Spider Silk Proteins Research Articles

Factors Influencing Properties of Spider Silk Coatings and Their Interactions within a Biological Environment.

Biomaterials are an indispensable part of biomedical research. However, although many materials display suitable application-specific properties, they provide only poor biocompatibility when implanted into a human/animal body leading to inflammation and rejection reactions. Coatings made of spider silk proteins are promising alternatives for various applications since they are biocompatible, non-toxic and anti-inflammatory. Nevertheless, the biological response toward a spider silk coating cannot be generalized. The properties of spider silk coatings are influenced by many factors, including silk source, solvent, the substrate to be coated, pre- and post-treatments and the processing technique. All these factors consequently affect the biological response of the environment and the putative application of the appropriate silk coating. Here, we summarize recently identified factors to be considered before spider silk processing as well as physicochemical characterization methods. Furthermore, we highlight important results of biological evaluations to emphasize the importance of adjustability and adaption to a specific application. Finally, we provide an experimental matrix of parameters to be considered for a specific application and a guided biological response as exemplarily tested with two different fibroblast cell lines.

Self-Assembly of RGD-Functionalized Recombinant Spider Silk Protein into Microspheres in Physiological Buffer and in the Presence of Hyaluronic Acid.

Biomaterials made of self-assembling protein building blocks are widely explored for biomedical applications, for example, as drug carriers, tissue engineering scaffolds, and functionalized coatings. It has previously been shown that a recombinant spider silk protein functionalized with a cell binding motif from fibronectin, FN-4RepCT (FN-silk), self-assembles into fibrillar structures at interfaces, i.e., membranes, fibers, or foams at liquid/air interfaces, and fibrillar coatings at liquid/solid interfaces. Recently, we observed that FN-silk also assembles into microspheres in the bulk of a physiological buffer (PBS) solution. Herein, we investigate the self-assembly process of FN-silk into microspheres in the bulk and how its progression is affected by the presence of hyaluronic acid (HA), both in solution and in a cross-linked HA hydrogel. Moreover, we characterize the size, morphology, mesostructure, and protein secondary structure of the FN-silk microspheres prepared in PBS and HA. Finally, we examine how the FN-silk microspheres can be used to mediate cell adhesion and spreading of human mesenchymal stem cells (hMSCs) during cell culture. These investigations contribute to our fundamental understanding of the self-assembly of silk protein into materials and demonstrate the use of silk microspheres as additives for cell culture applications.

Wet spinning is employed to produce spider silk with high elasticity

Spider silk exhibits exceptional strength and elasticity in its natural form. Over the course of several decades, researchers have been working on artificially spinning recombinant spider silk proteins (spidroin) in order to replicate the remarkable mechanical properties of natural spider silk. In this study, we utilized the wet spinning method to investigate the relationship between the concentration of the coagulation bath and fiber performance. We discovered that the concentration of methanol plays a crucial role in determining the continuity, diameter, and mechanical properties of the fibers. Lower concentrations of methanol favor the production of continuous, thinner fibers with higher strain. Additionally, secondary stretching during the spinning process contributes to the production of silk fibers with stable mechanical properties and thermal stability. By employing different concentrations of methanol and applying additional stretching, we successfully produced silk fibers with a high strain of 2.1652 ± 0.3871 mm/mm. Furthermore, these wet-spun fibers demonstrated the ability to promote the growth of Schwann cells, indicating their potential application in the field of biomedical engineering. Hence, the exceptional mechanical properties and the ability to promote cellular growth make the obtained spider silk fibers highly promising for various biomedical applications.

Spider dragline silk fibres maintain rectangular columnar liquid crystalline phase

ABSTRACT The liquid crystalline phase of spider silk is thought to enable for the silk protein to be stored at high concentration in the gland without gelation, facilitating the instant fibre formation at the spinning duct. Nevertheless, it has never been revealed whether the liquid crystalline state of the silk protein maintains after the fibre formation. In this study, we conducted the X-ray liquid crystal structure analysis to reveal the details of molecular assemblies of the native spider silk proteins. Based on the wide- and small angle X-ray scattering data using the uniaxial bundle of spider draglines, the spider dragline silk fibres maintain a rectangular columnar liquid crystalline phase with a P21/a (p2gg) symmetry. The liquid crystalline state of the spider silk fibre is not nematic, cholesteric, nor hexagonal columnar, as previously proposed, but rectangularly packed columnar liquid crystal. The movable silk molecular chains along the long fibre axis in the rectangular columnar liquid crystalline structure help to explain the mechanical strength of the native spider dragline silk fibres. The results obtained in this study will contribute to understanding the biological basis underlying the silk fibre formation and will be useful to the biomimicry of spider silk fibres.

