Recombinant Silk Proteins with Additional Polyalanine Have Excellent Mechanical Properties

This paper explores the structures of exogenous protein molecules that can effectively improve the mechanical properties of silkworm silk. Several transgenic vectors fused with the silkworm fibroin light chain and type 3 repeats in different multiples of the ampullate dragline silk protein 1 (MaSp1) from black widow spider with different lengths of the polyalanine motifs were constructed for this study. Transgenic silkworms were successfully obtained by piggyBac-mediated microinjection. Molecular detection showed that foreign proteins were successfully secreted and contained within the cocoon shells. According to the prediction of PONDR^® VSL2 and PONDR^® VL-XT, the type 3 repeats and the polyalanine motif of the MaSp1 protein were amorphous. The results of FTIR analysis showed that the content of β-sheets in the silk of transgenic silkworms engineered with transgenic vectors with additional polyalanine was significantly higher than that of wild-type silkworm silk. Additionally, silk with a higher β-sheet content had better fracture strength and Young’s modulus. The mechanical properties of silk with longer chains of exogenous proteins were improved. In general, our results provide theoretical guidance and technical support for the large-scale production of excellent bionic silk.     

Comparison of Silks from Pseudoips prasinana and Bombyx mori Shows Molecular Convergence in Fibroin Heavy Chains but Large Differences in Other Silk Components

Many lepidopteran larvae produce silk feeding shelters and cocoons to protect themselves and the developing pupa. As caterpillars evolved, the quality of the silk, shape of the cocoon, and techniques in forming and leaving the cocoon underwent a number of changes. The silk of Pseudoips prasinana has previously been studied using X-ray analysis and classified in the same category as that of Bombyx mori, suggesting that silks of both species have similar properties despite their considerable phylogenetic distance. In the present study, we examined P. prasinana silk using ‘omics’ technology, including silk gland RNA sequencing (RNA-seq) and a mass spectrometry-based proteomic analysis of cocoon proteins. We found that although the central repetitive amino acid sequences encoding crystalline domains of fibroin heavy chain molecules are almost identical in both species, the resulting fibers exhibit quite different mechanical properties. Our results suggest that these differences are most probably due to the higher content of fibrohexamerin and fibrohexamerin-like molecules in P. prasinana silk. Furthermore, we show that whilst P. prasinana cocoons are predominantly made of silk similar to that of other Lepidoptera, they also contain a second, minor silk type, which is present only at the escape valve.     

The Role of Filippi’s Glands in the Silk Moths Cocoon Construction

Filippi’s glands (FGs), formerly also called Lyonet’s glands, are accessory secretory structures of the labial (silk) glands of lepidopteran caterpillars, which were implicated to play an important role in the maturation of the silk material and the construction of the cocoon. In our previous study, we have identified several species of giant silk moths that completely lack the FGs. Interestingly, the absence of FGs in these species correlates with the construction of a loose cocoon architecture. We investigated the functions of FGs by their surgical extirpation in the last instar larvae of the silkworm, Bombyx mori. We found that the absence of FGs altered the structure of the resulting cocoon, in which the different layers of silk were separated. In further experiments, we found no effects of the absence of FGs on larval cocoon formation behavior or on changes in cocoon mass or lipid content. Differential proteomic analysis revealed no significant contribution of structural proteins from FGs to silk cocoon material, but we identified several low abundance proteins that may play a role in posttranslational modifications of some silk proteins. Proteomic analysis also revealed a difference in phosphorylation of the N-terminal sequence of fibroin-heavy chain molecule. Thus, FGs appear to affect silk stickiness during spinning by regulating posttranslational modifications. This could also explain the link that exists between the absence of these glands and the formation of loose cocoons in some giant silk moth species.     

Silk Spinning in Silkworms and Spiders

Spiders and silkworms spin silks that outcompete the toughness of all natural and manmade fibers. Herein, we compare and contrast the spinning of silk in silkworms and spiders, with the aim of identifying features that are important for fiber formation. Although spiders and silkworms are very distantly related, some features of spinning silk seem to be universal. Both spiders and silkworms produce large silk proteins that are highly repetitive and extremely soluble at high pH, likely due to the globular terminal domains that flank an intermediate repetitive region. The silk proteins are produced and stored at a very high concentration in glands, and then transported along a narrowing tube in which they change conformation in response primarily to a pH gradient generated by carbonic anhydrase and proton pumps, as well as to ions and shear forces. The silk proteins thereby convert from random coil and alpha helical soluble conformations to beta sheet fibers. We suggest that factors that need to be optimized for successful production of artificial silk proteins capable of forming tough fibers include protein solubility, pH sensitivity, and preservation of natively folded proteins throughout the purification and initial spinning processes.     

