Mori Silkworm Research Articles

Preparation and characterization of silk fibroin as a biomaterial with potential for drug delivery.

BackgroundDegummed silk fibroin from Bombyx mori (silkworm) has potential carrier capabilities for drug delivery in humans; however, the processing methods have yet to be comparatively analyzed to determine the differential effects on the silk protein properties, including crystalline structure and activity.MethodsIn this study, we treated degummed silk with four kinds of calcium-alcohol solutions, and performed secondary structure measurements and enzyme activity test to distinguish the differences between the regenerated fibroins and degummed silk fibroin.ResultsGel electrophoresis analysis revealed that Ca(NO3)2-methanol, Ca(NO3)2-ethanol, or CaCl2-methanol treatments produced more lower molecular weights of silk fibroin than CaCl2-ethanol. X-ray diffraction and Fourier-transform infrared spectroscopy showed that CaCl2-ethanol produced a crystalline structure with more silk I (α-form, type II β-turn), while the other treatments produced more silk II (β-form, anti-parallel β-pleated sheet). Solid-State 13C cross polarization and magic angle spinning-nuclear magnetic resonance measurements suggested that regenerated fibroins from CaCl2-ethanol were nearly identical to degummed silk fibroin, while the other treatments produced fibroins with significantly different chemical shifts. Finally, enzyme activity test indicated that silk fibroins from CaCl2-ethanol had higher activity when linked to a known chemotherapeutic drug, L-asparaginase, than the fibroins from other treatments.ConclusionsCollectively, these results suggest that the CaCl2-ethanol processing method produces silk fibroin with biomaterial properties that are appropriate for drug delivery.

Production of scFv-Conjugated Affinity Silk Powder by Transgenic Silkworm Technology

Bombyx mori (silkworm) silk proteins are being utilized as unique biomaterials for medical applications. Chemical modification or post-conjugation of bioactive ligands expand the applicability of silk proteins; however, the processes are elaborate and costly. In this study, we used transgenic silkworm technology to develop single-chain variable fragment (scFv)-conjugated silk fibroin. The cocoons of the transgenic silkworm contain fibroin L-chain linked with scFv as a fusion protein. After dissolving the cocoons in lithium bromide, the silk solution was dialyzed, concentrated, freeze-dried, and crushed into powder. Immunoprecipitation analyses demonstrate that the scFv domain retains its specific binding activity to the target molecule after multiple processing steps. These results strongly suggest the promise of scFv-conjugated silk fibroin as an alternative affinity reagent, which can be manufactured using transgenic silkworm technology at lower cost than traditional affinity carriers.

Extraction and application of natural silk protein sericin from Bombyx mori as antimicrobial finish for cotton fabrics

In this study, we developed an effective technology for the extraction of sericin from the cocoons of Bombyx mori silk worms. Sericin was extracted with ice cold ethanol to obtain crude extract. Sericin extract was coated onto cotton fabric by a pad–dry–cure method. FTIR characterization of the sericin-coated cotton fabric showed distinct amide peaks. The test organisms that were used in the study to assess the antimicrobial activity of sericin were Escherichia coli and Staphylococcus aureus according to AATCC standard. The antimicrobial activity of the sericin thus extracted was assessed by both qualitative (agar diffusion and parallel streak method) and quantitative (percentage reduction test) methods. An inhibition zone of 28 mm and 30 mm for E. coli and S. aureus by agar diffusion method and a zone of 40 mm and 42 mm for E. coli and S. aureus by parallel streak method were obtained. Quantitative assessment by percentage reduction test showed a reduction percentage of 89.4% and 81% for S. aureus and E. coli, respectively. Results suggested that sericin might be a valuable ingredient for the development of antimicrobial textiles.

