots of silk proteins. Then we spun these proteins into fibers using a method similar to the one spiders use. The result is a strong, environmentally friendly fiber. To control the silk fibers without touching them, we mixed the silk proteins with tiny magnetic particles. The resulting fibers are both strong and magnetic, making them perfect candidates for the advanced textiles of the future.

Combining spider silk and magnetic nanostructures is a key challenge for advancing technological fields such as soft robotics, magnetosurgery, and smart textiles. It is essential to devise approaches that also aim to expand fundamental knowledge on the feasibility of coupling the protein-based fiber with the inorganic magnetic phase. An original strategy is presented to create magneto-responsive artificial spider silk fibers by coating them with thin layers (nominal thickness 5nm, 10nm, and 100nm) of ferromagnetic and magnetostrictive FeCo alloy via sputtering deposition. The mechanical strength and elasticity of the produced fibers remain close to those of the uncoated material. A relationship is established between the magnetic properties of the FeCo coating and its structural characteristics, which are strongly influenced by the underlying fiber morphology. The magnetomechanical response of the FeCo coating of single fibers under strain is evaluated using magneto-optical Kerr effect magnetometry. Experimental data, interpreted through the shear-lag model, reveal excellent silk-metal coupling and highlight how the coating thickness governs stress transmission and the fragmentation process. The reversibility of the magnetomechanical response is investigated, providing further insights into the silk/metal coupling. These findings contribute to clarifying how the interplay between organic and inorganic components determines the composite's overall properties.

Spider silk formation involves tightly regulated protein assembly influenced by pH and the presence of ions. Kosmotropic salts induce phase separation of spidroins; however, their exact role in assembly is not clear. Here, we investigate how sodium and potassium phosphate affect spidroin interactions via the single-molecule method of mass photometry. We observed that spidroin oligomerization occurs at low nanomolar protein concentrations. Potassium ions were found to stabilize a compact conformation of individual spidroins and slow down pH-induced β-sheet aggregation, consistent with its more kosmotropic nature. Microfluidic MP showed that pre-assembly of the protein through salt-induced phase separation reduced the number and size of oligomeric intermediates that form upon acidification. Together, the findings suggest that spidroins have an inherent ability to self-assemble, blurring the line between one- and two-phase status. Subtle differences in ion composition are sufficient to change spidroin stability and assembly, potentially contributing to silk spinning in vivo by balancing storage stability with rapid fiber formation. Spider silk formation involves tightly regulated protein assembly, influenced by pH and the presence of ions. Here, single-molecule mass photometry reveals that mini-spidroin interactions are influenced by millimolar concentrations of sodium and potassium, which may control the formation of spidroin clusters.

Abstract Recombinant spider silk proteins (spidroins) are emerging as a promising feedstock for biomaterial production due to their inherent ability to form hydrogels at 37 °C. However, their broader application as a robust cell culture platform has been hindered by slow gelation kinetics, CO 2 -induced turbidity, unknown long-term stability, and the use of Tris-HCl buffers that are suboptimal for most mammalian cells. In this study, we aimed to accelerate gelation kinetics of mini-spidroin-based hydrogels, reduce their turbidity, and improve gel stability under physiological conditions. Systematic evaluation of protein pre-treatments and buffer compositions identified parameters governing conformational behavior, gelation dynamics, and structural stability. Multimodal characterization, including turbidity measurements, circular dichroism spectroscopy, Fourier-transform infrared spectroscopy, mechanical assessment, transmission electron microscopy, Thioflavin T assays, and in vitro studies, enabled the formulation of a cytocompatible buffer system optimized for mini-spidroin hydrogels. The formulation improves transparency and accelerates gelation, while maintaining experimental simplicity, thereby advancing the utility of mini-spidroin hydrogels as cell culture platforms.

Spider silk is an extraordinary natural material that combines strength, extensibility, and toughness in a lightweight, protein-based fiber. While the recombinant production of spidroins has advanced, the creation of silk fibers with additional intrinsic functionalities, such as color, remains a major challenge. Here, we report the rational design, expression, and spinning of intrinsically red-colored artificial silk fibers. A mini-spidroin variant was engineered as a fusion with the red fluorescent protein mCherry, expressed at high yields (20 g/L) in E. coli fed-batch fermentations, and purified under native conditions. Although the presence of the mCherry globular domain reduced spinnability when used alone, blending with wild-type mini-spidroins enabled continuous wet spinning into robust, fluorescent fibers. Our biomimetic spinning approach preserved the correct folding of the mCherry domain within the fiber, resulting in stable red coloration and fluorescence, while maintaining mechanical properties comparable to those of other recombinant silks. This work establishes a scalable, sustainable strategy for fabricating colored bio-based fibers, opening avenues for environmentally friendly textiles and functional biomaterials that may reduce reliance on synthetic and chemically dyed fibers. This paper reports the design and spinning of intrinsically red-colored artificial silk fibers. A mini-spidroin variant is engineered as a fusion with the red fluorescent protein mCherry, expressed at high yields in E. coli fed-batch fermentations.

