Single honeybee silk protein mimics properties of multi-protein silk

Honeybee silk is composed of four fibrous proteins that, unlike other silks, are readily synthesized at full-length and high yield. The four silk genes have been conserved for over 150 million years in all investigated bee, ant and hornet species, implying a distinct functional role for each protein. However, the amino acid composition and molecular architecture of the proteins are similar, suggesting functional redundancy. In this study we compare materials generated from a single honeybee silk protein to materials containing all four recombinant proteins or to natural honeybee silk. We analyse solution conformation by dynamic light scattering and circular dichroism, solid state structure by Fourier Transform Infrared spectroscopy and Raman spectroscopy, and fiber tensile properties by stress-strain analysis. The results demonstrate that fibers artificially generated from a single recombinant silk protein can reproduce the structural and mechanical properties of the natural silk. The importance of the four protein complex found in natural silk may lie in biological silk storage or hierarchical self-assembly. The finding that the functional properties of the mature material can be achieved with a single protein greatly simplifies the route to production for artificial honeybee silk.     

In vitro production of Bombyx mori silk fibroin by organ culture of the posterior silk glands; isotope labeling and fluorination of the silk fibroin

An in vitro silk fibroin production system has been developed by culture of posterior silk glands from Bombyx mori. A large amount of the silk fibroin was produced continuously and effectively with a rotation culture procedure. Modified Grace's insect medium was used, and oxygen bubbling in the medium was performed. In addition, half of the medium was replaced with fresh medium every 6 h. The production yield of silk fibroin produced after 100 h culture was 81 mg/g wet weight of posterior silk gland. This culture system was used successfully for efficient (15)N isotope labeling of silk fibroin, which is required for (15)N solid state nuclear magnetic resonance (NMR) analysis of silk fibroin. Moreover, the introduction of fluorinated amino acids into silk fibroin was also carried out using this culture system. (c) 1993 John Wiley & Sons, Inc.     

Localization of the genes for silk fibroin in silk gland cells of Bombyx mori.

Radioactive iodinated silk fibroin messenger RNA and ribosomal RNA have been used as probes to localize their genes in tissue sections of Bombyx mori by in situ hybridization. From filter hybridization experiments it is inferred that the majority of the grains produced by in situ hybridization with fibroin mRNA represents specific hybridization to fibroin genes. Sections of the posterior silk gland where silk is synthesized have been compared with those of the middle gland which does not synthesize fibroin. Glands have been analyzed from the second through the fifth (last) larval instar during feeding and moulting periods. During later stages when the gland cells increase their DNA content by polyploidization, serial sections were required to follow the distribution of grains through entire nuclei. At all stages, both ribosomal DNA and fibroin genes are distributed randomly throughout the nuclei without a preferred relationship to any nuclear structure.     

U6 snRNA variants isolated from the posterior silk gland of the silk moth Bombyx mori

Five U6 small nuclear RNA (snRNA) isoforms were detected and characterized from the posterior silk gland (PSG) of the silk moth Bombyx mori (Nistari strain). Using the currently accepted U6 secondary structure model as a basis for comparison, the variants were analyzed for nucleotide differences across the sequence with a focus on known functional domains. Differences were observed primarily in single-stranded areas of which sixty percent were found in the highly conserved U4-U6 binding sites. In the Nistari strain, the U6A variant was found to be approximately four times more abundant as part of high molecular weight spliceosomal complexes when compared with U6A in the total unfractionated PSG cell lysate. Additionally, the European 703 B. mori strain total cell lysate U6 snRNA was analyzed and only the dominant U6A isoform initially identified in Nistari was found. Due to U6's essential role in pre-mRNA processing, variants may modulate assemblage of the catalytic core and in doing so potentially affect the rate of splicing. Phylogenetic analysis of the U6 snRNA sequences indicate an ancient divergence of U6 from the self-splicing group II intron module and a high degree of evolutionary conservation across species possibly due to functional constraints on the gene. Using in silico analysis, 35 full-length U6 variants were observed in the recently released Whole Genome Shotgun (WGS) database of the p50T strain. The consensus sequence of these U6 genes from p50T is identical to U6A identified in the Nistari strain. Furthermore p50T variant 1, which is represented in 14 genes, is equivalent to Nistari U6A.     

Ras1(CA) overexpression in the posterior silk gland improves silk yield

Sericulture has been greatly advanced by applying hybrid breeding techniques to the domesticated silkworm, Bombyx mori, but has reached a plateau during the last decades. For the first time, we report improved silk yield in a GAL4/UAS transgenic silkworm. Overexpression of the Ras1(CA) oncogene specifically in the posterior silk gland improved fibroin production and silk yield by 60%, while increasing food consumption by only 20%. Ras activation by Ras1(CA) overexpression in the posterior silk gland enhanced phosphorylation levels of Ras downstream effector proteins, up-regulated fibroin mRNA levels, increased total DNA content, and stimulated endoreplication. Moreover, Ras1 activation increased cell and nuclei sizes, enriched subcellular organelles related to protein synthesis, and stimulated ribosome biogenesis for mRNA translation. We conclude that Ras1 activation increases cell size and protein synthesis in the posterior silk gland, leading to silk yield improvement.     

