High-Toughness Silk Produced by a Transgenic Silkworm Expressing Spider (Araneus ventricosus) Dragline Silk Protein

Spider dragline silk is a natural fiber that has excellent tensile properties; however, it is difficult to produce artificially as a long, strong fiber. Here, the spider (Araneus ventricosus) dragline protein gene was cloned and a transgenic silkworm was generated, that expressed the fusion protein of the fibroin heavy chain and spider dragline protein in cocoon silk. The spider silk protein content ranged from 0.37 to 0.61% w/w (1.4–2.4 mol%) native silkworm fibroin. Using a good silk-producing strain, C515, as the transgenic silkworm can make the raw silk from its cocoons for the first time. The tensile characteristics (toughness) of the raw silk improved by 53% after the introduction of spider dragline silk protein; the improvement depended on the quantity of the expressed spider dragline protein. To demonstrate the commercial feasibility for machine reeling, weaving, and sewing, we used the transgenic spider silk to weave a vest and scarf; this was the first application of spider silk fibers from transgenic silkworms.     

Fifty years later: The sequence, structure and function of lacewing cross-beta silk

Classic studies of protein structure in the 1950s and 1960s demonstrated that green lacewing egg stalk silk possesses a rare native cross-beta sheet conformation. We have identified and sequenced the silk genes expressed by adult females of a green lacewing species. The two encoded silk proteins are 109 and 67 kDa in size and rich in serine, glycine and alanine. Over 70% of each protein sequence consists of highly repetitive regions with 16-residue periodicity. The repetitive sequences can be fitted to an elegant cross-beta sheet structural model with protein chains folded into regular 8-residue long beta strands. This model is supported by wide-angle X-ray scattering data and tensile testing from both our work and the original papers. We suggest that the silk proteins assemble into stacked beta sheet crystallites bound together by a network of cystine cross-links. This hierarchical structure gives the lacewing silk high lateral stiffness nearly threefold that of silkworm silk, enabling the egg stalks to effectively suspend eggs and protect them from predators.     

A fibroin secretion-deficient silkworm mutant, Nd-sD, provides an efficient system for producing recombinant proteins

The silkworm Nd-s(D) mutant is silk fibroin-secretion deficient. In the mutant, a disulfide linkage between the heavy (H) and light (L) chains, which is essential for the intracellular transport and secretion of fibroin, is not formed because of a partial deletion of the L-chain gene. To utilize the inactivity of the mutant L-chain, we investigated the possibility of using the Nd-s(D) mutant for the efficient production of recombinant proteins in the silkworm. A germ line transformation of the mutant with a normal L-chain-GFP fusion gene was performed. In the transgenic mutant, normal development of the posterior silk gland (PSG) was restored and it formed a normal cocoon. The biochemical analysis showed that the transgenic silkworms expressed the introduced gene in PSG cells, produced a large amount of the recombinant protein, secreted it into the PSG lumen, and used it to construct the cocoon. The molar ratio of silk proteins, H-chain:L-chain-GFP:fibrohexamerin, in the lumen and cocoon in the transgenic silkworm was 6:6:1, and the final product of the fusion gene formed about 10% of the cocoon silk. This indicates that the transgenic mutant silkworm possesses the capacity to produce and secrete the recombinant proteins in a molar ratio equal to that of the fibroin H-chain, contributing around half molecules of the total PSG silk proteins.     

Review structure of silk by raman spectromicroscopy: from the spinning glands to the fibers

Raman spectroscopy has long been proved to be a useful tool to study the conformation of protein-based materials such as silk. Thanks to recent developments, linearly polarized Raman spectromicroscopy has appeared very efficient to characterize the molecular structure of native single silk fibers and spinning dopes because it can provide information relative to the protein secondary structure, molecular orientation, and amino acid composition. This review will describe recent advances in the study of the structure of silk by Raman spectromicroscopy. A particular emphasis is put on the spider dragline and silkworm cocoon threads, other fibers spun by orb-weaving spiders, the spinning dope contained in their silk glands and the effect of mechanical deformation. Taken together, the results of the literature show that Raman spectromicroscopy is particularly efficient to investigate all aspects of silk structure and production. The data provided can lead to a better understanding of the structure of the silk dope, transformations occurring during the spinning process, and structure and mechanical properties of native fibers.     