Suitable electrospinning approaches for recombinant spider silk proteins

Among the versatile opportunities of processing recombinant spider silk proteins into coatings, hydrogels, particles, fibrils and foams, electrospinning provides a further highly important morphology: submicron- and nanofibers. These fibers display a high surface-to-volume ratio, which, in combination with the silk's biocompatibility, flexibility and functionality allows various applications including drug-delivery, tissue engineering, wound dressings as well as particle filtration from liquid or gas. Based on the chosen application, individual spider silk proteins can be spun out of a choice of solvents using different spinning techniques in order to achieve the desired properties of the fibers. In this perspective article so far used electrospinning solvents, techniques and collectors are displayed and brought into context with their possible application.

Global analysis of kinetics reveals the role of secondary nucleation in recombinant spider silk self-assembly.

Recombinant spider silk proteins can be prepared in scalable fermentation processes and have been proven as sources of biomaterials for biomedical and technical applications. Nanofibrils, formed through the self-assembly of these proteins, possess unique structural and mechanical properties, serving as fundamental building blocks for the fabrication of micro- and nanostructured scaffolds. Despite significant progress in utilizing nanofibrils-based morphologies of recombinant spider silk proteins, a comprehensive understanding of the molecular mechanisms of nanofibrils self-assembly remains a challenge. Here, a detailed kinetic study of nanofibril formation from a recombinant spider silk protein eADF4(C16) in dependence on the protein concentration, seeding and temperature is provided. For the global fitting of kinetic data obtained during the fibril formation, we utilized the online platform AmyloFit. Evaluation of the data revealed that the self-assembly mechanism of recombinant spider silk is dominated by secondary nucleation. Thermodynamic analyses show that both primary and secondary nucleations, as well as the elongation step of the eADF4(C16), are endothermic processes. This article is protected by copyright. All rights reserved.

Spider Silk Protein Forms Amyloid-Like Nanofibrils through a Non-Nucleation-Dependent Polymerization Mechanism.

Amyloid fibrils-nanoscale fibrillar aggregates with high levels of order-are pathogenic in some today incurable human diseases; however, there are also many physiologically functioning amyloids in nature. The process of amyloid formation is typically nucleation-elongation-dependent, as exemplified by the pathogenic amyloid-β peptide (Aβ) that is associated with Alzheimer's disease. Spider silk, one of the toughest biomaterials, shares characteristics with amyloid. In this study, it is shown that forming amyloid-like nanofibrils is an inherent property preserved by various spider silk proteins (spidroins). Both spidroins and Aβ capped by spidroin N- and C-terminal domains, can assemble into macroscopic spider silk-like fibers that consist of straight nanofibrils parallel to the fiber axis as observed in native spider silk. While Aβ forms amyloid nanofibrils through a nucleation-dependent pathway and exhibits strong cytotoxicity and seeding effects, spidroins spontaneously and rapidly form amyloid-like nanofibrils via a non-nucleation-dependent polymerization pathway that involves lateral packing of fibrils. Spidroin nanofibrils share amyloid-like properties but lack strong cytotoxicity and the ability to self-seed or cross-seed human amyloidogenic peptides. These results suggest that spidroins´ unique primary structures have evolved to allow functional properties of amyloid, and at the same time direct their fibrillization pathways to avoid formation of cytotoxic intermediates.

Rheological and Biological Impact of Printable PCL-Fibers as Reinforcing Fillers in Cell-Laden Spider-Silk Bio-Inks.