Bacterial cellulose (BC) has emerged as a promising sustainable biomaterial with potential across multiple fields. However, its intrinsic stiffness and limited toughness restrict its use in applications such as textiles or wearable devices. Spider silk is one of nature's toughest materials, combining high strength and extensibility. The recombinant mini-spidroin (A3I)3-A14, a minimized synthetic variant of a spider silk protein, can be spun under mild conditions into ductile and tough fibers. Here, we present a bioinspired strategy to toughen BC with (A3I)3-A14 by tuning the incorporation route and protein conformation to achieve lower stiffness, higher extensibility, and improved energy dissipation. A motile strain, E. coli JM109 (DE3), was used as a heterologous production host and active delivery vehicle to introduce the mini-spidroin within the BC hydrogel. Guided by glucose-gradient-driven colonization of the porous BC nanofiber network, followed by controlled sodium dodecyl sulfate (SDS)-alkali-mediated cell lysis, this approach enabled deeper in situ incorporation of (A3I)3-A14 within the BC matrix. The resulting BC-silk composites exhibited an >240% increase in toughness, primarily driven by a significant enhancement of the strain at break. This balanced combination of softness, strain tolerance, and energy absorption highlights the potential of BC-silk composites as crease-resistant bio-based films.

Abstract We present an active mixing toolhead for extrusion bioprinting. The tool enables the programmable fabrication of tumor microenvironment gradient arrays, through controlled deposition of mixed hydrogel precursor formulations into 384-well plates, pre-seeded with tumor cells. It operates on an open-source bioprinter and can actively mix arbitrary ratios of two hydrogel precursors prior to extrusion. These concentration gradient arrays are compatible with quantitative image analysis of cell viability and morphological responses to hydrogels conditioned with drug or extracellular matrix (ECM) proteins. The tools capacity to mix and print hydrogel precursor gradients was demonstrated using alginate and highly concentrated mCherry-conjugated mini-spidroin solutions. Hydrogel precursor stocks contained fluorescent reporters to facilitate quantifications of mixing efficiency, and as proxies for drug and ECM protein concentrations. The tool was applied to generate hydrogel-based gradients of the apoptosis-inducer staurosporine, from which concentration-dependent MDA-MB-231 breast cancer cell death responses were quantified. Gradient arrays of the ECM protein laminin-511, implicated in breast cancer tumorigenesis, were generated and revealed that increasing laminin-511 concentrations potentiated staurosporine-induced cell death. The study demonstrates the utility of this active mixing toolhead for producing hydrogel gradient arrays, and demonstrates the relevance of studying drug-responses in tumor microenvironment models that account for disease-specific ECM components.

ABSTRACT The creation of protein‐based magnetic fibers is a strategic issue in the field of advanced biocompatible materials, particularly relevant for technological sectors such as soft robotics and smart medicine. Here, we endow artificial spider silk fibers, which outperform many man‐made fibers in terms of mechanical properties, with magnetic functionality through the incorporation of magnetic nanoparticles. We present two novel composite fibers, containing magnetite nanoparticles coated with aminopropylsilane and dextran, and compare them with a third fiber type, which was made, following an approach previously developed by us, using magnetite nanoparticles coated with dimercaptosuccinic acid. The nanoparticles also differ in their mean size, varying between 9 and 32 nm. The fibers are produced by wet spinning, with a nominal magnetite concentration in the 0.2–20 wt.% range. However, the coating rules the colloidal stability of the nanoparticles in the spinning dope and their tendency to agglomerate. Therefore, the actual magnetite concentration and the degree of dispersion of the nanoparticles in the fibers are different in the different composites, as revealed by magnetic analyses. All fibers, even those with the highest magnetite content, remain ductile, whereas the mechanical strength is only slightly reduced compared to the fiber without nanoparticles, hence without magnetic functionality.

