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Sample records for silk sericin protein

  1. Non-Mulberry and Mulberry Silk Protein Sericins as Potential Media Supplement for Animal Cell Culture

    PubMed Central

    Sahu, Neety; Pal, Shilpa; Sapru, Sunaina; Kundu, Joydip; Talukdar, Sarmistha; Singh, N. Ibotambi; Yao, Juming

    2016-01-01

    Silk protein sericins, in the recent years, find application in cosmetics and pharmaceuticals and as biomaterials. We investigate the potential of sericin, extracted from both mulberry Bombyx mori and different non-mulberry sources, namely, tropical tasar, Antheraea mylitta; muga, Antheraea assama; and eri, Samia ricini, as growth supplement in serum-free culture medium. Sericin supplemented media containing different concentrations of sericins from the different species are examined for attachment, growth, proliferation, and morphology of fibrosarcoma cells. The optimum sericin supplementation seems to vary with the source of sericins. The results indicate that all the sericins promote the growth of L929 cells in serum-free culture media; however, S. ricini sericin seems to promote better growth of cells amongst other non-mulberry sericins. PMID:27517047

  2. Non-Mulberry and Mulberry Silk Protein Sericins as Potential Media Supplement for Animal Cell Culture.

    PubMed

    Sahu, Neety; Pal, Shilpa; Sapru, Sunaina; Kundu, Joydip; Talukdar, Sarmistha; Singh, N Ibotambi; Yao, Juming; Kundu, Subhas C

    2016-01-01

    Silk protein sericins, in the recent years, find application in cosmetics and pharmaceuticals and as biomaterials. We investigate the potential of sericin, extracted from both mulberry Bombyx mori and different non-mulberry sources, namely, tropical tasar, Antheraea mylitta; muga, Antheraea assama; and eri, Samia ricini, as growth supplement in serum-free culture medium. Sericin supplemented media containing different concentrations of sericins from the different species are examined for attachment, growth, proliferation, and morphology of fibrosarcoma cells. The optimum sericin supplementation seems to vary with the source of sericins. The results indicate that all the sericins promote the growth of L929 cells in serum-free culture media; however, S. ricini sericin seems to promote better growth of cells amongst other non-mulberry sericins. PMID:27517047

  3. Interactions between fibroin and sericin proteins from Antheraea pernyi and Bombyx mori silk fibers.

    PubMed

    Du, Shan; Zhang, Jin; Zhou, Wei T; Li, Quan X; Greene, George W; Zhu, Hai J; Li, Jing L; Wang, Xun G

    2016-09-15

    Silkworm silk fibers are core-shell composites of fibroin and sericin proteins. Studying the interactions between fibroin and sericin is essential for understanding the properties of these composites. It is observed that compared to the domestic silk cocoon Bombyx mori (B. mori), the adhesion between fibroin and sericin from the wild silk cocoon, Antheraea pernyi (A. pernyi), is significantly stronger with a higher degree of heterogeneity. The adsorption of A. pernyi sericin on its fibroin is almost twice the value for B. mori sericin on fibroin, both showing a monolayer Langmuir adsorption. (1)H NMR and FTIR studies demonstrate on a molecular level the stronger interactions and the more intensive complex formation between A. pernyi fibroin and sericin, facilitated by the hydrogen bonding between glycine and serine. The findings of this study may help the design of composites with superior interfacial adhesion between different components. PMID:27314644

  4. The silk protein, sericin, protects against cell death caused by acute serum deprivation in insect cell culture.

    PubMed

    Takahashi, Masakazu; Tsujimoto, Kazuhisa; Yamada, Hideyuki; Takagi, Hiroshi; Nakamori, Shigeru

    2003-11-01

    Sericin is the silk protein that covers fibroin fibers and functions as a 'glue' in the cocoons of silkworms, and its most abundant component, Ser1, contains repeats of Ser- and Thr-rich 38 amino acid residues. The viability of Sf9 insect cells was 20, 57 and 49% on the fifth day and 41, 91 and 70% on the ninth day after serum deprivation in the presence of no additives, 3000 microg sericin hydrolysate and 350 microg SerD (the peptide containing the two repetitive units) ml(-1), respectively. Thus, the sericin samples were useful in preventing cell death and promoting cellular growth after acute serum deprivation. PMID:14677702

  5. Sericin Composition in the Silk of Antheraea yamamai.

    PubMed

    Zurovec, Michal; Yonemura, Naoyuki; Kludkiewicz, Barbara; Sehnal, František; Kodrik, Dalibor; Vieira, Ligia Cota; Kucerova, Lucie; Strnad, Hynek; Konik, Peter; Sehadova, Hana

    2016-05-01

    The silks produced by caterpillars consist of fibroin proteins that form two core filaments, and sericin proteins that seal filaments into a fiber and conglutinate fibers in the cocoon. Sericin genes are well-known in Bombyx mori (Bombycidae) but have received little attention in other insects. This paper shows that Antheraea yamamai (Saturniidae) contains five sericin genes very different from the three sericin genes of B. mori. In spite of differences, all known sericins are characterized by short exons 1 and 2 (out of 3-12 exons), expression in the middle silk gland section, presence of repeats with high contents of Ser and charged amino acid residues, and secretion as a sticky silk component soluble in hot water. The B. mori sericins represent tentative phylogenetic lineages (I) BmSer1 and orthologs in Saturniidae, (II) BmSer2, and (III) BmSer3 and related sericins of Saturniidae and of the pyralid Galleria mellonella. The lineage (IV) seems to be limited to Saturniidae. Concerted evolution of the sericin genes was apparently associated with gene amplifications as well as gene loses. Differences in the silk fiber morphology indicate that the cocktail of sericins linking the filaments and coating the fiber is modified during spinning. Silks are composite biomaterials of conserved function in spite of great diversity of their composition. PMID:27049111

  6. Maturation and fertilisation of sheep oocytes cultured in serum-free medium containing silk protein sericin.

    PubMed

    Yasmin, Cut; Otoi, Takeshige; Setiadi, Mohamad Agus; Karja, Ni Wayan Kurniani

    2015-03-01

    Sericin is a water-soluble component of silk and has been used as a biomaterial due to its antibacterial and ultraviolet radiation-resistant properties. This study was designed to evaluate the effect of sericin supplementation in a maturation medium on the meiotic competence and fertilisability of sheep oocytes. Cumulus-oocyte complexes (COCs) were cultured in TCM199 supplemented with sericin at various concentrations of 0 (control), 0.1, 0.25 and 0.5%, either with or without bovine serum albumin (BSA). When the COCs were matured without BSA, the supplementation of 0.1% sericin significantly increased the rates of maturation to metaphase II and the total fertilisation of oocytes compared with the other concentrations of sericin. When the COCs were matured with BSA, the beneficial effects of 0.1% sericin supplementation on the maturation and fertilisation of oocytes were not observed. Our findings indicate that supplementation with 0.1% sericin during maturation culture may improve the nuclear maturation and fertilisability of sheep oocytes. Moreover, it may be possible to replace BSA with sericin in chemically defined media without the risk of disease transmission. PMID:25655418

  7. The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway.

    PubMed

    Eidet, J R; Reppe, S; Pasovic, L; Olstad, O K; Lyberg, T; Khan, A Z; Fostad, I G; Chen, D F; Utheim, T P

    2016-01-01

    Restoration of the retinal pigment epithelial (RPE) cells to prevent further loss of vision in patients with age-related macular degeneration represents a promising novel treatment modality. Development of RPE transplants, however, requires up to 3 months of cell differentiation. We explored whether the silk protein sericin can induce maturation of primary human retinal pigment epithelial (hRPE) cells. Microarray analysis demonstrated that sericin up-regulated RPE-associated transcripts (RPE65 and CRALBP). Upstream analysis identified the NF-κB pathway as one of the top sericin-induced regulators. ELISA confirmed that sericin stimulates the main NF-κB pathway. Increased levels of RPE-associated proteins (RPE65 and the pigment melanin) in the sericin-supplemented cultures were confirmed by western blot, spectrophotometry and transmission electron microscopy. Sericin also increased cell density and reduced cell death following serum starvation in culture. Inclusion of NF-κB agonists and antagonists in the culture medium showed that activation of the NF-κB pathway appears to be necessary, but not sufficient, for sericin-induced RPE pigmentation. We conclude that sericin promotes pigmentation of cultured primary hRPE cells by activating the main NF-κB pathway. Sericin's potential role in culture protocols for rapid differentiation of hRPE cells derived from embryonic or induced pluripotent stem cells should be investigated. PMID:26940175

  8. The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway

    PubMed Central

    Eidet, J. R.; Reppe, S.; Pasovic, L.; Olstad, O. K.; Lyberg, T.; Khan, A. Z.; Fostad, I. G.; Chen, D. F.; Utheim, T. P.

    2016-01-01

    Restoration of the retinal pigment epithelial (RPE) cells to prevent further loss of vision in patients with age-related macular degeneration represents a promising novel treatment modality. Development of RPE transplants, however, requires up to 3 months of cell differentiation. We explored whether the silk protein sericin can induce maturation of primary human retinal pigment epithelial (hRPE) cells. Microarray analysis demonstrated that sericin up-regulated RPE-associated transcripts (RPE65 and CRALBP). Upstream analysis identified the NF-κB pathway as one of the top sericin-induced regulators. ELISA confirmed that sericin stimulates the main NF-κB pathway. Increased levels of RPE-associated proteins (RPE65 and the pigment melanin) in the sericin-supplemented cultures were confirmed by western blot, spectrophotometry and transmission electron microscopy. Sericin also increased cell density and reduced cell death following serum starvation in culture. Inclusion of NF-κB agonists and antagonists in the culture medium showed that activation of the NF-κB pathway appears to be necessary, but not sufficient, for sericin-induced RPE pigmentation. We conclude that sericin promotes pigmentation of cultured primary hRPE cells by activating the main NF-κB pathway. Sericin’s potential role in culture protocols for rapid differentiation of hRPE cells derived from embryonic or induced pluripotent stem cells should be investigated. PMID:26940175

  9. Processing and characterization of silk sericin from Bombyx mori and its application in biomaterials and biomedicines.

    PubMed

    Cao, Ting-Ting; Zhang, Yu-Qing

    2016-04-01

    Bombyx mori silk is composed of 60-80% fibroin, 15-35% sericin and 1-5% non-sericin component including wax, pigments, sugars and other impurities. For two decades, the protein-based silk fibroin was extensively used in the research and development of medical biomaterials and biomedicines. Sericin is frequently ignored and abandoned as a byproduct or waste in the processing of traditional silk fabrics, silk floss or modern silk biomaterials. However, similar to fibroin, sericin is not only a highly useful biological material, but also a lot of biological activity. Moreover, the non-sericin component present with sericin in the cocoon shell also has a strong biological activity. In this review, the extraction and recovery methods of sericin and the non-sericin component from the cocoon layer are reported, and their composition, properties and biological activity are described to produce a comprehensive report on biomedical materials and biological drugs. In addition, related problems or concerns present in the research and development of sericin are discussed, and a potential application of sericin in sustainable development is also presented. PMID:26838924

  10. Exploring natural silk protein sericin for regenerative medicine: an injectable, photoluminescent, cell-adhesive 3D hydrogel

    PubMed Central

    Wang, Zheng; Zhang, Yeshun; Zhang, Jinxiang; Huang, Lei; Liu, Jia; Li, Yongkui; Zhang, Guozheng; Kundu, Subhas C.; Wang, Lin

    2014-01-01

    Sericin, a major component of silk, has a long history of being discarded as a waste during silk processing. The value of sericin for tissue engineering is underestimated and its potential application in regenerative medicine has just begun to be explored. Here we report the successful fabrication and characterization of a covalently-crosslinked 3D pure sericin hydrogel for delivery of cells and drugs. This hydrogel is injectable, permitting its implantation through minimally invasive approaches. Notably, this hydrogel is found to exhibit photoluminescence, enabling bioimaging and in vivo tracking. Moreover, this hydrogel system possesses excellent cell-adhesive capability, effectively promoting cell attachment, proliferation and long-term survival of various types of cells. Further, the sericin hydrogel releases bioactive reagents in a sustained manner. Additionally, this hydrogel demonstrates good elasticity, high porosity, and pH-dependent degradation dynamics, which are advantageous for this sericin hydrogel to serve as a delivery vehicle for cells and therapeutic drugs. With all these unique features, it is expected that this sericin hydrogel will have wide utility in the areas of tissue engineering and regenerative medicine. PMID:25412301

  11. Exploring natural silk protein sericin for regenerative medicine: an injectable, photoluminescent, cell-adhesive 3D hydrogel.

    PubMed

    Wang, Zheng; Zhang, Yeshun; Zhang, Jinxiang; Huang, Lei; Liu, Jia; Li, Yongkui; Zhang, Guozheng; Kundu, Subhas C; Wang, Lin

    2014-01-01

    Sericin, a major component of silk, has a long history of being discarded as a waste during silk processing. The value of sericin for tissue engineering is underestimated and its potential application in regenerative medicine has just begun to be explored. Here we report the successful fabrication and characterization of a covalently-crosslinked 3D pure sericin hydrogel for delivery of cells and drugs. This hydrogel is injectable, permitting its implantation through minimally invasive approaches. Notably, this hydrogel is found to exhibit photoluminescence, enabling bioimaging and in vivo tracking. Moreover, this hydrogel system possesses excellent cell-adhesive capability, effectively promoting cell attachment, proliferation and long-term survival of various types of cells. Further, the sericin hydrogel releases bioactive reagents in a sustained manner. Additionally, this hydrogel demonstrates good elasticity, high porosity, and pH-dependent degradation dynamics, which are advantageous for this sericin hydrogel to serve as a delivery vehicle for cells and therapeutic drugs. With all these unique features, it is expected that this sericin hydrogel will have wide utility in the areas of tissue engineering and regenerative medicine. PMID:25412301

  12. Exploring natural silk protein sericin for regenerative medicine: an injectable, photoluminescent, cell-adhesive 3D hydrogel

    NASA Astrophysics Data System (ADS)

    Wang, Zheng; Zhang, Yeshun; Zhang, Jinxiang; Huang, Lei; Liu, Jia; Li, Yongkui; Zhang, Guozheng; Kundu, Subhas C.; Wang, Lin

    2014-11-01

    Sericin, a major component of silk, has a long history of being discarded as a waste during silk processing. The value of sericin for tissue engineering is underestimated and its potential application in regenerative medicine has just begun to be explored. Here we report the successful fabrication and characterization of a covalently-crosslinked 3D pure sericin hydrogel for delivery of cells and drugs. This hydrogel is injectable, permitting its implantation through minimally invasive approaches. Notably, this hydrogel is found to exhibit photoluminescence, enabling bioimaging and in vivo tracking. Moreover, this hydrogel system possesses excellent cell-adhesive capability, effectively promoting cell attachment, proliferation and long-term survival of various types of cells. Further, the sericin hydrogel releases bioactive reagents in a sustained manner. Additionally, this hydrogel demonstrates good elasticity, high porosity, and pH-dependent degradation dynamics, which are advantageous for this sericin hydrogel to serve as a delivery vehicle for cells and therapeutic drugs. With all these unique features, it is expected that this sericin hydrogel will have wide utility in the areas of tissue engineering and regenerative medicine.

  13. One-step synthesis of natural silk sericin-based microcapsules with bionic structures.

    PubMed

    Liu, Zhaogang; Cai, Yurong; Jia, Yaru; Liu, Lin; Kong, Xiangdong; Kundu, Subhas C; Yao, Juming

    2014-10-01

    Different techniques are being developed for fabricating microcapsules; it is still a challenge to fabricate them in an efficient and environment-friendly process. Here, a one-step green route to synthesize silk protein sericin-based microcapsules without any assistance of organic solvents is reported. By carefully changing the concentration of calcium ions accompanied with stirring, the morphology of the microcapsules can easily be regulated to form either discoidal, biconcave, cocoon-like, or tubular structures. The chelation of Ca(2+) and shearing force from agitation may induce the conformational transformation of sericin, which possibly results in the formation of microcapsules through the self-assembly of the protein subsequently. The as-prepared cocoon-like microcapsules exhibit pH-dependent stability. A potential application of microcapsules being fabricated from natural water-soluble silk protein sericin for controlled bioactive molecules loading and release system by a pH-triggered manner is quite feasible. PMID:25168858

  14. A silk sericin/silicone nerve guidance conduit promotes regeneration of a transected sciatic nerve.

    PubMed

    Xie, Hongjian; Yang, Wen; Chen, Jianghai; Zhang, Jinxiang; Lu, Xiaochen; Zhao, Xiaobo; Huang, Kun; Li, Huili; Chang, Panpan; Wang, Zheng; Wang, Lin

    2015-10-28

    Peripheral nerve gap defects lead to significant loss of sensory or motor function. Tissue engineering has become an important alternative to nerve repair. Sericin, a major component of silk, is a natural protein whose value in tissue engineering has just begun to be explored. Here, the first time use of sericin in vivo is reported as a long-term implant for peripheral nerve regeneration. A sericin nerve guidance conduit is designed and fabricated. This conduit is highly porous with mechanical strength matching peripheral nerve tissue. It supports Schwann cell proliferation and is capable of up-regulating the transcription of glial cell derived neurotrophic factor and nerve growth factor in Schwann cells. The sericin conduit wrapped with a silicone conduit (sericin/silicone double conduits) is used for bridging repair of a 5 mm gap in a rat sciatic nerve transection model. The sericin/silicone double conduits achieve functional recovery comparable to that of autologous nerve grafting as evidenced by drastically improved nerve function and morphology. Importantly, this improvement is mainly attributed to the sericin conduit as the silicone conduit alone only produces marginal functional recovery. This sericin/silicone-double-conduit strategy offers an efficient and valuable alternative to autologous nerve grafting for repairing damaged peripheral nerve. PMID:26332703

  15. Production of silk sericin/silk fibroin blend nanofibers

    NASA Astrophysics Data System (ADS)

    Zhang, Xianhua; Tsukada, Masuhiro; Morikawa, Hideaki; Aojima, Kazuki; Zhang, Guangyu; Miura, Mikihiko

    2011-08-01

    Silk sericin (SS)/silk fibroin (SF) blend nanofibers have been produced by electrospinning in a binary SS/SF trifluoroacetic acid (TFA) solution system, which was prepared by mixing 20 wt.% SS TFA solution and 10 wt.% SF TFA solution to give different compositions. The diameters of the SS/SF nanofibers ranged from 33 to 837 nm, and they showed a round cross section. The surface of the SS/SF nanofibers was smooth, and the fibers possessed a bead-free structure. The average diameters of the SS/SF (75/25, 50/50, and 25/75) blend nanofibers were much thicker than that of SS and SF nanofibers. The SS/SF (100/0, 75/25, and 50/50) blend nanofibers were easily dissolved in water, while the SS/SF (25/75 and 0/100) blend nanofibers could not be completely dissolved in water. The SS/SF blend nanofibers could not be completely dissolved in methanol. The SS/SF blend nanofibers were characterized by Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry, and differential thermal analysis. FTIR showed that the SS/SF blend nanofibers possessed a random coil conformation and ß-sheet structure.

  16. Production of silk sericin/silk fibroin blend nanofibers

    PubMed Central

    2011-01-01

    Silk sericin (SS)/silk fibroin (SF) blend nanofibers have been produced by electrospinning in a binary SS/SF trifluoroacetic acid (TFA) solution system, which was prepared by mixing 20 wt.% SS TFA solution and 10 wt.% SF TFA solution to give different compositions. The diameters of the SS/SF nanofibers ranged from 33 to 837 nm, and they showed a round cross section. The surface of the SS/SF nanofibers was smooth, and the fibers possessed a bead-free structure. The average diameters of the SS/SF (75/25, 50/50, and 25/75) blend nanofibers were much thicker than that of SS and SF nanofibers. The SS/SF (100/0, 75/25, and 50/50) blend nanofibers were easily dissolved in water, while the SS/SF (25/75 and 0/100) blend nanofibers could not be completely dissolved in water. The SS/SF blend nanofibers could not be completely dissolved in methanol. The SS/SF blend nanofibers were characterized by Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry, and differential thermal analysis. FTIR showed that the SS/SF blend nanofibers possessed a random coil conformation and ß-sheet structure. PMID:21867508

  17. Silk sericin ameliorates wound healing and its clinical efficacy in burn wounds.

    PubMed

    Aramwit, Pornanong; Palapinyo, Sirinoot; Srichana, Teerapol; Chottanapund, Suthat; Muangman, Pornprom

    2013-09-01

    The aim of this study was to evaluate the effect of silk sericin, a protein from silkworm cocoon, on scratch wound healing in vitro. For applicable result in clinical use, we also study the efficacy of sericin added to a standard antimicrobial cream, silver zinc sulfadiazine, for open wound care in the treatment of second-degree burn wounds. In vitro scratch assays show that sericin at concentration 100 μg/mL can promote the migration of fibroblast L929 cells similar to epidermal growth factor (positive control) at 100 μg/mL. After 1 day of treatment, the length of scratch in wounds treated with sericin was significantly shorter than the length of negative control wounds (culture medium without sericin). For clinical study, a total of 29 patients with 65 burn wounds which covered no less than 15 % of total body surface area were randomly assigned to either control (wounds treated with silver zinc sulfadiazine cream) or treatment (wounds treated with silver zinc sulfadiazine with added sericin cream) group in this randomized, double-blind, standard-controlled study. The results showed that the average time to reach 70 % re-epithelialization of the burned surface and complete healing in the treatment group was significantly shorter, approximately 5-7 days, than in the control group. Regarding time for complete healing, control wounds took approximately 29.28 ± 9.27 days, while wounds treated with silver zinc sulfadiazine with added sericin cream took approximately 22.42 ± 6.33 days, (p = 0.001). No infection or severe reaction was found in any wounds. This is the first clinical study to show that silk sericin is safe and beneficial for burn wound treatment when it is added to silver sulfadiazine cream. PMID:23748948

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

    PubMed

    Ki, Chang Seok; Kim, Jong Wook; Oh, Han Jin; Lee, Ki Hoon; Park, Young Hwan

    2007-08-01

    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. PMID:17573107

  19. Tuning Molecular Weights of Bombyx mori (B. mori) Silk Sericin to Modify Its Assembly Structures and Materials Formation

    PubMed Central

    2015-01-01

    Bombyx mori (B. mori) silk sericin is a protein with features desirable as a biomaterial, such as increased hydrophilicity and biodegradation, as well as resistance to oxidation, bacteria, and ultraviolet light. In contrast to other widely studied B. mori silk proteins such as fibroin, sericin is still unexplored as a building block for fabricating biomaterial, and thus a facile technique of processing it into a material is needed. Here, electrospinning technology was used to fabricate it into biomaterials from two forms of B. mori silk sericin with different molecular weights, one is a low (12.0 kDa) molecular sericin (LS) form and another is a high (66.0 kDa) molecular weight sericin (HS) form. Circular dichroism (CD) spectra showed that LS in hexafluoroacetone (HFA) solvent adopted a predominantly random coil conformation, whereas HS tended to form a β-sheet structure along with a large content of random coils. In addition, LS and HS in HFA solvent were found to form cylinder-like smaller nanoparticles and larger irregular aggregates before electrospinning, respectively. As a result, biomaterials based on microparticles and nanofibers were successfully fabricated by electrospinning of LS and HS dissolved in HFA, respectively. The cell viability and differentiation assay indicated that nanofibers and microparticles improved cell adhesion, growth, and differentiation, proving that the scaffolds electrospun from sericin are biocompatible regardless of its molecular weight. The microparticles, not common in electrospinning of silk proteins reported previously, were found to promote the osteogenic differentiation of mesenchymal stem cells in comparison to the nanofibers. This study suggested that molecular weight of sericin mediates its secondary structure and assembly structure, which in turn leads to a control of final morphology of the electrospun materials. The microparticles and nanofibers of sericin can be potentially used as building blocks for fabricating

  20. Effect of residual sericin on the structural characteristics and properties of regenerated silk films.

    PubMed

    Lee, Ji Hye; Song, Dae Woong; Park, Young Hwan; Um, In Chul

    2016-08-01

    Regenerated silk film has been increasingly attracting the research community's attention for biomedical applications due to its good biocompatibility and excellent cyto-compatibility. However, some limitations regarding its mechanical properties, such as brittleness, have restricted the use of silk films for industrial biomedical applications. In this study, regenerated silk films with different residual sericin content were prepared applying controlled degumming conditions to evaluate the effect of sericin content on the structure and properties of the films generated. When the residual sericin content increased to 0.6%, crystallinity index and breaking strength of silk films were increased. Above this value, these parameters then decreased. A 1.5 fold increase of silk film elongation properties was obtained when incorporating 16% sericin. Regardless of sericin content, all regenerated silk films showed excellent cyto-compatibility, comparable to the one obtained with tissue culture plates. PMID:27126168

  1. Silk sericin loaded alginate nanoparticles: Preparation and anti-inflammatory efficacy.

    PubMed

    Khampieng, Thitikan; Aramwit, Pornanong; Supaphol, Pitt

    2015-09-01

    In this study, silk sericin loaded alginate nanoparticles were prepared by the emulsification method followed by internal crosslinking. The effects of various silk sericin loading concentration on particle size, shape, thermal properties, and release characteristics were investigated. The initial silk sericin loadings of 20, 40, and 80% w/w to polymer were incorporated into these alginate nanoparticles. SEM images showed a spherical shape and small particles of about 71.30-89.50 nm. TGA analysis showed that thermal stability slightly increased with increasing silk sericin loadings. FTIR analysis suggested interactions between alginate and silk sericin in the nanoparticles. The release study was performed in acetate buffer at normal skin conditions (pH 5.5; 32 °C). The release profiles of silk sericin exhibited initial rapid release, consequently with sustained release. These silk sericin loaded alginate nanoparticles were further incorporated into topical hydrogel and their anti-inflammatory properties were studied using carrageenan-induced paw edema assay. The current study confirms the hypothesis that the application of silk sericin loaded alginate nanoparticle gel can inhibit inflammation induced by carrageenan. PMID:26188300

  2. The effect of sterilization methods on the physical properties of silk sericin scaffolds.

    PubMed

    Siritientong, Tippawan; Srichana, Teerapol; Aramwit, Pornanong

    2011-06-01

    Protein-based biomaterials respond differently to sterilization methods. Since protein is a complex structure, heat, or irradiation may result in the loss of its physical or biological properties. Recent investigations have shown that sericin, a degumming silk protein, can be successfully formed into a 3-D scaffolds after mixing with other polymers which can be applied in skin tissue engineering. The objective of this study was to investigate the effectiveness of ethanol, ethylene oxide (EtO) and gamma irradiation on the sterilization of sericin scaffolds. The influence of these sterilization methods on the physical properties such as pore size, scaffold dimensions, swelling and mechanical properties, as well as the amount of sericin released from sericin/polyvinyl alcohol/glycerin scaffolds, were also investigated. Ethanol treatment was ineffective for sericin scaffold sterilization whereas gamma irradiation was the most effective technique for scaffold sterilization. Moreover, ethanol also caused significant changes in pore size resulting from shrinkage of the scaffold. Gamma-irradiated samples exhibited the highest swelling property, but they also lost the greatest amount of weight after immersion for 24 h compared with scaffolds obtained from other sterilization methods. The results of the maximum stress test and Young's modulus showed that gamma-irradiated and ethanol-treated scaffolds are more flexible than the EtO-treated and untreated scaffolds. The amount of sericin released, which was related to its collagen promoting effect, was highest from the gamma-irradiated scaffold. The results of this study indicate that gamma irradiation should have the greatest potential for sterilizing sericin scaffolds for skin tissue engineering. PMID:21671201

  3. Effect of Sericin on Mechanical Behavior of Composite Material Reinforced by Silk Woven Fabric

    NASA Astrophysics Data System (ADS)

    Kimura, Teruo; Ino, Haruhiro; Hanada, Koji; Katori, Sigetaka

    Recent, attention has been given to shift from glass fibers and carbon fibers to natural fibers for FRP composites for the goal of protecting the environment. This paper concerned with the application of silk fabric for composite materials. Polypropylene (PP) was used for the matrix material and the silk fabric composites were molded using a compression molding method. Especially, the effect of sericin on mechanical behaviors of composite materials was discussed. Good adhesion between silk and PP was obtained by removing the sericin existing around the fibroin. The tensile modulus of composite decreased with decreasing the sericin because of the flexibility of silk fibers without sericin. In particular, the higher Izod impact value was obtained for the composites containing the silk fibers without sericin.

  4. Biomimetic Nucleation of Hydroxyapatite Crystals Mediated by Antheraea pernyi Silk Sericin Promotes Osteogenic Differentiation of Human Bone Marrow Derived Mesenchymal Stem Cells

    PubMed Central

    2015-01-01

    Biomacromolecules have been used as templates to grow hydroxyapatite crystals (HAps) by biomineralization to fabricate mineralized materials for potential application in bone tissue engineering. Silk sericin is a protein with features desirable as a biomaterial, such as increased hydrophilicity and biodegradation. Mineralization of the silk sericin from Antheraea pernyi (A. pernyi) silkworm has rarely been reported. Here, for the first time, nucleation of HAps on A. pernyi silk sericin (AS) was attempted through a wet precipitation method and consequently the cell viability and osteogenic differentiation of BMSCs on mineralized AS were investigated. It was found that AS mediated the nucleation of HAps in the form of nanoneedles while self-assembling into β-sheet conformation, leading to the formation of a biomineralized protein based biomaterial. The cell viability assay of BMSCs showed that the mineralization of AS stimulated cell adhesion and proliferation, showing that the resultant AS biomaterial is biocompatible. The differentiation assay confirmed that the mineralized AS significantly promoted the osteogenic differentiation of BMSCs when compared to nonmineralized AS as well as other types of sericin (B. mori sericin), suggesting that the resultant mineralized AS biomaterial has potential in promoting bone formation. This result represented the first work proving the osteogenic differentiation of BMSCs directed by silk sericin. Therefore, the biomineralization of A. pernyi silk sericin coupled with seeding BMSCs on the resultant mineralized biomaterials is a useful strategy to develop the potential application of this unexplored silk sericin in the field of bone tissue engineering. This study lays the foundation for the use of A. pernyi silk sericin as a potential scaffold for tissue engineering. PMID:24666022

  5. Effects of Silk Sericin on Incision Wound Healing in a Dorsal Skin Flap Wound Healing Rat Model

    PubMed Central

    Ersel, Murat; Uyanikgil, Yigit; Akarca, Funda Karbek; Ozcete, Enver; Altunci, Yusuf Ali; Karabey, Fatih; Cavusoglu, Turker; Meral, Ayfer; Yigitturk, Gurkan; Cetin, Emel Oyku

    2016-01-01

    Background The wound healing process is complex and still poorly understood. Sericin is a silk protein synthesized by silk worms (Bombyx mori). The objective of this study was to evaluate in vivo wound healing effects of a sericin-containing gel formulation in an incision wound model in rats. Material/Methods Twenty-eight Wistar-Albino rats were divided into 4 groups (n=7). No intervention or treatment was applied to the Intact control group. For other groups, a dorsal skin flap (9×3 cm) was drawn and pulled up with sharp dissection. The Sham operated group received no treatment. The Placebo group received placebo gel without sericin applied to the incision area once a day from day 0 to day 9. The Sericin Group 3 received 1% sericin gel applied to the incision area once a day from day 0 to day 9. Hematoxylin and eosin stain was applied for histological analysis and Mallory-Azan staining was applied for histoimmunochemical analysis of antibodies and iNOS (inducible nitric oxide synthase), and desmin was applied to paraffin sections of skin wound specimens. Parameters of oxidative stress were measured in the wound area. Results Epidermal thickness and vascularization were increased, and hair root degeneration, edema, cellular infiltration, collagen discoloration, and necrosis were decreased in Sericin group in comparison to the Placebo group and the Sham operated group. Malonyldialdehyde (MDA) levels were decreased, but superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities were increased in the sericin group. Conclusions We found that sericin had significant positive effects on wound healing and antioxidant activity. Sericin-based formulations can improve healing of incision wounds. PMID:27032876

  6. Effects of Silk Sericin on Incision Wound Healing in a Dorsal Skin Flap Wound Healing Rat Model.

    PubMed

    Ersel, Murat; Uyanikgil, Yigit; Karbek Akarca, Funda; Ozcete, Enver; Altunci, Yusuf Ali; Karabey, Fatih; Cavucoglu, Turker; Meral, Ayfer; Yigitturk, Gurkan; Oyku Cetin, Emel

    2016-01-01

    BACKGROUND The wound healing process is complex and still poorly understood. Sericin is a silk protein synthesized by silk worms (Bombyx mori). The objective of this study was to evaluate in vivo wound healing effects of a sericin-containing gel formulation in an incision wound model in rats. MATERIAL AND METHODS Twenty-eight Wistar-Albino rats were divided into 4 groups (n=7). No intervention or treatment was applied to the Intact control group. For other groups, a dorsal skin flap (9×3 cm) was drawn and pulled up with sharp dissection. The Sham operated group received no treatment. The Placebo group received placebo gel without sericin applied to the incision area once a day from day 0 to day 9. The Sericin Group 3 received 1% sericin gel applied to the incision area once a day from day 0 to day 9. Hematoxylin and eosin stain was applied for histological analysis and Mallory-Azan staining was applied for histoimmunochemical analysis of antibodies and iNOS (inducible nitric oxide synthase), and desmin was applied to paraffin sections of skin wound specimens. Parameters of oxidative stress were measured in the wound area. RESULTS Epidermal thickness and vascularization were increased, and hair root degeneration, edema, cellular infiltration, collagen discoloration, and necrosis were decreased in Sericin group in comparison to the Placebo group and the Sham operated group. Malonyldialdehyde (MDA) levels were decreased, but superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities were increased in the sericin group. CONCLUSIONS We found that sericin had significant positive effects on wound healing and antioxidant activity. Sericin-based formulations can improve healing of incision wounds. PMID:27032876

  7. Fibroin and Sericin from Bombyx mori Silk Stimulate Cell Migration through Upregulation and Phosphorylation of c-Jun

    PubMed Central

    García-Vizcaíno, Eva María; Alcaraz, Antonia; Cenis, José Luis; Nicolás, Francisco José

    2012-01-01

    Wound healing is a biological process directed to the restoration of tissue that has suffered an injury. An important phase of wound healing is the generation of a basal epithelium able to wholly replace the epidermis of the wound. A broad range of products derived from fibroin and sericin from Bombyx mori silk are used to stimulate wound healing. However, so far the molecular mechanism underlying this phenomenon has not been elucidated. The aim of this work was to determine the molecular basis underlying wound healing properties of silk proteins using a cell model. For this purpose, we assayed fibroin and sericin in a wound healing scratch assay using MDA-MB-231 and Mv1Lu cells. Both proteins stimulated cell migration. Furthermore, treatment with sericin and fibroin involved key factors of the wound healing process such as upregulation of c-Jun and c-Jun protein phosphorylation. Moreover, fibroin and sericin stimulated the phosphorylation of ERK 1/2 and JNK 1/2 kinases. All these experiments were done in the presence of specific inhibitors for some of the cell signalling pathways referred above. The obtained results revealed that MEK, JNK and PI3K pathways are involved in fibroin and sericin stimulated cells migration. Inhibition of these three kinases prevented c-Jun upregulation and phosphorylation by fibroin or sericin. Fibroin and sericin were tested in the human keratinocyte cell line, HaCaT, with similar results. Altogether, our results showed that fibroin and sericin initiate cell migration by activating the MEK, JNK and PI3K signalling pathways ending in c-Jun activation. PMID:22860103

  8. Effect of silk sericin on morphology and structure of calcium carbonate crystal

    NASA Astrophysics Data System (ADS)

    Zhao, Rui-Bo; Han, Hua-Feng; Ding, Shao; Li, Ze-Hao; Kong, Xiang-Dong

    2013-06-01

    In this paper, silk sericin was employed to regulate the mineralization of calcium carbonate (CaCO3). CaCO3 composite particles were prepared by the precipitation reaction of sodium carbonate with calcium chloride solution in the presence of silk sericin. The as-prepared samples were collected at different reaction time to study the crystallization process of CaCO3 by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and X-ray diffraction (XRD). The results showed that silk sericin significantly affected the morphology and crystallographic polymorph of CaCO3. With increasing the reaction time, the crystal phase of CaCO3 transferred from calcite dominated to vaterite dominated mixtures, while the morphology of CaCO3 changed from disk-like calcite crystal to spherical vaterite crystal. These studies showed the potential of silk sericin used as a template molecule to control the growth of inorganic crystal.

