Spider silks from plants – a challenge to create native-sized spidroins

Silk threads from spiders exhibit extraordinary mechanical properties, such as superior toughness and elasticity. Spider silks consist of several different large repetitive proteins that act as the basic materials responsible for these outstanding features. The production of spider silk protein variants in plants opens up new horizons in the production and functional investigation that enable the use of spider silks in innovative material development, nanotechnology and biomedicine in the future. This review summarizes and discusses production of spider silk protein variants in plants, especially with regards to plant expression systems, purification strategies, and characteristics of spider silk variants. Furthermore, the challenge of producing native-sized recombinant spidroins in planta is outlined, presenting three different strategies for achieving these high repetitive proteins with the help of non-repetitive C-terminal domains, crosslinking transglutaminase, and self-linking inteins. The potential of these fascinating proteins in medicine is also highlighted.     

Spider silks: recombinant synthesis,assembly, spinning,and engineering of synthetic proteins

Since thousands of years humans have utilized insect silks for their own benefit and comfort. The most famous example is the use of reeled silkworm silk from Bombyx mori to produce textiles. In contrast, despite the more promising properties of their silk, spiders have not been domesticated for large-scale or even industrial applications, since farming the spiders is not commercially viable due to their highly territorial and cannibalistic nature. Before spider silks can be copied or mimicked, not only the sequence of the underlying proteins but also their functions have to be resolved. Several attempts to recombinantly produce spider silks or spider silk mimics in various expression hosts have been reported previously. A new protein engineering approach, which combines synthetic repetitive silk sequences with authentic silk domains, reveals proteins that closely resemble silk proteins and that can be produced at high yields, which provides a basis for cost-efficient large scale production of spider silk-like proteins.     

蜘蛛丝蛋白研究进展

由于蜘蛛丝蛋白分子高度重复的一级结构、特殊的溶解特性和分子折叠行为以及具有形成非凡力学特性丝纤维的能力而引人注目。本文从蛛丝蛋白基因、天然蛛丝形成过程、蛛丝蛋白的基因工程生产及蛛丝蛋白的应用前景等几个方面着重介绍了近20年来对蛛丝蛋白的研究进展。围绕蛛丝蛋白展开的研究将有助于揭示蛋白质一级结构、蛋白质分子折叠与蛋白质大分子特性之间的内在联系。     

Environmentally induced post‐spin property changes in spider silks: influences of web type,spidroin composition and ecology

Many spiders use silk to construct webs that must function for days at a time, whereas many other species renew their webs daily. The mechanical properties of spider silk can change after spinning under environmental stress, which could influence web function. We hypothesize that spiders spinning longer‐lasting webs produce silks composed of proteins that are more resistant to environmental stresses. The major ampullate (MA) silks of orb web spiders are principally composed of a combination of two proteins (spidroins) called MaSp1 and MaSp2. We expected spider MA silks dominated by MaSp1 to have the greatest resistance to post‐spin property change because they have high concentrations of stable crystalline β‐sheets. Some orb web spiders that spin three‐dimensional orb webs, such as Cyrtophora, have MA silks that are predominantly composed of MaSp1. Hence, we expected that the construction of three‐dimensional orb webs might also coincide with MA silk resistance to post‐spin property change. Alternatively, the degree of post‐spin mechanical property changes in different spider silks may be explained by factors within the spider's ecosystem, such as exposure to solar radiation. We exposed the MA silks of ten spider species from five genera (Nephila, Cyclosa, Leucauge, Cyrtophora, and Argiope) to ecologically high temperatures and low humidity for 4 weeks, and compared the mechanical properties of these silks with unexposed silks. Using species pairs enabled us to assess the influence of web dimensionality and MaSp composition both with and without phylogenetic influences being accounted for. We found neither the MaSp composition nor the three‐dimensionality of the orb web to be associated with the degree of post‐spin mechanical property changes in MA silk. The MA silks in Leucauge spp. are dominated by MaSp2, which we found to have the least resistance to post‐spin property change. The MA silk in Argiope spp. is also dominated by MaSp2, but has high resistance to post‐spin property change. The ancestry of Argiope is unresolved, but it is largely a tropical genus inhabiting hot, open regions that present similar stressors to silk as those of our experiment. Ecological factors thus appear to influence the vulnerability of orb web spider MA silks to post‐spin property change. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106 , 580–588.     