The development of bio-inks capable of being 3D-printed into cell-containing bio-fabricates with sufficient shape fidelity is highly demanding. Structural integrity and favorable mechanical properties can be achieved by applying high polymer concentrations in hydrogels. Unfortunately, this often comes at the expense of cell performance since cells may become entrapped in the dense matrix. This drawback can be addressed by incorporating fibers as reinforcing fillers that strengthen the overall bio-ink structure and provide a second hierarchical micro-structure to which cells can adhere and align, resulting in enhanced cell activity. In this work, the potential impact of collagen-coated short polycaprolactone-fibers on cells after being printed in a hydrogel is systematically studied. The matrix is composed of eADF4(C16), a recombinant spider silk protein that is cytocompatible but non-adhesive for cells. Consequently, the impact of fibers could be exclusively examined, excluding secondary effects induced by the matrix. Applying this model system, a significant impact of such fillers on rheology and cell behavior is observed. Strikingly, it could be shown that fibers reduce cell viability upon printing but subsequently promote cell performance in the printed construct, emphasizing the need to distinguish between in-print and post-print impact of fillers in bio-inks.

Enhanced production of difficult-to-express proteins through knocking down rnpA gene expression.

Escherichia coli has been employed as a workhorse for the efficient production of recombinant proteins. However, some proteins were found to be difficult to produce in E. coli. The stability of mRNA has been considered as one of the important factors affecting recombinant protein production. Here we report a generally applicable and simple strategy for enhancing mRNA stability, and consequently improving recombinant protein production in E. coli. RNase P, a ribozyme comprising an RNA subunit (RnpB) and a protein subunit (RnpA), is involved in tRNA maturation. Based on the finding that purified RnpA can digest rRNA and mRNA in vitro, it was reasoned that knocking down the level of RnpA might enhance recombinant protein production. For this, the synthetic small regulatory RNA-based knockdown system was applied to reduce the expression level of RnpA. The developed RnpA knockdown system allowed successful overexpression of 23 different recombinant proteins of various origins and sizes, including Cas9 protein, antibody fragment, and spider silk protein. Notably, a 284.9-kDa ultra-high molecular weight, highly repetitive glycine-rich spider silk protein, which is one of the most difficult proteins to produce, could be produced to 1.38gL-1 , about two-fold higher than the highest value previously achieved, by a fed-batch culture of recombinant E. coli strain employing the RnpA knockdown system. The RnpA-knockdown strategy reported here will be generally useful for the production of recombinant proteins including those that have been difficult to produce.

Tuneable Recombinant Spider Silk Protein Hydrogels for Drug Release and 3D Cell Culture.

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.

In vitro Blood–Brain barrier model based on recombinant spider silk protein nanomembranes for evaluation of transcytosis capability of biomolecules

The blood–brain barrier (BBB) limits the uptake of central nervous system (CNS)-targeting drugs into the brain. Engineering molecular shuttles for active transportation across the barrier has thus potential for improving the efficacy of such drugs. In vitro assessment of potential transcytosis capability for engineered shuttle proteins facilitates ranking and the selection of promising candidates during development. Herein, the development of an assay based on brain endothelial cells cultured on permeable recombinant silk nanomembranes for screening of transcytosis capability of biomolecules is described. The silk nanomembranes supported growth of brain endothelial cells to form confluent monolayers with relevant cell morphology, and induced expression of tight-junction proteins. Evaluation of the assay using an established BBB shuttle antibody showed transcytosis over the membranes with an apparent permeability that significantly differed from the isotype control antibody.

PH-dependent self-assembly mechanism of a single repetitive domain from a spider silk protein

Spider silk is self-assembled from full-length silk proteins, and some silk protein fragments can also form silk-like fibers in vitro. However, the mechanism underlying the silk fiber formation is not understood well. In this study, we investigated the fiber formation of a single repetitive domain (RP) from a minor ampullate silk protein (MiSp). Our findings revealed that pH and salt concentration affect not only the stability of MiSp-RP but also its self-assembly into fibers and aggregates. Using nuclear magnetic resonance (NMR) spectroscopy, we solved the three-dimensional (3D) structure of MiSp RP in aqueous solution. On the basis of the structure and mutagenesis, we revealed that charge-dipole interactions are responsible for the pH- and salt-dependent properties of MiSp-RP. Our results indicate that fiber formation is regulated by a delicate balance between intermolecular and intramolecular interactions, rather than by the protein stability alone. These findings have implications for the design of silk proteins for mass production of spider silk.