Thermally induced alpha-helix to beta-sheet transition in regenerated silk fibers and films

The structure of thin films cast from regenerated solutions of Bombyx mori cocoon silk in hexafluoroisopropyl alcohol (HFIP) was studied by synchrotron X-ray diffraction during heating. A solid-state conformational transition from an alpha-helical structure to the well-known beta-sheet silk II structure occurred at a temperature of approximately 140 degrees C. The transition appeared to be homogeneous, as both phases do not coexist within the resolution of the current study. Modulated differential scanning calorimetry (DSC) of the films showed an endothermic melting peak followed by an exothermic crystallization peak, both occurring near 140 degrees C. Oriented fibers were also produced that displayed this helical molecular conformation. Subsequent heating above the structural transition temperature produced oriented beta-sheet fibers very similar in structure to B. mori cocoon fibers. Heat treatment of silk films at temperatures well below their degradation temperature offers a controllable route to materials with well-defined structures and mechanical behavior.     

A carotenoid-binding protein (CBP) plays a crucial role in cocoon pigmentation of silkworm (Bombyx mori) larvae

We examined the role of carotenoid-binding protein (CBP) in yellow cocoon pigmentation. First, using yellow or white cocoon races, we investigated the linkage between the yellow pigmentation and CBP expression. CBP was expressed only in the silk gland of the yellow cocoon races, which utilize carotenoids for cocoon pigmentation. Furthermore, CBP expression in the silk glands of day 1-7 fifth instar larvae matched the period of carotenoid uptake into the silk gland. Finally, we gave double-stranded CBP RNA to Bombyx mori (B. mori) larvae to induce RNA interference. The significantly reduced expression of CBP in the silk gland of fifth instar larva was confirmed on day 4 and a decrease in yellow pigmentation was observed in the cocoon. We showed that CBP plays a key role in the yellow cocoon pigmentation caused by carotenoids.     

Toxicity of Fipronil to the Predatory Lacewing Chrysoperla carnea (Neuroptera: Chrysopidae)

All the developmental stages of Chrysoperla carnea (Stephens) were treated with fipronil using different modes of exposure under laboratory conditions. Eggs were dipped in an aqueous range of concentrations and no effects were recorded, except at the highest concentration. Pupae treated topically on the silk cocoon moulted to healthy adults, without any deleterious effects on their reproduction. In contrast, larvae and adults were killed by the compound, irrespective of the mode of treatment, even at rates below the maximum field recommended rate in Spain (30 g c.p./ha). Sublethal concentrations of fipronil did not affect the fecundity or fertility of survivors. We conclude that fipronil is very toxic under laboratory conditions to this predatory lacewing.     

New silk protein: modification of silk protein by gene engineering for production of biomaterials

The interest in silk fibroin morphology and structure have increased due to its attractiveness for bio-related applications. Silk fibers have been used as sutures for a long time in the surgical field, due to the biocompatibility of silk fibroin fibers with human living tissue. In addition, it has been demonstrated that silk can be used as a substrate for enzyme immobilization in biosensors. A more complete understanding of silk structure would provide the possibility to further exploit silk fibroin for a wide range of new uses, such as the production of oxygen-permeable membranes and biocompatible materials. Silk fibroin-based membranes could be utilized as soft tissue compatible polymers. Baculovirus-mediated transgenesis of the silkworm allows specific alterations in a target sequence. Homologous recombination of a foreign gene downstream from a powerful promoter, such as the fibroin promoter, would allow the constitutive production of a useful protein in the silkworm and the modification of the character of silk protein. A chimeric protein consisted of fibroin and green fluorescent protein was expressed under the control of fibroin in the posterior silk gland and the gene product was spun into the cocoon layer. This technique, gene targeting, will lead to the modification and enhancement of physicochemical properties of silk protein.     

New silk protein: modification of silk protein by gene engineering for production of biomaterials.