Silks: Properties and uses of natural and designed variants

Silks: Properties and uses of natural and designed variants

Quantitative analysis of three commonly-used insect cell-specific promoters’ activities by transient and baculovirus-mediated expression

The activities of three insect cell-specific promoters were compared and analyzed here, including the cytoplasmic Actin 3 (A3) promoter from Bombox mori (silkworm), immediately early 2 (ie2) promoter from Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) and the very late polyhedrin (polh) promoter from Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Results of transient expression assays showed the activity of ie2 promoter was approximately forty fold higher in Spodoptera frugiperda 9 (Sf9) cells and thirty fold higher in ovarian cell line of Bombyx mori (BmN) than that of A3 promoter. However, it showed reverse effect in baculovirus system that the activity of A3 promoter became approximately ten fold higher than that of ie2 promoter in the late stage of baculovirus infection. We constructed recombinant baculoviruses with enhanced green fluorescent protein gene (EGFP) reporter gene controlled by individual A3, ie2 and polh promoter. The analysis result of EGFP expression level showed the activity of polh promoter was nearly 50 fold higher than that of ie2, and 5 times higher than that of A3 at 72 h post infection in Sf9 cells. The systemic studies on the activities of the three promoters indicate they are ideal drivers for the construction of a time sequential baculovirus expression system. Key words: Promoter activity, immediately early promoter, very late promoter, transient expression, recombinant baculovirus.

Potential of Silkworm in Biotechnology

Silkworms have been in use for human needs for centuries. The silk derived from the silkworm is of great economical value. Lately insects and in particular silkworms have attracted the attention for expressing proteins of high value, which need post-translational modifications for human applications or research. At 1985 Dr. Maeda demonstrated that silkworm larvae could produce functional interferon [1]. Since then, to express desired protein of interest in silkworms various techniques have been developed, and among them Baculovirus based expression system (BES) comes foremost. Baculovirus being the ideal vector carrying desired cDNA of interest does not infect nor cause any known disease to humans or other livestock other than its host, thus dismaying any biohazard concerns. Thus it suits the purpose and with the advent of bacmid technology, the ability to produce recombinant baculovirus has grown many folds; both Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV). BmNPV based expression system for expressing proteins was improvised using BmNPV bacmid [2]. BmNPV bacmid is capable of replicating in both Escherichia coli and expressing protein of interest in Bombyx mori derived cell lines or silkworms. This method utilizes the advantage of a bacmid, it can be easily prepared and screened in E. coli to produce sufficient DNA for subsequent expression in silkworms. Today’s biotechnology based industry faces a bottleneck towards further advancement owing to the lack of proper gene expression systems. In spite of rapid growth of bioscience, there are still many proteins of which structures are unidentified. Scientists concerned in the field of life science desired technique, which can express protein in large quantities, and purify protein in form having a biological function quickly. Thus said, with the completion of Human Genome Project, we have come across a herculean task of trying to understand the function of the proteins being coded by the genes. Many researchers proved silkworm biotechnology is one of solutions. Up to now, α-interferon, HBeAg, sugar-modified eukaryotic proteins, such as carbohydrate transferases and cell surface receptors, have been produced using B. mori silkworm larvae. This work demonstrates great potential advantages of the BmNPV bacmid system for the large-scale expression of foreign genes in the silkworm. Furthermore, recent reports showed that the recombinant baculovirus containing mammalian cell-active promoter can exhibit efficient gene transfer of potential vaccines into mammalian cells. This would be a great breakthrough in production of recombinant eukaryotic proteins and viruses, which will be a powerful tool in a new proteome era. This special issue aims at contributing in the field of life science, for example gene expression system, silkworm research directionality, and commercialized proteins from silkworm expression system, and focusing on genetics of the silkworm, developing new specialized silkworm for protein expression, application of proteins to medicine, transgenic silkworm, and the commercialized proteins in Japan.