Molecular studies of a novel dragline silk from a nursery web spider, Euprosthenops sp. (Pisauridae)

Various spider species produce dragline silks with different mechanical properties. The primary structure of silk proteins is thought to contribute to the elasticity and strength of the fibres. Previously published work has demonstrated that the dragline silk of Euprosthenops sp. is stiffer then comparable silk of Nephila edulis, Araneus diadematus and Latrodectus mactans. Our studies of Euprosthenops dragline silk at the molecular level have revealed that nursery web spider fibroin has the highest polyalanine content among previously characterised silks and this is likely to contribute to the superior qualities of pisaurid dragline.     

Blueprint for a high-performance biomaterial: full-length spider dragline silk genes

Spider dragline (major ampullate) silk outperforms virtually all other natural and manmade materials in terms of tensile strength and toughness. For this reason, the mass-production of artificial spider silks through transgenic technologies has been a major goal of biomimetics research. Although all known arthropod silk proteins are extremely large (>200 kiloDaltons), recombinant spider silks have been designed from short and incomplete cDNAs, the only available sequences. Here we describe the first full-length spider silk gene sequences and their flanking regions. These genes encode the MaSp1 and MaSp2 proteins that compose the black widow's high-performance dragline silk. Each gene includes a single enormous exon (>9000 base pairs) that translates into a highly repetitive polypeptide. Patterns of variation among sequence repeats at the amino acid and nucleotide levels indicate that the interaction of selection, intergenic recombination, and intragenic recombination governs the evolution of these highly unusual, modular proteins. Phylogenetic footprinting revealed putative regulatory elements in non-coding flanking sequences. Conservation of both upstream and downstream flanking sequences was especially striking between the two paralogous black widow major ampullate silk genes. Because these genes are co-expressed within the same silk gland, there may have been selection for similarity in regulatory regions. Our new data provide complete templates for synthesis of recombinant silk proteins that significantly improve the degree to which artificial silks mimic natural spider dragline fibers.     

The elaborate structure of spider silk

Biomaterials, having evolved over millions of years, often exceed man?made materials in their properties. Spider silk is one outstanding fibrous biomaterial which consists almost entirely of large proteins. Silk fibers have tensile strengths comparable to steel and some silks are nearly as elastic as rubber on a weight to weight basis. In combining these two properties, silks reveal a toughness that is two to three times that of synthetic fibers like Nylon or Kevlar. Spider silk is also antimicrobial, hypoallergenic and completely biodegradable.

This article focuses on the structure?function relationship of the characterized highly repetitive spider silk spidroins and their conformational conversion from solution into fibers. Such knowedge is of crucial importance to understanding the intrinsic properties of spider silk and to get insight into the sophisticated assembly processes of silk proteins. This review further outlines recent progress in recombinant production of spider silk proteins and their assembly into distinct polymer materials as a basis for novel products.     