The interest in silk fibroin morphology and structure have increased due to its attractiveness for bio-related applications. Silk fibers have been used as sutures for a long time in the surgical field, due to the biocompatibility of silk fibroin fibers with human living tissue. In addition, it has been demonstrated that silk can be used as a substrate for enzyme immobilization in biosensors. A more complete understanding of silk structure would provide the possibility to further exploit silk fibroin for a wide range of new uses, such as the production of oxygen-permeable membranes and biocompatible materials. Silk fibroin-based membranes could be utilized as soft tissue compatible polymers. Baculovirus-mediated transgenesis of the silkworm allows specific alterations in a target sequence. Homologous recombination of a foreign gene downstream from a powerful promoter, such as the fibroin promoter, would allow the constitutive production of a useful protein in the silkworm and the modification of the character of silk protein. A chimeric protein consisted of fibroin and green fluorescent protein was expressed under the control of fibroin in the posterior silk gland and the gene product was spun into the cocoon layer. This technique, gene targeting, will lead to the modification and enhancement of physicochemical properties of silk protein.     

家蚕不同茧色品种中cbp基因的结构和表达分析

类胡萝卜素结合蛋白(CBP)是唯一已被确认与家蚕黄色茧形成儡切相关的主要蛋白质。文章选择12个有色茧和白茧蚕品种, 调查了cbp基因结构、转录产物mRNA类型和丝腺类胡萝卜素紫外可见光吸收特征与其茧色的关系。结果表明: 黄色茧蚕品种含有2种或3种cbp基因结构, 同时转录具有CBP功能性的完整 mRNA和缺少第2外显子的mRNA; 绿茧品种间cbp基因结构存在差异, 转录缺少第2外显子的mRNA; 白茧蚕品种cbp基因只有1种结构, 转录缺少第2外显子的mRNA。文章在黄茧品种中新发现的cbp基因第1内含子序列可能具有茧色品种特异性, 家蚕黄茧品种丝腺的紫外可见吸收光谱特征显著区别于绿茧和白茧品种, cbp基因的结构和表达特征与家蚕茧色密切相关。     

A comparison of convergently evolved insect silks that share β‐sheet molecular structure

Raspy crickets produce silk webs that are used to build shelters. These webs have been found to consist of both fiber and film components. Raman spectra obtained from both components were found to be very similar for a given species. The protein structure of the fibers and films produced by both species was predominately β‐sheet with lesser amounts of β‐turns, unordered and α‐helical protein also detected. The orientation of the β‐sheet backbone in the fiber was determined to be parallel to the fiber axis. Compared to cocoon and dragline silk the orientation distribution exhibits a significant randomly orientated protein component. Amino acid analysis confirmed the presence of glycine, serine, and alanine in both species, which are known to form antiparallel β‐sheet structures. Both species, although at significantly different concentrations, where found to contain proline. This amino acid is uncommon in insect silks, and likely involved in increasing fiber elasticity. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 630–639, 2014.     

Transgenic silkworms that weave recombinant proteins into silk cocoons

As a result of breeding for more than 4,000 years, the silkworm, Bombyx mori, has acquired the ability to synthesize bulk amounts of silk proteins in its silk glands. To utilize this capacity for mass production of useful proteins, transgenic silkworms were generated that synthesized recombinant proteins in the silk gland and secreted them into the silk cocoon. The silk gland is classified into two main regions: the posterior (PSG) and the middle silk gland (MSG). By controlling the expressed regions of the recombinant protein gene in the silk gland, we were able to control the localization of the synthesized protein in the silk thread. Expression in the PSG or MSG led to localization in the insoluble fibroin core or hydrophilic outer sericin layer, respectively. This review focuses on the expression of recombinant protein in the MSG of transgenic silkworms. The recombinant protein secreted in the sericin layer is extractable from the cocoon with only a small amount of endogenous silk protein contamination by soaking the cocoon in mild aqueous solutions. The possibility of utilizing transgenic silkworms as a valuable tool for the mass production of therapeutic and industrially relevant recombinant proteins is discussed.     