A new silk: Mechanical, compositional, and morphological characterization of leafhopper (Kahaono montana) silk

The mechanical properties, amino acid composition, internal morphology, and solvent-induced interaction of silk produced by the endemic Australian leafhopper, Kahaono montana Evans (Hemiptera: Cicadellidae) were studied. Ion plasma etching/scanning electron microscopy examination of the internal morphology revealed a skin-core structure, with bands in the core region aligned regularly in a transverse direction to the fibre axis, separated by a nominal spacing of 100 nm. The internal structure of the silk was compared with those from spider Eriophora transmarina (Keyserling) (Araneida: Araneidae) radial thread and silkworm (Bombyx mori). The amino acid composition of K. montana silk was determined using HPLC, and was found to be dominated by small amino acids: Serine, alanine and glycine. The silk-solvent interaction was tested using selected aqueous, organic and surfactant solutions, and the solubility of the silk was found depend primarily on the pH and ionic strength of the solvent. Tensile tests showed that the silk has considerably weaker mechanical properties than spider silk and silkworm silk. The differences in mechanical properties of K. montana silk compared with spider and silkworm silk are attributed to the distinction in amino acid composition ratio and internal morphology, and are likely to reflect the functions of the silks in these species.     

Evidence of Decoupling Protein Structure from Spidroin Expression in Spider Dragline Silks

The exceptional strength and extensibility of spider dragline silk have been thought to be facilitated by two spidroins, major ampullate spidroin 1 (MaSp1) and major ampullate spidroin 2 (MaSp2), under the assumption that protein secondary structures are coupled with the expressed spidroins. We tested this assumption for the dragline silk of three co-existing Australian spiders, Argiope keyserlingi, Latrodectus hasselti and Nephila plumipes. We found that silk amino acid compositions did not differ among spiders collected in May. We extended these analyses temporally and found the amino acid compositions of A. keyserlingi silks to differ when collected in May compared to November, while those of L. hasselti did not. To ascertain whether their secondary structures were decoupled from spidroin expression, we performed solid-state nuclear magnetic resonance spectroscopy (NMR) analysis on the silks of all spiders collected in May. We found the distribution of alanine toward β-sheet and _3,10helix/random coil conformations differed between species, as did their relative crystallinities, with A. keyserlingi having the greatest _3,10helix/random coil composition and N. plumipes the greatest crystallinity. The protein secondary structures correlated with the mechanical properties for each of the silks better than the amino acid compositions. Our findings suggested that a differential distribution of alanine during spinning could decouple secondary structures from spidroin expression ensuring that silks of desirable mechanical properties are consistently produced. Alternative explanations include the possibility that other spidroins were incorporated into some silks.     

Expression of redesigned mussel silk-like protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Silks have been used widely for human beings due to their several extraordinary properties. Until now, the studies on silk proteins have mainly focused on spiders and silkworms. Because silk properties are organism-dependent, novel silk protein types can be found and developed through investigation of new silk-bearing organisms. We noticed that marine mussel has silk-like domains containing many repeats with abundance of glycine and alanine. In the present work, we redesigned mussel-derived silk-like gene sequence which contains alternating repeated and nonrepeated regions with optimized codons for Escherichia coli. For successful expression of recombinant mussel silk-like protein in E. coli cells, we employed several experimental strategies, including use of strong promoter, cold shock expression, and genetic fusions. We observed significant repression on cell growths by even low expression levels of soluble mussel silk-like proteins in cold shock- and glutathione s-transferase (GST) fusion-based systems. Thus, we finally used baculoviral polyhedrin protein as a fusion partner and successfully expressed insoluble mussel silk-like protein with relatively high expression level and without cell growth repression in E. coli.     

A PRELIMINARY STUDY OF STEAM DRYING OF SILKWORM COCOONS

ABSTRACT

An experimental study was conducted to explore the feasibility of silkworm cocoon drying with superheated steam. The influence of steam drying on the drying kinetics and the technological characteristics of the dried cocoons is discussed. A promising new technology of cocoon drying is suggested to improve cocoon quality and decrease raw cocoon consumption in silk production.     