Despite impressive progress in the field, there are still several major bottlenecks in producing fibers from recombinantly produced spider-silk-like proteins to replicate the extraordinary mechanical properties of spider major ampullate silk. The conventional artificial fiber spinning processes rely primarily on organic solvents to coagulate proteins into fibers and require complex post-treatments to obtain fibers with valuable properties. This is due to challenges in obtaining soluble silk proteins, but also because the native silk spinning process leading to the hierarchical organization of the silk proteins is not fully understood and is hard to replicate in a manner applicable to industrial settings. Here, recombinant spider-silk fusion proteins are efficiently produced and processed into as-spun fibers with a toughness modulus of 120 MJ m-3 and extensibility of 255% using solely aqueous solutions. The spider-silk fusion proteins assemble in a manner similar to that reported for native spider silk: they phase separate induced by salting out, followed by alignment and a secondary structure transition triggered by shear forces and dehydration. Finally, the design of the fusion silk proteins enables straightforward functionalization of the fibers under mild all-aqueous conditions via a simple biomolecular click reaction both pre- and post-spinning. A sustainable aqueous wet spinning method enables an efficient production of artificial spider silk fibers which show excellent extensibility and remarkable toughness. A highly specific and efficient biomolecular click reaction is employed to functionalize these fibers under mild conditions, which offers a promising method to construct multi-functional 1D materials. image

Recent biotechnological advancements in protein production and development of biomimetic spinning procedures make artificial spider silk a promising alternative to petroleum-based fibers. To enhance the competitiveness of artificial silk in terms of mechanical properties, refining the spinning techniques is imperative. One potential strategy involves the integration of post-spin stretching, known to improve fiber strength and stiffness while potentially offering additional advantages. Here, we demonstrate that post-spin stretching not only enhances the mechanical properties of artificial silk fibers but also restores a higher and more uniform alignment of the protein chains, leading to a higher fiber toughness. Additionally, fiber properties may be reduced by processes, such as aging, that cause increased network entropy. Post-spin stretching was found to partially restore the initial properties of fibers exposed aging. Finally, we propose to use the degree of necking as a simple measure of fiber quality in the development of spinning procedures for biobased fibers.

Flexible magnetic materials have great potential for biomedical and soft robotics applications, but they need to be mechanically robust. An extraordinary material from a mechanical point of view is spider silk. Recently, methods for producing artificial spider silk fibers in a scalable and all-aqueous-based process have been developed. If endowed with magnetic properties, such biomimetic artificial spider silk fibers would be excellent candidates for making magnetic actuators. In this study, we introduce magnetic artificial spider silk fibers, comprising magnetite nanoparticles coated with meso-2,3-dimercaptosuccinic acid. The composite fibers can be produced in large quantities, employing an environmentally friendly wet-spinning process. The nanoparticles were found to be uniformly dispersed in the protein matrix even at high concentrations (up to 20

Spider silk is renowned for its exceptional mechanical properties, surpassing those of other natural and many synthetic fibers. Yet, replicating its remarkable properties through synthetic production remains a challenge. The variability in the mechanical properties of synthetic spider silks lacking protective coatings, exacerbated by factors such as spinning conditions and humidity levels, poses an additional challenge, impacting their application potential. Bioconjugation offers a versatile synthetic method to modify protein structures, enhancing their pharmacokinetics, solubility, stability, and immune response. In particular, polyethylene glycol (PEG)-ylation has emerged as a successful strategy with numerous marketed PEG-protein conjugates. This study introduces synthetic spider silk-multiarm PEG bioconjugates, facilitating spidroin crosslinking, and chemical functionalization while retaining a biomimetic spinning approach. Two different examples demonstrate the potential of this approach to improve the fiber's tensile strength and extensibility, respectively, both leading to an increased toughness modulus. Furthermore, the approach could allow the tuning of fiber mechanical properties without developing a new mini-spidroin construct and fiber coating with lipids attached to multiarm PEG, potentially mitigating the impact of environmental conditions on synthetic spider silk fibers. Bioconjugation of recombinant mini-spidroins with multiarm polyethylene glycol (PEG) is employed to produce artificial spider silk, which can be crosslinked and chemically functionalized while maintaining a biomimetic spinning approach. The best fibers exhibit enhanced tensile strength and/or extensibility, both contributing to an increased toughness modulus. These spidroin-PEG bioconjugate fibers may be suitable for use as anti-adhesive coatings and scar-minimizing biomaterials. image