Spider silk as a resource for future biotechnologies

Insect silks have been used by mankind for millennia to produce textiles and in particular, the cocoon silk of Bombyx mori was the base of one of the most important industries in history. In fact, B. mori is probably the only domesticated insect if not invertebrate in its true and strict sense, comparable to cattle and other livestock that humans have known and bred since the Neolithic period. In contrast, reports regarding the use of spider silk throughout history have the character of travellers’ tales or anecdotes, and serious attempts to exploit these biomaterials on a large scale have not been undertaken until recently. Indeed, the cannibalism of these carnivores makes their farming difficult and the production of significant yields of spider silk virtually impossible. Only today, with recombinant technologies available, does this problem seem to have been overcome. But why use spider silk at all – if we have the infrastructure to produce significant yields of silk from Bombyx? In contrast to most insects, spiders do not spin from labial glands, and many spiders possess different types of gland, most of them active throughout the whole lifespan. Typical orb‐weavers (Araneoidea) for instance possess up to seven different types of silk gland to produce different silk fibers and glues. Each of these products has evolved for a particular use and the respective material properties are highly adapted to that use. As the group of Araneae is about 400 million years old, the oldest fossil orb‐weaver is dated about 150 million years, and the use of silk is crucial to a spider's survival, we can expect that evolution will have “squeezed out every iota” to achieve optimum performance at minimum cost. Indeed, some dragline silks such as the major ampullate silks of some Nephila species show amazing mechanical properties that, in terms of toughness, are far superior to Bombyx silk. Labels like “stronger than steel” or “even better than Kevlar” were attached to them, and the Canadian‐based biotech company Nexia created the trademark “bio‐steel” for their prospective product. The discovery of these exceptional mechanical properties of those protein fibers triggered intense research on spider silk, with the goal of their commercial exploitation. But there is more to Arachne's weave and science is beginning to pick up those threads.     

Construction of Synthetic Genes for Analogs of Spider Silk Spidroin 1 and Their Expression in Tobacco Plants

To obtain transgenic tobacco plants expressing recombinant analogs of spider dragline silk spidroin 1, artificial 1f5 and 1f9 coding for spidroin 1 analogs were 3"-fused in-frame with the reporter lichenase gene. The Tr2" weak constitutive promoter of Agrobacterium tumefaciens T-DNA and the strong constitutive promoter of the cauliflower mosaic virus 35S RNA gene were used as regulatory elements. The expression cassettes were used to transform agrobacteria and then introduced in tobacco leaf disks. On evidence of Southern hybridization, transgenic plants each carried a single copy of a hybrid gene, which corresponded in size to the constructed one. Zymography and Western blotting revealed full-length hybrid proteins in leaf extracts of transgenic plants. The results testified that plants can maintain and express synthetic genes for spider silks and, consequently, may be used as a convenient producer of recombinant silk analogs.     

Phase separation and mechanical properties of an elastomeric biomaterial from spider wrapping silk and elastin block copolymers

Elastin and silk spidroins are fibrous, structural proteins with elastomeric properties of extension and recoil. While elastin is highly extensible and has excellent recovery of elastic energy, silks are particularly strong and tough. This study describes the biophysical characterization of recombinant polypeptides designed by combining spider wrapping silk and elastin‐like sequences as a strategy to rationally increase the strength of elastin‐based materials while maintaining extensibility. We demonstrate a thermo‐responsive phase separation and spontaneous colloid‐like droplet formation from silk‐elastin block copolymers, and from a 34 residue disordered region of Argiope trifasciata wrapping silk alone, and measure a comprehensive suite of tensile mechanical properties from cross‐linked materials. Silk‐elastin materials exhibited significantly increased strength, toughness, and stiffness compared to an elastin‐only material, while retaining high failure strains and low energy loss upon recoil. These data demonstrate the mechanical tunability of protein polymer biomaterials through modular, chimeric recombination, and provide structural insights into mechanical design. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 693–703, 2016.     