Liquid-Liquid Phase Separation Primes Spider Silk Proteins for Fiber Formation via a Conditional Sticker Domain.

Many protein condensates can convert to fibrillar aggregates, but the underlying mechanisms are unclear. Liquid-liquid phase separation (LLPS) of spider silk proteins, spidroins, suggests a regulatory switch between both states. Here, we combine microscopy and native mass spectrometry to investigate the influence of protein sequence, ions, and regulatory domains on spidroin LLPS. We find that salting out-effects drive LLPS via low-affinity stickers in the repeat domains. Interestingly, conditions that enable LLPS simultaneously cause dissociation of the dimeric C-terminal domain (CTD), priming it for aggregation. Since the CTD enhances LLPS of spidroins but is also required for their conversion into amyloid-like fibers, we expand the stickers and spacers-model of phase separation with the concept of folded domains as conditional stickers that represent regulatory units.

Design of Recombinant Spider Silk Proteins for Cell Type Specific Binding (Adv. Healthcare Mater. 9/2023)

Cell-Selective Spider Silk Proteins In article number 2202660, Vanessa Tanja Trossmann and Thomas Scheibel design recombinant spider silk proteins to guide cell-specific materials interactions. The fusion of spider silk proteins with short peptides from extracellular matrix proteins such as KGD yield cell type selective binding, which enables the development of hierarchically organized tissues.

Highly Hydrophobic Films of Engineered Silk Proteins by a Simple Deposition Method.

Molecular engineering of protein structures offers a uniquely versatile route for novel functionalities in materials. Here, we describe a method to form highly hydrophobic thin films using genetically engineered spider silk proteins. We used structurally engineered protein variants containing ADF3 and AQ12 spider silk sequences. Wetting properties were studied using static and dynamic contact angle measurements. Solution conditions and the surrounding humidity during film preparation were key parameters to obtain high hydrophobicity, as shown by contact angles in excess of 120°. Although the surface layer was highly hydrophobic, its structure was disrupted by the added water droplets. Crystal-like structures were found at the spots where water droplets had been placed. To understand the mechanism of film formation, different variants of the proteins, the topography of the films, and secondary structures of the protein components were studied. The high contact angle in the films demonstrates that the conformations that silk proteins take in the protein layer very efficiently expose their hydrophobic segments. This work reveals a highly amphiphilic nature of silk proteins and contributes to an understanding of their assembly mechanisms. It will also help in designing diverse technical uses for recombinant silk.

Two-in-One Spider Silk Protein with Combined Mechanical Features in All-Aqueous Spun Fibers.

Major ampullate (MA) spider silk reveals outstanding mechanical properties in terms of a unique combination of high tensile strength and extensibility, unmatched by most other known native or synthetic fiber materials. MA silk contains at least two spider silk proteins (spidroins), and here, a novel two-in-one (TIO) spidroin was engineered, resembling amino acid sequences of such two of the European garden spider. The combination of mechanical and chemical features of both underlying proteins facilitated the hierarchical self-assembly into β-sheet-rich superstructures. Due to the presence of native terminal dimerization domains, highly concentrated aqueous spinning dopes could be prepared from recombinant TIO spidroins. Subsequently, fibers were spun in a biomimetic, aqueous wet-spinning process, yielding mechanical properties at least twice as high as fibers spun from individual spidroins or blends. The presented processing route holds great potential for future applications using ecological green high-performance fibers.

Evonik to make spider silk for AMSilk

Evonik Industries has signed a manufacturing contract to supply AMSilk, a German developer of synthetic spider silk proteins, with product made from the fermentation of renewable raw materials. The agreement is for industrial quantities of the protein, which can be produced as a powder, hydrogel, fiber, or coating. Applications include clothing and coatings for medical implants. Evonik will produce the proteins at its fermentation facility in Slovakia. In 2021, AMSilk raised $35 million in series C funding.

Silk Assembly against Hydrophobic Surfaces─Modeling and Imaging of Formation of Nanofibrils.