Silk-based materials are sought after across various industries due to their remarkable properties, including high strength and flexibility. However, their practical application depends largely on how well these properties are maintained under different environmental conditions. Despite significant advancements in the large-scale production of artificial silk fibers, the effects of temperature on their mechanical behavior are understudied. In this study, the mechanical properties of artificial spider silk fibers between -80 and +120 degrees C are examined and compared to both synthetic and natural silk fibers. The findings reveal that artificial silk fibers maintain their strength up to +120 degrees C, though the strain at break slightly decreases, remaining above 60%. At -80 degrees C, the fibers exhibit increased strength, but the strain at break is reduced. While these artificial fibers closely mimic the behavior of natural silk, they show a noticeable reduction in extensibility at low temperatures. Complementing experimental data, differential scanning calorimetry, and thermogravimetric analysis are also conducted, proposing a simple physical model to explain the observed temperature-induced softening. Encouragingly, the degradation temperature of artificial silk is comparable to that of native silkworm and spider silk. This study underscores the importance of enhancing the mechanical robustness of artificial silk to expand its applications.

Spiders can produce up to seven different types of silk, each with unique mechanical properties that stem from variations in the repetitive regions of spider silk proteins (spidroins). Artificial spider silk can be made from mini-spidroins in an all-aqueous-based spinning process, but the strongest fibers seldom reach more than 25% of the strength of native silk fibers. With the aim to improve the mechanical properties of silk fibers made from mini-spidroins and to understand the relationship between the protein design and the mechanical properties of the fibers, we designed 16 new spidroins, ranging from 31.7 to 59.5 kDa, that feature the globular spidroin N- and C-terminal domains, but harbor different repetitive sequences. We found that more than 50% of these constructs could be spun by extruding them into low-pH aqueous buffer and that the best fibers were produced from proteins whose repeat regions were derived from major ampullate spidroin 4 (MaSp4) and elastin. The mechanical properties differed between fiber types but did not correlate with the expected properties based on the origin of the repeats, suggesting that additional factors beyond protein design impact the properties of the fibers.

Hydrogels are useful drug release systems and tissue engineering scaffolds. However, synthetic hydrogels often require harsh gelation conditions and can contain toxic by-products while naturally derived hydrogels can transmit pathogens and in general have poor mechanical properties. Thus, there is a need for a hydrogel that forms under ambient conditions, is non-toxic, xeno-free, and has good mechanical properties. A recombinant spider silk protein-derived hydrogel that rapidly forms at 37 °C is recently developed. The temperature and gelation times are well-suited for an injectable in situ polymerising hydrogel, as well as a 3D cell culture scaffold. Here, it is shown that the diffusion rate and the mechanical properties can be tuned by changing the protein concentration and that human fetal mesenchymal stem cells encapsulated in the hydrogels show high survival and viability. Furthermore, mixtures of recombinant spider silk proteins and green fluorescent protein (GFP) form gels from which functional GFP is gradually released, indicating that bioactive molecules are easily included in the gels, maintain activity and can diffuse through the gel. Interestingly, encapsulated ARPE-19 cells are viable and continuously produce the growth factor progranulin, which is detected in the cell culture medium over the study period of 31 days.

Silk fibers have unique mechanical properties, and many studies of silk aim at understanding how these properties are related to secondary structure content, which often is determined by infrared spectroscopy. We report significant method-induced irreversible structural changes to both natural and synthetic spider silk fibers, derived from the widely used attenuated total reflection Fourier-transform infrared (ATR-FTIR) technique. By varying the force used to bring fibers into contact with the internal reflection elements of ATR-FTIR accessories, we observed correlated and largely irreversible changes in the secondary structure, with shape relaxation under pressure occurring within minutes. Fitting of spectral components shows that these changes agree with transformations from the alpha-helix to the beta-sheet secondary structure with possible contributions from other secondary structure elements. We further confirm the findings with IR microspectroscopy, where similar differences were seen between the pressed and unaffected regions of spider silk fibers. Our findings show that ATR-FTIR spectroscopy requires care in its use and in the interpretation of the results.

Spider silk fibers are of scientific and industrial interest because of their extraordinary mechanical properties. These properties are normally determined by tensile tests, but the values obtained are dependent on the morphology of the fibers, the test conditions, and the methods by which stress and strain are calculated. Because of this, results from many studies are not directly comparable, which has led to widespread misconceptions in the field. Here, we critically review most of the reports from the past 50 years on spider silk mechanical performance and use artificial spider silk and native silks as models to highlight the effect that different experimental setups have on the fibers' mechanical properties. The results clearly illustrate the importance of carefully evaluating the tensile test methods when comparing the results from different studies. Finally, we suggest a protocol for how to perform tensile tests on silk and biobased fibers.