Quasi one-dimensional assembly of gold nanoparticles templated by a pH-sensitive peptide amphiphile from silk fibroin

One dimensional (1-D) assembly of gold nanoparticles (AuNPs) plays critical roles in fabricating linear optical and electronic devices. Herein, a novel pH-sensitive template formed by C12-GAGAGAGY, a peptide amphiphile based on the sequence of Bombxy mori silkworm silk fibroin, was employed to generate gold nanoparticles (AuNPs) in situ and direct their aggregation. Due to the incorporation of tyrosine, C12-GAGAGAGY was able to reduce Au3+ to AuNPs and further stabilize them at pH 11 without any external reducing or capping reagents. In addition, these AuNPs could reversibly assemble/disassemble by varying pH due to the pH-sensitive assembly of the template, C12-GAGAGAGY. These templates were cylindrical nanofibers at pH 11 and stacking nanoribbons at pH 4. AuNPs were well-dispersed among the networks formed by cylindrical nanofibers of C12-GAGAGAGY at pH 11, while they aggregated on both sides of the stacking nanoribbons at pH 4. Interestingly, unlike traditional pre-defined templates, the pH-induced assembly of C12-GAGAGAGY and assembly/disassembly of AuNPs occurred simultaneously. These results present us with the potential to utilize such smart peptide amphiphile templates for the fabrication of 1-D inorganic nanostructures with promising applications in nano-scale optical devices.

Does Thermal Annealing Affect the Mechanical Properties of Silkworm (Bombyx mori) Cocoon Silk?

ABSTRACTIt has been reported [1] that microwave radiation can enhance many of the mechanical properties of Bombyx mori silkworm cocoon silk, as measured in constant strain rate tensile tests to failure and in stress relaxation tests. The consequences of microwave radiation will affect decisions about the use of silk in settings subjected to significant microwave exposure – for example, as a reinforcing fiber in an epoxy matrix composite that may be microwave cured, or as a component in aircraft radomes.There are two possible mechanisms by which microwave radiation may affect a material [2]: (i) the radiation may enable chemical and/or microstructural changes – and therefore property changes – in the same way that conventional heating would, or (ii) the high heating rates that are achievable by microwaving may selectively favor changes that would be masked under conventional conditions, where heating rates are low enough to give preference to changes that have a lower activation energy. Here we explore the former possibility for silk.We characterized several mechanical properties of degummed and subsequently annealed B. mori silk, and compared them to the corresponding properties of degummed B. mori silk that was not annealed. The annealing treatment was carried out at 140 °C for 7 hours (conditions that optimally increased crystal size in an unrelated study of B. mori silk [3]), and then the fibers were allowed to cool gradually to room temperature over the course of an hour. Comparison of mechanical properties revealed no differences between the materials that we tested. Thus, for annealed silk, we do not observe the enhancements that can be achieved by microwaving. We conclude that in cases where microwaving affects the properties of silk, those changes are not a simple consequence of annealing by the microwaves.

The Intrinsic Ability of Silk Fibroin to Direct the Formation of Diverse Aragonite Aggregates

AbstractAs an analogue of the main protein contained in naturally formed nacre, reconstituted silk fibroin (SF) from the Bombyx mori silkworm silk shows a strong preference for the formation of the aragonite form of CaCO3 crystals and allows fine control over their size and morphology. The aragonite phase could be generated via two different routes: direct growth or dissolution and recrystallization, depending on the concentration of Ca2+ and SF. Generally, lower concentrations of Ca2+ and SF favor the formation of aragonite needles and their aggregates, of which the lattice structure of the precursor is similar to that of the organic matrix in natural shell. Higher concentrations lead to the formation of aragonite aggregates via a dissolution and recrystallization process through intermediates of lens‐like vaterite. Molecular modeling shows that the β‐strand conformers of silk fibroin molecules has an excellent match with the ionic spacing in the aragonite (010) plane, which can promote growth along the (001) long axis of aragonite crystals. This synergy between silk fibroin and the aragonite phase may help our understanding of the function of organic matrices involved in the biomineralization process, and facilitate the fabrication of synthetic materials with the potential for high performance mechanical properties.