The effect of residual silk sericin on the structure and mechanical property of regenerated silk filament

In this study, we elucidated the effect of residual silk sericin (SS) on structure and mechanical properties of regenerated silk filament as well as on fiber formation. The dope viscosity markedly increased with increasing residual SS content in dope solution which was prepared by dissolving the silk protein in formic acid. As a result of FTIR, (13)C NMR, and XRD, a small amount of SS (9.6%) contained in the filament showed highest content of beta-sheet conformation and maximum crystallinity. It seems that the SS affects the structural change of SF up to a certain level by inducing the beta-transition easily. The tenacity of the filaments, containing 9.6-18.9% SS, was in the range of 2.1-2.4 gf/d, which was about 50% higher than the filament without SS (pure SF). Consequently, with the enhancement of spinnability in wet spinning process, the SS can play an important role for developing the crystalline structure of SF as well as for improving mechanical properties of the regenerated silk fiber.     

The synthesis of RNA and silk protein in Galleria mellonella silk glands

The silk protein synthesis in silk glands of Galleria mellonella is preceded by the increase of total RNA content. The levels of the main RNA classes: 28S and 18S rRNA, tRNA as well as poly(A) + RNA change proportionally to the total RNA pool of glands. The fibroin-like silk protein of molecular weight of about 240 000 is characterized by the high content of four amino acids: glycine, alanine, serine and leucine, which account for more than 70% of amino acid residues. This fibroin-like protein is present in the posterior, middle and anterior parts of silk gland of the last instar larvae.     

Polarized light microscopy, variability in spider silk diameters, and the mechanical characterization of spider silk

Abstract. Spider silks possess a remarkable combination of high tensile strength and extensibility that makes them among the toughest materials known. Despite the potential exploitation of these properties in biotechnology, very few silks have ever been characterized mechanically. This is due in part to the difficulty of measuring the thin diameters of silk fibers. The largest silk fibers are only 5–10 μm in diameter and some can be as fine as 50 nm in diameter. Such narrow diameters, coupled with the refraction of light due to the anisotropic nature of crystalline regions within silk fibers, make it difficult to determine the size of silk fibers. Here, we report upon a technique that uses polarized light microscopy (PLM) to accurately and precisely characterize the diameters of spider silk fibers. We found that polarized light microscopy is as precise as scanning electron microscopy (SEM) across repeated measurements of individual samples of silk and resulted in mean diameters that were ～0.10 μm larger than those from SEM. Furthermore, we demonstrate that thread diameters within webs of individual spiders can vary by as much as 600%. Therefore, the ability of PLM to non‐invasively characterize the diameters of each individual silk fiber used in mechanical tests can provide a crucial control for natural variation in silk diameters, both within webs and among spiders.     

Spider minor ampullate silk proteins are constituents of prey wrapping silk in the cob weaver Latrodectus hesperus

Spiders spin high performance fibers with diverse biological functions and mechanical properties. Molecular and biochemical studies of spider prey wrapping silks have revealed the presence of the aciniform silk fibroin AcSp1-like. In our studies we demonstrate the presence of a second distinct polypeptide present within prey wrapping silk. Combining matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry and reverse genetics, we have isolated a novel gene called MiSp1-like and demonstrate that its protein product is a constituent of prey wrap silks from the black widow spider, Latrodectus hesperus. BLAST searches of the NCBInr protein database using the amino acid sequence of MiSp1-like revealed similarity to the conserved C-terminal domain of silk family members. In particular, MiSp1-like showed the highest degree of sequence similarity to the nonrepetitive C-termini of published orb-weaver minor ampullate fibroin molecules. Analysis of the internal amino acid sequence of the black widow MiSp1-like revealed polyalanine stretches interrupted by glycine residues and glycine-alanine couplets within MiSp1-like as well as repeats of the heptameric sequence AGGYGQG. Real-time quantitative PCR analysis demonstrates that the MiSp1-like gene displays a minor ampullate gland-restricted pattern of expression. Furthermore, amino acid composition analysis, coupled with scanning electron microscopy of raw wrapping silk, supports the assertion that minor ampullate silks are important constituents of black widow spider prey wrap silk. Collectively, our findings provide direct molecular evidence for the involvement of minor ampullate fibroins in swathing silks and suggest composite materials play an important role in the wrap attack process for cob-weavers.     

Studies on the posterior silk gland of the silkworm Bombix mori. IV. Ultracentrifugal analyses of native silk proteins, especially fibroin extracted from the middle silk gland of the mature silkworm

Ultracentrifugal analyses of the native silk proteins extracted from the various parts of the middle silk gland of the mature silkworm have revealed that there exist four components with S°_20,w values of 10S, 9–10S, 9S, and 4S in the extract. It is suggested that the fastest 10S component is the native fibroin synthesized in the posterior silk gland and transferred to the middle silk gland to be stored there, while the slower three components probably correspond to inner, middle, and outer sericins which were synthesized in the posterior, middle, and anterior portion of the middle silk gland, respectively. Native fibroin solution was prepared from the most posterior part of the middle silk gland. Ultracentrifugal analyses have shown that the solution contains considerable amounts of aggregates in addition to the main 10S component. Treatment with lithium bromide (LiBr), urea, or guanidine hydrochloride solution up to 6 M all have failed to dissociate the 10S component. From the sedimentation equilibrium analyses and partial specific volume of 0.71₆, the molecular weight of the 10S component of the native fibroin solution was found to be between 3.2 – 4.2 x 10⁵, with a tendency to lie fairly close to 3.7 x 10⁵.