  9. Controlled Fabrication of Silk Protein Sericin Mediated Hierarchical Hybrid Flowers and Their Excellent Adsorption Capability of Heavy Metal Ions of Pb(II), Cd(II) and Hg(II).

    PubMed

    Koley, Pradyot; Sakurai, Makoto; Aono, Masakazu

    2016-01-27

    Fabrication of protein-inorganic hybrid materials of innumerable hierarchical patterns plays a major role in the development of multifunctional advanced materials with their improved features in synergistic way. However, effective fabrication and applications of the hybrid structures is limited due to the difficulty in control and production cost. Here, we report the controlled fabrication of complex hybrid flowers with hierarchical porosity through a green and facile coprecipitation method by using industrial waste natural silk protein sericin. The large surface areas and porosity of the microsize hybrid flowers enable water purification through adsorption of different heavy metal ions. The high adsorption capacity depends on their morphology, which is changed largely by sericin concentration in their fabrication. Superior adsorption and greater selectivity of the Pb(II) ions have been confirmed by the characteristic growth of needle-shaped nanowires on the hierarchical surface of the hybrid flowers. These hybrid flowers show excellent thermal stability even after complete evaporation of the protein molecules, significantly increasing the porosity of the flower petals. A simple, cost-effective and environmental friendly fabrication method of the porous flowers will lead to a new solution to water pollution required in the modern industrial society. PMID:26736132

  10. Sericin removal from raw Bombyx mori silk scaffolds of high hierarchical order.

    PubMed

    Teuschl, Andreas Herbert; van Griensven, Martijn; Redl, Heinz

    2014-05-01

    Silk fibroin has previously been described as a promising candidate for ligament tissue engineering (TE) approaches. For biocompatibility reasons, silkworm silk requires removal of sericin, which can elicit adverse immune responses in the human body. One disadvantage of the required degumming process is the alteration of the silk fiber structural properties, which can hinder textile engineering of high order hierarchical structures. Therefore, the aim of this study was to find a way to remove sericin from a compact and highly ordered raw silk fiber matrix. The wire rope design of the test model scaffold comprises several levels of geometric hierarchy. Commonly used degumming solutions fail in removing sericin in this wire rope design. Weight loss measurements, picric acid and carmine staining as well as scanning electron microscopy demonstrated that the removal of sericin from the model scaffold of a wire rope design can be achieved through a borate buffer-based system. Furthermore, the borate buffer degummed silks were shown to be nontoxic and did not alter cell proliferation behavior. The possibility to remove sericin after the textile engineering process has taken place eases the production of highly ordered scaffold structures and may expand the use of silk as scaffold material in further TE and regenerative medicine applications. PMID:24066942

  11. Bioconjugation of neutral protease on silk fibroin nanoparticles and application in the controllable hydrolysis of sericin.

    PubMed

    Zhu, Lin; Hu, Ren-Ping; Wang, Hai-Yan; Wang, Yuan-Jing; Zhang, Yu-Qing

    2011-09-28

    Bombyx mori silk fibroin is a protein-based macromolecular biopolymer with remarkable biocompatibility. Silk fiber was degummed and subjected to a series of treatments, including dissolution and dialysis, to yield an aqueous solution of silk fibroin, which was introduced rapidly into excess acetone to produce crystalline silk fibroin nanoparticles (SFNs). The SFNs were conjugated covalently with a neutral protease (NP) using glutaraldehyde as the cross-linking reagent. The objective of this study was to determine the optimal conditions for biosynthesis of the SFN-NP bioconjugates. First, SFN-NP was obtained by covalent cross-linking of SFN and NP at an SFN/NP ratio of 5-8 mg:1 IU with 0.75% glutaraldehyde for 6 h at 25 °C. When adding 50 IU of the enzyme, the residual activity of biological conjugates was increased to 31.45%. Studies on the enzyme activity of SFN-NP and its kinetics showed that the stability of SFN-NP bioconjugates was greater than that of the free enzyme, the optimum reactive temperature range was increased by 5-10 °C, and the optimum pH value range was increased to 6.5-8.0. Furthermore, the thermal stability was improved to some extent. A controlled hydrolysis test using the poorly water-soluble protein sericin as a substrate and SFN-NP as the enzyme showed that the longer the reaction time (within 1 h), the smaller the molecular mass (<30 kDa) is of the sericin peptide produced. The SFN-NP bioconjugate is easily recovered by centrifugation and can be used repeatedly. The highly efficient processing technology and the use of SFN as a novel vector for a protease has great potential for research and the development of food processing. PMID:21846144

  12. Silk sericin: A versatile material for tissue engineering and drug delivery.

    PubMed

    Lamboni, Lallepak; Gauthier, Mario; Yang, Guang; Wang, Qun

    2015-12-01

    Sericin is an inexpensive glycoprotein obtained as a by-product in the silk industry. Its variable amino acid composition and diverse functional groups confer upon it attractive bioactive properties, which are particularly interesting for biomedical applications. Because of its antioxidant character, moisturizing ability, and mitogenic effect on mammalian cells, sericin is useful in cell culture and tissue engineering. Its positive effects on keratinocytes and fibroblasts have led to the development of sericin-based biomaterials for skin tissue repair, mainly as wound dressings. Additionally, sericin can be used for bone tissue engineering owing to its ability to induce nucleation of bone-like hydroxyapatite. Stable silk sericin biomaterials, such as films, sponges, and hydrogels, are prepared by cross-linking, ethanol precipitation, or blending with other polymers. Sericin may also be employed for drug delivery because its chemical reactivity and pH-responsiveness facilitate the fabrication of nano- and microparticles, hydrogels, and conjugated molecules, improving the bioactivity of drugs. Here, we summarized the recent advancements in the study of silk sericin for application in tissue engineering and drug delivery. PMID:26523781

  13. Effect of silk protein processing on drug delivery from silk films.

    PubMed

    Pritchard, Eleanor M; Hu, Xiao; Finley, Violet; Kuo, Catherine K; Kaplan, David L

    2013-03-01

    Sericin removal from the core fibroin protein of silkworm silk is a critical first step in the use of silk for biomaterial-related applications, but degumming can affect silk biomaterial properties, including molecular weight, viscosity, diffusivity and degradation behavior. Increasing the degumming time (10, 30, 60, and 90 min) decreases the average molecular weight of silk protein in solution, silk solution viscosity, and silk film glass-transition temperature, and increases the rate of degradation of a silk film by protease. Model compounds spanning a range of physical-chemical properties generally show an inverse relationship between degumming time and release rate through a varied degumming time silk coating. Degumming provides a useful control point to manipulate silk's material properties. PMID:23349062

  14. Preparation of Silk Sericin/Lignin Blend Beads for the Removal of Hexavalent Chromium Ions.

    PubMed

    Kwak, Hyo Won; Shin, Munju; Yun, Haesung; Lee, Ki Hoon

    2016-01-01

    In the present study, novel adsorbents having high adsorption capability and reusability were prepared using agricultural by-products: silk sericin and lignin. Silk sericin and lignin blend beads were successfully prepared using simple coagulation methods for the removal of hexavalent chromium (Cr(VI)) from aqueous solution. A 1 M lithium chloride (LiCl)/dimethyl sulfoxide (DMSO) solvent system successfully dissolved both sericin and lignin and had sufficient viscosity for bead preparation. Compared to the conventional sericin bead adsorbent, sericin/lignin blend beads showed higher Cr(VI) adsorption capacity. The amount of lignin added to the adsorbent greatly affected the adsorption capacity of the beads, and a 50:50 sericin/lignin blend ratio was optimal. Adsorption behavior followed the Freundlich isotherm, which means the adsorption of Cr(VI) occurred on the heterogeneous surface. Cr(VI) adsorption capability increased with temperature because of thermodynamic-kinetic effects. In addition, over 90% of Cr(VI) ions were recovered from the Cr(VI) adsorbed sericin/lignin beads in a 1 M NaOH solution. The adsorption-desorption recycling process was stable for more than seven cycles, and the recycling efficiency was 82%. It is expected that the sericin/lignin beads could be successfully applied in wastewater remediation especially for hazardous Cr(VI) ions in industrial wastewater. PMID:27598142

  15. Biomedical Applications of Mulberry Silk and its Proteins: A Review

    NASA Astrophysics Data System (ADS)

    Nivedita, S.; Sivaprasad, V.

    2014-04-01

    Silk is a natural fibre used mainly for aesthetic purposes. It has also been used for making surgical sutures for centuries. The recent rediscovery of silk's biological properties have led to new areas of research and utilization in cosmetic, health and medical fields. The silk proteins, fibroin and sericin are processed into biomaterials because of bio-compatibility, bio-degradability, excellent mechanical properties, thermo tolerance and UV protective properties. Silk proteins could be obtained as pure liquids and regenerated in different forms suitable for tissue engineering applications. This paper presents some of the biomedical products and biomaterials made from native, degraded and regenerated silk and their fabrication techniques.

  16. Vibrational spectroscopic study of sulphated silk proteins

    NASA Astrophysics Data System (ADS)

    Monti, P.; Freddi, G.; Arosio, C.; Tsukada, M.; Arai, T.; Taddei, P.

    2007-05-01

    Degummed Bombyx mori ( B. m.) silk fibroin fabric and mutant naked pupa cocoons (Nd-s) consisting of almost pure silk sericin were treated with chlorosulphonic acid in pyridine and investigated by FT-IR and FT-Raman spectroscopies. Untreated silk fibroin and sericin displayed typical spectral features due to characteristic amino acid composition and molecular conformation (prevailing β-sheet with a less ordered structure in sericin). Upon sulphation, the degree of molecular disorder increased in both proteins and new bands appeared. The IR bands at 1049 and 1014 cm -1 were attributed to vibrations of sulphate salts and that at 1385 cm -1 to the νasSO 2 mode of organic covalent sulphates. In the 1300-1180 cm -1 range various contributions of alkyl and aryl sulphate salts, sulphonamides, sulphoamines and organic covalent sulphates, fell. Fibroin covalently bound sulphate groups through the hydroxyl groups of tyrosine and serine, while sericin through the hydroxyl groups of serine, since the δOH vibrations at 1399 cm -1 in IR and at 1408 cm -1 in Raman disappeared almost completely. Finally, the increase of the I850/ I830 intensity ratio of Raman tyrosine doublet in fibroin suggested a change towards a more exposed state of tyrosine residues, in good agreement with the more disordered conformation taken upon sulphation.

  17. Preparation of regenerated silk fibroin/silk sericin fibers by coaxial electrospinning.

    PubMed

    Hang, Yichun; Zhang, Yaopeng; Jin, Yuan; Shao, Huili; Hu, Xuechao

    2012-12-01

    The coaxial electrospinning using the regenerated silk fibroin (SF) and silk sericin (SS) aqueous solutions as the core and shell spinning dopes, respectively, was carried out to prepare regenerated SF/SS composite fibers with components and core-shell structure similar to the natural silkworm silks. It was found from the scanning electron microscope (SEM) and transmission electron microscope (TEM) results that the core dope (SF aqueous solution) flow rate (Q(c)) and the applied voltage (V) had some effects on the morphology of the composite fiber. With an increase in Q(c), the diameter nonuniformity and eccentricity of the core fiber became serious, while the increasing V played an inverse role. In this work, the suitable Q(c) for the fiber formation with better electrospinnability was about 6 μL/min, and the corresponding optimum V was 40 kV. Moreover, the results from Raman spectra analysis, modulated differential scanning calorimetry (MDSC), thermogravimetry (TG) measurement and mechanical property test showed that, compared with the pure SF fiber, the coaxially electrospun SF/SS fiber had more β-sheet conformation, better thermostability and mechanical properties. This was probably because that SS played significant roles in dehydrating SF molecules and inducing the conformational transition of SF to β-sheet structure. PMID:22935694

  18. Self-assembled silk sericin/poloxamer nanoparticles as nanocarriers of hydrophobic and hydrophilic drugs for targeted delivery

    NASA Astrophysics Data System (ADS)

    Mandal, Biman B.; Kundu, S. C.

    2009-09-01

    In recent times self-assembled micellar nanoparticles have been successfully employed in tissue engineering for targeted drug delivery applications. In this review, silk sericin protein from non-mulberry Antheraea mylitta tropical tasar silk cocoons was blended with pluronic F-127 and F-87 in the presence of solvents to achieve self-assembled micellar nanostructures capable of carrying both hydrophilic (FITC-inulin) and hydrophobic (anticancer drug paclitaxel) drugs. The fabricated nanoparticles were subsequently characterized for their size distribution, drug loading capability, cellular uptake and cytotoxicity. Nanoparticle sizes ranged between 100 and 110 nm in diameter as confirmed by dynamic light scattering. Rapid uptake of these particles into cells was observed in in vitro cellular uptake studies using breast cancer MCF-7 cells. In vitro cytotoxicity assay using paclitaxel-loaded nanoparticles against breast cancer cells showed promising results comparable to free paclitaxel drugs. Drug-encapsulated nanoparticle-induced apoptosis in MCF-7 cells was confirmed by FACS and confocal microscopic studies using Annexin V staining. Up-regulation of pro-apoptotic protein Bax, down-regulation of anti-apoptotic protein Bcl-2 and cleavage of regulatory protein PARP through Western blot analysis suggested further drug-induced apoptosis in cells. This study projects silk sericin protein as an alternative natural biomaterial for fabrication of self-assembled nanoparticles in the presence of poloxamer for successful delivery of both hydrophobic and hydrophilic drugs to target sites.

  19. Effect of silk protein surfactant on silk degumming and its properties.

    PubMed

    Wang, Fei; Cao, Ting-Ting; Zhang, Yu-Qing

    2015-10-01

    The silk protein surfactant (SPS) first used as a silk degumming agent in this study is an amino acid-type anionic surfactant that was synthesized using silk fibroin amino acids and lauroyl chloride. We studied it systematically in comparison with the traditional degumming methods such as sodium carbonate (Na2CO3) and neutral soap (NS). The experimental results showed that the sericin can be completely removed from the silk fibroin fiber after boiling the fibers three times for 30 min and using a bath ratio of 1:80 (g/mL) and a concentration of 0.2% SPS in an aqueous solution. The results of the tensile properties, thermal analysis, and SEM all show that SPS is similar to the NS, far superior to Na2CO3. In short, SPS may be used as an environmentally friendly silk degumming/refining agent in the silk textile industry and in the manufacture of silk floss quilts. PMID:26117747

  20. The effects of Bombyx mori silk strain and extraction time on the molecular and biological characteristics of sericin.

    PubMed

    Siritientong, Tippawan; Bonani, Walter; Motta, Antonella; Migliaresi, Claudio; Aramwit, Pornanong

    2016-01-01

    Sericin was extracted from three strains of Thai Bombyx mori silk cocoons (white shell Chul1/1, greenish shell Chul3/2, and yellow shell Chul4/2) by a high-pressure and high-temperature technique. The characteristics of sericin extracted from different fractions (15, 45, and 60 min extraction process) were compared. No differences in amino acid composition were observed among the three fractions. For all silk strains, sericin extracted from a 15-min process presented the highest molecular weight. The biological potential of the different sericin samples as a bioadditive for 3T3 fibroblast cells was assessed. When comparing sericin extracted from three silk strains, sericin fractions extracted from Chul4/2 improved cell proliferation, while sericin from Chul 1/1 activated Type I collagen production to the highest extent. This study allows the natural variability of sericin obtained from different sources and extraction conditions to be addressed and provides clues for the selection of sericin sources. PMID:26399155

  1. Comparing the properties of Bombyx mori silk cocoons against sericin-fibroin regummed biocomposite sheets.

    PubMed

    Morin, Alexander; Alam, Parvez

    2016-08-01

    This paper considers the utility of sericin, a degumming waste product, in the regumming of Bombyx mori silk fibroin fibres to form sericin-fibroin biocomposites. Regummed biocomposites have a chemical character that is somewhat closer to fibroin than sericin, though sericin presence is confirmed through FT-IR spectroscopy. Using direct measurements we further find the weight fractions of sericin in the regummed biocomposites and the native cocoons differ by only 5%. Mechanically, B. mori cocoons exhibit brittle stress-strain characteristics, failing at strengths of X̅= 16.6MPa and at strains of X̅= 13%. Contrarily, aligning fibroin fibres to a unidirectional axis in the regummed biocomposites causes them to exhibit characteristics of strain hardening, which is itself a typical characteristic of silk fibre pulled in tension. Though they are half as strong (X̅= 7.2MPa), regummed biocomposites are able to absorb five times more mechanical energy (X̅= 5.6MJm(-3)) than the B. mori cocoons (X̅= 1.1MJm(-3)) and are furthermore able to elongate to more than ten times (X̅= 180%) that of the native cocoons prior to failure. Our research shows that degummed B. mori cocoons can be regummed into sheets that have potential for use as load bearing engineering biocomposites. PMID:27157746

  2. The role of glycerol and water in flexible silk sericin film.

    PubMed

    Yun, Haesung; Kim, Moo Kon; Kwak, Hyo Won; Lee, Jeong Yun; Kim, Min Hwa; Lee, Ki Hoon

    2016-01-01

    Silk sericin (SS) can be obtained as a byproduct during the silk fiber process, but its application has been limited due to the brittleness of the SS film. To enhance the flexibility of the SS film, glycerol (Glc) has been added as a plasticizer. The addition of Glc enhanced the elongation property of the SS film when the Glc content was 50-70 wt% of SS. Glc also induced the structural transition of SS from a random coil structure to a β-sheet structure. The inconsistent increase of elongation and β-sheet structure of the SS/Glc film were explained by the content of moisture in the SS/Glc film. The moisture content of the SS/Glc film increased proportionally when the Glc content was higher than 50 wt% of SS, which was the same Glc content range that exhibited the plasticizing effect. Therefore, the plasticizing effect on the SS film may occur not only because of Glc but also because of water. Furthermore, water also contributed to the increase in the β-sheet structure development. Our results suggest that the moisture content in the plasticized protein film may play an important role when the plasticizer has hygroscopic properties. PMID:26562547

  3. Bioengineered silk proteins to control cell and tissue functions.

    PubMed

    Preda, Rucsanda C; Leisk, Gary; Omenetto, Fiorenzo; Kaplan, David L

    2013-01-01

    Silks are defined as protein polymers that are spun into fibers by some lepidoptera larvae such as silkworms, spiders, scorpions, mites, and flies. Silk proteins are usually produced within specialized glands in these animals after biosynthesis in epithelial cells that line the glands, followed by secretion into the lumen of the gland prior to spinning into fibers.The most comprehensively characterized silks are from the domesticated silkworm (Bombyx mori) and from some spiders (Nephila clavipes and Araneus diadematus). Silkworm silk has been used commercially as biomedical sutures for decades and in textile production for centuries. Because of their impressive mechanical properties, silk proteins provide an important set of material options in the fields of controlled drug release, and for biomaterials and scaffolds for tissue engineering. Silkworm silk from B. mori consists primarily of two protein components, fibroin, the structural protein of silk fibers, and sericins, the water-soluble glue-like proteins that bind the fibroin fibers together. Silk fibroin consists of heavy and light chain polypeptides linked by a disulfide bond. Fibroin is the protein of interest for biomedical materials and it has to be purified/extracted from the silkworm cocoon by removal of the sericin. Characteristics of silks, including biodegradability, biocompatibility, controllable degradation rates, and versatility to generate different material formats from gels to fibers and sponges, have attracted interest in the field of biomaterials. Cell culture and tissue formation using silk-based biomaterials have been pursued, where appropriate cell adhesion, proliferation, and differentiation on or in silk biomaterials support the regeneration of tissues. The relative ease with which silk proteins can be processed into a variety of material morphologies, versatile chemical functionalization options, processing in water or solvent, and the related biological features of biocompatibility and

  4. Preparation and mechanical properties of layers made of recombinant spider silk proteins and silk from silk worm

    NASA Astrophysics Data System (ADS)

    Junghans, F.; Morawietz, M.; Conrad, U.; Scheibel, T.; Heilmann, A.; Spohn, U.

    2006-02-01

    Layers of recombinant spider silks and native silks from silk worms were prepared by spin-coating and casting of various solutions. FT-IR spectra were recorded to investigate the influence of the different mechanical stress occurring during the preparation of the silk layers. The solubility of the recombinant spider silk proteins SO1-ELP, C16, AQ24NR3, and of the silk fibroin from Bombyx mori were investigated in hexafluorisopropanol, ionic liquids and concentrated salt solutions. The morphology and thickness of the layers were determined by Atomic Force Microscopy (AFM) or with a profilometer. The mechanical behaviour was investigated by acoustic impedance analysis by using a quartz crystal microbalance (QCMB) as well as by microindentation. The density of silk layers (d<300 nm) was determined based on AFM and QCMB measurements. At silk layers thicker than 300 nm significant changes of the half-band-half width can be correlated with increasing energy dissipation. Microhardness measurements demonstrate that recombinant spider silk and sericine-free Bombyx mori silk layers achieve higher elastic penetration modules EEP and Martens hardness values HM than those of polyethylenterephthalate (PET) and polyetherimide (PEI) foils.

  5. Viability and proliferation of L929, tumour and hybridoma cells in the culture media containing sericin protein as a supplement or serum substitute.

    PubMed

    Cao, Ting-Ting; Zhang, Yu-Qing

    2015-09-01

    Cell cultures often require the addition of animal serum and other supplements. In this study, silk sericin, a bioactive protein, recovered from the waste of silk floss production was hydrolysed into three pepsin-degraded sericin peptides with different ranges of molecular mass. Normal animal cells, tumour cells and hybridoma cells were cultured systematically in FBS culture media containing sericin as a supplement or serum substitute. The culture test and microscopic observation of L929 cells showed that the smaller molecular weight of the degraded sericin is most suitable for cell culture. The cell culture results showed that with the degradation of sericin, for normal mouse fibroblast L929 cells, addition of 0.75 % sericin into FBS culture medium yields cell viability that is superior to FBS culture medium alone. When all serum was replaced by sericin, cell viability in the sericin medium could reach about one half of that in FBS medium. When in a medium containing a mixture of FBS: sericin (6:4, v/v), the cell culture effect is about 80 %. For the cultures of four tumour and one hybridoma cells, regardless of the molecular weight range, these degraded sericin peptides could substitute all serum in FBS media. The cell viability and proliferation of these tumour and hybridoma cells are equivalent or superior to that in FBS medium. In other words, cell viability and proliferation of these tumour and hybridoma cells in sericin media are more preferable to serum media. The mechanism of the sericin protein to promote cell growth and proliferation will be further investigated later. PMID:25895088

  6. Biopatterning of Silk Proteins for Soft Micro-optics.

    PubMed

    Pal, Ramendra K; Kurland, Nicholas E; Wang, Congzhou; Kundu, Subhas C; Yadavalli, Vamsi K

    2015-04-29

    Silk proteins from spiders and silkworms have been proposed as outstanding candidates for soft micro-optic and photonic applications because of their optical transparency, unique biological properties, and mechanical robustness. Here, we present a method to form microstructures of the two constituent silk proteins, fibroin and sericin for use as an optical biomaterial. Using photolithography, chemically modified silk protein photoresists are patterned in 2D arrays of periodic patterns and Fresnel zone plates. Angle-dependent iridescent colors are produced in these periodic micropatterns because of the Bragg diffraction. Silk protein photolithography can used to form patterns on different substrates including flexible sheets with features of any shape with high fidelity and resolution over large areas. Finally, we show that these mechanically stable and transparent iridescent architectures are also completely biodegradable. This versatile and scalable technique can therefore be used to develop biocompatible, soft micro-optic devices that can be degraded in a controlled manner. PMID:25853731

  7. Silk sericin-alginate-chitosan microcapsules: hepatocytes encapsulation for enhanced cellular functions.

    PubMed

    Nayak, Sunita; Dey, Sanchareeka; Kundu, Subhas C

    2014-04-01

    The encapsulation based technology permits long-term delivery of desired therapeutic products in local regions of body without the need of immunosuppressant drugs. In this study microcapsules composed of sericin and alginate micro bead as inner core and with an outer chitosan shell are prepared. This work is proposed for live cell encapsulation for potential therapeutic applications. The sericin protein is obtained from cocoons of non-mulberry silkworm Antheraea mylitta. The sericin-alginate micro beads are prepared via ionotropic gelation under high applied voltage. The beads further coated with chitosan and crosslinked with genipin. The microcapsules developed are nearly spherical in shape with smooth surface morphology. Alamar blue assay and confocal microscopy indicate high cell viability and uniform encapsulated cell distribution within the sericin-alginate-chitosan microcapsules indicating that the microcapsules maintain favourable microenvironment for the cells. The functional analysis of encapsulated cells demonstrates that the glucose consumption, urea secretion rate and intracellular albumin content increased in the microcapsules. The study suggests that the developed sericin-alginate-chitosan microcapsule contributes towards the development of cell encapsulation model. It also offers to generate enriched population of metabolically and functionally active cells for the future therapeutics especially for hepatocytes transplantation in acute liver failure. PMID:24486492

  8. Comparative proteomic analysis of the silkworm middle silk gland reveals the importance of ribosome biogenesis in silk protein production.

    PubMed

    Li, Jian-ying; Ye, Lu-peng; Che, Jia-qian; Song, Jia; You, Zheng-ying; Yun, Ki-chan; Wang, Shao-hua; Zhong, Bo-xiong

    2015-08-01

    The silkworm middle silk gland (MSG) is the sericin synthesis and secretion unique sub-organ. The molecular mechanisms of regulating MSG protein synthesis are largely unknown. Here, we performed shotgun proteomic analysis on the three MSG subsections: the anterior (MSG-A), middle (MSG-M), and posterior (MSG-P) regions. The results showed that more strongly expressed proteins in the MSG-A were involved in multiple processes, such as silk gland development and silk protein protection. The proteins that were highly expressed in the MSG-M were enriched in the ribosome pathway. MSG-P proteins with stronger expression were mainly involved in the oxidative phosphorylation and citrate cycle pathways. These results suggest that the MSG-M is the most active region in the sericin synthesis. Furthermore, comparing the proteome of the MSG with the posterior silk gland (PSG) revealed that the specific and highly expressed proteins in the MSG were primarily involved in the ribosome and aminoacyl-tRNA biosynthesis pathways. These results indicate that silk protein synthesis is much more active as a result of the enhancement of translation-related pathways in the MSG. These results also suggest that enhancing ribosome biogenesis is important to the efficient synthesis of silk proteins. PMID:26051239

  9. Preliminary Characterization of Genipin-Cross-Linked Silk Sericin/Poly(vinyl alcohol) Films as Two-Dimensional Wound Dressings for the Healing of Superficial Wounds

    PubMed Central

    Siritientong, Tippawan; Ratanavaraporn, Juthamas; Srichana, Teerapol; Aramwit, Pornanong

    2013-01-01

    The genipin-cross-linked silk sericin/poly(vinyl alcohol) (PVA) films were developed aiming to be applied as two-dimensional wound dressings for the treatment of superficial wounds. The effects of genipin cross-linking concentration on the physical and biological properties of the films were investigated. The genipin-cross-linked silk sericin/PVA films showed the increased surface density, tensile strength, and percentage of elongation, but decreased percentage of light transmission, water vapor transmission rate, and water swelling, compared to the non-cross-linked films. This explained that the cross-linking bonds between genipin and silk sericin would reduce the mobility of molecular chains within the films, resulting in the more rigid molecular structure. Silk sericin was released from the genipin-cross-linked films in a sustained manner. In addition, either L929 mouse fibroblast or HaCat keratinocyte cells showed high percentage of viability when cultured on the silk sericin/PVA films cross-linked with 0.075 and 0.1% w/v genipin. The in vivo safety test performed according to ISO 10993-6 confirmed that the genipin-cross-linked silk sericin/PVA films were safe for the medical usages. The efficacy of the films for the treatment of superficial skin wounds will be further investigated in vivo and clinically. The genipin-cross-linked silk sericin/PVA films would be promising choices of two-dimensional wound dressings for the treatment of superficial wounds. PMID:24106722

  10. In vitro development of OPU-derived bovine embryos cultured either individually or in groups with the silk protein sericin and the viability of frozen-thawed embryos after transfer.