Comparative Characterization of Chitinases from Silkworm (Bombyx mori) and Bollworm (Helicoverpa armigera)

Chitinases play an important role in the degradation of the cuticular chitin during the process of ecdysis. In this study, we compared the chitinases of two insect species, Bombyx mori (silkworm) and Helicoverpa armigera (bollworm), to assess the relation between characteristics and chitinase patterns. Differences between two chitinases were observed after purification using ammonium sulfate precipitation, affinity chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) assay. Although the specific activities of the purified enzymes were different, the purification yields were similar. One band of 88 kDa was observed for B. mori, and the other band of 75 kDa was detected for H. armigera. When a range of properties was tested, it was found that the optimum temperatures of B. mori and H. armigera chitinases were 45 and 50°C, respectively; the optimum pH value was 6.0 for both chitinases. Mn(2+) played catalytic role while Cu(2+) and SDS strongly inhibited activities of both enzymes. Between two chitinases, differences in K (M) were also observed. K (M) of chitinase from silkworm and bollworm was found to be 22.3 and 41.0 μmol/l, respectively. Both the chitinases significantly inhibited the spore germination of two fungal species, Saccharomyces cerevisiae and Penicillium.

The properties of native silk fibroin (SF) solution/gel from bombyx mori silkworms during the full fifth instar larval stage

The properties of native silk fibroin (SF) solution in the gland of silkworms during the full fifth instar larval stage were examined in an attempt to elucidate the mechanism of natural silk spinning in the silkworm. The flow and gelation behavior, birefringence phenomenon, rheological properties, specific viscosity and conformation of SF solutions from the gland of silkworms were measured by polarized light microscope, HAKKE rheometer, Ubbelohde viscometer and Solid-state 13C NMR, respectively. After comparing their properties with regenerated SF solutions from natural silk fibers, it is believed that there exists a progressive maturation process favorable to spin silk fibers with excellent properties from native SF solution and a weak bonded and highly oriented SF gel network with SF molecules partly extended in α-helix conformation is formed in the middle section of the gland of silkworms. This suggests that a biomimetic maturation process for making spinnable solution might be necessary for artificial silk fiber spinning in order to obtain improved fiber properties.

Production of Silk Sericin Nanofibers

Electrospinning is a scientifically attractive spinning technique, since it can produce fibers with the diameters ranging from the sub-micron to nano dimensional scale. The fiber diameters and their standard deviation primarily depend on the polymer solution properties and electrospinning process variables. Silk sericin powder was obtained from the raw silk fiber of Bombyx mori silkworm. The solution was prepared by dissolving silk sericin powder in water at 85 °C for 30 minutes. In the electrospinning process, voltage 20 kV was applied and the inner diameter of the syringe needle was 0.3 mm. The distance from the syringe tip to the target was 15 cm. The sericin nanofibers with smooth surfaces can not be produced at 40 wt%. However, fine nanofibers were produced at the concentrations of 50 wt% and 60 wt%. It was suggested that the average diameters of the nanofibers increased with increasing solution concentration and the optimum concentration was 50 wt% for best nanofibers. Then solution 50 wt% was electrospun with different voltages from 13 kV to 25 kV, the working distance is 10 cm, the as-spun silk nanofibers were produced and exhibited fine nanofibers with smooth surfaces. The average diameters and diameter distributions of the nanofibers decreased with increasing voltage.

Production of recombinant Bombyx mori nucleopolyhedrovirus in silkworm by intrahaemocoelic injection with invasive diaminopimelate auxotrophic Escherichia coli containing BmNPV–Bacmid

The present study elaborates a cost-effective and transfectant-free method for generating recombinant Bombyx mori (silkworm) nucleopolyhedrovirus in silkworm larvae and pupae by injecting invasive Escherichia coli carrying BmBacmid [BmNPV (B. mori nucleopolyhedrovirus)-Bacmid] into larval haemocoel. Up to 109 PFU (plaque-forming units)/ml of infective recombinant baculovirus was generated in the silkworm by intrahaemocoelic injection with 106 DAP (diaminopimelic acid) auxotrophic and BmBacmid containing E. coli cells expressing both invasin and listeriolysin. Thus 1ml of overnight culture of E. coli is sufficient to inject more than 2000 larvae, while DAP costing up to $1 is enough to inject about 4000 larvae. Recombinant proteins can be controlled to be expressed mainly in pupae by adjusting the injection dose, too. In this new method, many original manipulations have been eliminated, including BmBacmid preparation and the subsequent complex transfection procedures. Hence it is a time- and cost-saving means for large-scale injection of B. mori for recombinant baculovirus production in comparison with the traditional transfection methods, which may play an important role in the industrial development of the BmNPV-silkworm bioreactor.