Conserved C-terminal domain of spider tubuliform spidroin 1 contributes to extensibility in synthetic fibers

Spider silk is renowned for its extraordinary mechanical properties, having a balance of high tensile strength and extensibility. To date, the majority of studies have focused on the production of dragline silks from synthetic spider silk gene products. Here we report the first mechanical analysis of synthetic egg case silk fibers spun from the Latrodectus hesperus tubuliform silk proteins, TuSp1 and ECP-2. We provide evidence that recombinant ECP-2 proteins can be spun into fibers that display mechanical properties similar to other synthetic spider silks. We also demonstrate that silks spun from recombinant thioredoxin-TuSp1 fusion proteins that contain the conserved C-terminal domain exhibit increased extensibility and toughness when compared to the identical fibers spun from fusion proteins lacking the C-terminus. Mechanical analyses reveal that the properties of synthetic tubuliform silks can be modulated by altering the postspin draw ratios of the fibers. Fibers subject to increased draw ratios showed elevated tensile strength and decreased extensibility but maintained constant toughness. Wide-angle X-ray diffraction studies indicate that postdrawn fibers containing the C-terminal domain of TuSp1 have more amorphous content when compared to fibers lacking the C-terminus. Taken together, these studies demonstrate that recombinant tubuliform spidroins that contain the conserved C-terminal domain with embedded protein tags can be effectively spun into fibers, resulting in similar tensile strength but increased extensibility relative to nontagged recombinant dragline silk proteins spun from equivalently sized proteins.     

In planta production of ELPylated spidroin-based proteins results in non-cytotoxic biopolymers

Background

Spider silk is a tear-resistant and elastic biopolymer that has outstanding mechanical properties. Additionally, exiguous immunogenicity is anticipated for spider silks. Therefore, spider silk represents a potential ideal biomaterial for medical applications. All known spider silk proteins, so-called spidroins, reveal a composite nature of silk-specific units, allowing the recombinant production of individual and combined segments.

Results

In this report, a miniaturized spidroin gene, named VSO1 that contains repetitive motifs of MaSp1 has been synthesized and combined to form multimers of distinct lengths, which were heterologously expressed as elastin-like peptide (ELP) fusion proteins in tobacco. The elastic penetration moduli of layered proteins were analyzed for different spidroin-based biopolymers. Moreover, we present the first immunological analysis of synthetic spidroin-based biopolymers. Characterization of the binding behavior of the sera after immunization by competitive ELISA suggested that the humoral immune response is mainly directed against the fusion partner ELP. In addition, cytocompatibility studies with murine embryonic fibroblasts indicated that recombinant spidroin-based biopolymers, in solution or as coated proteins, are well tolerated.

Conclusion

The results show that spidroin-based biopolymers can induce humoral immune responses that are dependent on the fusion partner and the overall protein structure. Furthermore, cytocompatibility assays gave no indication of spidroin-derived cytotoxicity, suggesting that recombinant produced biopolymers composed of spider silk-like repetitive elements are suitable for biomedical applications.

Electronic supplementary material

The online version of this article (doi:10.1186/s12896-015-0123-2) contains supplementary material, which is available to authorized users.     

Functionalized silk assembled from a recombinant spider silk fusion protein (Z‐4RepCT) produced in the methylotrophic yeast Pichia pastoris

Functional biological materials are a growing research area with potential applicability in medicine and biotechnology. Using genetic engineering, the possibility to introduce additional functions into spider silk‐based materials has been realized. Recently, a recombinant spider silk fusion protein, Z‐4RepCT, was produced intracellularly in Escherichia coli and could after purification self‐assemble into silk‐like fibers with ability to bind antibodies via the IgG‐binding Z domain. In this study, the use of the methylotrophic yeast Pichia pastoris for production of Z‐4RepCT has been investigated. Temperature, pH and production time were influencing the amount of soluble Z‐4RepCT retrieved from the extracellular fraction. Purification of secreted Z‐4RepCT resulted in a mixture of full‐length and degraded silk proteins that failed to self‐assemble into fibers. A position in the C‐terminal domain of 4RepCT was identified as being subjected to proteolytic cleavage by proteases in the Pichia culture supernatant. Moreover, the C‐terminal domain was subjected to glycosylation during production in P. pastoris. These observed alterations of the CT domain are suggested to contribute to the failure in fiber assembly. As alternative approach, Z‐4RepCT retrieved from the intracellular fraction, which was less degraded, was used and shown to retain ability to assemble into silk‐like fibers after enzymatic deglycosylation.     