    PubMed

    Isobe, Tomohiro; Ikebata, Yoshihisa; Do, Lanh Thi Kim; Tanihara, Fuminori; Taniguchi, Masayasu; Otoi, Takeshige

    2015-07-01

    The optimization of single-embryo culture conditions is very important, particularly in the in vitro production of bovine embryos using the ovum pick-up (OPU) procedure. The purpose of this study was to examine the development of embryos derived from oocytes obtained by OPU that were cultured either individually or in groups in medium supplemented with or without sericin and to investigate the viability of the frozen-thawed embryos after a direct transfer. When two-cell-stage embryos were cultured either individually or in groups for 7 days in CR1aa medium supplemented with or without 0.5% sericin, the rates of development to blastocysts and freezable blastocysts were significantly lower for the embryos cultured individually without sericin than for the embryos cultured in groups with or without sericin. Moreover, the rate of development to freezable blastocysts of the embryos cultured individually with sericin was significantly higher than that of the embryos cultured without sericin. When the frozen-thawed embryos were transferred directly to recipients, the rates of pregnancy, abortion, stillbirth and normal calving in the recipients were similar among the groups, irrespective of the culture conditions and sericin supplementation. Our findings indicate that supplementation with sericin during embryo culture improves the quality of the embryos cultured individually but not the viability of the frozen-thawed embryos after transfer to recipients. PMID:25488699

  11. Effects of solvent on the solution properties, structural characteristics and properties of silk sericin.

    PubMed

    Jo, Yoon Nam; Um, In Chul

    2015-07-01

    Sericin films have attracted much attention from researchers in biomedical and cosmetic fields because of its unique properties, including good cytocompatibility and its promotion of wound healing. However, poor mechanical properties of sericin films have restricted its application in these fields. In this study, a new solvent, formic acid, was used to fabricate sericin solutions and films. The effects of formic acid on the structural characteristics and mechanical properties of the sericin solutions and films were examined and compared with water. The sericin/formic acid solution showed fewer aggregated sericin molecules, resulting in a lower turbidity than that of the sericin/water solution. In addition, the gelation of the sericin solution was retarded in formic acid compared to that of water. Sericin films cast from the formic acid solution exhibited a much higher crystallinity index than that produced from water. The tensile strength and elongation of the sericin films cast from the formic acid solution were more than double that of the sericin films cast from water. It is expected that the more stable sericin solution and high-crystallinity sericin films, which have significantly improved mechanical properties, produced by using formic acid as the solvent could be utilized in biomedical and cosmetic applications. PMID:25869308

  12. Effect of degumming condition on the solution properties and electrospinnablity of regenerated silk solution.

    PubMed

    Ko, Jae Sang; Yoon, Kyunghwan; Ki, Chang Seok; Kim, Hyun Ju; Bae, Do Gyu; Lee, Ki Hoon; Park, Young Hwan; Um, In Chul

    2013-04-01

    The application of silk on tissue engineering scaffolds has been studied intensively because silk has an electrospinning technique using a good blood compatibility, excellent cytocompatibility and biodegradability. Silk consists of two polymers, fibroin and sericin. In spite of importance of sericin, most studies were focused on the fibroin only and the effect of residual sericin on the electrospinning performance of silk has not been considered. In this study, regenerated silk with different residual sericin contents was prepared by controlling the degumming conditions. The effects of the degumming conditions on the solution properties and electrospinning performance of silk were examined. The fast protein liquid chromatography (FPLC) measurements confirmed that the molecular weight of the regenerated silk decreased slightly with increasing residual sericin content. More molecular aggregation of silk occurred with increasing sericin content, resulting in an increase in the solution turbidity of formic acid. All silk formic acid solutions exhibited almost Newtonian fluid behavior and the viscosity increased with increasing sericin content. Interestingly, the dope solution viscosity of silk increased remarkably at sericin contents <1% (or degumming ratio >25%) leading to significant improvements in electrospinnability and an increase in the fiber diameter of the silk web. PMID:23295206

  13. Green synthesis of silk sericin-capped silver nanoparticles and their potent anti-bacterial activity

    NASA Astrophysics Data System (ADS)

    Aramwit, Pornanong; Bang, Nipaporn; Ratanavaraporn, Juthamas; Ekgasit, Sanong

    2014-02-01

    In this study, a `green chemistry' approach was introduced to synthesize silk sericin (SS)-capped silver nanoparticles (AgNPs) under an alkaline condition (pH 11) using SS as a reducing and stabilizing agent instead of toxic chemicals. The SS-capped AgNPs were successfully synthesized at various concentrations of SS and AgNO3, but the yields were different. A higher yield of SS-capped AgNPs was obtained when the concentrations of SS and AgNO3 were increased. The SS-capped AgNPs showed a round shape and uniform size with diameter at around 48 to 117 nm. The Fourier transform infrared (FT-IR) spectroscopy result proved that the carboxylate groups obtained from alkaline degradation of SS would be a reducing agent for the generation of AgNPs while COO- and NH2 + groups stabilized the AgNPs and prevented their precipitation or aggregation. Furthermore, the SS-capped AgNPs showed potent anti-bacterial activity against various gram-positive bacteria (minimal inhibitory concentration (MIC) 0.008 mM) and gram-negative bacteria (MIC ranging from 0.001 to 0.004 mM). Therefore, the SS-capped AgNPs would be a safe candidate for anti-bacterial applications.

  14. The development of non-toxic ionic-crosslinked chitosan-based microspheres as carriers for the controlled release of silk sericin.

    PubMed

    Aramwit, Pornanong; Ekasit, Sanong; Yamdech, Rungnapha

    2015-10-01

    Silk sericin is recently shown to possess various biological activities for biomedical applications. While various sericin carriers were developed for drug delivery system, very few researches considered sericin as a bioactive molecule itself. In this study, sericin incorporated in the chitosan-based microspheres was introduced as a bioactive molecule and bioactive carrier at the same time. The chitosan/sericin (CH/SS) microspheres at different composition (80/20, 70/30, 60/40, and 50/50) were successfully fabricated using anhydroustri-polyphosphate (TPP) as a polyanionic crosslinker. The microspheres with an average size of 1-4 μm and narrow size distribution were obtained. From FT-IR spectra, the presence of both chitosan and sericin in the microspheres confirmed the occurrence of ionic interaction that crosslink them within the microspheres. We also found that the CH/SS microspheres prepared at 50/50 could encapsulate sericin at the highest percentage (37.28%) and release sericin in the most sustained behavior, possibly due to the strong ionic interaction of the positively charged chitosan and the negatively charged sericin. On the other hand, the composition of CH/SS had no effect on the degradation rate of microspheres. All microspheres continuously degraded and remained around 20% after 14 days of enzymatic degradation. This explained that the ionic crosslinkings between chitosan and sericin could be demolished by the enzyme and hydrolysis. Furthermore, we have verified that all CH/SS microspheres at any concentrations showed non-toxicity to L929 mouse fibroblast cells. Therefore, we suggested that the non-toxic ionic-crosslinked CH/SS microspheres could be incorporated in wound dressing material to achieve the sustained release of sericin for accelerated wound healing. PMID:26233725

  15. Production and properties of electrosprayed sericin nanopowder

    NASA Astrophysics Data System (ADS)

    Hazeri, Najmeh; Tavanai, Hossein; Moradi, Ali Reza

    2012-06-01

    Sericin is a proteinous substrate that envelops fibroin (silk) fiber, and its recovery provides significant economical and social benefits. Sericin is an antibacterial agent that resists oxidation and absorbs moisture and UV light. In powder form, sericin has a wide range of applications in food, cosmetics and drug delivery. Asides from other techniques of producing powder, such as precipitation and spray drying, electrospraying can yield solid nanoparticles, particularly in the submicron range. Here, we report the production of sericin nanopowder by electrospraying. Sericin sponge was recovered from Bombyx mori cocoons through a high-temperature, high-pressure process, followed by centrifugation and freeze drying of the sericin solution. The electrospraying solution was prepared by dissolving the sericin sponge in dimethyl sulfoxide. We demonstrate that electrospraying is capable of producing sericin nanopowder with an average particle size of 25 nm, which is by far smaller than the particles produced by other techniques. The electrosprayed sericin nanopowder consists of small crystallites and exhibits a high moisture absorbance.

  16. Recombinant DNA production of spider silk proteins

    PubMed Central

    Tokareva, Olena; Michalczechen-Lacerda, Valquíria A; Rech, Elíbio L; Kaplan, David L

    2013-01-01

    Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks. PMID:24119078

  17. A novel poly(γ-glutamic acid)/silk-sericin hydrogel for wound dressing: Synthesis, characterization and biological evaluation.

    PubMed

    Shi, Lu; Yang, Ning; Zhang, Hao; Chen, Li; Tao, Lei; Wei, Yen; Liu, Hui; Luo, Ying

    2015-03-01

    A novel multifunctional poly(γ-glutamic acid)/silk sericin (γ-PGA/SS) hydrogel has been developed and used as wound dressing. The physical and chemical properties of the γ-PGA/SS gels were systemically investigated. Furthermore, these γ-PGA/SS gels have been found to promote the L929 fibroblast cells proliferate, and in the in vivo study, significant stimulatory effects were also observed on granulation and capillary formation on day 9 in H-2-treated wounds, indicating that this new complex hydrogel could maintain a moist healing environment, protect the wound from bacterial infection, absorb excess exudates, and promote cell proliferation to reconstruct damaged tissue. Considering the simple preparation process and excellent biological property, this γ-PGA/SS hydrogel might have a wide range of applications in biomedical and clinical areas. PMID:25579954

  18. Advanced silk material spun by a transgenic silkworm promotes cell proliferation for biomedical application.

    PubMed

    Wang, Feng; Xu, Hanfu; Wang, Yuancheng; Wang, Riyuan; Yuan, Lin; Ding, Huan; Song, Chunnuan; Ma, Sanyuan; Peng, Zhixin; Peng, Zhangchuan; Zhao, Ping; Xia, Qingyou

    2014-12-01

    Natural silk fiber spun by the silkworm Bombyx mori is widely used not only for textile materials, but also for biofunctional materials. In the present study, we genetically engineered an advanced silk material, named hSFSV, using a transgenic silkworm, in which the recombinant human acidic fibroblast growth factor (hFGF1) protein was specifically synthesized in the middle silk gland and secreted into the sericin layer to surround the silk fiber using our previously optimized sericin1 expression system. The content of the recombinant hFGF1 in the hSFSV silk was estimated to be approximate 0.07% of the cocoon shell weight. The mechanical properties of hSFSV raw silk fiber were enhanced slightly compared to those of the wild-type raw silk fiber, probably due to the presence of the recombinant of hFGF1 in the sericin layer. Remarkably, the hSFSV raw silk significantly stimulated the cell growth and proliferation of NIH/3T3 mouse embryonic fibroblast cells, suggesting that the mitogenic activity of recombinant hFGF1 was well maintained and functioned in the sericin layer of hSFSV raw silk. These results show that the genetically engineered raw silk hSFSV could be used directly as a fine biomedical material for mass application. In addition, the strategy whereby functional recombinant proteins are expressed in the sericin layer of silk might be used to create more genetically engineered silks with various biofunctions and applications. PMID:24980060

  19. Antioxidant activities of two sericin proteins extracted from cocoon of silkworm (Bombyx mori) measured by DPPH, chemiluminescence, ORAC and ESR methods

    PubMed Central

    TAKECHI, TAYORI; WADA, RITSUKO; FUKUDA, TSUBASA; HARADA, KAZUKI; TAKAMURA, HITOSHI

    2014-01-01

    Recent efforts have focused on the use of sericin proteins extracted from cocoons of silkworm as a healthy food source for human consumption. In this study, we focused on the antioxidative properties of sericin proteins. The antioxidative properties were measured in sericin proteins extracted from the shell of the cocoon, designated hereafter as white sericin protein and yellow-green sericin protein, as well as bread without sericin protein and bread to which white sericin powder had been added using four measurement methods: 1,1-Diphenyl-2-picrylhydrazyl (DPPH), chemiluminescence, oxygen radical absorbance capacity (ORAC) and electron spin resonance (ESR). High antioxidative properties of sericin proteins were indicated by all four methods. A comparison of the two types of sericin proteins revealed that yellow-green sericin protein exhibited high antioxidative properties as indicated by the DPPH, chemiluminescence and ORAC methods. By contrast, a higher antioxidative property was determined in white sericin protein by the ESR method. Consequently, our findings confirmed that sericin proteins have antioxidative properties against multiple radicals. In addition, the antioxidative property of bread was enhanced by the addition of sericin powder to the bread. Therefore, findings of this study suggest that sericin proteins may be efficiently used as beneficial food for human health. PMID:24748975

  20. Thermal crystallization mechanism of silk fibroin protein

    NASA Astrophysics Data System (ADS)

    Hu, Xiao

    In this thesis, the thermal crystallization mechanism of silk fibroin protein from Bombyx mori silkworm, was treated as a model for the general study of protein based materials, combining theories from both biophysics and polymer physics fields. A systematic and scientific path way to model the dynamic beta-sheet crystallization process of silk fibroin protein was presented in the following sequence: (1) The crystallinity, fractions of secondary structures, and phase compositions in silk fibroin proteins at any transition stage were determined. Two experimental methods, Fourier transform infrared spectroscopy (FTIR) with Fourier self-deconvolution, and specific reversing heat capacity, were used together for the first time for modeling the static structures and phases in the silk fibroin proteins. The protein secondary structure fractions during the crystallization were quantitatively determined. The possibility of existence of a "rigid amorphous phase" in silk protein was also discussed. (2) The function of bound water during the crystallization process of silk fibroin was studied using heat capacity, and used to build a silk-water dynamic crystallization model. The fundamental concepts and thermal properties of silk fibroin with/without bound water were discussed. Results show that intermolecular bound water molecules, acting as a plasticizer, will cause silk to display a water-induced glass transition around 80°C. During heating, water is lost, and the change of the microenvironment in the silk fibroin chains induces a mesophase prior to thermal crystallization. Real time FTIR during heating and isothermal holding above Tg show the tyrosine side chain changes only during the former process, while beta sheet crystallization occurs only during the latter process. Analogy is made between the crystallization of synthetic polymers according to the four-state scheme of Strobl, and the crystallization process of silk fibroin, which includes an intermediate precursor

  1. Genipin-cross-linked thermosensitive silk sericin/poly(N-isopropylacrylamide) hydrogels for cell proliferation and rapid detachment.

    PubMed

    Zhang, Qingsong; Dong, Panpan; Chen, Li; Wang, Xiaozhao; Lu, Si

    2014-01-01

    To overcome release of silk sericin (SS) from semi-interpenetrating polymer network (semi-IPN) SS/poly(N-isopropylacrylamide) (PNIPAm) hydrogels, natural biocompatible genipin (GNP) was adopted as cross-linking agent of SS. The GNP/SS/PNIPAm hydrogels with various GNP contents were prepared by radical polymerization. Depending on GNP content, the resultant hydrogels present white, yellow, or dark blue. Required time of color change for GNP/SS mixture solution shortened with increasing GNP ratio. The GNP/SS/PNIPAm hydrogels present good oscillatory shrinking-swelling behavior between 20 and 37°C. The behaviors of L929 cell proliferation, desorption, and transshipment on the surface of hydrogels and tissue culture polystyrene were investigated by 3-(4,5-dimethy thioazol-2-yl)-2,5-di-phenytetrazoliumromide and scanning electron microscopy method. In comparison with pure SS/PNIPAm hydrogels, the introduction of certain GNP can accelerate cell adhesion and proliferation. Due to reversible change between hydrophobicity and hydrophilicity, by lowering temperature to 4°C from 37°C, L929 cells could spontaneously detach from the surface of hydrogels without the need for trypsin or ethylenediaminetetraacetic acid. The detached cells could subsequently be recultured. The prepared hydrogel and detached cells have potential applications in biomedical fields, such as organs or tissue regeneration and cancer or disease therapy. PMID:23606462

  2. A Hox Gene, Antennapedia, Regulates Expression of Multiple Major Silk Protein Genes in the Silkworm Bombyx mori.

    PubMed

    Tsubota, Takuya; Tomita, Shuichiro; Uchino, Keiro; Kimoto, Mai; Takiya, Shigeharu; Kajiwara, Hideyuki; Yamazaki, Toshimasa; Sezutsu, Hideki

    2016-03-25

    Hoxgenes play a pivotal role in the determination of anteroposterior axis specificity during bilaterian animal development. They do so by acting as a master control and regulating the expression of genes important for development. Recently, however, we showed that Hoxgenes can also function in terminally differentiated tissue of the lepidopteranBombyx mori In this species,Antennapedia(Antp) regulates expression of sericin-1, a major silk protein gene, in the silk gland. Here, we investigated whether Antpcan regulate expression of multiple genes in this tissue. By means of proteomic, RT-PCR, and in situ hybridization analyses, we demonstrate that misexpression of Antpin the posterior silk gland induced ectopic expression of major silk protein genes such assericin-3,fhxh4, and fhxh5 These genes are normally expressed specifically in the middle silk gland as is Antp Therefore, the evidence strongly suggests that Antpactivates these silk protein genes in the middle silk gland. The putativesericin-1 activator complex (middle silk gland-intermolt-specific complex) can bind to the upstream regions of these genes, suggesting that Antpdirectly activates their expression. We also found that the pattern of gene expression was well conserved between B. moriand the wild species Bombyx mandarina, indicating that the gene regulation mechanism identified here is an evolutionarily conserved mechanism and not an artifact of the domestication of B. mori We suggest that Hoxgenes have a role as a master control in terminally differentiated tissues, possibly acting as a primary regulator for a range of physiological processes. PMID:26814126

  3. Structure, evolution, and expression of antimicrobial silk proteins, seroins in Lepidoptera.

    PubMed

    Dong, Zhaoming; Song, Qianru; Zhang, Yan; Chen, Shiyi; Zhang, Xiaolu; Zhao, Ping; Xia, Qingyou

    2016-08-01

    The silks of silkworm and waxworm contain abundant antimicrobial proteins, including protease inhibitors and seroins. Protease inhibitors have antifungal activities, whereas seroins have antiviral and antibacterial activities. In order to obtain insights into the structure, evolution, and expression of seroins, we performed an extensive survey based on the available genome, transcriptome, and expressed sequence tags datasets. Sixty-four seroins were identified in 32 lepidopteran species. The phylogenetic and structural analyses revealed that seroins can be classified into five subfamilies: seroin 1, seroin 2, seroin 3, seroin 2 + 1, and seroin 3 + 3. It is interesting that seroin 2 + 1 contains two tandem seroin domains, seroin 2 and seroin 1, whereas seroin 3 + 3 has two tandem seroin 3 domains. Each seroin domain contains a proline-rich N-terminal motif and a conserved C-terminal motif. The transcriptome and EST data indicated that seroin 1 and seroin 2 genes were expressed in the silk gland but seroin 3 genes were not. Semi-quantitative RT-PCR and western blot analyses suggested that seroin 1 and seroin 2 were constantly accumulated in the silk gland of silkworm during the fifth instar, and then secreted into cocoon silk during spinning. Immunofluorescence analyses indicated that seroin 1 was secreted into the fibroin and sericin layers, whereas seroin 2 protein was only secreted into the sericin layer. However, the antimicrobial activity of seroin 2 was more effective than that of seroin 1. The presence of seroin 1 in the fibroin layer suggested that this protein not only acts as an antimicrobial protein, but might also play a role in the assembly and secretion of fibroins. Seroin 3, which was first identified here, might be related to pheromone synthesis or recognition, as it was highly expressed in male antennae and in the pheromone gland. PMID:27180727

  4. Designing Silk-silk Protein Alloy Materials for Biomedical Applications

    PubMed Central

    Hu, Xiao; Duki, Solomon; Forys, Joseph; Hettinger, Jeffrey; Buchicchio, Justin; Dobbins, Tabbetha; Yang, Catherine

    2014-01-01

    Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi) and domestic mulberry silk (Bombyx mori) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys. PMID:25145602

  5. In vivo bioresponses to silk proteins.

    PubMed

    Thurber, Amy E; Omenetto, Fiorenzo G; Kaplan, David L

    2015-12-01

    Silks are appealing materials for numerous biomedical applications involving drug delivery, tissue engineering, or implantable devices, because of their tunable mechanical properties and wide range of physical structures. In addition to the functionalities needed for specific clinical applications, a key factor necessary for clinical success for any implanted material is appropriate interactions with the body in vivo. This review summarizes our current understanding of the in vivo biological responses to silks, including degradation, the immune and inflammatory response, and tissue remodeling with particular attention to vascularization. While we focus in this review on silkworm silk fibroin protein due to the large quantity of in vivo data thanks to its widespread use in medical materials and consumer products, spider silk information is also included if available. Silk proteins are degraded in the body on a time course that is dependent on the method of silk fabrication and can range from hours to years. Silk protein typically induces a mild inflammatory response that decreases within a few weeks of implantation. The response involves recruitment and activation of macrophages and may include activation of a mild foreign body response with the formation of multinuclear giant cells, depending on the material format and location of implantation. The number of immune cells present decreases with time and granulation tissue, if formed, is replaced by endogenous, not fibrous, tissue. Importantly, silk materials have not been demonstrated to induce mineralization, except when used in calcified tissues. Due to its ability to be degraded, silk can be remodeled in the body allowing for vascularization and tissue ingrowth with eventual complete replacement by native tissue. The degree of remodeling, tissue ingrowth, or other specific cell behaviors can be modulated with addition of growth or other signaling factors. Silk can also be combined with numerous other materials

  6. Enhancer activity of Helitron in sericin-1 gene promoter from Bombyx mori.

    PubMed

    Huang, Ke; Li, Chun-Feng; Wu, Jie; Wei, Jun-Hong; Zou, Yong; Han, Min-Jin; Zhou, Ze-Yang

    2016-06-01

    Sericin is a kind of water-soluble protein expressed specifically in the middle silk gland of Bombyx mori. When the sericin-1 gene promoter was cloned and a transgenic vector was constructed to express a foreign protein, a specific Helitron, Bmhel-8, was identified in the sericin-1 gene promoter sequence in some genotypes of Bombyx mori and Bombyx mandarina. Given that the Bmhel-8 Helitron transposon was present only in some genotypes, it could be the source of allelic variation in the sericin-1 promoter. The length of the sericin-1 promoter sequence is approximately 1063 or 643 bp. The larger size of the sequence or allele is ascribed to the presence of Bmhel-8. Silkworm genotypes can be homozygous for either the shorter or larger promoter sequence or heterozygous, containing both alleles. Bmhel-8 in the sericin-1 promoter exhibits enhancer activity, as demonstrated by a dual-luciferase reporter system in BmE cell lines. Furthermore, Bmhel-8 displays enhancer activity in a sericin-1 promoter-driven gene expression system but does not regulate the tissue-specific expression of sericin-1. PMID:27067405

  7. Silk protein aggregation kinetics revealed by Rheo-IR.

    PubMed

    Boulet-Audet, Maxime; Terry, Ann E; Vollrath, Fritz; Holland, Chris

    2014-02-01

    The remarkable mechanical properties of silk fibres stem from a multi-scale hierarchical structure created when an aqueous protein "melt" is converted to an insoluble solid via flow. To directly relate a silk protein's structure and function in response to flow, we present the first application of a Rheo-IR platform, which couples cone and plate rheology with attenuated total reflectance infrared spectroscopy. This technique provides a new window into silk processing by linking shear thinning to an increase in molecular alignment, with shear thickening affecting changes in the silk protein's secondary structure. Additionally, compared to other static characterization methods for silk, Rheo-IR proved particularly useful at revealing the intrinsic difference between natural (native) and reconstituted silk feedstocks. Hence Rheo-IR offers important novel insights into natural silk processing. This has intrinsic academic merit, but it might also be useful when designing reconstituted silk analogues alongside other polymeric systems, whether natural or synthetic. PMID:24200713

  8. Photoprotection by silk cocoons.

    PubMed

    Kaur, Jasjeet; Rajkhowa, Rangam; Tsuzuki, Takuya; Millington, Keith; Zhang, Jin; Wang, Xungai

    2013-10-14

    A silk cocoon protects a silkworm during its pupal stage from various threats. We systematically investigated the role of fiber, sericin, and embedded crystals in the UV protection of a silk cocoon. Diffuse reflectance and UV absorbance were measured and free radicals generated during exposure to UV radiation were quantified using photoinduced chemiluminescence (PICL). We identified the response to both UV-A and UV-B radiations by silk materials and found that sericin was primarily responsible for UV-A absorption. When sericin was removed, the photoinduced chemiluminescence intensity increased significantly, indicating higher UV-A-induced reactions of cocoons in the absence of sericin. There is progressively higher sericin content toward the outer part of the cocoon shell that allows an effective shield to pupae from UV radiation and resists photodegradation of silk fibers. The study will inspire development of advanced organic photoprotective materials and designing silk-based, free-radical-scavenging antioxidants. PMID:24000973

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

    PubMed Central

    Kuwana, Yoshihiko; Sezutsu, Hideki; Nakajima, Ken-ichi; Tamada, Yasushi; Kojima, Katsura

    2014-01-01

    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. PMID:25162624

  10. Enhancing effects of sericin on corneal wound healing in rat debrided corneal epithelium.

    PubMed

    Nagai, Noriaki; Murao, Takatoshi; Ito, Yoshimasa; Okamoto, Norio; Sasaki, Masahiro

    2009-05-01

    The protein sericin is the main constituent of silk. We demonstrate the effects of sericin on corneal wound healing in rat debrided corneal epithelium. We also determined the effects of sericin on cell adhesion and proliferation in a human cornea epithelial cell line (HCE-T). Epithelium was removed from the corneas of rats with a BD Micro-Sharp, and wounded corneas were dyed with a 1% fluorescein solution. The corneal wounds were monitored using a fundus camera TRC-50X equipped with a digital camera. The corneal wound of rats instilled with saline was approximately 10% healing at 12 h, and approximately 65% healing at 24 h after corneal epithelial abrasion. The corneal wounds of rats instilled with saline showed almost complete healing by 36 h after corneal epithelial abrasion. On the other hand, the corneal healing rate of rats instilled with sericin solution was higher than that of rats instilled with saline, and the corneal healing rate constant increased with increasing sericin concentration. In addition, the adhesion and proliferation of HCE-T cells treated with 0.01-0.5% sericin solutions were enhanced, reaching a maximum at treatments with 0.2 and 0.1% sericin solutions, respectively. The present study demonstrates that the instillation of sericin solution has a potent effect in promoting wound healing and wound-size reduction in rats, probably caused by increasing cell movement and proliferation. PMID:19420767

  11. Recombinant protein blends: silk beyond natural design.

    PubMed

    Dinjaski, Nina; Kaplan, David L

    2016-06-01

    Recombinant DNA technology and new material concepts are shaping future directions in biomaterial science for the design and production of the next-generation biomaterial platforms. Aside from conventionally used synthetic polymers, numerous natural biopolymers (e.g., silk, elastin, collagen, gelatin, alginate, cellulose, keratin, chitin, polyhydroxyalkanoates) have been investigated for properties and manipulation via bioengineering. Genetic engineering provides a path to increase structural and functional complexity of these biopolymers, and thereby expand the catalog of available biomaterials beyond that which exists in nature. In addition, the integration of experimental approaches with computational modeling to analyze sequence-structure-function relationships is starting to have an impact in the field by establishing predictive frameworks for determining material properties. Herein, we review advances in recombinant DNA-mediated protein production and functionalization approaches, with a focus on hybrids or combinations of proteins; recombinant protein blends or 'recombinamers'. We highlight the potential biomedical applications of fibrous protein recombinamers, such as Silk-Elastin Like Polypeptides (SELPs) and Silk-Bacterial Collagens (SBCs). We also discuss the possibility for the rationale design of fibrous proteins to build smart, stimuli-responsive biomaterials for diverse applications. We underline current limitations with production systems for these proteins and discuss the main trends in systems/synthetic biology that may improve recombinant fibrous protein design and production. PMID:26686863

  12. Systematic evaluation of sericin protein as a substitute for fetal bovine serum in cell culture.

    PubMed

    Liu, Liyuan; Wang, Jinhuan; Duan, Shengchang; Chen, Lei; Xiang, Hui; Dong, Yang; Wang, Wen

    2016-01-01

    Fetal bovine serum (FBS) shows obvious deficiencies in cell culture, such as low batch to batch consistency, adventitious biological contaminant risk, and high cost, which severely limit the development of the cell culture industry. Sericin protein derived from the silkworm cocoon has become increasingly popular due to its diverse and beneficial cell culture characteristics. However, systematic evaluation of sericin as a substitute for FBS in cell culture medium remains limited. In this study, we conducted cellular morphological, physiological, and transcriptomic evaluation on three widely used mammalian cells. Compared with cells cultured in the control, those cultured in sericin-substitute medium showed similar cellular morphology, similar or higher cellular overall survival, lower population doubling time (PDT), and a higher percentage of S-phase with similar G2/G1 ratio, indicating comparable or better cell growth and proliferation. At the transcriptomic level, differentially expressed genes between cells in the two media were mainly enriched in function and biological processes related to cell growth and proliferation, reflecting that genes were activated to facilitate cell growth and proliferation. The results of this study suggest that cells cultured in sericin-substituted medium perform as well as, or even better than, those cultured in FBS-containing medium. PMID:27531556

  13. Systematic evaluation of sericin protein as a substitute for fetal bovine serum in cell culture

    PubMed Central

    Liu, Liyuan; Wang, Jinhuan; Duan, Shengchang; Chen, Lei; Xiang, Hui; Dong, Yang; Wang, Wen

    2016-01-01

    Fetal bovine serum (FBS) shows obvious deficiencies in cell culture, such as low batch to batch consistency, adventitious biological contaminant risk, and high cost, which severely limit the development of the cell culture industry. Sericin protein derived from the silkworm cocoon has become increasingly popular due to its diverse and beneficial cell culture characteristics. However, systematic evaluation of sericin as a substitute for FBS in cell culture medium remains limited. In this study, we conducted cellular morphological, physiological, and transcriptomic evaluation on three widely used mammalian cells. Compared with cells cultured in the control, those cultured in sericin-substitute medium showed similar cellular morphology, similar or higher cellular overall survival, lower population doubling time (PDT), and a higher percentage of S-phase with similar G2/G1 ratio, indicating comparable or better cell growth and proliferation. At the transcriptomic level, differentially expressed genes between cells in the two media were mainly enriched in function and biological processes related to cell growth and proliferation, reflecting that genes were activated to facilitate cell growth and proliferation. The results of this study suggest that cells cultured in sericin-substituted medium perform as well as, or even better than, those cultured in FBS-containing medium. PMID:27531556

  14. Cell proliferation by silk gut incorporating FGF-2 protein microcrystals.

    PubMed

    Kotani, Eiji; Yamamoto, Naoto; Kobayashi, Isao; Uchino, Keiro; Muto, Sayaka; Ijiri, Hiroshi; Shimabukuro, Junji; Tamura, Toshiki; Sezutsu, Hideki; Mori, Hajime

    2015-01-01

    Silk gut processed from the silk glands of the silkworm could be an ideal biodegradable carrier for cell growth factors. We previously demonstrated that polyhedra, microcrystals of Cypovirus 1 polyhedrin, can serve as versatile carrier proteins. Here, we report the generation of a transgenic silkworm that expresses polyhedrin together with human basic fibroblast growth factor (FGF-2) in its posterior silk glands to utilize silk gut as a proteinaceous carrier to protect and slowly release active cell growth factors. In the posterior silk glands, polyhedrin formed polyhedral microcrystals, and FGF-2 became encapsulated within the polyhedra due to a polyhedron-immobilization signal. Silk gut powder prepared from posterior silk glands containing polyhedron-encapsulated FGF-2 stimulated the phosphorylation of p44/p42 MAP kinase and induced the proliferation of serum-starved NIH3T3 cells by releasing bioactive FGF-2. Even after a one-week incubation at 25 °C, significantly higher biological activity of FGF-2 was observed for silk gut powder incorporating polyhedron-encapsulated FGF-2 relative to silk gut powder with non-encapsulated FGF-2. Our results demonstrate that posterior silk glands incorporating polyhedron-encapsulated FGF-2 are applicable to the preparation of biodegradable silk gut, which can protect and release FGF-2 that is produced in a virus- and serum-free expression system with significant application potential. PMID:26053044

  15. Cell proliferation by silk gut incorporating FGF-2 protein microcrystals

    PubMed Central

    Kotani, Eiji; Yamamoto, Naoto; Kobayashi, Isao; Uchino, Keiro; Muto, Sayaka; Ijiri, Hiroshi; Shimabukuro, Junji; Tamura, Toshiki; Sezutsu, Hideki; Mori, Hajime

    2015-01-01

    Silk gut processed from the silk glands of the silkworm could be an ideal biodegradable carrier for cell growth factors. We previously demonstrated that polyhedra, microcrystals of Cypovirus 1 polyhedrin, can serve as versatile carrier proteins. Here, we report the generation of a transgenic silkworm that expresses polyhedrin together with human basic fibroblast growth factor (FGF-2) in its posterior silk glands to utilize silk gut as a proteinaceous carrier to protect and slowly release active cell growth factors. In the posterior silk glands, polyhedrin formed polyhedral microcrystals, and FGF-2 became encapsulated within the polyhedra due to a polyhedron-immobilization signal. Silk gut powder prepared from posterior silk glands containing polyhedron-encapsulated FGF-2 stimulated the phosphorylation of p44/p42 MAP kinase and induced the proliferation of serum-starved NIH3T3 cells by releasing bioactive FGF-2. Even after a one-week incubation at 25 °C, significantly higher biological activity of FGF-2 was observed for silk gut powder incorporating polyhedron-encapsulated FGF-2 relative to silk gut powder with non-encapsulated FGF-2. Our results demonstrate that posterior silk glands incorporating polyhedron-encapsulated FGF-2 are applicable to the preparation of biodegradable silk gut, which can protect and release FGF-2 that is produced in a virus- and serum-free expression system with significant application potential. PMID:26053044

  16. Sericin supplementation improves semen freezability of buffalo bulls by minimizing oxidative stress during cryopreservation.