Recent analyses of the excavated textile find from Grave 35 HTR73, Kerameikos cemetery, Athens, Greece

The following study presents the results of instrumental analyses performed on the excavated textile find HTR73 from the Kerameikos cemetery in Athens. In Greece, preservation of excavated textiles is rare, mainly due to the unfavourable environmental conditions prevailing. The Kerameikos textile find is from the 5th century BC, and has been preserved in association with copper, which is the most common type of preservation in Greece. Past analyses had indicated the presence of silk fibres, of the Bombyx mori silkworm, the use of which was not common in Classical Greece. In the present study, non-destructive instrumental analytical techniques were applied, namely ESEM and FTIR microspectroscopy, for the purpose of material identification. None of the techniques applied confirmed the presence of silk. However, cellulosic bast and possibly cotton fibres were identified. The presence of cotton is an equally rare phenomenon for the Classical period in Greece.

Biomaterials from Ultrasonication-Induced Silk Fibroin−Hyaluronic Acid Hydrogels

We report formation of biocompatible hydrogels using physically cross-linked biopolymers. Gelation of silk fibroin (from B. mori silkworm) aqueous solution was effected by ultrasonication and used to entrap blended, un-cross-linked, hyaluronic acid (HA) without chemical cross-linking. HA was formed into silk/HA blended hydrogels with different mixing ratios, forming homogeneous materials with stable swelling behavior when the HA content was less than 40 wt %. This is a novel approach to HA hydrogel systems, which otherwise require chemical cross-linking. Further, these systems exploit the beneficial material and biological properties of both polymers. Differential scanning calorimetry (DSC), temperature modulated DSC, and thermal gravimetric analysis were used to show that well-blended silk/HA hydrogel systems formed without macrophase separation. Fourier transform infrared spectroscopy was used to determine secondary structures from the amide I region of silk protein by spectral subtraction and Fourier-self-deconvolution. The β-sheet crystal fraction of the silk protein increased with increase of HA content (26-35 wt %), which resulted in stable, crystalline features in the blend hydrogel materials, favorable features to support human mesenchymal stem cell attachment and proliferation. Scanning electron microscopy was used to characterize morphology. β-Sheet content controlled the stability of the silk/HA hydrogel systems, with a minimum crystalline content needed to maintain a stable hydrogel system of ∼26 wt %. This value is close to the β-sheet content in pure silk fibroin hydrogels. These novel nonchemically cross-linked blend hydrogels may be useful for biomedical applications due to biocompatibility and the widespread utility of hydrogel systems. The attributes of HA in combination with the features of silk, offer a useful suite of properties, combining the mechanical integrity and slow degradation of silk with the control of water interactions and biological signaling of HA.

Production of scFv-displaying BmNPV in silkworm larvae and its efficient purification

Baculovirus-display technology utilizing the gp64 envelope protein has been developed. A simple and efficient process to separate the virus from the majority of the protein contaminants may be needed for the future demand of pure and functional baculovirus vectors ideal for vaccine- and gene-delivery applications. In the present study, using Bombyx mori (silkworm) larvae as a host, scFv (single-chain variable fragment)-surface displaying recombinant baculovirus production and its purification from silkworm larval haemolymph by SEC (size-exclusion chromatography) were demonstrated. The amounts of scFv were 4-8 μg/ml in the haemolymph. The scFv-gp64 fusion protein was confirmed to be incorporated into the cell membrane and the BmNPV (B. mori nucleopolyhedrovirus) surface by immunofluorescence microscopy and Western blotting. rBmNPV (recombinant BmNPV) was purified to higher purity by SEC using Sephacryl S-1000 column chromatography than by sucrose-density-gradient centrifugation. The recovery of purified rBmNPV was 22.2%, and the virus purity in the SEC fraction was increased 269-fold compared with its purity in haemolymph. Judging from the results of ELISA, approx. 0.9% of the total baculovirus-particle proteins were occupied by scFv on their surface. A BmNPV-based silkworm-larval system is suitable for large-scale production of baculovirus-surface-displayed proteins or peptides in comparison with a cell-culture system. The present study will be useful for future BmNPV-application studies for gene delivery and vaccine trials.