Silkworm silk as an engineering material

While silk exhibits high values of tensile strength and stiffness, these properties are compromised by their poor reproducibility. We present the results of experiments aimed at characterizing the variability of tensile properties exhibited by cocoon silk from Bombyx mori silkworms. Scanning electron microscopy is used to measure an average diameter for individual test specimens; the interspecimen variability of diameter is quantified and found to be inadequately represented by standard deviation. When load-extension data are converted into stress-strain curves, a marked improvement in reproducibility is realized if each specimen cross-section is calculated from diameter measurements specific to that specimen. Nevertheless, a significant variability in fracture stress remains; a Weibull analysis reveals that silkworm silk has a failure predictability comparable with that of glass and nonengineering ceramics. Unloading/reloading tests demonstrate that stiffness is not significantly affected by cumulative deformation, and the stress–strain relationship is not sensitive to strain rate. © 1998 John Wiley & Sons, Inc. J Appl Polym Sci 70: 2439–2447, 1998     

红外干燥不能提高蚕茧的干燥速率

通过大量实验,测定了蚕蛹、茧层的红外吸收图谱,选择相应波长范围的红外涂料和红外灯泡,在不同温度和不同堆放厚度条件下测定了蚕茧的干燥曲线,并与不采用红外线的干燥曲线进行了比较,结果表明红外线未能够提高蚕茧的干燥速率。实验还测定了0.25～25μm范围内单片茧层的红外线透过曲线,结果表明,在所测范围内,红外线的透过率为零,这说明红外线无法穿透茧层。茧层的水分含量很低(约10%),蚕蛹的水分含量很高(约80%),蚕茧干燥的主要目的是除掉蚕蛹中的水分,但茧层包含着蚕蛹,红外线无法穿透茧层对蛹体进行快速干燥。而且,茧层的红外吸收率很低,也不能通过茧层快速吸收红外线热能,然后传给蚕蛹来提高干燥速率,这便是红外线不能提高蚕茧干燥速率的原因。这一结论对国内外蚕业界探讨蚕茧干燥的理论与实践有重要的参考意义。     

Structure and morphology of electrospun silk nanofibers

Nanoscale fibers of natural silks of Bombyx mori and Nephila clavipes were produced from solutions in hexafluoro-2-propanol. The electrospun fibers were observed by optical, scanning electron, and transmission electron microscopy. These nanofibers showed optical retardation, appeared to have a circular cross-section, and were thermally stable under nitrogen to 280 °C (N. clavipes) and to 245 °C (B. mori). The diameter of the fibers ranged from approximately 6.5-200 nm making them orders of magnitudes smaller than the natural silks spun by most silkworms and spiders. The smallest fiber diameters correspond to 200 molecules in the cross section of the N. clavipes fibers and 150 in B. mori. Electron diffraction patterns of annealed electrospun fibers of B. mori and N. clavipes exhibit diffraction peaks demonstrating orientational and crystalline order comparable to that of naturally spun silks.     

Natural and unnatural silks

Natural silk is an important biopolymer with huge potential as it combines superb mechanical properties with environmentally sensitive production methods. Native silk dope taken straight from the gland can easily and without chemical assistance be drawn into strong fibres. Artificial silk fibres, on the other hand, rely on spinning dopes typically ‘reconstituted’ from natural silk fibres by strong chaotropic agents. Such fibres do not form readily, and often require chemical post-spin treatment for stabilisation. In addition these fibres tend to be brittle, and so far have been unable to match native fibres. Here we present novel rheometric data to argue that native and reconstituted silkworm silk dope differ in kind, not just in degree. While native silks behave like typical molten polymers, reconstituted silks do not. We conclude that rheology provides a powerful tool in the quest to learn from the Nature's polymer fibre technology.     

The Development of Fibers That Mimic the Core–Sheath and Spindle‐Knot Morphology of Artificial Silk Using Microfluidic Devices

Spider and silkworm produce diverse silk fibers from spinning dopes through smart spinnerets. Spider's capture silk is composed of core thread and periodic spindle‐knots, while silkworm silk consists of fibroin core and sericin outer layer. To mimic the morphologies of natural heterostructured silks, artificial fibers are dry‐spun using a multichannel microfluidic chip, served with a highly viscous core solution of regenerated silk fibroin and low viscosity sheath solution of sericin. Silk fibers with core–sheath, groove, and spindle‐knot structures are obtained by controlling the flow rates and viscosities of the two microfluids depending on the laminar flow, Kelvin–Helmholtz instability, or Plateau–Rayleigh instability.     