    PubMed

    Kumar, Pradeep; Kumar, Dharmendra; Sikka, P; Singh, P

    2015-01-01

    The variety of mammalian cells has been successfully cryopreserved by use of the silk protein sericin due to its strong free-radical-scavenging and potent antioxidant activity. The present study was conducted to examine the protective role of sericin on buffalo spermatozoa during cryopreservation. Semen of four breeding bulls was collected twice a week using artificial vagina technique. The ejaculates of four bulls were pooled, divided into five equal fractions, diluted with the extender supplemented with different concentrations of sericin (0, 0.25, 0.5, 1.5 and 2%) and then cryopreserved. Post-thawed motility was objectively assessed by computer assisted sperm analyzer. Sperm plasma membrane integrity was assessed by hypo-osmotic swelling test (HOST). Malondialdehyde (MDA) concentration, glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were determined in frozen-thawed extended seminal plasma by spectrophotometry. The extender supplemented with 0.25, 0.5 and 1% sericin resulted in the higher sperm motility and GPx acivity. Furthermore, plasma membrane integrity and SOD activity were found to be higher (P<0.05) in group supplemented with 0.25 and 0.5% sericin (P<0.05). The MDA concentration was found to be significantly lower (P<0.05) in 0.25 and 0.5% sericin treated groups than control and other treated groups. In conclusion, the supplementation of 0.25-0.5% sericin in semen extender improves frozen-thawed semen quality through protecting sperm from oxidative stress. PMID:25497424

  17. Design and performance of a sericin-alginate interpenetrating network hydrogel for cell and drug delivery.

    PubMed

    Zhang, Yeshun; Liu, Jia; Huang, Lei; Wang, Zheng; Wang, Lin

    2015-01-01

    Although alginate hydrogels have been extensively studied for tissue engineering applications, their utilization is limited by poor mechanical strength, rapid drug release, and a lack of cell adhesive ability. Aiming to improve these properties, we employ the interpenetrating hydrogel design rationale. Using alginate and sericin (a natural protein with many unique properties and a major component of silkworm silk), we develop an interpenetrating polymer network (IPN) hydrogel comprising interwoven sericin and alginate double networks. By adjusting the sericin-to-alginate ratios, IPNs' mechanical strength can be adjusted to meet stiffness requirements for various tissue repairs. The IPNs with high sericin content show increased stability during degradation, avoiding pure alginate's early collapse. These IPNs have high swelling ratios, benefiting various applications such as drug delivery. The IPNs sustain controlled drug release with the adjustable rates. Furthermore, these IPNs are adhesive to cells, supporting cell proliferation, long-term survival and migration. Notably, the IPNs inherit sericin's photoluminescent property, enabling bioimaging in vivo. Together, our study indicates that the sericin-alginate IPN hydrogels may serve as a versatile platform for delivering cells and drugs, and suggests that sericin may be a building block broadly applicable for generating IPN networks with other biomaterials for diverse tissue engineering applications. PMID:26205586

  18. Protein-protein nanoimprinting of silk fibroin films

    PubMed Central

    Brenckle, Mark A; Tao, Hu; Kim, Sunghwan; Paquette, Mark; Kaplan, David L; Omenetto, Fiorenzo G

    2013-01-01

    Control of the interface between biological tissue and high technology materials is paramount for the development of future applications in biomedicine, especially in the case of implantable integrated devices for signal transduction.[1]-[3] Such work requires careful materials design to develop devices that can efficiently perform technological functions while retaining biocompatibility and biological integration. Silk fibroin protein from the Bombyx mori silkworm has come of considerable interest in this context, owing to its attractive mechanical,[4]-[7] biological, [8][9] and optical properties.[10][11] Recent work has shown adaptation of common micro- and nano-fabrication tools to silk films,[12]-[18] as well as silk protein secondary structure patterning techniques,[19] leading to biocompatible and degradable electronic and photonic devices which can simultaneously act as a carrier and stabilizer for protein pharmaceuticals and other bioactive reagents.[20]-[23] In particular, silk based nanoscale photonic devices face the challenge of sub-wavelength resolution fabrication on a soft polymeric substrate.[15][24] Previous work introduced the possibility of direct, rapid nanoimprinting in silk for the fabrication of photonic structures by leveraging the material properties of this protein.[25] PMID:23483712

  19. Metal nanoparticles triggered persistent negative photoconductivity in silk protein hydrogels

    NASA Astrophysics Data System (ADS)

    Gogurla, Narendar; Sinha, Arun K.; Naskar, Deboki; Kundu, Subhas C.; Ray, Samit K.

    2016-03-01

    Silk protein is a natural biopolymer with intriguing properties, which are attractive for next generation bio-integrated electronic and photonic devices. Here, we demonstrate the negative photoconductive response of Bombyx mori silk protein fibroin hydrogels, triggered by Au nanoparticles. The room temperature electrical conductivity of Au-silk hydrogels is found to be enhanced with the incorporation of Au nanoparticles over the control sample, due to the increased charge transporting networks within the hydrogel. Au-silk lateral photoconductor devices show a unique negative photoconductive response under an illumination of 325 nm, with excitation energy higher than the characteristic metal plasmon resonance band. The enhanced photoconductance yield in the hydrogels over the silk protein is attributed to the photo-oxidation of amino groups in the β-pleated sheets of the silk around the Au nanoparticles followed by the breaking of charge transport networks. The Au-silk nanocomposite does not show any photoresponse under visible illumination because of the localization of excited charges in Au nanoparticles. The negative photoconductive response of hybrid Au-silk under UV illumination may pave the way towards the utilization of silk for future bio-photonic devices using metal nanoparticle platforms.

  20. Silks produced by insect labial glands.

    PubMed

    Sehnal, Frantisek; Sutherland, Tara

    2008-01-01

    Insect silks are secreted from diverse gland types; this chapter deals with the silks produced by labial glands of Holometabola (insects with pupa in their life cycle). Labial silk glands are composed of a few tens or hundreds of large polyploid cells that secrete polymerizing proteins which are stored in the gland lumen as a semi-liquid gel. Polymerization is based on weak molecular interactions between repetitive amino acid motifs present in one or more silk proteins; cross-linking by disulfide bonds may be important in the silks spun under water. The mechanism of long-term storage of the silk dope inside the glands and its conversion into the silk fiber during spinning is not fully understood. The conversion occurs within seconds at ambient temperature and pressure, under minimal drawing force and in some cases under water. The silk filament is largely built of proteins called fibroins and in Lepidoptera and Trichoptera coated by glue-type proteins known as sericins. Silks often contain small amounts of additional proteins of poorly known function. The silk components controlling dope storage and filament formation seem to be conserved at the level of orders, while the nature of polymerizing motifs in the fibroins, which determine the physical properties of silk, differ at the level of family and even genus. Most silks are based on fibroin beta-sheets interrupted with other structures such as alpha-helices but the silk proteins of certain sawflies have predominantly a collagen-like or polyglycine II arrangement and the silks of social Hymenoptera are formed from proteins in a coiled coil arrangement. PMID:19221523

  1. The Potential of Silk and Silk-Like Proteins as Natural Mucoadhesive Biopolymers for Controlled Drug Delivery.

    PubMed

    Brooks, Amanda E

    2015-01-01

    Drug delivery across mucus membranes is a particularly effective route of administration due to the large surface area. However, the unique environment present at the mucosa necessitates altered drug formulations designed to (1) deliver sensitive biologic molecules, (2) promote intimate contact between the mucosa and the drug, and (3) prolong the drug's local residence time. Thus, the pharmaceutical industry has an interest in drug delivery systems formulated around the use of mucoadhesive polymers. Mucoadhesive polymers, both synthetic and biological, have a history of use in local drug delivery. Prominently featured in the literature are chitosan, alginate, and cellulose derivatives. More recently, silk and silk-like derivatives have been explored for their potential as mucoadhesive polymers. Both silkworms and spiders produce sticky silk-like glue substances, sericin and aggregate silk respectively, that may prove an effective, natural matrix for drug delivery to the mucosa. This mini review will explore the potential of silk and silk-like derivatives as a biocompatible mucoadhesive polymer matrix for local controlled drug delivery. PMID:26636069

  2. The Potential of Silk and Silk-Like Proteins as Natural Mucoadhesive Biopolymers for Controlled Drug Delivery

    PubMed Central

    Brooks, Amanda E.

    2015-01-01

    Drug delivery across mucus membranes is a particularly effective route of administration due to the large surface area. However, the unique environment present at the mucosa necessitates altered drug formulations designed to (1) deliver sensitive biologic molecules, (2) promote intimate contact between the mucosa and the drug, and (3) prolong the drug's local residence time. Thus, the pharmaceutical industry has an interest in drug delivery systems formulated around the use of mucoadhesive polymers. Mucoadhesive polymers, both synthetic and biological, have a history of use in local drug delivery. Prominently featured in the literature are chitosan, alginate, and cellulose derivatives. More recently, silk and silk-like derivatives have been explored for their potential as mucoadhesive polymers. Both silkworms and spiders produce sticky silk-like glue substances, sericin and aggregate silk respectively, that may prove an effective, natural matrix for drug delivery to the mucosa. This mini review will explore the potential of silk and silk-like derivatives as a biocompatible mucoadhesive polymer matrix for local controlled drug delivery. PMID:26636069

  3. The potential of silk and silk-like proteins as natural mucoadhesive biopolymers for controlled drug delivery

    NASA Astrophysics Data System (ADS)

    Brooks, Amanda

    2015-11-01

    Drug delivery across mucus membranes is a particularly effective route of administration due to the large surface area. However, the unique environment present at the mucosa necessitates altered drug formulations designed to (1) deliver sensitive biologic molecules, (2) promote intimate contact between the mucosa and the drug, and (3) prolong the drug’s local residence time. Thus, the pharmaceutical industry has an interest in drug delivery systems formulated around the use of mucoadhesive polymers. Mucoadhesive polymers, both synthetic and biological, have a history of use in local drug delivery. Prominently featured in the literature are chitosan, alginate, and cellulose derivatives. More recently, silk and silk-like derivatives have been explored for their potential as mucoadhesive polymers. Both silkworms and spiders produce sticky silk-like glue substances, sericin and aggregate silk respectively, that may prove an effective, natural matrix for drug delivery to the mucosa. This mini review will explore the potential of silk and silk-like derivatives as a biocompatible mucoadhesive polymer matrix for local controlled drug delivery.

  4. [Processing and Modification of Recombinant Spider Silk Proteins].

    PubMed

    Liu, Bin; Wang, Tao; Liu, Xiaobing; Luo, Yongen

    2015-08-01

    Due to its special sequence structure, spider silk protein has unique physical and chemical properties, mechanical properties and excellent biological properties. With the expansion of the application value of spider silk in many fields as a functional material, progress has been made in the studies on the expression of recombinant spider silk proteins through many host systems by gene recombinant techniques. Recombinant spider silk proteins can be processed into high performance fibers, and a wide range of nonfibrous morphologies. Moreover, for their excellent biocompatibility and low immune response they are ideal for biomedical applications. Here we review the process and mechanism of preparation in vitro, chemistry and genetic engineering modification on recombinant spider silk protein. PMID:26710473

  5. Investigation of Natural Bombyx mori Silk Fibroin Proteins Using INS

    NASA Astrophysics Data System (ADS)

    Crain, Christopher; Strange, Nicholas; Larese, J. Z.

    The mechanical properties of many protein comprised biomaterials are a direct reflection of non-covalent (i.e. weak) interacting ions such as F-actin in muscles, tubulin in the cytoskeleton of cells, viral capsids, and silk. Porter and Vollrath underscored the two main factors that are critical for understanding the high mechanical strength of silks: the nanoscale semi-crystalline folding structure, which gives it exceptional toughness and strength, and the degree of hydration of the disordered fraction, which acts to modify these properties. Understanding and controlling these two principal factors are the key to the functionality of protein elastomers, and render silk an ideal model protein for (bio)material design. We will describe our investigation of electrospun silk of the Bombyx mori (silk worm), using Inelastic Neutron Scattering (INS). These techniques were used to investigate the microscopic dynamics of the dry and hydrated protein.

  6. Purification and biochemical characterization of a 70 kDa sericin from tropical tasar silkworm, Antheraea mylitta.

    PubMed

    Dash, Rupesh; Ghosh, Sudip K; Kaplan, David L; Kundu, S C

    2007-05-01

    Sericin isolated from the cocoon of the tropical tasar silkmoth Antheraea mylitta showed three major bands, with the lowest 70 kDa. This band was purified by anion exchange chromatography. Immunoblotting with concanavalin-A suggests a glycoprotein and CD analysis of secondary structure includes beta-sheet. Amino acid analysis shows that the protein is enriched in glycine and serine while the mole percentages of these two amino acids are different from sericin of mulberry silkworm. An anti A. mylitta sericin antibody was able to cross-react with sericin from A. assamensis but not the sericin of Bombyx mori and Philosamia ricini. Immunoblot analysis with proteins isolated from middle silk gland of A. mylitta at different developmental stages of larva showed that the 70 kDa sericin is developmentally regulated. These data extend the range of biochemical features found in this unusual family of proteins and may help in developing an improved understanding of their role in forming environmentally stable fibroin fiber-sericin composite structures (cocoons). PMID:17350301

  7. Solution structure of eggcase silk protein and its implications for silk fiber formation

    PubMed Central

    Lin, Zhi; Huang, Weidong; Zhang, Jingfeng; Fan, Jing-Song; Yang, Daiwen

    2009-01-01

    Spider silks are renowned for their excellent mechanical properties and biomimetic and industrial potentials. They are formed from the natural refolding of water-soluble fibroins with α-helical and random coil structures in silk glands into insoluble fibers with mainly β-structures. The structures of the fibroins at atomic resolution and silk formation mechanism remain largely unknown. Here, we report the 3D structures of individual domains of a ≈366-kDa eggcase silk protein that consists of 20 identical type 1 repetitive domains, one type 2 repetitive domain, and conserved nonrepetitive N- and C-terminal domains. The structures of the individual domains in solution were determined by using NMR techniques. The domain interactions were investigated by NMR and dynamic light-scattering techniques. The formation of micelles and macroscopic fibers from the domains was examined by electron microscopy. We find that either of the terminal domains covalently linked with at least one repetitive domain spontaneously forms micelle-like structures and can be further transformed into fibers at ≥37 °C and a protein concentration of >0.1 wt%. Our biophysical and biochemical experiments indicate that the less hydrophilic terminal domains initiate the assembly of the proteins and form the outer layer of the micelles whereas the more hydrophilic repetitive domains are embedded inside to ensure the formation of the micelle-like structures that are the essential intermediates in silk formation. Our results establish the roles of individual silk protein domains in fiber formation and provide the basis for designing miniature fibroins for producing artificial silks. PMID:19458259

  8. Bioengineered Chimeric Spider Silk-Uranium Binding Proteins

    PubMed Central

    Krishnaji, Sreevidhya Tarakkad; Kaplan, David L.

    2014-01-01

    Heavy metals constitute a source of environmental pollution. Here, novel functional hybrid biomaterials for specific interactions with heavy metals are designed by bioengineering consensus sequence repeats from spider silk of Nephila clavipes with repeats of a uranium peptide recognition motif from a mutated 33-residue of calmodulin protein from Paramecium tetraurelia. The self-assembly features of the silk to control nanoscale organic/inorganic material interfaces provides new biomaterials for uranium recovery. With subsequent enzymatic digestion of the silk to concentrate the sequestered metals, options can be envisaged to use these new chimeric protein systems in environmental engineering, including to remediate environments contaminated by uranium. PMID:23212989

  9. Design and performance of a sericin-alginate interpenetrating network hydrogel for cell and drug delivery

    NASA Astrophysics Data System (ADS)

    Zhang, Yeshun; Liu, Jia; Huang, Lei; Wang, Zheng; Wang, Lin

    2015-07-01

    Although alginate hydrogels have been extensively studied for tissue engineering applications, their utilization is limited by poor mechanical strength, rapid drug release, and a lack of cell adhesive ability. Aiming to improve these properties, we employ the interpenetrating hydrogel design rationale. Using alginate and sericin (a natural protein with many unique properties and a major component of silkworm silk), we develop an interpenetrating polymer network (IPN) hydrogel comprising interwoven sericin and alginate double networks. By adjusting the sericin-to-alginate ratios, IPNs’ mechanical strength can be adjusted to meet stiffness requirements for various tissue repairs. The IPNs with high sericin content show increased stability during degradation, avoiding pure alginate’s early collapse. These IPNs have high swelling ratios, benefiting various applications such as drug delivery. The IPNs sustain controlled drug release with the adjustable rates. Furthermore, these IPNs are adhesive to cells, supporting cell proliferation, long-term survival and migration. Notably, the IPNs inherit sericin’s photoluminescent property, enabling bioimaging in vivo. Together, our study indicates that the sericin-alginate IPN hydrogels may serve as a versatile platform for delivering cells and drugs, and suggests that sericin may be a building block broadly applicable for generating IPN networks with other biomaterials for diverse tissue engineering applications.

  10. Design and performance of a sericin-alginate interpenetrating network hydrogel for cell and drug delivery

    PubMed Central

    Zhang, Yeshun; Liu, Jia; Huang, Lei; Wang, Zheng; Wang, Lin

    2015-01-01

    Although alginate hydrogels have been extensively studied for tissue engineering applications, their utilization is limited by poor mechanical strength, rapid drug release, and a lack of cell adhesive ability. Aiming to improve these properties, we employ the interpenetrating hydrogel design rationale. Using alginate and sericin (a natural protein with many unique properties and a major component of silkworm silk), we develop an interpenetrating polymer network (IPN) hydrogel comprising interwoven sericin and alginate double networks. By adjusting the sericin-to-alginate ratios, IPNs’ mechanical strength can be adjusted to meet stiffness requirements for various tissue repairs. The IPNs with high sericin content show increased stability during degradation, avoiding pure alginate’s early collapse. These IPNs have high swelling ratios, benefiting various applications such as drug delivery. The IPNs sustain controlled drug release with the adjustable rates. Furthermore, these IPNs are adhesive to cells, supporting cell proliferation, long-term survival and migration. Notably, the IPNs inherit sericin’s photoluminescent property, enabling bioimaging in vivo. Together, our study indicates that the sericin-alginate IPN hydrogels may serve as a versatile platform for delivering cells and drugs, and suggests that sericin may be a building block broadly applicable for generating IPN networks with other biomaterials for diverse tissue engineering applications. PMID:26205586

  11. Chimeric spider silk proteins mediated by intein result in artificial hybrid silks.

    PubMed

    Lin, Senzhu; Chen, Gefei; Liu, Xiangqin; Meng, Qing

    2016-07-01

    Hybrid silks hold a great potential as specific biomaterials due to its controlled mechanical properties. To produce fibers with tunable properties, here we firstly made chimeric proteins in vitro, called W2C4CT and W2C8CT, with ligation of MaSp repetitive modules (C) with AcSp modules (W) by intein trans splicing technology from smaller precursors without final yield reduction. Intein mediated chimeric proteins form fibers at a low concentration of 0.4 mg/mL in 50 mM K3 PO4 pH 7.5 just drawn by hand. Hybrid fibers show smoother surface, and also have stronger chemical resistance as compared with fibers from W2CT (W fibers) and mixture of W2CT/C8CT (MHF8 fibers). Fibers from chimeric protein W2C4CT (HFH4) have improved mechanical properties than W fibers; however, with more C modules W2C8CT fibers (HFH8) properties decreased, indicates the length proportion of various modules is very important and should be optimized for fibers with specific properties. Generally, hybrid silks generated via chimeric proteins, which can be simplified by intein trans splicing, has greater potential to produce fibers with tunable properties. Our research shows that intein mediated directional protein ligation is a novel way to make large chimeric spider silk proteins and hybrid silks. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 385-392, 2016. PMID:26948769

  12. Nanostructure and molecular mechanics of spider dragline silk protein assemblies

    PubMed Central

    Keten, Sinan; Buehler, Markus J.

    2010-01-01

    Spider silk is a self-assembling biopolymer that outperforms most known materials in terms of its mechanical performance, despite its underlying weak chemical bonding based on H-bonds. While experimental studies have shown that the molecular structure of silk proteins has a direct influence on the stiffness, toughness and failure strength of silk, no molecular-level analysis of the nanostructure and associated mechanical properties of silk assemblies have been reported. Here, we report atomic-level structures of MaSp1 and MaSp2 proteins from the Nephila clavipes spider dragline silk sequence, obtained using replica exchange molecular dynamics, and subject these structures to mechanical loading for a detailed nanomechanical analysis. The structural analysis reveals that poly-alanine regions in silk predominantly form distinct and orderly beta-sheet crystal domains, while disorderly regions are formed by glycine-rich repeats that consist of 31-helix type structures and beta-turns. Our structural predictions are validated against experimental data based on dihedral angle pair calculations presented in Ramachandran plots, alpha-carbon atomic distances, as well as secondary structure content. Mechanical shearing simulations on selected structures illustrate that the nanoscale behaviour of silk protein assemblies is controlled by the distinctly different secondary structure content and hydrogen bonding in the crystalline and semi-amorphous regions. Both structural and mechanical characterization results show excellent agreement with available experimental evidence. Our findings set the stage for extensive atomistic investigations of silk, which may contribute towards an improved understanding of the source of the strength and toughness of this biological superfibre. PMID:20519206

  13. A sericin-derived peptide protects sf9 insect cells from death caused by acute serum deprivation.

    PubMed

    Takahashi, Masakazu; Tsujimoto, Kazuhisa; Kato, Youichi; Yamada, Hideyuki; Takagi, Hiroshi; Nakamori, Shigeru

    2005-07-01

    Sericin is the silk protein enveloping fibroin fibers in cocoons. Sericin hydrolysate protects cultured Sf9 insect cells from death caused by serum deprivation; the activity depends on the repeats of 38 amino acids. A partial peptide from the 38 residues, SGGSSTYGYS, inhibited serum-deprivation death as well. Cell viabilities in the presence of 10% (v/v) foetal calf serum, no additives and 1 mM: SGGSSTYGYS were 96, 12 and 31% on the third day after inoculation, respectively. Aromatic residues seemed to be important because SGGSSTWGWS had the same activity as SGGSSTYGYS but SGGSSTAGAS had no activity. PMID:16091882

  14. Environmental conditions impinge on dragline silk protein composition.

    PubMed

    Guehrs, K-H; Schlott, B; Grosse, F; Weisshart, K

    2008-09-01

    The silk formed in the major ampullate (MA) gland of the orb weaving spider Nephila clavipes is composed of two silk fibroins, which are called major ampullate spidroins 1 (MaSp1) and 2 (MaSp2). Analysis of proteolytic peptides and reactivity to spidroin type specific antibodies indicated that MaSp2 constituted only a minor part in the spinning dope as well as in the spun filaments. Upon starvation, a change in the silk's characteristic features was observed that was concomitant of a decrease in the contribution of MaSp2. The silk became less elastic and stiffer, which will better tailor its usability for the safety line, albeit at the expense of its employment as the web frame threads. In addition, since MaSp2 production requires greater ATP consumption, such a shift in the protein ratio cuts down on the energy costs to produce the silk. From this change in protein composition the spider might therefore benefit twice, by synthesizing 'cheaper' silk that into the bargain has properties that potentially can better support foraging in times of food shortage. PMID:18828841

  15. Stem cell-based tissue engineering with silk biomaterials.

    PubMed

    Wang, Yongzhong; Kim, Hyeon-Joo; Vunjak-Novakovic, Gordana; Kaplan, David L

    2006-12-01

    Silks are naturally occurring polymers that have been used clinically as sutures for centuries. When naturally extruded from insects or worms, silk is composed of a filament core protein, termed fibroin, and a glue-like coating consisting of sericin proteins. In recent years, silk fibroin has been increasingly studied for new biomedical applications due to the biocompatibility, slow degradability and remarkable mechanical properties of the material. In addition, the ability to now control molecular structure and morphology through versatile processability and surface modification options have expanded the utility for this protein in a range of biomaterial and tissue-engineering applications. Silk fibroin in various formats (films, fibers, nets, meshes, membranes, yarns, and sponges) has been shown to support stem cell adhesion, proliferation, and differentiation in vitro and promote tissue repair in vivo. In particular, stem cell-based tissue engineering using 3D silk fibroin scaffolds has expanded the use of silk-based biomaterials as promising scaffolds for engineering a range of skeletal tissues like bone, ligament, and cartilage, as well as connective tissues like skin. To date fibroin from Bombyx mori silkworm has been the dominant source for silk-based biomaterials studied. However, silk fibroins from spiders and those formed via genetic engineering or the modification of native silk fibroin sequence chemistries are beginning to provide new options to further expand the utility of silk fibroin-based materials for medical applications. PMID:16890988

  16. Conformation transition kinetics of Bombyx mori silk protein.

    PubMed

    Chen, Xin; Shao, Zhengzhong; Knight, David P; Vollrath, Fritz

    2007-07-01

    Time-resolved FTIR analysis was used to monitor the conformation transition induced by treating regenerated Bombyx mori silk fibroin films and solutions with different concentrations of ethanol. The resulting curves showing the kinetics of the transition for both films and fibroin solutions were influenced by the ethanol concentration. In addition, for silk fibroin solutions the protein concentration also had an effect on the kinetics. At low ethanol concentrations (for example, less than 40% v/v in the case of film), films and fibroin solutions showed a phase in which beta-sheets slowly formed at a rate dependent on the ethanol concentration. Reducing the concentration of the fibroin in solutions also slowed the formation of beta-sheets. These observations suggest that this phase represents a nucleation step. Such a nucleation phase was not seen in the conformation transition at ethanol concentrations > 40% in films or > 50% in silk fibroin solutions. Our results indicate that the ethanol-induced conformation transition of silk fibroin in films and solutions is a three-phase process. The first phase is the initiation of beta-sheet structure (nucleation), the second is a fast phase of beta-sheet growth while the third phase represents a slow perfection of previously formed beta-sheet structure. The nucleation step can be very fast or relatively slow, depending on factors that influence protein chain mobility and intermolecular hydrogen bond formation. The findings give support to the previous evidence that natural silk spinning in silkworms is nucleation-dependent, and that silkworms (like spiders) use concentrated silk protein solutions, and careful control of the pH value and metallic ion content of the processing environment to speed up the nucleation step to produce a rapid conformation transition to convert the water soluble spinning dope to a tough solid silk fiber. PMID:17436322

  17. Acid extraction and purification of recombinant spider silk proteins.

    PubMed

    Mello, Charlene M; Soares, Jason W; Arcidiacono, Steven; Butler, Michelle M

    2004-01-01

    A procedure has been developed for the isolation of recombinant spider silk proteins based upon their unique stability and solubilization characteristics. Three recombinant silk proteins, (SpI)7, NcDS, and [(SpI)4/(SpII)1]4, were purified by extraction with organic acids followed by affinity or ion exchange chromatography resulting in 90-95% pure silk solutions. The protein yield of NcDS (15 mg/L culture) and (SpI)7 (35 mg/L) increased 4- and 5-fold, respectively, from previously reported values presumably due to a more complete solubilization of the expressed recombinant protein. [(SpI)4/(SpII)1]4, a hybrid protein based on the repeat sequences of spidroin I and spidroin II, had a yield of 12.4 mg/L. This method is an effective, reproducible technique that has broad applicability for a variety of silk proteins as well as other acid stable biopolymers. PMID:15360297

  18. Analysis of proteome dynamics inside the silk gland lumen of Bombyx mori

    PubMed Central

    Dong, Zhaoming; Zhao, Ping; Zhang, Yan; Song, Qianru; Zhang, Xiaolu; Guo, Pengchao; Wang, Dandan; Xia, Qingyou

    2016-01-01

    The silk gland is the only organ where silk proteins are synthesized and secreted in the silkworm, Bombyx mori. Silk proteins are stored in the lumen of the silk gland for around eight days during the fifth instar. Determining their dynamic changes is helpful for clarifying the secretion mechanism of silk proteins. Here, we identified the proteome in the silk gland lumen using liquid chromatography–tandem mass spectrometry, and demonstrated its changes during two key stages. From day 5 of the fifth instar to day 1 of wandering, the abundances of fibroins, sericins, seroins, and proteins of unknown functions increased significantly in different compartments of the silk gland lumen. As a result, these accumulated proteins constituted the major cocoon components. In contrast, the abundances of enzymes and extracellular matrix proteins decreased in the silk gland lumen, suggesting that they were not the structural constituents of silk. Twenty-five enzymes may be involved in the regulation of hormone metabolism for proper silk gland function. In addition, the metabolism of other non-proteinous components such as chitin and pigment were also discussed in this study. PMID:27102218

  19. Analysis of proteome dynamics inside the silk gland lumen of Bombyx mori.

    PubMed

    Dong, Zhaoming; Zhao, Ping; Zhang, Yan; Song, Qianru; Zhang, Xiaolu; Guo, Pengchao; Wang, Dandan; Xia, Qingyou

    2016-01-01

    The silk gland is the only organ where silk proteins are synthesized and secreted in the silkworm, Bombyx mori. Silk proteins are stored in the lumen of the silk gland for around eight days during the fifth instar. Determining their dynamic changes is helpful for clarifying the secretion mechanism of silk proteins. Here, we identified the proteome in the silk gland lumen using liquid chromatography-tandem mass spectrometry, and demonstrated its changes during two key stages. From day 5 of the fifth instar to day 1 of wandering, the abundances of fibroins, sericins, seroins, and proteins of unknown functions increased significantly in different compartments of the silk gland lumen. As a result, these accumulated proteins constituted the major cocoon components. In contrast, the abundances of enzymes and extracellular matrix proteins decreased in the silk gland lumen, suggesting that they were not the structural constituents of silk. Twenty-five enzymes may be involved in the regulation of hormone metabolism for proper silk gland function. In addition, the metabolism of other non-proteinous components such as chitin and pigment were also discussed in this study. PMID:27102218

  20. Characterization of silk gland ribosomes from a bivoltine caddisfly, Stenopsyche marmorata: translational suppression of a silk protein in cold conditions.

    PubMed

    Nomura, Takaomi; Ito, Miho; Kanamori, Mai; Shigeno, Yuta; Uchiumi, Toshio; Arai, Ryoichi; Tsukada, Masuhiro; Hirabayashi, Kimio; Ohkawa, Kousaku

    2016-01-01

    Larval Stenopsyche marmorata constructs food capture nets and fixed retreats underwater using self-produced proteinaceous silk fibers. In the Chikuma River (Nagano Prefecture, Japan) S. marmorata has a bivoltine life cycle; overwintering larvae grow slowly with reduced net spinning activity in winter. We recently reported constant transcript abundance of S. marmorata silk protein 1 (Smsp-1), a core S. marmorata silk fiber component, in all seasons, implying translational suppression in the silk gland during winter. Herein, we prepared and characterized silk gland ribosomes from seasonally collected S. marmorata larvae. Ribosomes from silk glands immediately frozen in liquid nitrogen (LN2) after dissection exhibited comparable translation elongation activity in spring, summer, and autumn. Conversely, silk glands obtained in winter did not contain active ribosomes and Smsp-1. Ribosomes from silk glands immersed in ice-cold physiological saline solution for approximately 4 h were translationally inactive, despite summer collection and Smsp-1 expression. The ribosomal inactivation occurs because of defects in the formation of 80S ribosomes, presumably due to splitting of 60S subunits containing 28S rRNA with central hidden break, in response to cold stress. These results suggest a novel-type ribosome-regulated translation control mechanism. PMID:26646291

  1. Role of pH and charge on silk protein assembly in insects and spiders

    NASA Astrophysics Data System (ADS)

    Foo, C. Wong Po; Bini, E.; Hensman, J.; Knight, D. P.; Lewis, R. V.; Kaplan, D. L.

    2006-02-01

    Silk fibers possess impressive mechanical properties, dependant, in part, on the crystalline β-sheets silk II conformation. The transition to silk II from soluble silk I-like conformation in silk glands, is thought to originate in the spinning ducts immediately before the silk is drawn down into a fiber. However the assembly process of these silk molecules into fibers, whether in silkworms or spiders, is not well understood. Extensional flow, protein concentration, pH and metal ion concentrations are thought to be most important in in vivo silk processing and in affecting structural conformations. We look at how parameters such as pH, [Ca2+], [K+], and [Cu2+], and water content, interact with the domain structure of silk proteins towards the successful storage and processing of these concentrated hydrophobic silk proteins. Our recent domain mapping studies of all known silk proteins, and 2D Raman spectroscopy, NMR, and DLS studies performed on sections of silkworm gland, suggest that low pH and gradual water removal promote intermolecular over intramolecular hydrogen bonding. This discussion helps to provide the necessary ground rules towards the design of silk protein analogues with specific hydrophobicity and charge profiles to optimize expression, solubility and assembly with implications in structural biology and material science.

  2. Characterization of the protein components of Nephila clavipes dragline silk.

    PubMed

    Sponner, Alexander; Schlott, Bernhard; Vollrath, Fritz; Unger, Eberhard; Grosse, Frank; Weisshart, Klaus

    2005-03-29

    Spider silk is predominantly composed of structural proteins called spider fibroins or spidroins. The major ampullate silk that forms the dragline and the cobweb's frame threads of Nephila clavipes is believed to be a composite of two spidroins, designated as Masp 1 and 2. Specific antibodies indeed revealed the presence of Masp 1 and 2 specific epitopes in the spinning dope and solubilized threads. In contrast, sequencing of specific peptides obtained from solubilized threads or gland urea extracts were exclusively homologous to segments of Masp 1, suggesting that this protein is more abundantly expressed in silk than Masp 2. The strength of immunoreactivities corroborated this finding. Polypeptides reactive against both Masp 1 and 2 specific antibodies were found to be expressed in the epithelia of the tail and different gland zones and accumulated in the gland secreted material. Both extracts of gland secretion and solubilized threads showed a ladder of polypeptides in the size range of 260-320 kDa in gel electrophoresis under reducing conditions, whereas gel filtration chromatography yielded molecular masses of the proteins of approximately 300-350 kDa. In the absence of a reducing agent, dimeric forms of the spidroins were observed with estimated molecular masses of 420-480 kDa according to gel electrophoresis and 550-650 kDa as determined by gel filtration chromatography. Depending on the preparation, some silk material readily underwent degradation, and polypeptides down to 20 kDa in size and less were detectable. PMID:15779899

  3. The advances and perspectives of recombinant protein production in the silk gland of silkworm Bombyx mori.

    PubMed

    Xu, Hanfu

    2014-10-01

    The silk gland of silkworm Bombyx mori, is one of the most important organs that has been fully studied and utilized so far. It contributes finest silk fibers to humankind. The silk gland has excellent ability of synthesizing silk proteins and is a kind tool to produce some useful recombinant proteins, which can be widely used in the biological, biotechnical and pharmaceutical application fields. It's a very active area to express recombinant proteins using the silk gland as a bioreactor, and great progress has been achieved recently. This review recapitulates the progress of producing recombinant proteins and silk-based biomaterials in the silk gland of silkworm in addition to the construction of expression systems. Current challenges and future trends in the production of valuable recombinant proteins using transgenic silkworms are also discussed. PMID:25113390

  4. A Materiomics Approach to Spider Silk: Protein Molecules to Webs

    NASA Astrophysics Data System (ADS)

    Tarakanova, Anna; Buehler, Markus J.