A detailed insight about the molecular organization behind spider silk assembly is valuable for the decoding of the unique properties of silk. The recombinant partial spider silk protein 4RepCT contains four poly-alanine/glycine-rich repeats followed by an amphiphilic C-terminal domain and has shown the capacity to self-assemble into fibrils on hydrophobic surfaces. We herein use molecular dynamic simulations to address the structure of 4RepCT and its different parts on hydrophobic versus hydrophilic surfaces. When 4RepCT is placed in a wing arrangement model and periodically repeated on a hydrophobic surface, β-sheet structures of the poly-alanine repeats are preserved, while the CT part is settled on top, presenting a fibril with a height of ∼7 nm and a width of ∼11 nm. Both atomic force microscopy and cryo-electron microscopy imaging support this model as a possible fibril formation on hydrophobic surfaces. These results contribute to the understanding of silk assembly and alignment mechanism onto hydrophobic surfaces.

Tyrosine's Unique Role in the Hierarchical Assembly of Recombinant Spider Silk Proteins: From Spinning Dope to Fibers.

Producing recombinant spider silk fibers that exhibit mechanical properties approaching native spider silk is highly dependent on the constitution of the spinning dope. Previously published work has shown that recombinant spider silk fibers spun from dopes with phosphate-induced pre-assembly (biomimetic dopes) display a toughness approaching native spider silks far exceeding the mechanical properties of fibers spun from dopes without pre-assembly (classical dopes). Dynamic light scattering experiments comparing the two dopes reveal that biomimetic dope displays a systematic increase in assembly size over time, while light microscopy indicates liquid-liquid-phase separation (LLPS) as evidenced by the formation of micron-scale liquid droplets. Solution nuclear magnetic resonance (NMR) shows that the structural state in classical and biomimetic dopes displays a general random coil conformation in both cases; however, some subtle but distinct differences are observed, including a more ordered state for the biomimetic dope and small chemical shift perturbations indicating differences in hydrogen bonding of the protein in the different dopes with notable changes occurring for Tyr residues. Solid-state NMR demonstrates that the final wet-spun fibers from the two dopes display no structural differences of the poly(Ala) stretches, but biomimetic fibers display a significant difference in Tyr ring packing in non-β-sheet, disordered helical domains that can be traced back to differences in dope preparations. It is concluded that phosphate pre-orders the recombinant silk protein in biomimetic dopes resulting in LLPS and fibers that exhibit vastly improved toughness that could be due to aromatic ring packing differences in non-β-sheet domains that contain Tyr.

Novel insights into construct toxicity, strain optimization, and primary sequence design for producing recombinant silk fibroin and elastin-like peptide in E. coli

Spider silk proteins (spidroins) are a remarkable class of biomaterials that exhibit a unique combination of high-value attributes and can be processed into numerous morphologies for targeted applications in diverse fields. Recombinant production of spidroins represents the most promising route towards establishing the industrial production of the material, however, recombinant spider silk production suffers from fundamental difficulties that includes low titers, plasmid instability, and translational inefficiencies. In this work, we sought to gain a deeper understanding of upstream bottlenecks that exist in the field through the production of a panel of systematically varied spidroin sequences in multiple E. coli strains. A restriction on basal expression and specific genetic mutations related to stress responses were identified as primary factors that facilitated higher titers of the recombinant silk constructs. Using these findings, a novel strain of E. coli was created that produces recombinant silk constructs at levels 4–33 times higher than standard BL21(DE3). However, these findings did not extend to a similar recombinant protein, an elastin-like peptide. It was found that the recombinant silk proteins, but not the elastin-like peptide, exert toxicity on the E. coli host system, possibly through their high degree of intrinsic disorder. Along with strain engineering, a bioprocess design that utilizes longer culturing times and attenuated induction was found to raise recombinant silk titers by seven-fold and mitigate toxicity. Targeted alteration to the primary sequence of the recombinant silk constructs was also found to mitigate toxicity. These findings identify multiple points of focus for future work seeking to further optimize the recombinant production of silk proteins and is the first work to identify the intrinsic disorder and subsequent toxicity of certain spidroin constructs as a primary factor related to the difficulties of production.