Web building and silk properties functionally covary among species of wolf spider

Although phylogenetic studies have shown covariation between the properties of spider major ampullate (MA) silk and web building, both spider webs and silks are highly plastic so we cannot be sure whether these traits functionally covary or just vary across environments that the spiders occupy. As MaSp2‐like proteins provide MA silk with greater extensibility, their presence is considered necessary for spider webs to effectively capture prey. Wolf spiders (Lycosidae) are predominantly non‐web building, but a select few species build webs. We accordingly collected MA silk from two web‐building and six non‐web‐building species found in semirural ecosystems in Uruguay to test whether the presence of MaSp2‐like proteins (indicated by amino acid composition, silk mechanical properties and silk nanostructures) was associated with web building across the group. The web‐building and non‐web‐building species were from disparate subfamilies so we estimated a genetic phylogeny to perform appropriate comparisons. For all of the properties measured, we found differences between web‐building and non‐web‐building species. A phylogenetic regression model confirmed that web building and not phylogenetic inertia influences silk properties. Our study definitively showed an ecological influence over spider silk properties. We expect that the presence of the MaSp2‐like proteins and the subsequent nanostructures improves the mechanical performance of silks within the webs. Our study furthers our understanding of spider web and silk co‐evolution and the ecological implications of spider silk properties.     

Microdissection of Black Widow Spider Silk-producing Glands

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of developing offspring ^1,2. Spiders accomplish these tasks by spinning several distinct fiber types that have diverse mechanical properties. Such specialization of fiber types has occurred through the evolution of different silk-producing glands, which function as small biofactories. These biofactories manufacture and store large quantities of silk proteins for fiber production. Through a complex series of biochemical events, these silk proteins are converted from a liquid into a solid material upon extrusion.Mechanical studies have demonstrated that spider silks are stronger than high-tensile steel ³. Analyses to understand the relationship between the structure and function of spider silk threads have revealed that spider silk consists largely of proteins, or fibroins, that have block repeats within their protein sequences ⁴. Common molecular signatures that contribute to the incredible tensile strength and extensibility of spider silks are being unraveled through the analyses of translated silk cDNAs. Given the extraordinary material properties of spider silks, research labs across the globe are racing to understand and mimic the spinning process to produce synthetic silk fibers for commercial, military and industrial applications. One of the main challenges to spinning artificial spider silk in the research lab involves a complete understanding of the biochemical processes that occur during extrusion of the fibers from the silk-producing glands.Here we present a method for the isolation of the seven different silk-producing glands from the cobweaving black widow spider, which includes the major and minor ampullate glands [manufactures dragline and scaffolding silk] ^5,6, tubuliform [synthesizes egg case silk] ^7,8, flagelliform [unknown function in cob-weavers], aggregate [makes glue silk], aciniform [synthesizes prey wrapping and egg case threads] ⁹ and pyriform [produces attachment disc silk] ¹⁰. This approach is based upon anesthetizing the spider with carbon dioxide gas, subsequent separation of the cephalothorax from the abdomen, and microdissection of the abdomen to obtain the silk-producing glands. Following the separation of the different silk-producing glands, these tissues can be used to retrieve different macromolecules for distinct biochemical analyses, including quantitative real-time PCR, northern- and western blotting, mass spectrometry (MS or MS/MS) analyses to identify new silk protein sequences, search for proteins that participate in the silk assembly pathway, or use the intact tissue for cell culture or histological experiments.     