    2012-02-01

    The exceptional mechanical properties of hierarchical self-assembling silk biopolymers have been extensively studied experimentally and in computational investigations. A series of recent studies has been conducted to examine structure-function relationships across different length scales in silk, ranging from atomistic models of protein constituents to the spider web architecture. Silk is an exemplary natural material because its superior properties stem intrinsically from the synergistic cooperativity of hierarchically organized components, rather than from the superior properties of the building blocks themselves. It is composed of beta-sheet nanocrystals interspersed within less orderly amorphous domains, where the underlying molecular structure is dominated by weak hydrogen bonding. Protein chains are organized into fibrils, which pack together to form threads of a spider web. In this article we survey multiscale studies spanning length scales from angstroms to centimeters, from the amino acid sequence defining silk components to an atomistically derived spider web model, with the aim to bridge varying levels of hierarchy to elucidate the mechanisms by which structure at each composite level contributes to organization and material phenomena at subsequent levels. The work demonstrates that the web is a highly adapted system where both material and hierarchical structure across all length scales is critical for its functional properties.

  5. Differential Scanning Fluorimetry provides high throughput data on silk protein transitions.

    PubMed Central

    Vollrath, Fritz; Hawkins, Nick; Porter, David; Holland, Chris; Boulet-Audet, Maxime

    2014-01-01

    Here we present a set of measurements using Differential Scanning Fluorimetry (DSF) as an inexpensive, high throughput screening method to investigate the folding of silk protein molecules as they abandon their first native melt conformation, dehydrate and denature into their final solid filament conformation. Our first data and analyses comparing silks from spiders, mulberry and wild silkworms as well as reconstituted ‘silk' fibroin show that DSF can provide valuable insights into details of silk denaturation processes that might be active during spinning. We conclude that this technique and technology offers a powerful and novel tool to analyse silk protein transitions in detail by allowing many changes to the silk solutions to be tested rapidly with microliter scale sample sizes. Such transition mechanisms will lead to important generic insights into the folding patterns not only of silks but also of other fibrous protein (bio)polymers. PMID:25004800

  6. Differential Scanning Fluorimetry provides high throughput data on silk protein transitions.

    NASA Astrophysics Data System (ADS)

    Vollrath, Fritz; Hawkins, Nick; Porter, David; Holland, Chris; Boulet-Audet, Maxime

    2014-07-01

    Here we present a set of measurements using Differential Scanning Fluorimetry (DSF) as an inexpensive, high throughput screening method to investigate the folding of silk protein molecules as they abandon their first native melt conformation, dehydrate and denature into their final solid filament conformation. Our first data and analyses comparing silks from spiders, mulberry and wild silkworms as well as reconstituted `silk' fibroin show that DSF can provide valuable insights into details of silk denaturation processes that might be active during spinning. We conclude that this technique and technology offers a powerful and novel tool to analyse silk protein transitions in detail by allowing many changes to the silk solutions to be tested rapidly with microliter scale sample sizes. Such transition mechanisms will lead to important generic insights into the folding patterns not only of silks but also of other fibrous protein (bio)polymers.

  7. Impact of Protein-Metal Ion Interactions on the Crystallization of Silk Fibroin Protein

    NASA Astrophysics Data System (ADS)

    Hu, Xiao; Lu, Qiang; Kaplan, David; Cebe, Peggy

    2009-03-01

    Proteins can easily form bonds with a variety of metal ions, which provides many unique biological functions for the protein structures, and therefore controls the overall structural transformation of proteins. We use advanced thermal analysis methods such as temperature modulated differential scanning calorimetry and quasi-isothermal TMDSC, combined with Fourier transform infrared spectroscopy, and scanning electron microscopy, to investigate the protein-metallic ion interactions in Bombyx mori silk fibroin proteins. Silk samples were mixed with different metal ions (Ca^2+, K^+, Ma^2+, Na^+, Cu^2+, Mn^2+) with different mass ratios, and compared with the physical conditions in the silkworm gland. Results show that all metallic ions can directly affect the crystallization behavior and glass transition of silk fibroin. However, different ions tend to have different structural impact, including their role as plasticizer or anti-plasticizer. Detailed studies reveal important information allowing us better to understand the natural silk spinning and crystallization process.

  8. Sericin Accelerates the Production of Hyaluronan and Decreases the Incidence of Polyspermy Fertilization in Bovine Oocytes During In Vitro Maturation

    PubMed Central

    HOSOE, Misa; YOSHIDA, Nao; HASHIYADA, Yutaka; TERAMOTO, Hidetoshi; TAKAHASHI, Toru; NIIMURA, Sueo

    2014-01-01

    Fetal bovine serum (FBS) has been widely used as a supplement in the maturation medium of bovine oocytes in vitro. However, serum contains many undefined factors and is potentially infectious to humans and animals. As a serum replacement, we evaluated the feasibility of using the silk protein, sericin, derived from the cocoons of silkworm. To examine the rates of oocyte maturation and fertilization, cumulus-oocyte complexes were cultured in TCM-199 supplemented with 0.01%, 0.05%, 0.1% or 0.15% sericin or 5% FBS. The sizes of the perivitelline space that might relate to polyspermy, the expressions of Has2 and CD44 mRNA, the amount of hyaluronan (hyaluronic acid: HA) contained in the oocytes and the rates of blastocyst formation following insemination were then compared between the oocytes cultured with 0.05% sericin and 5% FBS, because the polyspermy rates in oocytes cultured with 0.05% sericin were significantly lower than in those cultured with 5% FBS. After in vitro maturation (IVM), the mean size of the perivitelline space was significantly greater in oocytes cultured with sericin than in those cultured with FBS, although the rates of nuclear maturation, fertilization and blastocyst formation of oocytes under both IVM conditions were not significantly different. The expression of HAS2 and CD44 mRNA and the amount of HA in the denuded oocytes cultured with 0.05% sericin were significantly greater than in those cultured with FBS. These results indicate the feasibility of sericin as an alternative protein supplement for IVM in bovine oocytes. PMID:24748396

  9. Fibroin silk proteins from the nonmulberry silkworm Philosamia ricini are biochemically and immunochemically distinct from those of the mulberry silkworm Bombyx mori.

    PubMed

    Ahmad, Raies; Kamra, Anita; Hasnain, Seyed Ehtesham

    2004-03-01

    Silk proteins were isolated from the cocoons of the nonmulberry silkworm, Philosamia ricini. Three polypeptides of 97, 66, and 45 kDa were identified. The 66-kDa molecule represented sericin, whereas the 97-kDa and the 45-kDa polypeptides linked together through a disulfide bond constituted the fibroin protein. Antibodies raised against the 97-kDa P. ricini fibroin heavy chain reacted specifically with this molecule and did not recognize fibroin heavy chain from another nonmulberry silkworm, Antheraea assama or from the mulberry silkworm, Bombyx mori, suggesting the presence of P. ricini species-specific determinants in this heavy chain. Antibodies generated against fibroin light chain of P. ricini also showed similar reactivity pattern. Immunoblot analysis with proteins isolated from the silk glands of P. ricini at different stages of larval development showed that the expression of fibroin heavy chain was developmentally and spatially regulated. The protein was most abundant in the 5th instar larva, and could be detected in the middle and the posterior but not the anterior silk glands. The amino acid composition of the 97-kDa fibroin protein showed abundance of glutamic acid and did not contain (Gly-Ala)(n) motifs, a characteristic feature of B. mori fibroin heavy chain. Our study reveals significant differences between the nonmulberry silkworm P. ricini and the mulberry silkworm B. mori in the biochemical composition and immunochemical characteristics of fibroin heavy chain. These differences might be responsible for the differences seen in the quality of silk produced by these two silkworms. PMID:15068584

  10. Physically Transient Resistive Switching Memory Based on Silk Protein.

    PubMed

    Wang, Hong; Zhu, Bowen; Ma, Xiaohua; Hao, Yue; Chen, Xiaodong

    2016-05-01

    Physically transient resistive switching devices based on silk protein are successfully demonstrated. The devices can be absolutely dissolved in deionized water or in phosphate-buffered saline in 2 h. At the same time, a reasonable resistance OFF/ON ratio of larger than 10(2) and a retention time of more than 10(4) s are achieved for nonvolatile memory applications. PMID:27028213

  11. Identification, recombinant production and structural characterization of four silk proteins from the Asiatic honeybee Apis cerana.

    PubMed

    Shi, Jiahai; Lua, Shixiong; Du, Ning; Liu, Xiangyang; Song, Jianxing

    2008-06-01

    Unlike silkworm and spider silks assembled from very large and repetitive fibrous proteins, the bee and ant silks were recently demonstrated to consist of four small and non-repetitive coiled-coil proteins. The design principle for this silk family remains largely unknown and so far no structural study is available on them in solution. The present study aimed to identify, express and characterize the Asiatic honeybee silk proteins using DLS, CD and NMR spectroscopy. Consequently, (1) four silk proteins are identified, with approximately 6, 10, 9 and 8% variations, respectively, from their European honeybee homologs. Strikingly, their recombinant forms can be produced in Escherichia coil with yields of 10-60 mg/l. (2) Despite containing approximately 65% coiled-coil sequences, four proteins have very low alpha-helix (9-27%) but unusually high random coil (45-56%) contents. Surprisingly, beta-sheet is also detected in four silk proteins (26-35%), implying the possible presence of beta-sheet in the bee and ant silks. (3) Four proteins lacking of the tight tertiary packing appear capable of interacting with each other weakly but this interaction triggers no significant formation of the tight tertiary packing. The study not only implies the promising potential to produce recombinant honeybee silk proteins for the development of various biomaterials; but also provides the first structural insight into the molecular mechanism underlying the formation of the coiled-coil silks. PMID:18394700

  12. Stability of Silk and Collagen Protein Materials in Space

    PubMed Central

    Hu, Xiao; Raja, Waseem K.; An, Bo; Tokareva, Olena; Cebe, Peggy; Kaplan, David L.

    2013-01-01

    Collagen and silk materials, in neat forms and as silica composites, were flown for 18 months on the International Space Station [Materials International Space Station Experiment (MISSE)-6] to assess the impact of space radiation on structure and function. As natural biomaterials, the impact of the space environment on films of these proteins was investigated to understand fundamental changes in structure and function related to the future utility in materials and medicine in space environments. About 15% of the film surfaces were etched by heavy ionizing particles such as atomic oxygen, the major component of the low-Earth orbit space environment. Unexpectedly, more than 80% of the silk and collagen materials were chemically crosslinked by space radiation. These findings are critical for designing next-generation biocompatible materials for contact with living systems in space environments, where the effects of heavy ionizing particles and other cosmic radiation need to be considered. PMID:24305951

  13. Stability of silk and collagen protein materials in space.

    PubMed

    Hu, Xiao; Raja, Waseem K; An, Bo; Tokareva, Olena; Cebe, Peggy; Kaplan, David L

    2013-01-01

    Collagen and silk materials, in neat forms and as silica composites, were flown for 18 months on the International Space Station [Materials International Space Station Experiment (MISSE)-6] to assess the impact of space radiation on structure and function. As natural biomaterials, the impact of the space environment on films of these proteins was investigated to understand fundamental changes in structure and function related to the future utility in materials and medicine in space environments. About 15% of the film surfaces were etched by heavy ionizing particles such as atomic oxygen, the major component of the low-Earth orbit space environment. Unexpectedly, more than 80% of the silk and collagen materials were chemically crosslinked by space radiation. These findings are critical for designing next-generation biocompatible materials for contact with living systems in space environments, where the effects of heavy ionizing particles and other cosmic radiation need to be considered. PMID:24305951

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

    PubMed

    Xu, Hanfu; Yuan, Lin; Wang, Feng; Wang, Yuancheng; Wang, Riyuan; Song, Chunnuan; Xia, Qingyou; Zhao, Ping

    2014-10-01

    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. PMID:25106848

  15. Sericin/Dextran Injectable Hydrogel as an Optically Trackable Drug Delivery System for Malignant Melanoma Treatment.

    PubMed

    Liu, Jia; Qi, Chao; Tao, Kaixiong; Zhang, Jinxiang; Zhang, Jian; Xu, Luming; Jiang, Xulin; Zhang, Yunti; Huang, Lei; Li, Qilin; Xie, Hongjian; Gao, Jinbo; Shuai, Xiaoming; Wang, Guobin; Wang, Zheng; Wang, Lin

    2016-03-01

    Severe side effects of cancer chemotherapy prompt developing better drug delivery systems. Injectable hydrogels are an effective site-target system. For most of injectable hydrogels, once delivered in vivo, some properties including drug release and degradation, which are critical to chemotherapeutic effects and safety, are challenging to monitor. Developing a drug delivery system for effective cancer therapy with in vivo real-time noninvasive trackability is highly desired. Although fluorescence dyes are used for imaging hydrogels, the cytotoxicity limits their applications. By using sericin, a natural photoluminescent protein from silk, we successfully synthesized a hydrazone cross-linked sericin/dextran injectable hydrogel. This hydrogel is biodegradable and biocompatible. It achieves efficient drug loading and controlled release of both macromolecular and small molecular drugs. Notably, sericin's photoluminescence from this hydrogel is directly and stably correlated with its degradation, enabling long-term in vivo imaging and real-time monitoring of the remaining drug. The hydrogel loaded with Doxorubicin significantly suppresses tumor growth. Together, the work demonstrates the efficacy of this drug delivery system, and the in vivo effectiveness of this sericin-based optical monitoring strategy, providing a potential approach for improving hydrogel design toward optimal efficiency and safety of chemotherapies, which may be widely applicable to other drug delivery systems. PMID:26900631

  16. Nanoscale control of silica particle formation via silk-silica fusion proteins for bone regeneration.

    PubMed

    Mieszawska, Aneta J; Nadkarni, Lauren D; Perry, Carole C; Kaplan, David L

    2010-10-26

    The biomimetic design of silk/silica fusion proteins was carried out, combining the self assembling domains of spider dragline silk (Nephila clavipes) and silaffin derived R5 peptide of Cylindrotheca fusiformis that is responsible for silica mineralization. Genetic engineering was used to generate the protein-based biomaterials incorporating the physical properties of both components. With genetic control over the nanodomain sizes and chemistry, as well as modification of synthetic conditions for silica formation, controlled mineralized silk films with different silica morphologies and distributions were successfully generated; generating 3D porous networks, clustered silica nanoparticles (SNPs), or single SNPs. Silk serves as the organic scaffolding to control the material stability and multiprocessing makes silk/silica biomaterials suitable for different tissue regenerative applications. The influence of these new silk-silica composite systems on osteogenesis was evaluated with human mesenchymal stem cells (hMSCs) subjected to osteogenic differentiation. hMSCs adhered, proliferated, and differentiated towards osteogenic lineages on the silk/silica films. The presence of the silica in the silk films influenced osteogenic gene expression, with the upregulation of alkaline phosphatase (ALP), bone sialoprotein (BSP), and collagen type 1 (Col 1) markers. Evidence for early bone formation as calcium deposits was observed on silk films with silica. These results indicate the potential utility of these new silk/silica systems towards bone regeneration. PMID:20976116

  17. Molecular Architecture and Evolution of a Modular Spider Silk Protein Gene

    NASA Astrophysics Data System (ADS)

    Hayashi, Cheryl Y.; Lewis, Randolph V.

    2000-02-01

    Spider flagelliform silk is one of the most elastic natural materials known. Extensive sequencing of spider silk genes has shown that the exons and introns of the flagelliform gene underwent intragenic concerted evolution. The intron sequences are more homogenized within a species than are the exons. This pattern can be explained by extreme mutation and recombination pressures on the internally repetitive exons. The iterated sequences within exons encode protein structures that are critical to the function of silks. Therefore, attributes that make silks exceptional biomaterials may also hinder the fixation of optimally adapted protein sequences.

  18. Structural Model for the Spider Silk Protein Spidroin-1.

    PubMed

    dos Santos-Pinto, José Roberto Aparecido; Arcuri, Helen Andrade; Priewalder, Helga; Salles, Heliana Clara; Palma, Mario Sergio; Lubec, Gert

    2015-09-01

    Most reports about the 3-D structure of spidroin-1 have been proposed for the protein in solid state or for individual domains of these proteins. A gel-based mass spectrometry strategy using collision-induced dissociation (CID) and electron-transfer dissociation (ETD) fragmentation methods was used to completely sequence spidroins-1A and -1B and to assign a series of post-translational modifications (PTMs) on to the spidroin sequences. A total of 15 and 16 phosphorylation sites were detected on spidroin-1A and -1B, respectively. In this work, we present the nearly complete amino acid sequence of spidroin-1A and -1B, including the nonrepetitive N- and C-terminal domains and a highly repetitive central core. We also described a fatty acid layer surrounding the protein fibers and PTMs in the sequences of spidroin-1A and -1B, including phosphorylation. Thus, molecular models for phosphorylated spidroins were proposed in the presence of a mixture fatty acids/water (1:1) and submitted to molecular dynamics simulation. The resulting models presented high content of coils, a higher percentage of α-helix, and an almost neglected content of 310-helix than the previous models. Knowledge of the complete structure of spidroins-1A and -1B would help to explain the mechanical features of silk fibers. The results of the current investigation provide a foundation for biophysical studies of the mechanoelastic properties of web-silk proteins. PMID:26211688

  19. Bombyx mori silk protein films microprocessing with a nanosecond ultraviolet laser and a femtosecond laser workstation: theory and experiments

    NASA Astrophysics Data System (ADS)

    Lazare, S.; Sionkowska, A.; Zaborowicz, M.; Planecka, A.; Lopez, J.; Dijoux, M.; Louména, C.; Hernandez, M.-C.

    2012-01-01

    Laser microprocessing of several biopolymers from renewable resources is studied. Three proteinic materials were either extracted from the extracellular matrix like Silk Fibroin/Sericin and collagen, or coming from a commercial source like gelatin. All can find future applications in biomedical experimentation, in particular for cell scaffolding. Films of ˜hundred of microns thick were made by aqueous solution drying and laser irradiation. Attention is paid to the properties making them processable with two laser sources: the ultraviolet and nanosecond (ns) KrF (248 nm) excimer and the infrared and femtosecond (fs) Yb:KGW laser. The UV radiation is absorbed in a one-photon resonant process to yield ablation and the surface foaming characteristics of a laser-induced pressure wave. To the contrary, resonant absorption of the IR photons of the fs laser is not possible and does not take place. However, the high field of the intense I>˜1012 W/cm2 femtosecond laser pulse ionizes the film by the multiphoton absorption followed by the electron impact mechanism, yielding a dense plasma capable to further absorb the incident radiation of the end of the pulse. The theoretical model of this absorption is described in detail, and used to discuss the presented experimental effects (cutting, ablation and foaming) of the fs laser. The ultraviolet laser was used to perform simultaneous multiple spots experiments in which energetic foaming yields melt ejection and filament spinning. Airborne nanosize filaments "horizontally suspended by both ends" (0.25 μm diameter and 10 μm length) of silk biopolymer were observed upon irradiation with large fluences.

  20. Spider silk-like proteins derived from transgenic Nicotiana tabacum.

    PubMed

    Peng, Congyue Annie; Russo, Julia; Gravgaard, Charlene; McCartney, Heather; Gaines, William; Marcotte, William R

    2016-08-01

    The high tensile strength and biocompatibility of spider dragline silk makes it a desirable material in many engineering and tissue regeneration applications. Here, we present the feasibility to produce recombinant proteins in transgenic tobacco Nicotiana tabacum with sequences representing spider silk protein building blocks . Recombinant mini-spidroins contain native N- and C-terminal domains of major ampullate spidroin 1 (rMaSp1) or rMaSp2 flanking an abbreviated number (8, 16 or 32) of consensus repeat domains. Two different expression plasmid vectors were tested and a downstream chitin binding domain and self-cleavable intein were included to facilitate protein purification. We confirmed gene insertion and RNA transcription by PCR and reverse-transcriptase PCR, respectively. Mini-spidroin production was detected by N-terminus specific antibodies. Purification of mini-spidroins was performed through chitin affinity chromatography and subsequent intein activation with reducing reagent. Mini-spidroins, when dialyzed and freeze-dried, formed viscous gelatin-like fluids. PMID:27026165

  1. Silk structure and degradation.

    PubMed

    Liu, Bin; Song, Yu-wei; Jin, Li; Wang, Zhi-jian; Pu, De-yong; Lin, Shao-qiang; Zhou, Chan; You, Hua-jian; Ma, Yan; Li, Jin-min; Yang, Li; Sung, K L Paul; Zhang, Yao-guang

    2015-07-01

    To investigate the structure of silk and its degradation properties, we have monitored the structure of silk using scanning electron microscopy and frozen sections. Raw silk and degummed raw silk were immersed in four types of degradation solutions for 156 d to observe their degradation properties. The subcutaneous implants in rats were removed after 7, 14, 56, 84, 129, and 145 d for frozen sectioning and subsequent staining with hematoxylin and eosin (H.E.), DAPI, Beta-actin and Collagen I immunofluorescence staining. The in vitro weight loss ratio of raw silk and degummed raw silk in water, PBS, DMEM and DMEM containing 10% FBS (F-DMEM) were, respectively, 14%/11%, 12.5%/12.9%, 11.1%/14.3%, 8.8%/11.6%. Silk began to degrade after 7 d subcutaneous implantation and after 145 d non-degraded silk was still observed. These findings suggest the immunogenicity of fibroin and sericin had no essential difference. In the process of in vitro degradation of silk, the role of the enzyme is not significant. The in vivo degradation of silk is related to phagocytotic activity and fibroblasts may be involved in this process to secrete collagen. This study also shows the developing process of cocoons and raw silk. PMID:25982316

  2. Tolerogenic responses of CD206+, CD83+, FOXP3+, and CTLA-4 to sericin/polyvinyl alcohol/glycerin scaffolds relevant to IL-33 and HSP60 activity.

    PubMed

    Ampawong, Sumate; Aramwit, Pornanong

    2016-09-01

    Silk sericin-releasing (sericin/polyvinyl alcohol (PVA)/glycerin) scaffolds have been designed for wound dressing applications using different fabrication techniques that influence scaffold antigenicity. The immunological tolerance of scaffolds depends on the balance of immunogenic and tolerogenic responses modulated by dendritic cells (DCs). An in vivo skin implantation model was used to compare the tolerogenic effect of sericin/PVA/glycerin scaffolds prepared by freeze-drying versus salt-leaching techniques, using an Allevyn® scaffold as a control. Immunohistochemical and histopathological studies were performed to evaluate tolerogenic DCs (CD206+), immunogenic DCs (CD83+), regulatory T-cells (FOXP3+ and CTLA-4), a proinflammatory cytokine (interleukin 33: IL-33), a stress marker (heat shock protein 60; HSP60), histopathological changes and related inflammatory cells. It was found that both sericin/PVA/glycerin scaffolds were tolerogenic and induced early activated Treg functions, while the Allevyn® scaffold was immunogenic. However, the tolerance of the freeze-dried sericin/PVA/glycerin scaffolds was not as consistent as the salt-leached sericin/PVA/glycerin scaffolds, indicated by the low level of CTLA-4 expression. This was probably due to molecular cross-linking and the morphological and mechanical properties of the freeze-drying technique, which would enhance the immune response. Severe inflammatory responses (including mast cell degranulation and foreign body giant cell accumulation) and histopathological changes (including fat infiltration and fibrosis formation) were mainly found with the Allevyn® scaffold, presumably from its architecture and chemical composition, especially polyurethane. The up-regulation of IL-33 and HSP60 with the Allevyn® scaffold was correlated with the inflammatory and pathological levels. Our findings suggested that salt-leached sericin/PVA/glycerin scaffolds were tolerogenic, induced a low inflammatory response and were

  3. Recombinant production and film properties of full-length hornet silk proteins.

    PubMed

    Kambe, Yusuke; Sutherland, Tara D; Kameda, Tsunenori

    2014-08-01

    Full-length versions of the four main components of silk cocoons of Vespa simillima hornets, Vssilk1-4, were produced as recombinant proteins in Escherichia coli. In shake flasks, the recombinant Vssilk proteins yielded 160-330mg recombinant proteinl(-1). Films generated from solutions of single Vssilk proteins had a secondary structure similar to that of films generated from native hornet silk. The films made from individual recombinant hornet silk proteins had similar or enhanced mechanical performance compared with films generated from native hornet silk, possibly reflecting the homogeneity of the recombinant proteins. The pH-dependent changes in zeta (ζ) potential of each Vssilk film were measured, and isoelectric points (pI) of Vssilk1-4 were determined as 8.9, 9.1, 5.0 and 4.2, respectively. The pI of native hornet silk, a combination of the four Vssilk proteins, was 4.7, a value similar to that of Bombyx mori silkworm silk. Films generated from Vssilk1 and 2 had net positive charge under physiological conditions and showed significantly higher cell adhesion activity. It is proposed that recombinant hornet silk is a valuable new material with potential for cell culture applications. PMID:24862540

  4. Dragline silk: a fiber assembled with low-molecular-weight cysteine-rich proteins.

    PubMed

    Pham, Thanh; Chuang, Tyler; Lin, Albert; Joo, Hyun; Tsai, Jerry; Crawford, Taylor; Zhao, Liang; Williams, Caroline; Hsia, Yang; Vierra, Craig

    2014-11-10

    Dragline silk has been proposed to contain two main protein constituents, MaSp1 and MaSp2. However, the mechanical properties of synthetic spider silks spun from recombinant MaSp1 and MaSp2 proteins have yet to approach natural fibers, implying the natural spinning dope is missing critical factors. Here we report the discovery of novel molecular constituents within the spinning dope that are extruded into dragline silk. Protein studies of the liquid spinning dope from the major ampullate gland, coupled with the analysis of dragline silk fibers using mass spectrometry, demonstrate the presence of a new family of low-molecular-weight cysteine-rich proteins (CRPs) that colocalize with the MA fibroins. Expression of the CRP family members is linked to dragline silk production, specifically MaSp1 and MaSp2 mRNA synthesis. Biochemical data support that CRP molecules are secreted into the spinning dope and assembled into macromolecular complexes via disulfide bond linkages. Sequence analysis supports that CRP molecules share similarities to members that belong to the cystine slipknot superfamily, suggesting that these factors may have evolved to increase fiber toughness by serving as molecular hubs that dissipate large amounts of energy under stress. Collectively, our findings provide molecular details about the components of dragline silk, providing new insight that will advance materials development of synthetic spider silk for industrial applications. PMID:25259849

  5. Use of immunoblot technique for detection of human IgE and IgG antibodies to individual silk proteins.

    PubMed

    Dewair, M; Baur, X; Ziegler, K

    1985-10-01

    Allergenic proteins were extracted from one silk batch that was imported to be used as filling material for bed mattresses and rugs. IgE and IgG antibodies to the extracted silk proteins were measured by RAST in sera of nine silk-sensitive persons as well as in sera of healthy control donors. Silk proteins were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into 12 polypeptides of molecular weights between 14 and 70 kilodaltons. By means of the immunoblot technique, IgE and IgG antibodies to the individual silk polypeptides could be detected. Sera of silk-sensitive persons contained high titers of IgE and low titers of IgG antibodies to the separated silk polypeptides. Sera of control donors contained low IgG antibody titers to a limited number of these polypeptides. PMID:4056241

  6. Genome editing of BmFib-H gene provides an empty Bombyx mori silk gland for a highly efficient bioreactor.

    PubMed

    Ma, Sanyuan; Shi, Run; Wang, Xiaogang; Liu, Yuanyuan; Chang, Jiasong; Gao, Jie; Lu, Wei; Zhang, Jianduo; Zhao, Ping; Xia, Qingyou

    2014-01-01

    Evolution has produced some remarkable creatures, of which silk gland is a fascinating organ that exists in a variety of insects and almost half of the 34,000 spider species. The impressive ability to secrete huge amount of pure silk protein, and to store proteins at an extremely high concentration (up to 25%) make the silk gland of Bombyx mori hold great promise to be a cost-effective platform for production of recombinant proteins. However, the extremely low production yields of the numerous reported expression systems greatly hindered the exploration and application of silk gland bioreactors. Using customized zinc finger nucleases (ZFN), we successfully performed genome editing of Bmfib-H gene, which encodes the largest and most abundant silk protein, in B. mori with efficiency higher than any previously reported. The resulted Bmfib-H knocked-out B. mori showed a smaller and empty silk gland, abnormally developed posterior silk gland cells, an extremely thin cocoon that contain only sericin proteins, and a slightly heavier pupae. We also showed that removal of endogenous Bmfib-H protein could significantly increase the expression level of exogenous protein. Furthermore, we demonstrated that the bioreactor is suitable for large scale production of protein-based materials. PMID:25359576

  7. Genome editing of BmFib-H gene provides an empty Bombyx mori silk gland for a highly efficient bioreactor

    PubMed Central

    Ma, Sanyuan; Shi, Run; Wang, Xiaogang; Liu, Yuanyuan; Chang, Jiasong; Gao, Jie; Lu, Wei; Zhang, Jianduo; Zhao, Ping; Xia, Qingyou

    2014-01-01

    Evolution has produced some remarkable creatures, of which silk gland is a fascinating organ that exists in a variety of insects and almost half of the 34,000 spider species. The impressive ability to secrete huge amount of pure silk protein, and to store proteins at an extremely high concentration (up to 25%) make the silk gland of Bombyx mori hold great promise to be a cost-effective platform for production of recombinant proteins. However, the extremely low production yields of the numerous reported expression systems greatly hindered the exploration and application of silk gland bioreactors. Using customized zinc finger nucleases (ZFN), we successfully performed genome editing of Bmfib-H gene, which encodes the largest and most abundant silk protein, in B. mori with efficiency higher than any previously reported. The resulted Bmfib-H knocked-out B. mori showed a smaller and empty silk gland, abnormally developed posterior silk gland cells, an extremely thin cocoon that contain only sericin proteins, and a slightly heavier pupae. We also showed that removal of endogenous Bmfib-H protein could significantly increase the expression level of exogenous protein. Furthermore, we demonstrated that the bioreactor is suitable for large scale production of protein-based materials. PMID:25359576

  8. A Neuroprotective Sericin Hydrogel As an Effective Neuronal Cell Carrier for the Repair of Ischemic Stroke.

    PubMed

    Wang, Zheng; Wang, Jian; Jin, Yang; Luo, Zhen; Yang, Wen; Xie, Hongjian; Huang, Kai; Wang, Lin

    2015-11-11

    Ischemic stroke causes extensive cellular loss that impairs brain functions, resulting in severe disabilities. No effective treatments are currently available for brain tissue regeneration. The need to develop effective therapeutic approaches for treating stroke is compelling. A tissue engineering approach employing a hydrogel carrying both cells and neurotrophic cytokines to damaged regions is an encouraging alternative for neuronal repair. However, this approach is often challenged by low in vivo cell survival rate, and low encapsulation efficiency and loss of cytokines. To address these limitations, we propose to develop a biomaterial that can form a matrix capable of improving in vivo survival of transplanted cells and reducing in vivo loss of cytokines. Here, we report that using sericin, a natural protein from silk, we have fabricated a genipin-cross-linked sericin hydrogel (GSH) with porous structure and mild swelling ratio. The GSH supports the effective attachment and growth of neurons in vitro. Strikingly, our data reveal that sericin protein is intrinsically neurotrophic and neuroprotective, promoting axon extension and branching as well as preventing primary neurons from hypoxia-induced cell death. Notably, these functions are inherited by the GSH's degradation products, which might spare a need of incorporating costly cytokines. We further demonstrate that this neurotrophic effect is dependent on the Lkb1-Nuak1 pathway, while the neuroprotective effect is realized through regulating the Bcl-2/Bax protein ratio. Importantly, when transplanted in vivo, the GSH gives a high cell survival rate and allows the cells to continuously proliferate. Together, this work unmasks the neurotrophic and neuroprotective functions for sericin and provides strong evidence justifying the GSH's suitability as a potential neuronal cell delivery vehicle for ischemic stroke repair. PMID:26478947

  9. Recombinant Minimalist Spider Wrapping Silk Proteins Capable of Native-Like Fiber Formation

    PubMed Central

    Xu, Lingling; Rainey, Jan K.; Meng, Qing; Liu, Xiang-Qin

    2012-01-01

    Spider silks are desirable biomaterials characterized by high tensile strength, elasticity, and biocompatibility. Spiders produce different types of silks for different uses, although dragline silks have been the predominant focus of previous studies. Spider wrapping silk, made of the aciniform protein (AcSp1), has high toughness because of its combination of high elasticity and tensile strength. AcSp1 in Argiope trifasciata contains a 200-aa sequence motif that is repeated at least 14 times. Here, we produced in E. coli recombinant proteins consisting of only one to four of the 200-aa AcSp1 repeats, designated W1 to W4. We observed that purified W2, W3 and W4 proteins could be induced to form silk-like fibers by shear forces in a physiological buffer. The fibers formed by W4 were ∼3.4 µm in diameter and up to 10 cm long. They showed an average tensile strength of 115 MPa, elasticity of 37%, and toughness of 34 J cm−3. The smaller W2 protein formed fewer fibers and required a higher protein concentration to form fibers, whereas the smallest W1 protein did not form silk-like fibers, indicating that a minimum of two of the 200-aa repeats was required for fiber formation. Microscopic examinations revealed structural features indicating an assembly of the proteins into spherical structures, fibrils, and silk-like fibers. CD and Raman spectral analysis of protein secondary structures suggested a transition from predominantly α-helical in solution to increasingly β-sheet in fibers. PMID:23209681

  10. The molecular structures of major ampullate silk proteins of the wasp spider, Argiope bruennichi: a second blueprint for synthesizing de novo silk.