The larval silk of Hypera spp. (Coleoptera: Curculionidae). A new example of the cross-β protein conformation in an insect silk

X-ray diffraction studies on the larval cocoon silk of the weevils Hypera postica and H. rumicis (Coleoptera: Curculionidae) indicate that it is a new example of the cross-β conformation in proteins. This silk is secreted by Malpighian tubules and stored in the rectum. Chemically it is more complex, with a greater range of constituent amino acids, than Chrysopa (Neuroptera) silk (the first well-documented example of a naturally occurring cross-β protein). There is a relatively high proline content, an unusual feature for an arthropod silk. X-ray data, and direct measurement of dispersed ribbons, suggest that the micellar width is about 3.0 compared to 2.5 nm in Chrysopa silk. This latter silk is the ‘type structure’ for naturally occurring cross-β protein conformations. Hypera silk can be considered the ‘next one up’ in a possible hierarchy of cross-β structures.     

A CD36-related Transmembrane Protein Is Coordinated with an Intracellular Lipid-binding Protein in Selective Carotenoid Transport for Cocoon Coloration

The transport pathway of specific dietary carotenoids from the midgut lumen to the silk gland in the silkworm, Bombyx mori, is a model system for selective carotenoid transport because several genetic mutants with defects in parts of this pathway have been identified that manifest altered cocoon pigmentation. In the wild-type silkworm, which has both genes, Yellow blood (Y) and Yellow cocoon (C), lutein is transferred selectively from the hemolymph lipoprotein to the silk gland cells where it is accumulated into the cocoon. The Y gene encodes an intracellular carotenoid-binding protein (CBP) containing a lipid-binding domain known as the steroidogenic acute regulatory protein-related lipid transfer domain. Positional cloning and transgenic rescue experiments revealed that the C gene encodes Cameo2, a transmembrane protein gene belonging to the CD36 family genes, some of which, such as the mammalian SR-BI and the fruit fly ninaD, are reported as lipoprotein receptors or implicated in carotenoid transport for visual system. In C mutant larvae, Cameo2 expression was strongly repressed in the silk gland in a specific manner, resulting in colorless silk glands and white cocoons. The developmental profile of Cameo2 expression, CBP expression, and lutein pigmentation in the silk gland of the yellow cocoon strain were correlated. We hypothesize that selective delivery of lutein to specific tissue requires the combination of two components: 1) CBP as a carotenoid transporter in cytosol and 2) Cameo2 as a transmembrane receptor on the surface of the cells.     

Gene expression analysis in the larval silk gland of the eri silkworm Samia ricini

Insects produce silk for a range of purposes. In the Lepidoptera, silk is utilized as a material for cocoon production and serves to protect larvae from adverse environmental conditions or predators. Species in the Saturniidae family produce an especially wide variety of cocoons, for example, large, golden colored cocoons and those with many small holes. Although gene expression in the silk gland of the domestic silkworm (Bombyx mori L.) has been extensively studied, considerably fewer investigations have focused on members of the saturniid family. Here, we established expression sequence tags from the silk gland of the eri silkworm (Samia ricini), a saturniid species, and used these to analyze gene expression. Although we identified the fibroin heavy chain gene in the established library, genes for other major silk proteins, such as fibroin light chain and fibrohexamerin, were absent. This finding is consistent with previous reports that these latter proteins are lacking in saturniid silk. Recently, a series of fibrohexamerin‐like genes were identified in the Bombyx genome. We used this information to conduct a detailed analysis of the library established here. This analysis identified putative homologues of these genes. We also found several genes encoding small silk protein molecules that are also present in the silk of other Lepidoptera. Gene expression patterns were compared between eri and domestic silkworm, and both conserved and nonconserved expression patterns were identified for the tested genes. Such differential gene expression might be one of the major causes of the differences in silk properties between these species. We believe that our study can be of value as a basic catalogue for silk gland gene expression, which will yield to the further understanding of silk evolution.     

Silk cocoon of Bombyx mori: Proteins and posttranslational modifications – heavy phosphorylation and evidence for lysine‐mediated cross links

Although silk is used to produce textiles and serves as a valuable biomaterial in medicine, information on silk proteins of the cocoon is limited. Scanning electron microscopy was applied to morphologically characterise the sample and the solubility of cocoon in lithium thiocyanate and 2‐DE was carried out with multi‐enzyme in‐gel digestion followed by MS identification of silk‐peptides. High‐sequence coverage of the silk cocoon proteins fibroin light and heavy chain, sericins and fibrohexamerins was revealed and PTMs as heavy phosphorylation of silk fibroin heavy chain; lysine hydroxylation and Lys‐>allysine formation have been observed providing evidence for lysine‐mediated cross linking of silk as found in collagens, which has not been reported so far. Tyrosine oxidation verified the presence of di‐tyrosine cross links. A high degree of sequence conflicts probably representing single‐nucleotide polymorphisms was observed. PTM and sequence conflicts may be modulating structure and physicochemical properties of silk.     