RGD-functionalized bioengineered spider dragline silk biomaterial

Spider silk fibers have remarkable mechanical properties that suggest the component proteins could be useful biopolymers for fabricating biomaterial scaffolds for tissue formation. Two bioengineered protein variants from the consensus sequence of the major component of dragline silk from Nephila clavipes were cloned and expressed to include RGD cell-binding domains. The engineered silks were characterized by CD and FTIR and showed structural transitions from random coil to insoluble beta-sheet upon treatment with methanol. The recombinant proteins were processed into films and fibers and successfully used as biomaterial matrixes to culture human bone marrow stromal cells induced to differentiate into bone-like tissue upon addition of osteogenic stimulants. The recombinant spider silk and the recombinant spider silk with RGD encoded into the protein both supported enhanced the differentiation of human bone marrow derived mesenchymal stem cells (hMSCs) to osteogenic outcomes when compared to tissue culture plastic. The recombinant spider silk protein without the RGD displayed enhanced bone related outcomes, measured by calcium deposition, when compared to the same protein with RGD. Based on comparisons to our prior studies with silkworm silks and RGD modifications, the current results illustrate the potential to bioengineer spider silk proteins into new biomaterial matrixes, while also highlighting the importance of subtle differences in silk sources and modes of presentation of RGD to cells in terms of tissue-specific outcomes.     

Purification of Spider Silk-elastin from Transgenic Plants and Application for Human Chondrocyte Proliferation

Research on spider silk proteins has led to the possibility of designing genetically engineered silks according to defined material properties. Here we show the efficient and stable production of spider silk-elastin fusion proteins in transgenic tobacco and potato plants by retention in the ER. The proteins were purified by a simple method, using heat treatment and 'inverse transition cycling'. Laboratory scale extraction of 1 kg tobacco leaf material leads to a yield of 80 mg pure recombinant spider silk-elastin protein. As a possible application, as well as to demonstrate biocompatibility, the growth of anchorage-dependent mammalian cells on spider silk-elastin coated culture plates was compared with conventional coatings such as collagen, fibronectin and poly-D-lysine. The anchorage-dependent chondrocytes showed similar growth behaviour and a rounded phenotype on collagen and on spider silk-elastin coated plates and the proliferation was remarkably superior to untreated polystyrene plates.     

Construction of the synthetic genes for protein analogs of spider silk carcass spidroin 1 and their expression in tobacco plants

To obtain transgenic tobacco plants expressing recombinant analogs of spider dragline silk spidroin 1, artificial 1f5 and 1f9 coding for spidroin 1 analogs were 3'-fused in-frame with the reporter lichenase gene. The Tr2' weak constitutive promoter of Agrobacterium tumefaciens T-DNA and the strong constitutive promoter of the cauliflower mosaic virus 35S RNA gene were used as regulatory elements. The expression cassettes were used to transform agrobacteria and then introduced in tobacco leaf disks. On evidence of Southern hybridization, transgenic plants each carried a single copy of a hybrid gene, which corresponded in size to the constructed one. Zymography and Western blotting revealed full-length hybrid proteins in leaf extracts of transgenic plants. The results testified that plants can maintain and express synthetic genes for spider silks and, consequently, may be used as a convenient producer of recombinant silk analogs.     

Novel In Silico Analyses of Repetitive Spider Silk Sequences to Understand the Evolution and Mechanical Properties of Fibrous Protein Materials

The mechanical properties of spider silks have diverged as spiders have diversely speciated. Because the main components of silks are proteins, it is valuable to investigate their sequences. However, silk sequences have been regarded as difficult information to analyze due to their imbalance and imperfect tandem repeats (ITR). Here, an in silico approach is applied to systemically analyze a group of silk sequences. It is found that every time new spider groups emerge, unique trimer motifs appear. These trimer motifs are used to find additional clues of evolution and to determine relationships with mechanical properties. For the first time, crucial evidence is provided that shows silk sequences coevolved with spider species and the mechanical properties of their fibers to adapt to new living environments. This novel approach can be used as a platform for analyzing other groups of ITR‐harboring proteins and to obtain information for the design of tailor‐made fibrous protein materials.