    PubMed

    Zhang, Yang; Zhao, Ai-Chun; Sima, Yang-Hu; Lu, Cheng; Xiang, Zhong-Huai; Nakagaki, Masao

    2013-03-01

    The dragline silk of orb-weaving spiders possesses extremely high tensile strength and elasticity. To date, full-length sequences of only two genes encoding major ampullate silk protein (MaSp) in Latrodectus hesperus have been determined. In order to further understand this gene family, we utilized in this study a variety of strategies to isolate full-length MaSp1 and MaSp2 cDNAs in the wasp spider Argiope bruennichi. A. bruennichi MaSp1 and MaSp2 are primarily composed of remarkably homogeneous ensemble repeats containing several complex motifs, and both have highly conserved C-termini and N-termini. Two novel amino acid motifs, GGF and SGR, were found in MaSp1 and MaSp2, respectively. Amino acid composition analysis of silk, luminal contents and predicted sequences indicates that MaSp1 and MaSp2 are two major components of major ampullate glands and that the ratio of MaSp1 to MaSp2 is approximately 3:2 in dragline silk. Furthermore, both the MaSp1:MaSp2 ratio and the conserved termini are closely linked with the production of high quality synthetic fibers. Our results make an important contribution to our understanding of major ampullate silk protein structure and provide a second blueprint for creating new composite silk which mimics natural spider dragline silk. PMID:23262065

  11. Bioinspired silicification of silica-binding peptide-silk protein chimeras: comparison of chemically and genetically produced proteins.

    PubMed

    Canabady-Rochelle, Laetitia L S; Belton, David J; Deschaume, Olivier; Currie, Heather A; Kaplan, David L; Perry, Carole C

    2012-03-12

    Novel protein chimeras constituted of "silk" and a silica-binding peptide (KSLSRHDHIHHH) were synthesized by genetic or chemical approaches and their influence on silica-silk based chimera composite formation evaluated. Genetic chimeras were constructed from 6 or 15 repeats of the 32 amino acid consensus sequence of Nephila clavipes spider silk ([SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQG](n)) to which one silica binding peptide was fused at the N terminus. For the chemical chimera, 28 equiv of the silica binding peptide were chemically coupled to natural Bombyx mori silk after modification of tyrosine groups by diazonium coupling and EDC/NHS activation of all acid groups. After silica formation under mild, biomaterial-compatible conditions, the effect of peptide addition on the properties of the silk and chimeric silk-silica composite materials was explored. The composite biomaterial properties could be related to the extent of silica condensation and to the higher number of silica binding sites in the chemical chimera as compared with the genetically derived variants. In all cases, the structure of the protein/chimera in solution dictated the type of composite structure that formed with the silica deposition process having little effect on the secondary structural composition of the silk-based materials. Similarly to our study of genetic silk based chimeras containing the R5 peptide (SSKKSGSYSGSKGSKRRIL), the role of the chimeras (genetic and chemical) used in the present study resided more in aggregation and scaffolding than in the catalysis of condensation. The variables of peptide identity, silk construct (number of consensus repeats or silk source), and approach to synthesis (genetic or chemical) can be used to "tune" the properties of the composite materials formed and is a general approach that can be used to prepare a range of materials for biomedical and sensor-based applications. PMID:22229696

  12. Sensitization to silk allergen among workers of silk filatures in India: a comparative study

    PubMed Central

    Gowda, Giriyanna; Vijayeendra, Anagha Manakari; Sarkar, Nivedita; Nagaraj, Chitra; Masthi, Nugehally Raju Ramesh

    2016-01-01

    Background Sericulture plays an eminent role in development of rural economy in India. Silk filature is a unit where silk is unwound from the cocoons and the strands are collected into skeins. During the process workers are exposed to the high molecular weight proteins like Sericin and Fibroin which are potent allergens leading to sensitization over a period of time and subsequently occupational related health disorders. Objective To identify and compare the magnitude of silk allergen sensitization in workers of silk filatures. Methods A community based comparative descriptive study was conducted for a period of 1 year at Ramanagara in south India. One hundred twenty subjects working in the silk filatures formed the study group. For comparison, 2 types of controls were selected viz.120 subjects who were not working in the silk filatures but resided in the same geographical area (control A) and 360 subjects who were not working in silk filatures as well not residing in the same geographical area (control B). Skin prick test was used to identify the silk allergen sensitization. Results Mean age was 34.14 ± 2.84 years in the study group. Mean age was 40.59 ± 14.40 years and 38.54 ± 12.20 years in control A and control B, respectively. There were 35 males (29.16%) and 85 females (70.84%) in the study group. There were 58 (48.34%) males and 62 (51.66%) females and 152 (42.2%) males and 208 females (57.8%) in control A and control B, respectively. Sensitization to silk allergen was 35.83% in the study group and 20.83% in the control group A and 11.11% in control group B. There was difference in the allergen sensitivity between the study group and control groups and it was statistically significant (chi-square = 38.08; p < 0.001). Conclusion There is high burden of silk allergen sensitization among silk filature workers. PMID:27141481

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

    PubMed

    Blamires, Sean J; Kasumovic, Michael M; Tso, I-Min; Martens, Penny J; Hook, James M; Rawal, Aditya

    2016-01-01

    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. PMID:27517909

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

    PubMed Central

    Blamires, Sean J.; Kasumovic, Michael M.; Tso, I-Min; Martens, Penny J.; Hook, James M.; Rawal, Aditya

    2016-01-01

    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. PMID:27517909

  15. Silk Fibroin Aqueous-Based Adhesives Inspired by Mussel Adhesive Proteins.

    PubMed

    Burke, Kelly A; Roberts, Dane C; Kaplan, David L

    2016-01-11

    Silk fibroin from the domesticated silkworm Bombyx mori is a naturally occurring biopolymer with charged hydrophilic terminal regions that end-cap a hydrophobic core consisting of repeating sequences of glycine, alanine, and serine residues. Taking inspiration from mussels that produce proteins rich in L-3,4-dihydroxyphenylalanine (DOPA) to adhere to a variety of organic and inorganic surfaces, the silk fibroin was functionalized with catechol groups. Silk fibroin was selected for its high molecular weight, tunable mechanical and degradation properties, aqueous processability, and wide availability. The synthesis of catechol-functionalized silk fibroin polymers containing varying amounts of hydrophilic polyethylene glycol (PEG, 5000 g/mol) side chains was carried out to balance silk hydrophobicity with PEG hydrophilicity. The efficiency of the catechol functionalization reaction did not vary with PEG conjugation over the range studied, although tuning the amount of PEG conjugated was essential for aqueous solubility. Adhesive bonding and cell compatibility of the resulting materials were investigated, where it was found that incorporating as little as 6 wt % PEG prior to catechol functionalization resulted in complete aqueous solubility of the catechol conjugates and increased adhesive strength compared with silk lacking catechol functionalization. Furthermore, PEG-silk fibroin conjugates maintained their ability to form β-sheet secondary structures, which can be exploited to reduce swelling. Human mesenchymal stem cells (hMSCs) proliferated on the silks, regardless of PEG and catechol conjugation. These materials represent a protein-based approach to catechol-based adhesives, which we envision may find applicability as biodegradable adhesives and sealants. PMID:26674175

  16. An improvement in the attaching capability of cryopreserved human hepatocytes by a proteinaceous high molecule, sericin, in the serum-free solution.

    PubMed

    Miyamoto, Yoshitaka; Teramoto, Naozumi; Hayashi, Shuji; Enosawa, Shin

    2010-01-01

    The methodology of cryopreservation of human hepatocytes remains unsatisfactory. Even when the viability of thawed cells is tolerable, the cells often lose the attaching capability to a culture dish, resulting in the cells' inability to survive. Previously, we described the effectiveness of maltose on the attachment of hepatocytes. This article demonstrates that a silk-derived high molecular protein, sericin, improves the cell-attaching capability in the serum-free freezing medium. When human hepatocytes [initial viability: 60.9 ± 3.1% (mean ± SD, n = 3)] were frozen with serum-free Dulbecco's modified Eagle medium (DMEM) containing 10% dimethyl sulfoxide (DMSO), the viability was 29.4 ± 3.2% and the cell-attaching capability 20.4 ± 4.1%. On the other hand, DMEM containing 10% DMSO and 1% sericin increased the values to 45.0 ± 0.8% and 26.2 ± 3.2%. Moreover, the addition of 0.1 mol/L maltose to the sericin-containing medium improved to 42.2 ± 3.2% and 51.1 ± 1.0%, as we demonstrated in a previous report. The present results indicated that sericin combined with maltose is a novel additive in the serum-free freezing medium for human hepatocytes. PMID:20525438

  17. Control of silicification by genetically engineered fusion proteins: silk-silica binding peptides.

    PubMed

    Zhou, Shun; Huang, Wenwen; Belton, David J; Simmons, Leo O; Perry, Carole C; Wang, Xiaoqin; Kaplan, David L

    2015-03-01

    In the present study, an artificial spider silk gene, 6mer, derived from the consensus sequence of Nephila clavipes dragline silk gene, was fused with different silica-binding peptides (SiBPs), A1, A3 and R5, to study the impact of the fusion protein sequence chemistry on silica formation and the ability to generate a silk-silica composite in two different bioinspired silicification systems: solution-solution and solution-solid. Condensed silica nanoscale particles (600-800 nm) were formed in the presence of the recombinant silk and chimeras, which were smaller than those formed by 15mer-SiBP chimeras, revealing that the molecular weight of the silk domain correlated to the sizes of the condensed silica particles in the solution system. In addition, the chimeras (6mer-A1/A3/R5) produced smaller condensed silica particles than the control (6mer), revealing that the silica particle size formed in the solution system is controlled by the size of protein assemblies in solution. In the solution-solid interface system, silicification reactions were performed on the surface of films fabricated from the recombinant silk proteins and chimeras and then treated to induce β-sheet formation. A higher density of condensed silica formed on the films containing the lowest β-sheet content while the films with the highest β-sheet content precipitated the lowest density of silica, revealing an inverse correlation between the β-sheet secondary structure and the silica content formed on the films. Intriguingly, the 6mer-A3 showed the highest rate of silica condensation but the lowest density of silica deposition on the films, compared with 6mer-A1 and -R5, revealing antagonistic crosstalk between the silk and the SiBP domains in terms of protein assembly. These findings offer a path forward in the tailoring of biopolymer-silica composites for biomaterial related needs. PMID:25462851

  18. The Widespread Prevalence and Functional Significance of Silk-Like Structural Proteins in Metazoan Biological Materials.

    PubMed

    McDougall, Carmel; Woodcroft, Ben J; Degnan, Bernard M

    2016-01-01

    In nature, numerous mechanisms have evolved by which organisms fabricate biological structures with an impressive array of physical characteristics. Some examples of metazoan biological materials include the highly elastic byssal threads by which bivalves attach themselves to rocks, biomineralized structures that form the skeletons of various animals, and spider silks that are renowned for their exceptional strength and elasticity. The remarkable properties of silks, which are perhaps the best studied biological materials, are the result of the highly repetitive, modular, and biased amino acid composition of the proteins that compose them. Interestingly, similar levels of modularity/repetitiveness and similar bias in amino acid compositions have been reported in proteins that are components of structural materials in other organisms, however the exact nature and extent of this similarity, and its functional and evolutionary relevance, is unknown. Here, we investigate this similarity and use sequence features common to silks and other known structural proteins to develop a bioinformatics-based method to identify similar proteins from large-scale transcriptome and whole-genome datasets. We show that a large number of proteins identified using this method have roles in biological material formation throughout the animal kingdom. Despite the similarity in sequence characteristics, most of the silk-like structural proteins (SLSPs) identified in this study appear to have evolved independently and are restricted to a particular animal lineage. Although the exact function of many of these SLSPs is unknown, the apparent independent evolution of proteins with similar sequence characteristics in divergent lineages suggests that these features are important for the assembly of biological materials. The identification of these characteristics enable the generation of testable hypotheses regarding the mechanisms by which these proteins assemble and direct the construction of

  19. The Widespread Prevalence and Functional Significance of Silk-Like Structural Proteins in Metazoan Biological Materials

    PubMed Central

    McDougall, Carmel; Woodcroft, Ben J.

    2016-01-01

    In nature, numerous mechanisms have evolved by which organisms fabricate biological structures with an impressive array of physical characteristics. Some examples of metazoan biological materials include the highly elastic byssal threads by which bivalves attach themselves to rocks, biomineralized structures that form the skeletons of various animals, and spider silks that are renowned for their exceptional strength and elasticity. The remarkable properties of silks, which are perhaps the best studied biological materials, are the result of the highly repetitive, modular, and biased amino acid composition of the proteins that compose them. Interestingly, similar levels of modularity/repetitiveness and similar bias in amino acid compositions have been reported in proteins that are components of structural materials in other organisms, however the exact nature and extent of this similarity, and its functional and evolutionary relevance, is unknown. Here, we investigate this similarity and use sequence features common to silks and other known structural proteins to develop a bioinformatics-based method to identify similar proteins from large-scale transcriptome and whole-genome datasets. We show that a large number of proteins identified using this method have roles in biological material formation throughout the animal kingdom. Despite the similarity in sequence characteristics, most of the silk-like structural proteins (SLSPs) identified in this study appear to have evolved independently and are restricted to a particular animal lineage. Although the exact function of many of these SLSPs is unknown, the apparent independent evolution of proteins with similar sequence characteristics in divergent lineages suggests that these features are important for the assembly of biological materials. The identification of these characteristics enable the generation of testable hypotheses regarding the mechanisms by which these proteins assemble and direct the construction of

  20. Functional Material Features of Bombyx mori Silk Light vs. Heavy Chain Proteins

    PubMed Central

    Zafar, Muhammad S.; Belton, David J.; Hanby, Benjamin; Kaplan, David L.; Perry, Carole C.

    2016-01-01

    Bombyx mori (BM) silk fibroin is composed of two different subunits; heavy chain and light chain fibroin linked by a covalent disulphide bond. Current methods of separating the two silk fractions is complicated and produces inadequate quantities of the isolated components for the study of the individual light and heavy chain silks with respect to new materials. We report a simple method of separating silk fractions using formic acid. The formic acid treatment partially releases predominately the light chain fragment (soluble fraction) and then the soluble fraction and insoluble fractions can be converted into new materials. The regenerated original (total) silk fibroin and the separated fractions (soluble vs. insoluble) had different molecular weights and showed distinctive pH stabilities against aggregation/precipitation based on particle charging. All silk fractions could be electrospun to give fibre mats with viscosity of the regenerated fractions being the controlling factor for successful electrospinning. The silk fractions could be mixed to give blends with different proportions of the two fractions to modify the diameter and uniformity of the electrospun fibres formed. The soluble fraction containing the light chain was able to modify the viscosity by thinning the insoluble fraction containing heavy chain fragments, perhaps analogous to its role in natural fibre formation where the light chain provides increased mobility and the heavy chain producing shear thickening effects. The simplicity of this new separation method should enable access to these different silk protein fractions and accelerate the identification of methods, modifications and potential applications of these materials in biomedical and industrial applications. PMID:25565556

  1. Influence of silk-silica fusion protein design on silica condensation in vitro and cellular calcification

    PubMed Central

    Plowright, Robyn; Dinjaski, Nina; Zhou, Shun; Belton, David J.; Kaplan, David L.; Perry, Carole C.

    2016-01-01

    Biomaterial design via genetic engineering can be utilized for the rational functionalization of proteins to promote biomaterial integration and tissue regeneration. Spider silk has been extensively studied for its biocompatibility, biodegradability and extraordinary material properties. As a protein-based biomaterial, recombinant DNA derived derivatives of spider silks have been modified with biomineralization domains which lead to silica deposition and potentially accelerated bone regeneration. However, the influence of the location of the R5 (SSKKSGSYSGSKGSKRRIL) silicifying domain fused with the spider silk protein sequence on the biosilicification process remains to be determined. Here we designed two silk-R5 fusion proteins that differed in the location of the R5 peptide, C- vs. N-terminus, where the spider silk domain consisted of a 15mer repeat of a 33 amino acid consensus sequence of the major ampullate dragline Spidroin 1 from Nephila clavipes (SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQGT). The chemical, physical and silica deposition properties of these recombinant proteins were assessed and compared to a silk 15mer control without the R5 present. The location of the R5 peptide did not have a significant effect on wettability and surface energies, while the C-terminal location of the R5 promoted more controlled silica precipitation, suggesting differences in protein folding and possibly different access to charged amino acids that drive the silicification process. Further, cell compatibility in vitro, as well as the ability to promote human bone marrow derived mesenchymal stem cell (hMSC) differentiation were demonstrated for both variants of the fusion proteins. PMID:26989487

  2. Rheology of reconstituted silk fibroin protein gels: the epitome of extreme mechanics.

    PubMed

    Tabatabai, A Pasha; Kaplan, David L; Blair, Daniel L

    2015-01-28

    In nature, silk fibroin proteins assemble into hierarchical structures with dramatic mechanical properties. With the hope of creating new classes of on demand silk-based biomaterials, Bombyx mori silk is reconstituted back into stable aqueous solutions that can be reassembled into functionalized materials; one strategy for reassembly is electrogelation. Electrogels (e-gels) are particularly versatile and can be produced using electrolysis with small DC electric fields. We characterize the linear and nonlinear rheological behavior of e-gels to provide fundamental insights into these distinct protein-based materials. We observe that e-gels form robust biopolymer networks that exhibit distinctive strain hardening and are recoverable from strains as large as γ=27, i.e. 2700%. We propose a simple microscopic model that is consistent with local restructuring of single proteins within the e-gel network. PMID:25489795

  3. Biomimetic spinning of silk fibers and in situ cell encapsulation.

    PubMed

    Cheng, Jie; Park, DoYeun; Jun, Yesl; Lee, JaeSeo; Hyun, Jinho; Lee, Sang-Hoon

    2016-07-01

    In situ embedding of sensitive materials (e.g., cells and proteins) in silk fibers without damage presents a significant challenge due to the lack of mild and efficient methods. Here, we report the development of a microfluidic chip-based method for preparation of meter-long silk fibroin (SF) hydrogel fibers by mimicking the silkworm-spinning process. For the spinning of SF fibers, alginate was used as a sericin-like material to induce SF phase separation and entrap liquid SFs, making it possible to shape the outline of SF-based fibers under mild physicochemical conditions. L929 fibroblasts were encapsulated in the fibric hydrogel and displayed excellent viability. Cell-laden SF fibric hydrogels prepared using our method offer a new type of SF-based biomedical device with potential utility in biomedicine. PMID:27296229

  4. Charge-Tunable Silk-Tropoelastin Protein Alloys That Control Neuron Cell Responses

    PubMed Central

    Hu, Xiao; Tang-Schomer, Min D.; Huang, Wenwen; Xia, Xiao-Xia; Weiss, Anthony S.

    2014-01-01

    Tunable protein composites are important for constructing extracellular matrix mimics of human tissues with control of biochemical, structural, and mechanical properties. Molecular interaction mechanisms between silk fibroin protein and recombinant human tropoelastin, based on charge, are utilized to generate a new group of multifunctional protein alloys (mixtures of silk and tropoelastin) with different net charges. These new biomaterials are then utilized as a biomaterial platform to control neuron cell response. With a +38 net charge in water, tropoelastin molecules provide extraordinary elasticity and selective interactions with cell surface integrins. In contrast, negatively charged silk fibroin protein (net charge −36) provides remarkable toughness and stiffness with morphologic stability in material formats via autoclaving-induced beta-sheet crystal physical crosslinks. The combination of these properties in alloy format extends the versatility of both structural proteins, providing a new biomaterial platform. The alloys with weak positive charges (silk/tropoelastin mass ratio 75/25, net charge around +16) significantly improved the formation of neuronal networks and maintained cell viability of rat cortical neurons after 10 days in vitro. The data point to these protein alloys as an alternative to commonly used poly-L-lysine (PLL) coatings or other charged synthetic polymers, particularly with regard to the versatility of material formats (e.g., gels, sponges, films, fibers). The results also provide a practical example of physically designed protein materials with control of net charge to direct biological outcomes, in this case for neuronal tissue engineering. PMID:25093018

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

    PubMed

    Jansson, Ronnie; Lau, Cheuk H; Ishida, Takuya; Ramström, Margareta; Sandgren, Mats; Hedhammar, My

    2016-05-01

    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. PMID:26814048

  6. Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber.

    PubMed

    Xia, Xiao-Xia; Qian, Zhi-Gang; Ki, Chang Seok; Park, Young Hwan; Kaplan, David L; Lee, Sang Yup

    2010-08-10

    Spider dragline silk is a remarkably strong fiber that makes it attractive for numerous applications. Much has thus been done to make similar fibers by biomimic spinning of recombinant dragline silk proteins. However, success is limited in part due to the inability to successfully express native-sized recombinant silk proteins (250-320 kDa). Here we show that a 284.9 kDa recombinant protein of the spider Nephila clavipes is produced and spun into a fiber displaying mechanical properties comparable to those of the native silk. The native-sized protein, predominantly rich in glycine (44.9%), was favorably expressed in metabolically engineered Escherichia coli within which the glycyl-tRNA pool was elevated. We also found that the recombinant proteins of lower molecular weight versions yielded inferior fiber properties. The results provide insight into evolution of silk protein size related to mechanical performance, and also clarify why spinning lower molecular weight proteins does not recapitulate the properties of native fibers. Furthermore, the silk expression, purification, and spinning platform established here should be useful for sustainable production of natural quality dragline silk, potentially enabling broader applications. PMID:20660779

  7. Design and Fabrication of Multifunctional Sericin Nanoparticles for Tumor Targeting and pH-Responsive Subcellular Delivery of Cancer Chemotherapy Drugs.

    PubMed

    Huang, Lei; Tao, Kaixiong; Liu, Jia; Qi, Chao; Xu, Luming; Chang, Panpan; Gao, Jinbo; Shuai, Xiaoming; Wang, Guobin; Wang, Zheng; Wang, Lin

    2016-03-01

    The severe cytotoxicity of cancer chemotherapy drugs limits their clinical applications. Various protein-based nanoparticles with good biocompatibility have been developed for chemotherapy drug delivery in hope of reducing drugs' side effects. Sericin, a natural protein from silk, has no immunogenicity and possesses diverse bioactivities that have prompted sericin's application studies. However, the potential of sericin as a multifunctional nanoscale vehicle for cancer therapy have not been fully explored. Here we report the successful fabrication and characterization of folate-conjugated sericin nanoparticles with cancer-targeting capability for pH-responsive release of doxorubicin (these nanoparticles are termed "FA-SND"). DOX is covalently linked to sericin through pH-sensitive hydrazone bonds that render a pH-triggered release property. The hydrophobicity of DOX and the hydrophilicity of sericin promote the self-assembly of sericin-DOX (SND) nanoconjugates. Folate (FA) is then covalently grafted to SND nanoconjugates as a binding unit for actively targeting cancer cells that overexpress folate receptors. Our characterization study shows that FA-SND nanoparticles exhibit negative surface charges that would reduce nonspecific clearance by circulation. These nanoparticles possess good cytotoxicity and hemocompatibiliy. Acidic environment (pH 5.0) triggers effective DOX release from FA-SND, 5-fold higher than does a neutral condition (pH 7.4). Further, FA-SND nanoparticles specifically target folate-receptor-rich KB cells, and endocytosed into lysosomes, an acidic organelle. The acidic microenvironment of lysosomes promotes a rapid release of DOX to nuclei, producing cancer specific chemo-cytotoxicity. Thus, FA-mediated cancer targeting and lysosomal-acidity promoting DOX release, two sequentially-occurring cellular events triggered by the designed components of FA-SND, form the basis for FA-SND to achieve its localized and intracellular chemo

  8. Gene Delivery Mediated by Recombinant Silk Proteins Containing Cationic and Cell Binding Motifs

    PubMed Central

    Numata, Keiji; Hamasaki, Juliana; Subramanian, Balajikarthick; Kaplan, David L

    2010-01-01

    Silk proteins are biodegradable and biocompatible, and can also be tailored to contain additional features via genetic engineering, suggesting utility for gene delivery. In the present study, novel silk-based block copolymers were bioengineered both with poly(L-lysine) domains to interact with plasmid DNA (pDNA) and RGD, to enhance cell-binding and transfection efficiency. Ionic complexes of these silk-polylysine-RGD block copolymers with pDNA were prepared, characterized and utilized for gene delivery to HeLa cells and human embryonic kidney (HEK) cells. The material systems were characterized by agarose gel electrophoresis, zeta-potentialmeter, atomic force microscopy, and dynamic light scattering. Sizes and charges of the pDNA complexes were regulated by the polymer/nucleotide molar ratio. Samples with 30-lysine residues and 11 RGD sequences, prepared at the ratio of number of amines/phosphates from pDNA (N/P) of 2, had an average solution diameter of 186 nm and showed the highest transfection efficiency. The intracellular distribution of complexes of Cy5-labeled pDNA was investigated by confocal laser scanning microscopy. The Cy5-labeled pDNA was distributed near the cell membrane and around the nuclei, indicating that the pDNA was transferred near the nucleus. The results demonstrated the potential of bioengineered silk proteins with additional functional features as a new family of highly tailored gene delivery systems. PMID:20457191

  9. Structural characterization of the major ampullate silk spidroin-2 protein produced by the spider Nephila clavipes.

    PubMed

    Santos-Pinto, José Roberto Aparecido Dos; Arcuri, Helen Andrade; Lubec, Gert; Palma, Mario Sergio

    2016-10-01

    Major ampullate spidroin-2 (MaSp2) is one of the most important spider silk protein, but up to now no information is available regarding the post-translational modifications (PTMs) of this protein. A gel-based mass spectrometry strategy using collision-induced dissociation (CID) and electron-transfer dissociation (ETD) fragmentation methods was used to sequence Nephila clavipes MaSp2 (including the N- and C-terminal non-repetitive domains, and the great part of the central core), and to assign a series of post-translational modifications (PTMs) on to the MaSp2 sequence. Two forms of this protein were identified, with different levels of phosphorylation along their sequences. These findings provide a basis for understanding mechanoelastic properties and can support the future design of recombinant spider silk proteins for biotechnological applications. PMID:27208434

  10. Reinforcing Silk Scaffolds with Silk Particles

    PubMed Central

    Rajkhowa, Rangam; Gil, Eun Seok; Kluge, Jonathan; Numata, Keiji; Wang, Lijing; Kaplan, David L.

    2014-01-01

    Silk fibroin is a useful protein polymer for biomaterials and tissue engineering. In this work, porogen leached scaffolds prepared from aqueous and HFIP silk solutions were reinforced through the addition of silk particles. This led to about 40 times increase in the specific compressive modulus and the yield strength of HFIP-based scaffolds. This increase in mechanical properties resulted from the high interfacial cohesion between the silk matrix and the reinforcing silk particles, due to partial solubility of the silk particles in HFIP. The porosity of scaffolds was reduced from ≈90% (control) to ≈75% for the HFIP systems containing 200% particle reinforcement, while maintaining pore interconnectivity. The presence of the particles slowed the enzymatic degradation of silk scaffolds. PMID:20166230

  11. [Construction, fermentation and purification of high polymer spider dragline silk protein containing RGD peptide].

    PubMed

    Ruan, Chao-Ran; Huang, Jing-Xing; Wei, Mei-Hong; Li, Min

    2007-09-01

    Spider silk is a natural protein fibroin with excellent character as it is light and tenacious. It has a wild potential applications in the biomedical field due to its good biocompatibility and degradation. Arginine-glycine-aspartic acid (RGD) is a highly conserved amino acid sequence of many adhesion protein. Biological materials binding with RGD peptide in the surface can promote cells adhesion, migration and proliferation. Our lab had constructed the 16 muhimers with the introduced RGD peptide codons which involve cell adhesion for the first time. It was found that the mechanical capability of the 16 mulimer protein was very limited because of the big gap in molecular weight with nature spider proteins when it was used to made biomaterial scaffold.In this paper,based on the 16 multimers of the highly, repetitive sequence of spider dragline silk and with RGD peptide condons which has been constructed by our lab forestall, it was used to construct the 32 and 64 multimers sequence of spider dragline silk by the strategy of "head to tail". The 32 and 64 multimers were ligated into prokaryotic expression vector pET-30a, and then the B121 (DE3) pLysS. The fragments were in agreement with the desired through digestion, agarose gel electrophoresis respectively. By registration into the GenBank data-base, the serial numbers of DQ469929 and DQ837297 were gained respectively. The expression of recombinant protein was introduced by the addition of IPTG. SDS-PAGE analysis shows that the molecular weight of products expressed here are 102 kD and 196.6kD in agreement with the desired respectively. It was the first time for the high polymer spider dragline silk protein expressed in prokaryotic biology. Furthermore, a larger quantity of synthetical proteins with high density fermentation were searched after, and a suit of high efficient purification methods for 32 multimers protein were established. PMID:18051865

  12. Structure and post-translational modifications of the web silk protein spidroin-1 from Nephila spiders.

    PubMed

    dos Santos-Pinto, José Roberto Aparecido; Lamprecht, Günther; Chen, Wei-Qiang; Heo, Seok; Hardy, John George; Priewalder, Helga; Scheibel, Thomas Rainer; Palma, Mario Sergio; Lubec, Gert

    2014-06-13

    Spidroin-1 is one of the major ampullate silk proteins produced by spiders for use in the construction of the frame and radii of orb webs, and as a dragline to escape from predators. Only partial sequences of spidroin-1 produced by Nephila clavipes have been reported up to now, and there is no information on post-translational modifications (PTMs). A gel-based mass spectrometry strategy with ETD and CID fragmentation methods were used to sequence and determine the presence/location of any PTMs on the spidroin-1. Sequence coverage of 98.06%, 95.05%, and 98.37% were obtained for N. clavipes, Nephila edulis and for Nephila madagascariensis, respectively. Phosphorylation was the major PTM observed with 8 phosphorylation sites considered reliable on spidroin-1 produced by N. clavipes, 4 in N. madagascariensis and 2 for N. edulis. Dityrosine and 3,4-dihydroxyphenylalanine (formed by oxidation of the spidroin-1) were observed, although the mechanism by which they are formed (i.e. exposure to UV radiation or to peroxidases in the major ampullate silk gland) is uncertain. Herein we present structural information on the spidroin-1 produced by three different Nephila species; these findings may be valuable for understanding the physicochemical properties of the silk proteins and moreover, future designs of recombinantly produced spider silk proteins. Biotechnological significance The present investigation shows for the first time spidroin structure and post-translational modifications observed on the major ampullate silk spidroin-1. The many site specific phosphorylations (localized within the structural motifs) along with the probably photoinduction of hydroxylations may be relevant for scientists in material science, biology, biochemistry and environmental scientists. Up to now all the mechanical properties of the spidroin have been characterized without any consideration about the existence of PTMs in the sequence of spidroins. Thus, these findings for major ampullate silk

  13. Carbonization of a stable β-sheet-rich silk protein into a pseudographitic pyroprotein

    PubMed Central

    Cho, Se Youn; Yun, Young Soo; Lee, Sungho; Jang, Dawon; Park, Kyu-Young; Kim, Jae Kyung; Kim, Byung Hoon; Kang, Kisuk; Kaplan, David L.; Jin, Hyoung-Joon

    2015-01-01

    Silk proteins are of great interest to the scientific community owing to their unique mechanical properties and interesting biological functionality. In addition, the silk proteins are not burned out following heating, rather they are transformed into a carbonaceous solid, pyroprotein; several studies have identified potential carbon precursors for state-of-the-art technologies. However, no mechanism for the carbonization of proteins has yet been reported. Here we examine the structural and chemical changes of silk proteins systematically at temperatures above the onset of thermal degradation. We find that the β-sheet structure is transformed into an sp2-hybridized carbon hexagonal structure by simple heating to 350 °C. The pseudographitic crystalline layers grew to form highly ordered graphitic structures following further heating to 2,800 °C. Our results provide a mechanism for the thermal transition of the protein and demonstrate a potential strategy for designing pyroproteins using a clean system with a catalyst-free aqueous wet process for in vivo applications. PMID:25990218

  14. Carbonization of a stable β-sheet-rich silk protein into a pseudographitic pyroprotein.

    PubMed

    Cho, Se Youn; Yun, Young Soo; Lee, Sungho; Jang, Dawon; Park, Kyu-Young; Kim, Jae Kyung; Kim, Byung Hoon; Kang, Kisuk; Kaplan, David L; Jin, Hyoung-Joon

    2015-01-01

    Silk proteins are of great interest to the scientific community owing to their unique mechanical properties and interesting biological functionality. In addition, the silk proteins are not burned out following heating, rather they are transformed into a carbonaceous solid, pyroprotein; several studies have identified potential carbon precursors for state-of-the-art technologies. However, no mechanism for the carbonization of proteins has yet been reported. Here we examine the structural and chemical changes of silk proteins systematically at temperatures above the onset of thermal degradation. We find that the β-sheet structure is transformed into an sp(2)-hybridized carbon hexagonal structure by simple heating to 350 °C. The pseudographitic crystalline layers grew to form highly ordered graphitic structures following further heating to 2,800 °C. Our results provide a mechanism for the thermal transition of the protein and demonstrate a potential strategy for designing pyroproteins using a clean system with a catalyst-free aqueous wet process for in vivo applications. PMID:25990218

  15. Carbonization of a stable β-sheet-rich silk protein into a pseudographitic pyroprotein

    NASA Astrophysics Data System (ADS)

    Cho, Se Youn; Yun, Young Soo; Lee, Sungho; Jang, Dawon; Park, Kyu-Young; Kim, Jae Kyung; Kim, Byung Hoon; Kang, Kisuk; Kaplan, David L.; Jin, Hyoung-Joon

    2015-05-01

    Silk proteins are of great interest to the scientific community owing to their unique mechanical properties and interesting biological functionality. In addition, the silk proteins are not burned out following heating, rather they are transformed into a carbonaceous solid, pyroprotein; several studies have identified potential carbon precursors for state-of-the-art technologies. However, no mechanism for the carbonization of proteins has yet been reported. Here we examine the structural and chemical changes of silk proteins systematically at temperatures above the onset of thermal degradation. We find that the β-sheet structure is transformed into an sp2-hybridized carbon hexagonal structure by simple heating to 350 °C. The pseudographitic crystalline layers grew to form highly ordered graphitic structures following further heating to 2,800 °C. Our results provide a mechanism for the thermal transition of the protein and demonstrate a potential strategy for designing pyroproteins using a clean system with a catalyst-free aqueous wet process for in vivo applications.