Overexpression of recombinant infectious bursal disease virus (IBDV) capsid protein VP2 in the middle silk gland of transgenic silkworm

Infectious bursal disease virus (IBDV) is the causative agent of a highly contagious disease affecting young chickens and causes serious economic losses to the poultry industry worldwide. Development of subunit vaccine using its major caspid protein, VP2, is one of the promising strategies to protect against IBDV. This study aim to test the feasibility of using silkworm to produce recombinant VP2 protein (rVP2) derived from a very virulent strain of IBDV (vvIBDV). A total of 16 transgenic silkworm lines harboring a codon-optimized VP2 gene driven by the sericin1 promoter were generated and analyzed. The results showed that the rVP2 was synthesized in the middle silk gland of all lines and secreted into their cocoons. The content of rVP2 in the cocoon of each line was ranged from 0.07 to 16.10 % of the total soluble proteins. The rVP2 was purified from 30 g cocoon powders with a yield of 3.33 mg and a purity >90 %. Further analysis indicated that the rVP2 was able to tolerate high temperatures up to 80 °C, and exhibited specific immunogenic activity in mice. To our knowledge, this is the first report of overexpressing rVP2 in the middle silk gland of transgenic silkworm, which demonstrates the capability of silkworm as an efficient tool to produce recombinant immunogens for use in new vaccines against animal diseases.     

Macroscopic fibers self-assembled from recombinant miniature spider silk proteins

Strength, elasticity, and biocompatibility make spider silk an attractive resource for the production of artificial biomaterials. Spider silk proteins, spidroins, contain hundreds of repeated poly alanine/glycine-rich blocks and are difficult to produce recombinantly in soluble form. Most previous attempts to produce artificial spider silk fibers have included solubilization steps in nonphysiological solvents. It is here demonstrated that a miniature spidroin from a protein in dragline silk of Euprosthenops australis can be produced in a soluble form in Escherichia coli when fused to a highly soluble protein partner. Although this miniature spidroin contains only four poly alanine/glycine-rich blocks followed by a C-terminal non-repetitive domain, meter-long fibers are spontaneously formed after proteolytic release of the fusion partner. The structure of the fibers is similar to that of dragline silks, and although self-assembled from recombinant proteins they are as strong as fibers spun from redissolved silk. Moreover, the fibers appear to be biocompatible because human tissue culture cells can grow on and attach to the fibers. These findings enable controlled production of high-performance biofibers at large scale under physiological conditions.     

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.     

Hemolin expression in the silk glands of Galleria mellonella in response to bacterial challenge and prior to cell disintegration

Hemolin, a member of the immunoglobulin protein superfamily, functions in Lepidoptera as an opsonin in defence against potential pathogens and seems to play a role in tissue morphogenesis. We show that hemolin gene is expressed in several organs of Galleria mellonella larvae, including the nervous system and the silk glands. The expression in the silk glands of the wandering larvae and their isolated abdomens is enhanced within 6 h after an injection of bacteria, lipopolysaccharides, or peptidoglycans. The magnitude of silk gland response to bacterial challenge is similar to that seen in the fat body. A profound rise of hemolin expression without bacterial inoculation occurs in the silk glands of isolated abdomens when they are induced to pupate by a topical application of 20-hydroxyecdysone (20E). The induction of pupation is associated with silk gland programming for disintegration by apoptosis and phagocytosis. Administration of a juvenile hormone agonist prevents pupation and abolishes the stimulatory 20E effect on the hemolin expression. Hemolin protein can be immunodetected in the silk glands as well as in the spun-out cocoon silk. The results suggest that silk glands are a component of the insect immune system and that hemolin may mark the apoptic cells for the elimination by hemocytes.