Selection of Suitable Endogenous Reference Genes for Relative Copy Number Detection in Sugarcane

Transgene copy number has a great impact on the expression level and stability of exogenous gene in transgenic plants. Proper selection of endogenous reference genes is necessary for detection of genetic components in genetically modification (GM) crops by quantitative real-time PCR (qPCR) or by qualitative PCR approach, especially in sugarcane with polyploid and aneuploid genomic structure. qPCR technique has been widely accepted as an accurate, time-saving method on determination of copy numbers in transgenic plants and on detection of genetically modified plants to meet the regulatory and legislative requirement. In this study, to find a suitable endogenous reference gene and its real-time PCR assay for sugarcane (Saccharum spp. hybrids) DNA content quantification, we evaluated a set of potential “single copy” genes including P4H, APRT, ENOL, CYC, TST and PRR, through qualitative PCR and absolute quantitative PCR. Based on copy number comparisons among different sugarcane genotypes, including five S. officinarum, one S. spontaneum and two S. spp. hybrids, these endogenous genes fell into three groups: ENOL-3—high copy number group, TST-1 and PRR-1—medium copy number group, P4H-1, APRT-2 and CYC-2—low copy number group. Among these tested genes, P4H, APRT and CYC were the most stable, while ENOL and TST were the least stable across different sugarcane genotypes. Therefore, three primer pairs of P4H-3, APRT-2 and CYC-2 were then selected as the suitable reference gene primer pairs for sugarcane. The test of multi-target reference genes revealed that the APRT gene was a specific amplicon, suggesting this gene is the most suitable to be used as an endogenous reference target for sugarcane DNA content quantification. These results should be helpful for establishing accurate and reliable qualitative and quantitative PCR analysis of GM sugarcane.     

Thermal decomposition behavior of sericin cocoon

The behavior on thermal degradation of the sericin cocoon consisting of a sericin and a few fibroin has been examined by means of evolved gas analysis (EGA), evolved gas detection (EGD), and differential thermal analysis (DTA). The sericin cocoons produced from the silkworm (Nd, Nd-s/Nd-s,Nd-s/+) and sericin stripped from the silk gland in the silkworm show two endothermic peaks at 220°C and 270°C according to differential thermal analysis. From the x-ray diffraction pattern, dynamic mechanical measurement, and the thermal gravity analysis (TG), the former peak occurs by the scission of the structural state and the change from crystalline to amorphous. Moreover, at 220°C, the weight changes markedly, the yellowness index (L/b) measured by the color difference meter abruptly decreases, and the gas (CO₂), evolved from the sericin cocoon, shows increases above 200°C.     

Genome-Wide Identification and Characterization of Carboxypeptidase Genes in Silkworm (Bombyx mori)

The silkworm (Bombyx mori) is an economically-important insect that can secrete silk. Carboxypeptidases have been found in various metazoan species and play important roles in physiological and biochemical reactions. Here, we analyzed the silkworm genome database and characterized 48 carboxypeptidases, including 34 metal carboxypeptidases (BmMCP1–BmMCP34) and 14 serine carboxypeptidases (BmSCP1–BmSCP14), to better understand their diverse functions. Compared to other insects, our results indicated that carboxypeptidases from silkworm have more family members. These silkworm carboxypeptidases could be divided into four families: Peptidase_M2 carboxypeptidases, Peptidase_M14 carboxypeptidases, Peptidase_S10 carboxypeptidases and Peptidase_S28 carboxypeptidases. Microarray analysis showed that the carboxypeptidases had distinct expression patterns, whereas quantitative real-time PCR demonstrated that the expression level of 13 carboxypeptidases significantly decreased after starvation and restored after re-feeding. Overall, our study provides new insights into the functional and evolutionary features of silkworm carboxypeptidases.     

Structure of the spinning apparatus of a wild silkworm Samia cynthia ricini and molecular dynamics calculation on the structural change of the silk fibroin

Silkworms have been developed over thousands years to optimize folding and crystallization of fibroin under highly controlled conditions which have resulted in their efficient fiber formation. In this paper, we reconstructed the three-dimensional architecture of the spinneret of a wild silkworm Samia cynthia ricini from approximately 1000 optical micrographs of the semi-thin cross sections. The chitin plates and muscles were observed in the silk press part together with large change in the diameter of the spinneret lumen at the press part by large shear stress. This is similar to the case of the spinneret of Bombyx mori silkworm, indicating that the structural change in the silk fibroin of S.c. ricini silkworm occurs exclusively at the silk press part due to large shear stresses. Molecular dynamics (MD) calculations were then performed to study the structural change that occurs in the crystalline region of S.c. ricini silk fibroin under shear stress. Namely, using the peptide AGGAGG(A)₁₂GGAGAG as a model of the crystalline part of the silk fibroin under different shear stresses in the presence of water molecules and followed by molecular mechanics (MM) calculation after removal of water molecules. The simulation indicates that the Ala residues in the model peptides adopt a predominantly β-sheet structure under shear stresses of above 1.0 GPa.