  16. Air Filter Devices Including Nonwoven Meshes of Electrospun Recombinant Spider Silk Proteins

    PubMed Central

    Lang, Gregor; Jokisch, Stephan; Scheibel, Thomas

    2013-01-01

    Based on the natural sequence of Araneus diadematus Fibroin 4 (ADF4), the recombinant spider silk protein eADF4(C16) has been engineered. This highly repetitive protein has a molecular weight of 48kDa and is soluble in different solvents (hexafluoroisopropanol (HFIP), formic acid and aqueous buffers). eADF4(C16) provides a high potential for various technical applications when processed into morphologies such as films, capsules, particles, hydrogels, coatings, fibers and nonwoven meshes. Due to their chemical stability and controlled morphology, the latter can be used to improve filter materials. In this protocol, we present a procedure to enhance the efficiency of different air filter devices, by deposition of nonwoven meshes of electrospun recombinant spider silk proteins. Electrospinning of eADF4(C16) dissolved in HFIP results in smooth fibers. Variation of the protein concentration (5-25% w/v) results in different fiber diameters (80-1,100 nm) and thus pore sizes of the nonwoven mesh. Post-treatment of eADF4(C16) electrospun from HFIP is necessary since the protein displays a predominantly α-helical secondary structure in freshly spun fibers, and therefore the fibers are water soluble. Subsequent treatment with ethanol vapor induces formation of water resistant, stable β-sheet structures, preserving the morphology of the silk fibers and meshes. Secondary structure analysis was performed using Fourier transform infrared spectroscopy (FTIR) and subsequent Fourier self-deconvolution (FSD). The primary goal was to improve the filter efficiency of existing filter substrates by adding silk nonwoven layers on top. To evaluate the influence of electrospinning duration and thus nonwoven layer thickness on the filter efficiency, we performed air permeability tests in combination with particle deposition measurements. The experiments were carried out according to standard protocols. PMID:23685883

  17. Sericin for resistance switching device with multilevel nonvolatile memory.

    PubMed

    Wang, Hong; Meng, Fanben; Cai, Yurong; Zheng, Liyan; Li, Yuangang; Liu, Yuanjun; Jiang, Yueyue; Wang, Xiaotian; Chen, Xiaodong

    2013-10-11

    Resistance switching characteristics of natural sericin protein film is demonstrated for nonvolatile memory application for the first time. Excellent memory characteristics with a resistance OFF/ON ratio larger than 10(6) have been obtained and a multilevel memory based on sericin has been achieved. The environmentally friendly high performance biomaterial based memory devices may hold a place in the future of electronic device development. PMID:23893500

  18. Self-assembly of spider silk proteins is controlled by a pH-sensitive relay.

    PubMed

    Askarieh, Glareh; Hedhammar, My; Nordling, Kerstin; Saenz, Alejandra; Casals, Cristina; Rising, Anna; Johansson, Jan; Knight, Stefan D

    2010-05-13

    Nature's high-performance polymer, spider silk, consists of specific proteins, spidroins, with repetitive segments flanked by conserved non-repetitive domains. Spidroins are stored as a highly concentrated fluid dope. On silk formation, intermolecular interactions between repeat regions are established that provide strength and elasticity. How spiders manage to avoid premature spidroin aggregation before self-assembly is not yet established. A pH drop to 6.3 along the spider's spinning apparatus, altered salt composition and shear forces are believed to trigger the conversion to solid silk, but no molecular details are known. Miniature spidroins consisting of a few repetitive spidroin segments capped by the carboxy-terminal domain form metre-long silk-like fibres irrespective of pH. We discovered that incorporation of the amino-terminal domain of major ampullate spidroin 1 from the dragline of the nursery web spider Euprosthenops australis (NT) into mini-spidroins enables immediate, charge-dependent self-assembly at pH values around 6.3, but delays aggregation above pH 7. The X-ray structure of NT, determined to 1.7 A resolution, shows a homodimer of dipolar, antiparallel five-helix bundle subunits that lack homologues. The overall dimeric structure and observed charge distribution of NT is expected to be conserved through spider evolution and in all types of spidroins. Our results indicate a relay-like mechanism through which the N-terminal domain regulates spidroin assembly by inhibiting precocious aggregation during storage, and accelerating and directing self-assembly as the pH is lowered along the spider's silk extrusion duct. PMID:20463740

  19. Facts and myths of antibacterial properties of silk.

    PubMed

    Kaur, Jasjeet; Rajkhowa, Rangam; Afrin, Tarannum; Tsuzuki, Takuya; Wang, Xungai

    2014-03-01

    Silk cocoons provide protection to silkworm from biotic and abiotic hazards during the immobile pupal phase of the lifecycle of silkworms. Protection is particularly important for the wild silk cocoons reared in an open and harsh environment. To understand whether some of the cocoon components resist growth of microorganisms, in vitro studies were performed using gram negative bacteria Escherichia coli (E. coli) to investigate antibacterial properties of silk fiber, silk gum, and calcium oxalate crystals embedded inside some cocoons. The results show that the previously reported antibacterial properties of silk cocoons are actually due to residues of chemicals used to isolate/purify cocoon elements, and properly isolated silk fiber, gum, and embedded crystals free from such residues do not have inherent resistance to E. coli. This study removes the uncertainty created by previous studies over the presence of antibacterial properties of silk cocoons, particularly the silk gum and sericin. PMID:23784754

  20. Changes in 30K protein synthesis during delayed degeneration of the silk gland by a caspase-dependent pathway in a Bombyx (silkworm) mutant.

    PubMed

    Wang, Huan; Wang, Yulong; Wu, Chengjia; Tao, Hui; Chen, Xuedong; Yin, Weimin; Sima, Yanghu; Wang, Yujun; Xu, Shiqing

    2016-08-01

    The silk gland in silkworm (Bombyx mori) is a highly specialized organ that specifically synthesizes silk proteins. A function shift to the synthesis of large quantities of 30K proteins occurs in the degenerating silk gland cells during larval-pupal metamorphosis. The posterior silk gland developmental mutant model of silkworm was used in this study and changes in the programmed cell death (PCD) regulatory signals and 30K protein synthesis during silk gland degeneration were investigated. The results showed that PCD induced by 20-hydroxyecdysone was initiated early during larval-pupal metamorphosis in the mutant, but PCD proceeded slowly, resulting in the degeneration process of the silk gland being extended and took almost twice the time compared with the wild type. Caspase-dependent pathway signals regulated by Dronc in the silk gland cells of the mutant were significantly reduced, while the PCD initiation signal regulated by the Atg family was not delayed or reduced, and PCD-related epigenetic modification such as lysine methylation, acetylation, and succinylation, and tyrosine phosphorylation changed significantly. During the degeneration process in the mutant, 30K proteins were efficiently synthesized in the silk gland cells in stage PP1 even when no caspase protein was detected. Degeneration of the silk gland is a PCD process in which autophagy and apoptosis may participate. The degeneration process was regulated by a caspase-dependent pathway, while the synthesis of 30K proteins along with silk gland degeneration may not be entirely dependent on caspase signals. PMID:27107566

  1. Spider dragline silk proteins in transgenic tobacco leaves: accumulation and field production.

    PubMed

    Menassa, Rima; Zhu, Hong; Karatzas, Costas N; Lazaris, Anthoula; Richman, Alex; Brandle, Jim

    2004-09-01

    Spider dragline silk is a unique biomaterial and represents nature's strongest known fibre. As it is almost as strong as many commercial synthetic fibres, it is suitable for use in many industrial and medical applications. The prerequisite for such a widespread use is the cost-effective production in sufficient quantities for commercial fibre manufacturing. Agricultural biotechnology and the production of recombinant dragline silk proteins in transgenic plants offer the potential for low-cost, large-scale production. The purpose of this work was to examine the feasibility of producing the two protein components of dragline silk (MaSp1 and MaSp2) from Nephila clavipes in transgenic tobacco. Two different promoters, the enhanced CaMV 35S promoter (Kay et al., 1987) and a new tobacco cryptic constitutive promoter, tCUP (Foster et al., 1999) were used, in conjunction with a plant secretory signal (PR1b), a translational enhancer (alfalfa mosaic virus, AMV) and an endoplasmic reticulum (ER) retention signal (KDEL), to express the MaSp1 and MaSp2 genes in the leaves of transgenic plants. Both genes expressed successfully and recombinant protein accumulated in transgenic plants grown in both greenhouse and field trials. PMID:17168889

  2. Harnessing disorder: onychophorans use highly unstructured proteins, not silks, for prey capture.

    PubMed

    Haritos, Victoria S; Niranjane, Ajay; Weisman, Sarah; Trueman, Holly E; Sriskantha, Alagacone; Sutherland, Tara D

    2010-11-01

    Onychophora are ancient, carnivorous soft-bodied invertebrates which capture their prey in slime that originates from dedicated glands located on either side of the head. While the biochemical composition of the slime is known, its unusual nature and the mechanism of ensnaring thread formation have remained elusive. We have examined gene expression in the slime gland from an Australian onychophoran, Euperipatoides rowelli, and matched expressed sequence tags to separated proteins from the slime. The analysis revealed three categories of protein present: unique high-molecular-weight proline-rich proteins, and smaller concentrations of lectins and small peptides, the latter two likely to act as protease inhibitors and antimicrobial agents. The predominant proline-rich proteins (200 kDa+) are composed of tandem repeated motifs and distinguished by an unusually high proline and charged residue content. Unlike the highly structured proteins such as silks used for prey capture by spiders and insects, these proteins lack ordered secondary structure over their entire length. We propose that on expulsion of slime from the gland onto prey, evaporative water loss triggers a glass transition change in the protein solution, resulting in adhesive and enmeshing thread formation, assisted by cross-linking of complementary charged and hydrophobic regions of the protein. Euperipatoides rowelli has developed an entirely new method of capturing prey by harnessing disordered proteins rather than structured, silk-like proteins. PMID:20519222

  3. Synthesis and study of sericin-g-PLA

    NASA Astrophysics Data System (ADS)

    Saetae, S.; Magaraphan, R.

    2015-05-01

    In this paper we present an experiment for bulk synthesis of the sericin-g-PLA by using Sn(Oct)2 as catalyst and study the effect of Thai silk cocoon species (Dok Bua, Luang Pirote, Nang Noi and Nang Lai) on properties of the sericin-g-PLA. We investigated the chemical structure of the grafted copolymers by using FTIR and GPC. Moreover, the grafting percentage was determined by soxhlet extaction. The IR spectra of extracted sample showed peaks at 1188 and 1215 cm-1 that assigned to the symmetric C-O-C stretching modes of the ester group. The methyl rocking stretching and C-CH3 vibration of polylactide appeared at 1130 and 1045 cm-1, respectively. The peak positioned 3440 cm-1 belonged to the hydroxyl group and the amino group of sericin which became less after polymerized with lactide. These evidences suggested that the lactide was reacted with sericin. Also, the molecular weight of the grafted copolymers were in range from 5.2 to 6.1 kg/mole. And Nang Lai-g-PLA showed the highest grafting percentage of the grafted copolymers.

  4. Differential polymerization of the two main protein components of dragline silk during fibre spinning.

    PubMed

    Sponner, Alexander; Unger, Eberhard; Grosse, Frank; Weisshart, Klaus

    2005-10-01

    Spider silks are some of the strongest materials found in nature. Achieving the high tensile strength and elasticity of the dragline of orb-weaving spiders, such as Nephila clavipes, is a principal goal in biomimetics research. The dragline has a composite nature and is predominantly made up by two proteins, the major ampullate spidroins 1 and 2 (refs 3, 6, 7), which can be considered natural block copolymers. On the basis of their molecular structures both spidroins are thought to contribute, in different ways, to the mechanical properties of dragline silk. The spinning process itself is also considered important for determining the observed features by shaping the hierarchical structure of the fibre. Here we study the heterogeneous distribution of proteins along the radial axis of the fibre. This heterogeneity is generated during the conversion of the liquid spinning dope into solid fibre. Whereas spidroin 1 is distributed almost uniformly within the fibre core, spidroin 2 is missing in the periphery and is tightly packed in certain core areas. Our findings suggest that the role of spidroin 2 in the spinning process could be to facilitate the formation of fibrils and contribute directly to the elasticity of the silk. PMID:16184170

  5. Structure and pH-induced alterations of recombinant and natural spider silk proteins in solution.

    PubMed

    Leclerc, Jérémie; Lefèvre, Thierry; Pottier, Fabien; Morency, Louis-Philippe; Lapointe-Verreault, Camille; Gagné, Stéphane M; Auger, Michèle

    2012-06-01

    The spinning process of spiders can modulate the mechanical properties of their silk fibers. It is therefore of primary importance to understand what are the key elements of the spider spinning process to develop efficient industrial spinning processes. We have exhaustively investigated the native conformation of major ampullate silk (MaS) proteins by comparing the content of the major ampullate gland of Nephila clavipes, solubilized MaS (SolMaS) fibers and the recombinant proteins rMaSpI and rMaSpII using (1) H solution NMR spectroscopy. The results indicate that the protein secondary structure is basically identical for the recombinant protein rMaSpI, SolMaS proteins, and the proteins in the dope, and corresponds to a disordered protein rich in 3(1) -helices. The data also show that glycine proton chemical shifts of rMaSpI and SolMaS are affected by pH, but that this change is not due to a modification of the secondary structure. Using a combination of NMR and dynamic light scattering, we have found that the spectral alteration of glycine is concomitant to a modification of the hydrodynamical diameter of recombinant and solubilized MaS. This led us to suggest new potential roles for the pH acidification in the spinning process of MaS proteins. PMID:21898365

  6. Reducing blood glucose levels in TIDM mice with an orally administered extract of sericin from hIGF-I-transgenic silkworm cocoons.

    PubMed

    Song, Zuowei; Zhang, Mengyao; Xue, Renyu; Cao, Guangli; Gong, Chengliang

    2014-05-01

    In previous studies, we reported that the blood glucose levels of mice with type I diabetes mellitus (TIDM) was reduced with orally administered silk gland powder from silkworms transgenic for human insulin-like growth factor-I (hIGF-I). However, potential safety hazards could not be eliminated because the transgenic silk gland powder contained heterologous DNA, including the green fluorescent protein (gfp) and neomycin resistance (neo) genes. These shortcomings might be overcome if the recombinant hIGF-I were secreted into the sericin layer of the cocoon. In this study, silkworm eggs were transfected with a novel piggyBac transposon vector, pigA3GFP-serHS-hIGF-I-neo, containing the neo, gfp, and hIGF-I genes controlled by the sericin-1 (ser-1) promoter with the signal peptide DNA sequence of the fibrin heavy chain (Fib-H) and a helper plasmid containing the piggyBac transposase sequence under the control of the Bombyx mori actin 3 (A3) promoter, using sperm-mediated gene transfer to generate the transformed silkworms. The hIGF-I content estimated by enzyme-linked immunosorbent assay was approximately 162.7 ng/g. To estimate the biological activity of the expressed hIGF-I, streptozotocin-induced TIDM mice were orally administered sericin from the transgenic silkworm. The blood glucose levels of the mice were significantly reduced, suggesting that the extract from the transgenic hIGF-I silkworm cocoons can be used as an orally administered drug. PMID:24632065

  7. A Complete Recombinant Silk-Elastinlike Protein-Based Tissue Scaffold

    PubMed Central

    Qiu, Weiguo; Huang, Yiding; Teng, Weibing; Cohn, Celine M.; Cappello, Joseph; Wu, Xiaoyi

    2010-01-01

    Due to their improved biocompatibility and specificity over synthetic materials, protein-based biomaterials, either derived from natural sources or genetically engineered, have been widely fabricated into nanofibrous scaffolds for tissue engineering applications. However, their inferior mechanical properties often require the reinforcement of protein-based tissue scaffolds using synthetic polymers. In this study, we report the electrospinning of a completely recombinant silk-elastinlike protein-based tissue scaffold with excellent mechanical properties and biocompatibility. In particular, SELP-47K containing tandemly repeated polypeptide sequences derived from native silk and elastin was electrospun into nanofibrous scaffolds, and stabilized via chemical vapor treatment and mechanical preconditioning. When fully hydrated in 1x PBS at 37 °C, mechanically preconditioned SELP-47K scaffolds displayed elastic moduli of 3.4 to 13.2 MPa, ultimate tensile strengths of 5.7 to 13.5 MPa, deformabilities of 100 to 130% strain, and resilience of 80.6 to 86.9%, closely matching or exceeding those of protein-synthetic blend polymeric scaffolds. Additionally, SELP-47K nanofibrous scaffolds promoted cell attachment and growth demonstrating their in vitro biocompatibility. PMID:21058633

  8. Potential mode of protection of silkworm pupae from environmental stress by harboring the bacterial biofilm on the surfaces of silk cocoons.

    PubMed

    Halder, Pranab K; Naskar, Deboki; Kumar, Akash; Yao, Juming; Kundu, Subhas C; Ghosh, Anindya S

    2015-02-01

    The silkworm forms cocoon to protect its pupa that survives for months inside the cocoon without being affected by various environmental stresses. To understand the possible mode of pupal survival within the cocoon encasement, we investigate the cause that protects the cocoon. During the end of the spinning process, we have isolated different bacterial species from the cocoon surface. These are identified using molecular techniques and checked for their abilities to form biofilm in vitro. The bacteria are able to form biofilm either individually or in consortia. Of which, Bacillus and Erwinia species are prominent biofilm formers. Interestingly, these bacteria have the ability to form biofilm on the cocoon mimetic surface of the silk protein Sericin Hope that contains only sericin. The origin and the behavior of the bacteria lead us to hypothesize the possible role of biofilm layer on the cocoon surface, which provides protection from adverse environmental conditions. PMID:25292249

  9. Autoclaving as a chemical-free process to stabilize recombinant silk-elastinlike protein polymer nanofibers

    NASA Astrophysics Data System (ADS)

    Qiu, Weiguo; Cappello, Joseph; Wu, Xiaoyi

    2011-06-01

    We report here that autoclaving is a chemical-free, physical crosslinking strategy capable of stabilizing electrospun recombinant silk-elastinlike protein (SELP) polymer nanofibers. Fourier transform infrared spectroscopy showed that the autoclaving of SELP nanofibers induced a conformational conversion of β-turns and unordered structures to ordered β-sheets. Tensile stress-strain analysis of the autoclaved SELP nanofibrous scaffolds in phosphate buffered saline at 37 °C revealed a Young's modulus of 1.02 ± 0.28 MPa, an ultimate tensile strength of 0.34 ± 0.04 MPa, and a strain at failure of 29% ± 3%.

  10. Electroresponsive Aqueous Silk Protein As “Smart” Mechanical Damping Fluid

    PubMed Central

    2015-01-01

    Here we demonstrate the effectiveness of an electroresponsive aqueous silk protein polymer as a smart mechanical damping fluid. The aqueous polymer solution is liquid under ambient conditions, but is reversibly converted into a gel once subjected to an electric current, thereby increasing or decreasing in viscosity. This nontoxic, biodegradable, reversible, edible fluid also bonds to device surfaces and is demonstrated to reduce friction and provide striking wear protection. The friction and mechanical damping coefficients are shown to modulate with electric field exposure time and/or intensity. Damping coefficient can be modulated electrically, and then preserved without continued power for longer time scales than conventional “smart” fluid dampers. PMID:24750065

  11. Silk as a Biomaterial

    PubMed Central

    Vepari, Charu

    2009-01-01

    Silks are fibrous proteins with remarkable mechanical properties produced in fiber form by silkworms and spiders. Silk fibers in the form of sutures have been used for centuries. Recently regenerated silk solutions have been used to form a variety of biomaterials, such as gels, sponges and films, for medical applications. Silks can be chemically modified through amino acid side chains to alter surface properties or to immobilize cellular growth factors. Molecular engineering of silk sequences has been used to modify silks with specific features, such as cell recognition or mineralization. The degradability of silk biomaterials can be related to the mode of processing and the corresponding content of beta sheet crystallinity. Several primary cells and cell lines have been successfully grown on different silk biomaterials to demonstrate a range of biological outcomes. Silk biomaterials are biocompatible when studied in vitro and in vivo. Silk scaffolds have been successfully used in wound healing and in tissue engineering of bone, cartilage, tendon and ligament tissues. PMID:19543442

  12. A new estimation of the total flavonoids in silkworm cocoon sericin layer through aglycone determination by hydrolysis-assisted extraction and HPLC-DAD analysis

    PubMed Central

    Zhao, Jin-Ge; Zhang, Yu-Qing

    2016-01-01

    Background Silk sericin and a few non-protein components isolated from the cocoon layer including two silk proteins in silkworm Bombyx mori has many bioactivities. The dietary sericin possess antinatural oxidation, anticancer, antihyperlipidemic, and antidiabetic activities. The non-protein components surrounding the sericin layer involve in wax, pigments mainly meaning flavonoids, sugars, and other impurities. However, very few investigations have reported the estimation of the total flavonoids derived from the cocoon layer. The flavonoids are commonly present in their glycosylated forms and mostly exist as quercetin glycosides in the sericin layers of silkworm cocoons. Objective The aim of this study was to find a more accurate method to estimate the level of the total flavonoids in silkworm cocoons. Design An efficient procedure of hydrolysis-assisted extraction (HAE) was first established to estimate the level of the total flavonoids through the determination of their aglycones, quercetin, and kaempferol. Then, a comparison was made between traditional colorimetric method and our method. In addition, the antioxidant activities of hydrolysis-assisted extract sample were determined. Results The average contents of quercetin and kaempferol were 1.98 and 0.42 mg/g in Daizo cocoon. Their recoveries were 99.56 and 99.17%. The total sum of quercetin and kaempferol was detected to be 2.40±0.07 mg/g by HAE-HPLC, while the total flavonoids (2.59±0.48 mg/g) estimated by the traditional colorimetric method were only equivalent to 1.28±0.04 mg/g of quercetin. The HAE sample also exhibits that IC50 values of scavenging ability of diphenyl picryl hydrazinyl (DPPH) radical and hydroxyl radical (HO·) are 243.63 µg/mL and 4.89 mg/mL, respectively. Conclusions These results show that the HAE-HPLC method is specificity of cocoon and far superior to the colorimetric method. Therefore, this study has profound significance for the comprehensive utilization of silkworm cocoon and

  13. A novel marine silk

    NASA Astrophysics Data System (ADS)

    Kronenberger, Katrin; Dicko, Cedric; Vollrath, Fritz

    2012-01-01

    The discovery of a novel silk production system in a marine amphipod provides insights into the wider potential of natural silks. The tube-building corophioid amphipod Crassicorophium bonellii produces from its legs fibrous, adhesive underwater threads that combine barnacle cement biology with aspects of spider silk thread extrusion spinning. We characterised the filamentous silk as a mixture of mucopolysaccharides and protein deriving from glands representing two distinct types. The carbohydrate and protein silk secretion is dominated by complex β-sheet structures and a high content of charged amino acid residues. The filamentous secretion product exits the gland through a pore near the tip of the secretory leg after having moved through a duct, which subdivides into several small ductules all terminating in a spindle-shaped chamber. This chamber communicates with the exterior and may be considered the silk reservoir and processing/mixing space, in which the silk is mechanically and potentially chemically altered and becomes fibrous. We assert that further study of this probably independently evolved, marine arthropod silk processing and secretion system can provide not only important insights into the more complex arachnid and insect silks but also into crustacean adhesion cements.

  14. A novel marine silk.

    PubMed

    Kronenberger, Katrin; Dicko, Cedric; Vollrath, Fritz

    2012-01-01

    The discovery of a novel silk production system in a marine amphipod provides insights into the wider potential of natural silks. The tube-building corophioid amphipod Crassicorophium bonellii produces from its legs fibrous, adhesive underwater threads that combine barnacle cement biology with aspects of spider silk thread extrusion spinning. We characterised the filamentous silk as a mixture of mucopolysaccharides and protein deriving from glands representing two distinct types. The carbohydrate and protein silk secretion is dominated by complex β-sheet structures and a high content of charged amino acid residues. The filamentous secretion product exits the gland through a pore near the tip of the secretory leg after having moved through a duct, which subdivides into several small ductules all terminating in a spindle-shaped chamber. This chamber communicates with the exterior and may be considered the silk reservoir and processing/mixing space, in which the silk is mechanically and potentially chemically altered and becomes fibrous. We assert that further study of this probably independently evolved, marine arthropod silk processing and secretion system can provide not only important insights into the more complex arachnid and insect silks but also into crustacean adhesion cements. PMID:22057952

  15. Silk-elastin-like protein biomaterials for the controlled delivery of therapeutics

    PubMed Central

    Huang, Wenwen; Rollett, Alexandra; Kaplan, David L.

    2015-01-01

    Introduction Genetically engineered biomaterials are useful for controlled delivery owing to their rational design, tunable structure-function, biocompatibility, degradability and target specificity. Silk-elastin-like proteins (SELPs), a family of genetically engineered recombinant protein polymers, possess these properties. Additionally, given the benefits of combining semicrystalline silk-blocks and elastomeric elastin-blocks, SELPs possess multi-stimuli responsive properties and tenability, thereby, becoming promising candidates for targeted cancer therapeutics delivery and controlled gene release. Areas covered An overview of SELP biomaterials for drug delivery and gene release is provided. Biosynthetic strategies used for SELP production, fundamental physicochemical properties, and self-assembly mechanisms are discussed. The review focuses on sequence-structure-function relationships, stimuli responsive features, and current and potential drug delivery applications. Expert opinion The tunable material properties allow SELPs to be pursued as promising biomaterials for nano-carriers and injectable drug release systems. Current applications of SELPs have focused on thermally-triggered biomaterial formats for the delivery of therapeutics, based on local hyperthermia in tumors or infections. Other prominent controlled release applications of SELPs as injectable hydrogels for gene release have also been pursued. Further biomedical applications that utilize other stimuli to trigger the reversible material responses of SELPs for targeted delivery, including pH, ionic strength, redox, enzymatic stimuli and electric field, are in progress. Exploiting these additional stimuli responsive features will provide a broader range of functional biomaterials for controlled therapeutics release and tissue regeneration. PMID:25476201

  16. Effect of sericin on diabetic hippocampal growth hormone/insulin-like growth factor 1 axis

    PubMed Central

    Chen, Zhihong; Yang, Songhe; He, Yaqiang; Song, Chengjun; Liu, Yongping

    2013-01-01

    Previous studies have shown that sericin extracted from silk cocoon significantly reduces blood glucose levels and protects the nervous system against diabetes mellitus. In this study, a rat type 2 diabetes mellitus model was established by intraperitoneal injection of 25 mg/kg streptozotocin for 3 successive days, following which the rats were treated with sericin for 35 days. After treatment, the blood glucose levels of the diabetic rats decreased significantly, the growth hormone level in serum and its expression in the hippocampus decreased significantly, while the insulin-like growth factor-1 level in serum and insulin-like growth factor-1 and growth hormone receptor expression in the hippocampus increased significantly. The experimental findings indicate that sericin improves disorders of the growth hormone/insulin-like growth factor 1 axis to alleviate hippocampal damage in diabetic rats. PMID:25206472

  17. Fabrication of Highly Uniform Nanoparticles from Recombinant Silk-Elastinlike Protein Polymers for Therapeutic Agent Delivery

    PubMed Central

    Anumolu, Rajasekhar; Gustafson, Joshua A.; Magda, Jules J.; Cappello, Joseph; Ghandehari, Hamidreza; Pease, Leonard F.

    2011-01-01

    Here we generate silk-elastinlike protein (SELP) polymeric nanoparticles and demonstrate precise control over their dimensions using an electrospray differential mobility analyzer (ES-DMA). Electrospray produces droplets encompassing several polymer strands. Evaporation ensues, leading polymer strands to accumulate at the droplet interface forming a hollow nanoparticle. The resulting nanoparticle size distributions which govern particle yield, depend on buffer concentration to the −1/3 power, polymer concentration to the 1/3 power, and ratio of silk to elastin blocks. Three recombinantly tuned ratios of silk to elastin blocks, 8:16, 4:8, and 4:16, respectively named SELP-815K, SELP-47K, and SELP-415K, are employed with the latter ratio resulting in a thinner shell and larger diameter for the nanoparticles than the former. The DMA narrows the size distribution by electrostatically classifying the aerosolized nanoparticles. These highly uniform nanoparticles have variations of 1.2 nm and 1.4 nm for 24.0 nm and 36.0 nm particles, respectively. Transmission electron microscopy reveals the nanoparticles to be faceted, as a buckling instability releases compression energy arising from evaporation after the shell has formed by bending it. A thermodynamic equilibrium exists between compression and bending energies, where the facet length is 1/2 the particle diameter, in agreement with experiments. Rod-like particles also formed from polymer stabilized filaments when the viscous length exceeds the jet radius at higher solution viscosities. The unusual uniformity in composition and dimension indicates the potential of these nanoparticles to deliver bioactive and imaging agents. PMID:21696150

  18. E-spun composite fibers of collagen and dragline silk protein: fiber mechanics, biocompatibility, and application in stem cell differentiation.