Efficient production of human Fas receptor extracellular domain–human IgG1 heavy chain Fc domain fusion protein using baculovirus/silkworm expression system

The fusion protein consisting of human Fas receptor extracellular domain and human IgG1 heavy chain Fc domain (hFasRECD-Fc) is a medically important protein that potentially has therapeutic uses. The fusion gene composed of a synthetic human Fas receptor extracellular domain gene and the cDNA encoding human IgG1 heavy chain Fc domain was investigated on the secretory expression using two baculovirus systems which employed either Spodoptera frugiperda 9 (Sf9) cell line or Bombyx mori (silkworm) larvae as the host organism. Both expression systems produced the functional hFasRECD-Fc as a dimer molecule linked by disulfide bridges. The secretion level per unit volume was much higher in the case of silkworm larvae as compared to Sf9 cell line, and was estimated to be more than 150 times. A substantially pure hFasRECD-Fc sample from silkworm larvae was obtained by single step Protein G-agarose affinity column chromatography. The affinity purified sample was further fractionated by anion-exchange chromatography with the final purification yield of 22.5 mg from 26 ml hemolymph. The hFasRECD-Fc from silkworm larvae and the tag-free human Fas ligand extracellular domain derivative from Pichia pastoris formed a stable complex in solution, which was verified by size-exclusion chromatography. This study demonstrated that the baculovirus/silkworm expression system provided the means for efficient production of highly pure hFasRECD-Fc with functional activity.

English

The present study was conducted to evaluate the entrance of Zn(II) into the food chain of Bombyx mori (silk worm) from mulberry plants irrigated using Zn(II) containing synthetic effluents. The soil, plant, silkworm and their excreta were sampled to determine Zn(II) amount by using atomic absorption spectrometry (AAS). The amount of Zn(II) deposited by synthetic effluent to soil was increased with pH of the effluent. However, the bioaccumaltion of Zn(II) in Morus alba leaves and B. mori larvae was high when the effluent pH was in the acidic range. B. mori excreted considerable amount of Zn(II) but still most of Zn(II) resided inside its body. The maximum Zn(II) amount detected in soil, leaves, larvae and faeces were 386.51 ± 0.03, 142.85 ± 0.001, 91.375 ± 0.019 and 42.13 ± 0.69 mg/kg, respectively. Zn(II) present in B. mori body was responsible for toxic effects on its life cycle. First instar of B. mori was most affected by Zn(II) toxicity. Body length, body weight of B. mori decreased with increase in bioaccumlated Zn(II) amount in larval body. Higher Zn(II) concentration in larval body increased B. mori death rate significantly.

Silk fibroin nanoparticles for cellular uptake and control release

Silk nanoparticles were prepared from silk fibroin solutions of domesticated Bombyx mori and tropical tasar silkworm Antheraea mylitta and investigated in respect to its particle size, surface charge, stability and morphology along with its cellular uptake and release of growth factors. The nanoparticles were stable, spherical, negatively charged, 150–170 nm in average diameter and exhibited mostly Silk II (β-sheet) structure and did not impose any overt toxicity. Cellular uptake studies showed the accumulation of fluorescence isothiocyanate conjugated silk nanoparticles in the cytosol of murine squamous cell carcinoma cells. In vitro VEGF release from the nanoparticles showed a significantly sustained release over 3 weeks, signifying the potential application as a growth factor delivery system.

Silk Fibroin Based Porous Materials

Silk from the Bombyx mori silkworm is a protein-based fiber. Bombyx mori silk fibroin (SF) is one of the most important candidates for biomedical porous material based on its superior machinability, biocompatibility, biodegradation, bioresorbability, and so on. In this paper, we have reviewed the key features of SF. Moreover we have focused on the morphous, technical processing, and biocompatibility of SF porous materials, followed by the application research. Finally, we provide a perspective the potential and problems of SF porous materials.