    PubMed

    Zhu, Bofan; Li, Wen; Lewis, Randolph V; Segre, Carlo U; Wang, Rong

    2015-01-12

    Biocomposite matrices with high mechanical strength, high stability, and the ability to direct matrix-specific stem cell differentiation are essential for the reconstruction of lesioned tissues in tissue engineering and cell therapeutics. Toward this end, we used the electrospinning technique to fabricate well-aligned composite fibers from collagen and spider dragline silk protein, obtained from the milk of transgenic goats, mimicking the native extracellular matrix (ECM) on a similar scale. Collagen and the dragline silk proteins were found to mix homogeneously at all ratios in the electrospun (E-spun) fibers. As a result, the ultimate tensile strength and elasticity of the fibers increased monotonically with silk percentage, whereas the stretchability was slightly reduced. Strikingly, we found that the incorporation of silk proteins to collagen dramatically increased the matrix stability against excessive fiber swelling and shape deformation in cell culture medium. When human decidua parietalis placental stem cells (hdpPSCs) were seeded on the collagen-silk matrices, the matrices were found to support cell proliferation at a similar rate as that of the pure collagen matrix, but they provided cell adhesion with reduced strengths and induced cell polarization at varied levels. Matrices containing 15 and 30 wt % silk in collagen (CS15, CS30) were found to induce a level of neural differentiation comparable to that of pure collagen. In particular, CS15 matrix induced the highest extent of cell polarization and promoted the development of extended 1D neural filaments strictly in-line with the aligned fibers. Taking the increased mechanical strength and fiber stability into consideration, CS15 and CS30 E-spun fibers offer better alternatives to pure collagen fibers as scaffolds that can be potentially utilized in neural tissue repair and the development of future nanobiodevices. PMID:25405355

  19. E-Spun Composite Fibers of Collagen and Dragline Silk Protein: Fiber Mechanics, Biocompatibility, and Application in Stem Cell Differentiation

    PubMed Central

    2015-01-01

    Biocomposite matrices with high mechanical strength, high stability, and the ability to direct matrix-specific stem cell differentiation are essential for the reconstruction of lesioned tissues in tissue engineering and cell therapeutics. Toward this end, we used the electrospinning technique to fabricate well-aligned composite fibers from collagen and spider dragline silk protein, obtained from the milk of transgenic goats, mimicking the native extracellular matrix (ECM) on a similar scale. Collagen and the dragline silk proteins were found to mix homogeneously at all ratios in the electrospun (E-spun) fibers. As a result, the ultimate tensile strength and elasticity of the fibers increased monotonically with silk percentage, whereas the stretchability was slightly reduced. Strikingly, we found that the incorporation of silk proteins to collagen dramatically increased the matrix stability against excessive fiber swelling and shape deformation in cell culture medium. When human decidua parietalis placental stem cells (hdpPSCs) were seeded on the collagen–silk matrices, the matrices were found to support cell proliferation at a similar rate as that of the pure collagen matrix, but they provided cell adhesion with reduced strengths and induced cell polarization at varied levels. Matrices containing 15 and 30 wt % silk in collagen (CS15, CS30) were found to induce a level of neural differentiation comparable to that of pure collagen. In particular, CS15 matrix induced the highest extent of cell polarization and promoted the development of extended 1D neural filaments strictly in-line with the aligned fibers. Taking the increased mechanical strength and fiber stability into consideration, CS15 and CS30 E-spun fibers offer better alternatives to pure collagen fibers as scaffolds that can be potentially utilized in neural tissue repair and the development of future nanobiodevices. PMID:25405355

  20. Self-assembly of silk-elastinlike protein polymers into three-dimensional scaffolds for biomedical applications

    NASA Astrophysics Data System (ADS)

    Zeng, Like

    Production of brand new protein-based materials with precise control over the amino acid sequences at single residue level has been made possible by genetic engineering, through which artificial genes can be developed that encode protein-based materials with desired features. As an example, silk-elastinlike protein polymers (SELPs), composed of tandem repeats of amino acid sequence motifs from Bombyx mori (silkworm) silk and mammalian elastin, have been produced in this approach. SELPs have been studied extensively in the past two decades, however, the fundamental mechanism governing the self-assembly process to date still remains largely unresolved. Further, regardless of the unprecedented success when exploited in areas including drug delivery, gene therapy, and tissue augmentation, SELPs scaffolds as a three-dimensional cell culture model system are complicated by the inability of SELPs to provide the embedded tissue cells with appropriate biochemical stimuli essential for cell survival and function. In this dissertation, it is reported that the self-assembly of silk-elastinlike protein polymers (SELPs) into nanofibers in aqueous solutions can be modulated by tuning the curing temperature, the size of the silk blocks, and the charge of the elastin blocks. A core-sheath model was proposed for nanofiber formation, with the silk blocks in the cores and the hydrated elastin blocks in the sheaths. The folding of the silk blocks into stable cores -- affected by the size of the silk blocks and the charge of the elastin blocks -- plays a critical role in the assembly of silk-elastin nanofibers. The assembled nanofibers further form nanofiber clusters on the microscale, and the nanofiber clusters then coalesce into nanofiber micro-assemblies, interconnection of which eventually leads to the formation of three-dimensional scaffolds with distinct nanoscale and microscale features. SELP-Collagen hybrid scaffolds were also fabricated to enable independent control over the

  1. Structure and applications of a temperature responsive recombinant protein hydrogel based on silk- and elastin-like amino acid motifs

    NASA Astrophysics Data System (ADS)

    Drummy, Lawrence; Tomczak, Melanie; Macauliffe, Joseph; Vaia, Richard; Naik, Rajesh

    2008-03-01

    Proteins form the main components of many natural materials, and they can be designed to offer tailored functionality and material properties. Silk elastin-like proteins (SELP)s come from a family of repeat sequence protein polymers based on Bombyx mori silk and mammalian elastin that are recombinantly expressed in E. coli. SELP gels are formed by heating the protein solutions in order to induce physical crosslinking of the silk β-sheet regions, they contain approximately 80-90% water by weight and they can be used for encapsulation of enzymes or nanoparticles. For example, horseradish peroxidase demonstrates added resistance to drying and heat treatment when encapsulated in the gel matrix. During gel formation, small angle X-ray scattering shows intensity increases in two distinct regions of reciprocal space, one reversible with temperature and one irreversible. By fitting the scattering data to a unified power-law/Gunier model, morphological parameters are extracted. The thermally reversible intensity changes are attributed to a hydrophilic/hydrophobic transition in the elastin segments, while the irreversible intensity change is due to the crystalline regions formed by the silk blocks.

  2. In situ gelling silk-elastinlike protein polymer for transarterial chemoembolization

    PubMed Central

    Poursaid, Azadeh; Price, Robert; Tiede, Andrea; Olson, Erik; Huo, Eugene; McGill, Lawrence; Ghandehari, Hamidreza; Cappello, Joseph

    2015-01-01

    Hepatocellular carcinoma annually affects over 700,000 people worldwide and trends indicate increasing prevalence. Patients ineligible for surgery undergo loco-regional treatments such as transarterial chemoembolization (TACE) to selectively target tumoral blood supply. Using a microcatheter, chemotherapeutics are infused followed by an embolic agent, or the drug is encapsulated by the embolic moiety; simultaneously inducing stasis while delivering localized chemotherapy. Presently, several products are used, but no universally accepted system is promoted because very disparate limitations exist. The goal of this investigation was to design and develop in situ gelling recombinant silk-elastinlike protein polymers (SELPs) for TACE. Two SELP compositions, SELP-47K and SELP-815K, with varying lengths of silk and elastin blocks, were investigated to formulate a new embolic that was injectable through commercially available microcatheters. The goal was to develop a composition providing maximal permeation of tumor vasculature while exhibiting effective embolic activity. The SELPs evaluated remain soluble until reaching 37°C, when irreversible tran sition ensues forming a solid hydrogel network. SELP-815K formulated at 12% w/w with shear processing demonstrated acceptable rheological properties and clear embolic capability under flow conditions in vitro. A rabbit model showed feasibility of embolization in vivo allowing selective occlusion of lobar hepatic arterial branches. PMID:25916502

  3. Microsecond folding and domain motions of a spider silk protein structural switch.

    PubMed

    Ries, Julia; Schwarze, Simone; Johnson, Christopher M; Neuweiler, Hannes

    2014-12-10

    Web spiders rapidly assemble protein monomers, so-called spidroins, into extraordinarily tough silk fibers. The process involves the pH-triggered self-association of the spidroin N-terminal domain (NTD), which contains a structural switch connecting spidroins to supermolecules. Single-molecule spectroscopy can detect conformational heterogeneity that is hidden to conventional methods, but motions of the NTD are beyond the resolution limit. Here, we engineered probes for 1 nm conformational changes based on the phenomenon of fluorescence quenching by photoinduced electron transfer into the isolated NTD of a spidroin from the nursery web spider Euprosthenops australis. Correlation analysis of single-molecule fluorescence fluctuations uncovered site-dependent nanosecond-to-microsecond movement of secondary and tertiary structure. Kinetic amplitudes were most pronounced for helices that are part of the association interface and where structural studies show large displacements between monomeric and dimeric conformations. A single tryptophan at the center of the five-helix bundle toggled conformations in ∼100 μs and in a pH-dependent manner. Equilibrium denaturation and temperature-jump relaxation experiments revealed cooperative and ultrafast folding in only 60 μs. We deduced a free-energy surface that exhibits native-state ruggedness with apparently similar barrier heights to folding and native motions. Observed equilibrium dynamics within the domain suggest a conformational selection mechanism in the rapid association of spidroins through their NTDs during silk synthesis by web spiders. PMID:25382060

  4. Biomimetic production of silk-like recombinant squid sucker ring teeth proteins.

    PubMed

    Ding, Dawei; Guerette, Paul A; Hoon, Shawn; Kong, Kiat Whye; Cornvik, Tobias; Nilsson, Martina; Kumar, Akshita; Lescar, Julien; Miserez, Ali

    2014-09-01

    The sucker ring teeth (SRT) of Humboldt squid exhibit mechanical properties that rival those of robust engineered synthetic polymers. Remarkably, these properties are achieved without a mineral phase or covalent cross-links. Instead, SRT are exclusively made of silk-like proteins called "suckerins", which assemble into nanoconfined β-sheet reinforced supramolecular networks. In this study, three streamlined strategies for full-length recombinant suckerin protein production and purification were developed. Recombinant suckerin exhibited high solubility and colloidal stability in aqueous-based solvents. In addition, the colloidal suspensions exhibited a concentration-dependent conformational switch, from random coil to β-sheet enriched structures. Our results demonstrate that recombinant suckerin can be produced in a facile manner in E. coli and processed from mild aqueous solutions into materials enriched in β-sheets. We suggest that recombinant suckerin-based materials offer potential for a range of biomedical and engineering applications. PMID:25068184

  5. Activation of the Ubiquitin Proteasome Pathway by Silk Fibroin Modified Chitosan Nanoparticles in Hepatic Cancer Cells

    PubMed Central

    Yang, Ming-Hui; Chung, Tze-Wen; Lu, Yi-Shan; Chen, Yi-Ling; Tsai, Wan-Chi; Jong, Shiang-Bin; Yuan, Shyng-Shiou; Liao, Pao-Chi; Lin, Po-Chiao; Tyan, Yu-Chang

    2015-01-01

    Silk fibroin (SF) is a protein with bulky hydrophobic domains and can be easily purified as sericin-free silk-based biomaterial. Silk fibroin modified chitosan nanoparticle (SF-CSNP), a biocompatible material, has been widely used as a potential drug delivery system. Our current investigation studied the bio-effects of the SF-CSNP uptake by liver cells. In this experiment, the characterizations of SF-CSNPs were measured by particle size analysis and protein assay. The average size of the SF-CSNP was 311.9 ± 10.7 nm, and the average zeta potential was +13.33 ± 0.3 mV. The SF coating on the SF-CSNP was 6.27 ± 0.17 μg/mL. Moreover, using proteomic approaches, several proteins involved in the ubiquitin proteasome pathway were identified by analysis of differential protein expressions of HepG2 cell uptake the SF-CSNP. Our experimental results have demonstrated that the SF-CSNP may be involved in liver cancer cell survival and proliferation. PMID:25588218

  6. Activation of the ubiquitin proteasome pathway by silk fibroin modified chitosan nanoparticles in hepatic cancer cells.

    PubMed

    Yang, Ming-Hui; Chung, Tze-Wen; Lu, Yi-Shan; Chen, Yi-Ling; Tsai, Wan-Chi; Jong, Shiang-Bin; Yuan, Shyng-Shiou; Liao, Pao-Chi; Lin, Po-Chiao; Tyan, Yu-Chang

    2015-01-01

    Silk fibroin (SF) is a protein with bulky hydrophobic domains and can be easily purified as sericin-free silk-based biomaterial. Silk fibroin modified chitosan nanoparticle (SF-CSNP), a biocompatible material, has been widely used as a potential drug delivery system. Our current investigation studied the bio-effects of the SF-CSNP uptake by liver cells. In this experiment, the characterizations of SF-CSNPs were measured by particle size analysis and protein assay. The average size of the SF-CSNP was 311.9 ± 10.7 nm, and the average zeta potential was +13.33 ± 0.3 mV. The SF coating on the SF-CSNP was 6.27 ± 0.17 μg/mL. Moreover, using proteomic approaches, several proteins involved in the ubiquitin proteasome pathway were identified by analysis of differential protein expressions of HepG2 cell uptake the SF-CSNP. Our experimental results have demonstrated that the SF-CSNP may be involved in liver cancer cell survival and proliferation. PMID:25588218

  7. High yield exogenous protein HPL production in the Bombyx mori silk gland provides novel insight into recombinant expression systems

    PubMed Central

    Wang, Huan; Wang, Lu; Wang, Yulong; Tao, Hui; Yin, Weimin; SiMa, Yanghu; Wang, Yujun; Xu, Shiqing

    2015-01-01

    The silk gland of Bombyx mori (BmSG) has gained significant attention by dint of superior synthesis and secretion of proteins. However, the application of BmSG bioreactor is still a controversial issue because of low yields of recombinant proteins. Here, a 3057 bp full-length coding sequence of Hpl was designed and transformed into the silkworm genome, and then the mutant (Hpl/Hpl) with specific expression of Hpl in posterior BmSG (BmPSG) was obtained. In the mutants, the transcription level of Fib-L and P25, and corresponding encoding proteins, did not decrease. However, the mRNA level of Fib-H was reduced by 71.1%, and Fib-H protein in the secreted fibroin was decreased from 91.86% to 71.01%. The mRNA level of Hpl was 0.73% and 0.74% of Fib-H and Fib-L, respectively, while HPL protein accounted for 18.85% of fibroin and 15.46% of the total amount of secreted silk protein. The exogenous protein was therefore very efficiently translated and secreted. Further analysis of differentially expressed gene (DEG) was carried out in the BmPSG cells and 891 DEGs were detected, of which 208 genes were related to protein metabolism. Reduced expression of endogenous silk proteins in the BmPSG could effectively improve the production efficiency of recombinant exogenous proteins. PMID:26370318

  8. Hydrogen bonding-assisted thermal conduction in β-sheet crystals of spider silk protein

    NASA Astrophysics Data System (ADS)

    Zhang, Lin; Chen, Teli; Ban, Heng; Liu, Ling

    2014-06-01

    Using atomistic simulations, we demonstrate that β-sheet, an essential component of spider silk protein, has a thermal conductivity 1-2 orders of magnitude higher than that of some other protein structures reported in the literature. In contrast to several other nanostructured materials of similar bundled/layered structures (e.g. few-layer graphene and bundled carbon nanotubes), the β-sheet is found to uniquely feature enhanced thermal conductivity with an increased number of constituting units, i.e. β-strands. Phonon analysis identifies inter-β-strand hydrogen bonding as the main contributor to the intriguing phenomenon, which prominently influences the state of phonons in both low- and high-frequency regimes. A thermal resistance model further verifies the critical role of hydrogen bonding in thermal conduction through β-sheet structures.Using atomistic simulations, we demonstrate that β-sheet, an essential component of spider silk protein, has a thermal conductivity 1-2 orders of magnitude higher than that of some other protein structures reported in the literature. In contrast to several other nanostructured materials of similar bundled/layered structures (e.g. few-layer graphene and bundled carbon nanotubes), the β-sheet is found to uniquely feature enhanced thermal conductivity with an increased number of constituting units, i.e. β-strands. Phonon analysis identifies inter-β-strand hydrogen bonding as the main contributor to the intriguing phenomenon, which prominently influences the state of phonons in both low- and high-frequency regimes. A thermal resistance model further verifies the critical role of hydrogen bonding in thermal conduction through β-sheet structures. Electronic supplementary information (ESI) available: Structure of the β-sheets, computational model, determination of area and temperature gradient, and additional phonon DOS results. See DOI: 10.1039/c4nr01195c

  9. Ancient Properties of Spider Silks Revealed by the Complete Gene Sequence of the Prey-Wrapping Silk Protein (AcSp1)

    PubMed Central

    Ayoub, Nadia A.; Garb, Jessica E.; Kuelbs, Amanda; Hayashi, Cheryl Y.

    2013-01-01

    Spider silk fibers have impressive mechanical properties and are primarily composed of highly repetitive structural proteins (termed spidroins) encoded by a single gene family. Most characterized spidroin genes are incompletely known because of their extreme size (typically >9 kb) and repetitiveness, limiting understanding of the evolutionary processes that gave rise to their unusual gene architectures. The only complete spidroin genes characterized thus far form the dragline in the Western black widow, Latrodectus hesperus. Here, we describe the first complete gene sequence encoding the aciniform spidroin AcSp1, the primary component of spider prey-wrapping fibers. L. hesperus AcSp1 contains a single enormous (∼19 kb) exon. The AcSp1 repeat sequence is exceptionally conserved between two widow species (∼94% identity) and between widows and distantly related orb-weavers (∼30% identity), consistent with a history of strong purifying selection on its amino acid sequence. Furthermore, the 16 repeats (each 371–375 amino acids long) found in black widow AcSp1 are, on average, >99% identical at the nucleotide level. A combination of stabilizing selection on amino acid sequence, selection on silent sites, and intragenic recombination likely explains the extreme homogenization of AcSp1 repeats. In addition, phylogenetic analyses of spidroin paralogs support a gene duplication event occurring concomitantly with specialization of the aciniform glands and the tubuliform glands, which synthesize egg-case silk. With repeats that are dramatically different in length and amino acid composition from dragline spidroins, our L. hesperus AcSp1 expands the knowledge base for developing silk-based biomimetic technologies. PMID:23155003

  10. Characterization of sericin powder prepared from citric acid-degraded sericin polypeptides of the silkworm, Bombyx Mori.

    PubMed

    Kurioka, Akira; Kurioka, Fujie; Yamazaki, Masayoshi

    2004-04-01

    Acid-degraded sericin powder (AC-SP) was prepared from aqueous solution containing citric acid-degraded sericin polypeptides of Bombyx mori. The morphological and biochemical properties of AC-SP were compared with those of alkali-degraded sericin powder (AL-SP) and hot-water degraded sericin powder (HW-SP). Based on an SEM analysis, AC-SP showed a thin film structure of 10-100 microm with good dispersity while AL-SP and HW-SP had a much larger thin film structure (<500 microm). The extract of AC-SP showed stronger trypsin inhibitor activity due to cocoon shell trypsin inhibitor (CSTI-IV) than that of HW-SP. The extract of AL-SP showed no CSTI-IV activity. It was found that AC-SP was a trypsin inhibitor complex powder and that the release of CSTI-IV from AC-SP depended on pH and ion strength. Similar powder materials were obtained when such organic acids as tartaric acid and succinic acid were used. These results suggest that the acid-degraded sericin polypeptides work as a protein matrix to which CSTI-IV may bind ionically. PMID:15118302

  11. Exploring the Properties of Genetically Engineered Silk-Elastin-Like Protein Films.

    PubMed

    Machado, Raul; da Costa, André; Sencadas, Vitor; Pereira, Ana Margarida; Collins, Tony; Rodríguez-Cabello, José Carlos; Lanceros-Méndez, Senentxu; Casal, Margarida

    2015-12-01

    Free standing films of a genetically engineered silk-elastin-like protein (SELP) were prepared using water and formic acid as solvents. Exposure to methanol-saturated air promoted the formation of aggregated β-strands rendering aqueous insolubility and improved the mechanical properties leading to a 10-fold increase in strain-to-failure. The films were optically clear with resistivity values similar to natural rubber and thermally stable up to 180 °C. Addition of glycerol showed to enhance the flexibility of SELP/glycerol films by interacting with SELP molecules through hydrogen bonding, interpenetrating between the polymer chains and granting more conformational freedom. This detailed characterization provides cues for future and unique applications using SELP based biopolymers. PMID:26214274

  12. Genetically Programmable Thermoresponsive Plasmonic Gold/Silk-Elastin Protein Core/Shell Nanoparticles

    PubMed Central

    2015-01-01

    The design and development of future molecular photonic/electronic systems pose the challenge of integrating functional molecular building blocks in a controlled, tunable, and reproducible manner. The modular nature and fidelity of the biosynthesis method provides a unique chemistry approach to one-pot synthesis of environmental factor-responsive chimeric proteins capable of energy conversion between the desired forms. In this work, facile tuning of dynamic thermal response in plasmonic nanoparticles was facilitated by genetic engineering of the structure, size, and self-assembly of the shell silk-elastin-like protein polymers (SELPs). Recombinant DNA techniques were implemented to synthesize a new family of SELPs, S4E8Gs, with amino acid repeats of [(GVGVP)4(GGGVP)(GVGVP)3(GAGAGS)4] and tunable molecular weight. The temperature-reversible conformational switching between the hydrophilic random coils and the hydrophobic β-turns in the elastin blocks were programmed to between 50 and 60 °C by site-specific glycine mutation, as confirmed by variable-temperature proton NMR and circular dichroism (CD) spectroscopy, to trigger the nanoparticle aggregation. The dynamic self-aggregation/disaggregation of the Au-SELPs nanoparticles was regulated in size and pattern by the β-sheet-forming, thermally stable silk blocks, as revealed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The thermally reversible, shell dimension dependent, interparticle plasmon coupling was investigated by both variable-temperature UV–vis spectroscopy and finite-difference time-domain (FDTD)-based simulations. Good agreement between the calculated and measured spectra sheds light on design and synthesis of responsive plasmonic nanostructures by independently tuning the refractive index and size of the SELPs through genetic engineering. PMID:24712906

  13. Genetically programmable thermoresponsive plasmonic gold/silk-elastin protein core/shell nanoparticles.

    PubMed

    Lin, Yinan; Xia, Xiaoxia; Wang, Ming; Wang, Qianrui; An, Bo; Tao, Hu; Xu, Qiaobing; Omenetto, Fiorenzo; Kaplan, David L

    2014-04-22

    The design and development of future molecular photonic/electronic systems pose the challenge of integrating functional molecular building blocks in a controlled, tunable, and reproducible manner. The modular nature and fidelity of the biosynthesis method provides a unique chemistry approach to one-pot synthesis of environmental factor-responsive chimeric proteins capable of energy conversion between the desired forms. In this work, facile tuning of dynamic thermal response in plasmonic nanoparticles was facilitated by genetic engineering of the structure, size, and self-assembly of the shell silk-elastin-like protein polymers (SELPs). Recombinant DNA techniques were implemented to synthesize a new family of SELPs, S4E8Gs, with amino acid repeats of [(GVGVP)4(GGGVP)(GVGVP)3(GAGAGS)4] and tunable molecular weight. The temperature-reversible conformational switching between the hydrophilic random coils and the hydrophobic β-turns in the elastin blocks were programmed to between 50 and 60 °C by site-specific glycine mutation, as confirmed by variable-temperature proton NMR and circular dichroism (CD) spectroscopy, to trigger the nanoparticle aggregation. The dynamic self-aggregation/disaggregation of the Au-SELPs nanoparticles was regulated in size and pattern by the β-sheet-forming, thermally stable silk blocks, as revealed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The thermally reversible, shell dimension dependent, interparticle plasmon coupling was investigated by both variable-temperature UV-vis spectroscopy and finite-difference time-domain (FDTD)-based simulations. Good agreement between the calculated and measured spectra sheds light on design and synthesis of responsive plasmonic nanostructures by independently tuning the refractive index and size of the SELPs through genetic engineering. PMID:24712906

  14. Proteomic Evidence for Components of Spider Silk Synthesis from Black Widow Silk Glands and Fibers.

    PubMed

    Chaw, Ro Crystal; Correa-Garhwal, Sandra M; Clarke, Thomas H; Ayoub, Nadia A; Hayashi, Cheryl Y

    2015-10-01

    Spider silk research has largely focused on spidroins, proteins that are the primary components of spider silk fibers. Although a number of spidroins have been characterized, other types of proteins associated with silk synthesis are virtually unknown. Previous analyses of tissue-specific RNA-seq libraries identified 647 predicted genes that were differentially expressed in silk glands of the Western black widow, Latrodectus hesperus. Only ∼5% of these silk-gland specific transcripts (SSTs) encode spidroins; although the remaining predicted genes presumably encode other proteins associated with silk production, this is mostly unverified. Here, we used proteomic analysis of multiple silk glands and dragline silk fiber to investigate the translation of the differentially expressed genes. We find 48 proteins encoded by the differentially expressed transcripts in L. hesperus major ampullate, minor ampullate, and tubuliform silk glands and detect 17 SST encoded proteins in major ampullate silk fibers. The observed proteins include known silk-related proteins, but most are uncharacterized, with no annotation. These unannotated proteins likely include novel silk-associated proteins. Major and minor ampullate glands have the highest overlap of identified proteins, consistent with their shared, distinctive ampullate shape and the overlapping functions of major and minor ampullate silks. Our study substantiates and prioritizes predictions from differential expression analysis of spider silk gland transcriptomes. PMID:26302244

  15. Characterization of a cysteine-rich protein specifically expressed in the silk gland of caddisfly Stenopsyche marmorata (Trichoptera; Stenopsychidae).

    PubMed

    Wang, Yujun; Wang, Hong; Zhao, Tianfu; Nakagaki, Masao

    2010-01-01

    A novel protein, Smsp-72k, was found to be selectively expressed in the silk gland of aquatic larvae of the Stenopsychid caddisfly (Stenopsyche marmorata). The protein was characterized by an abundance of cysteine (13.97%) and charged residues (47.21%). Amino acids with hydroxyl side-chains accounted for an additional 10% of the Smsp-72k protein, with serine at 4.4% and threonine at 5.6%. A cysteine-rich repetitive sequence is common to many potential and known underwater adhesive/cement proteins and cell-cell adhesion molecules. We hypothesized that Smsp-72k is an adhesive/cement protein that increases the adhesiveness of the silk fiber of S. marmorata. The hydroxyl groups of Smsp-72k might form a link with the heavy chain fibroin of S. marmorata, removing the weak boundary-water layer and allowing the spreading of the silk protein onto the surface of the substratum during the process of adhesion. PMID:20057124

  16. Ubiquitous distribution of salts and proteins in spider glue enhances spider silk adhesion

    PubMed Central

    Amarpuri, Gaurav; Chaurasia, Vishal; Jain, Dharamdeep; Blackledge, Todd A.; Dhinojwala, Ali

    2015-01-01

    Modern orb-weaving spiders use micron-sized glue droplets on their viscid silk to retain prey in webs. A combination of low molecular weight salts and proteins makes the glue viscoelastic and humidity responsive in a way not easily achieved by synthetic adhesives. Optically, the glue droplet shows a heterogeneous structure, but the spatial arrangement of its chemical components is poorly understood. Here, we use optical and confocal Raman microscopy to show that salts and proteins are present ubiquitously throughout the droplet. The distribution of adhesive proteins in the peripheral region explains the superior prey capture performance of orb webs as it enables the entire surface area of the glue droplet to act as a site for prey capture. The presence of salts throughout the droplet explains the recent Solid-State NMR results that show salts directly facilitate protein mobility. Understanding the function of individual glue components and the role of the droplet's macro-structure can help in designing better synthetic adhesives for humid environments. PMID:25761668

  17. Ubiquitous distribution of salts and proteins in spider glue enhances spider silk adhesion

    NASA Astrophysics Data System (ADS)

    Amarpuri, Gaurav; Chaurasia, Vishal; Jain, Dharamdeep; Blackledge, Todd A.; Dhinojwala, Ali

    2015-03-01

    Modern orb-weaving spiders use micron-sized glue droplets on their viscid silk to retain prey in webs. A combination of low molecular weight salts and proteins makes the glue viscoelastic and humidity responsive in a way not easily achieved by synthetic adhesives. Optically, the glue droplet shows a heterogeneous structure, but the spatial arrangement of its chemical components is poorly understood. Here, we use optical and confocal Raman microscopy to show that salts and proteins are present ubiquitously throughout the droplet. The distribution of adhesive proteins in the peripheral region explains the superior prey capture performance of orb webs as it enables the entire surface area of the glue droplet to act as a site for prey capture. The presence of salts throughout the droplet explains the recent Solid-State NMR results that show salts directly facilitate protein mobility. Understanding the function of individual glue components and the role of the droplet's macro-structure can help in designing better synthetic adhesives for humid environments.

  18. Toward spinning artificial spider silk.

    PubMed

    Rising, Anna; Johansson, Jan

    2015-05-01

    Spider silk is strong and extensible but still biodegradable and well tolerated when implanted, making it the ultimate biomaterial. Shortcomings that arise in replicating spider silk are due to the use of recombinant spider silk proteins (spidroins) that lack native domains, the use of denaturing conditions under purification and spinning and the fact that the understanding of how spiders control silk formation is incomplete. Recent progress has unraveled the molecular mechanisms of the spidroin N- and C-terminal nonrepetitive domains (NTs and CTs) and revealed the pH and ion gradients in spiders' silk glands, clarifying how spidroin solubility is maintained and how silk is formed in a fraction of a second. Protons and CO2, generated by carbonic anhydrase, affect the stability and structures of the NT and CT in different ways. These insights should allow the design of conditions and devices for the spinning of recombinant spidroins into native-like silk. PMID:25885958

  19. Clinical Application of a Silk Fibroin Protein Biologic Scaffold for Abdominal Wall Fascial Reinforcement

    PubMed Central

    Downey, Susan; Agullo, Frank; Lehfeldt, Max R.; Kind, Gabriel M.; Palladino, Humberto; Marshall, Deirdre; Jewell, Mark L.; Mathur, Anshu B.; Bengtson, Bradley P.

    2014-01-01

    Background: Preclinical studies have demonstrated that macroporous silk fibroin protein scaffolds are capable of promoting physiologically durable supportive tissue, which favors application of these engineered tissues for clinical implantation. The safety and effectiveness of a long-lasting, transitory, 510(k)-cleared purified silk fibroin biologic scaffold (SBS) are investigated for soft-tissue support and repair of the abdominal wall. Methods: We conducted a multicenter retrospective review of all consecutive patients who underwent abdominal wall soft-tissue reinforcement with an SBS device between 2011 and 2013. Indications, comorbid conditions, surgical technique, complications, and outcomes were evaluated. Results: We reviewed the records of 172 consecutive patients who received an SBS for soft-tissue support. Of those, 77 patients underwent abdominal wall fascial repair, with a mean follow-up of 18.4 ± 7.5 months. Procedures using an SBS included reinforcement of an abdominal-based flap donor site (31.2%), ventral hernia repair (53.2%), and abdominoplasty (15.6%). The overall complication rate was 6.5%, consisting of 2 wound dehiscences, 1 with device exposure, 1 seroma, 1 infection with explantation, and a perioperative bulge requiring reoperation. There were no reports of hernia. Conclusions: Postoperative complication rates after 18 months were low, and most surgical complications were managed nonoperatively on an outpatient basis without mesh removal. To our knowledge, this is the only series to report on a long-lasting, transitory SBS for abdominal wall repair and reinforcement. Procedure-specific outcome studies are warranted to delineate optimal patient selection and define potential device characteristic advantages. PMID:25506529

  20. More than just fibers: an aqueous method for the production of innovative recombinant spider silk protein materials.

    PubMed

    Jones, Justin A; Harris, Thomas I; Tucker, Chauncey L; Berg, Kyle R; Christy, Stacia Y; Day, Breton A; Gaztambide, Danielle A; Needham, Nate J C; Ruben, Ashley L; Oliveira, Paula F; Decker, Richard E; Lewis, Randolph V

    2015-04-13

    Spider silk is a striking and robust natural material that has an unrivaled combination of strength and elasticity. There are two major problems in creating materials from recombinant spider silk proteins (rSSps): expressing sufficient quantities of the large, highly repetitive proteins and solvating the naturally self-assembling proteins once produced. To address the second problem, we have developed a method to rapidly dissolve rSSps in water in lieu of traditional organic solvents and accomplish nearly 100% solvation and recovery of the protein. Our method involves generating pressure and temperature in a sealed vial by using short, repetitive bursts from a conventional microwave. The method is scalable and has been successful with all rSSps used to date. From these easily generated aqueous solutions of rSSps, a wide variety of materials have been produced. Production of fibers, films, hydrogels, lyogels, sponges, and adhesives and studies of their mechanical and structural properties are reported. To our knowledge, ours is the only method that is cost-effective and scalable for mass production. This solvation method allows a choice of the physical form of product to take advantage of spider silks' mechanical properties without using costly and problematic organic solvents. PMID:25789668

  1. Seed-Specific Expression of Spider Silk Protein Multimers Causes Long-Term Stability.

    PubMed

    Weichert, Nicola; Hauptmann, Valeska; Helmold, Christine; Conrad, Udo

    2016-01-01

    Seeds enable plants to germinate and to grow in situations of limited availability of nutrients. The stable storage of different seed proteins is a remarkable presumption for successful germination and growth. These strategies have been adapted and used in several molecular farming projects. In this study, we explore the benefits of seed-based expression to produce the high molecular weight spider silk protein FLAG using intein-based trans-splicing. Multimers larger than 460 kDa in size are routinely produced, which is above the native size of the FLAG protein. The storage of seeds for 8 weeks and 1 year at an ambient temperature of 15°C does not influence the accumulation level. Even the extended storage time does not influence the typical pattern of multimerized bands. These results show that seeds are the method of choice for stable accumulation of products of complex transgenes and have the capability for long-term storage at moderate conditions, an important feature for the development of suitable downstream processes. PMID:26858734

  2. Seed-Specific Expression of Spider Silk Protein Multimers Causes Long-Term Stability

    PubMed Central

    Weichert, Nicola; Hauptmann, Valeska; Helmold, Christine; Conrad, Udo

    2016-01-01

    Seeds enable plants to germinate and to grow in situations of limited availability of nutrients. The stable storage of different seed proteins is a remarkable presumption for successful germination and growth. These strategies have been adapted and used in several molecular farming projects. In this study, we explore the benefits of seed-based expression to produce the high molecular weight spider silk protein FLAG using intein-based trans-splicing. Multimers larger than 460 kDa in size are routinely produced, which is above the native size of the FLAG protein. The storage of seeds for 8 weeks and 1 year at an ambient temperature of 15°C does not influence the accumulation level. Even the extended storage time does not influence the typical pattern of multimerized bands. These results show that seeds are the method of choice for stable accumulation of products of complex transgenes and have the capability for long-term storage at moderate conditions, an important feature for the development of suitable downstream processes. PMID:26858734

  3. A silk hydrogel-based delivery system of bone morphogenetic protein for the treatment of large bone defects.

    PubMed

    Diab, Tamim; Pritchard, Eleanor M; Uhrig, Brent A; Boerckel, Joel D; Kaplan, David L; Guldberg, Robert E

    2012-07-01

    The use of tissue grafting for the repair of large bone defects has numerous limitations including donor site morbidity and the risk of disease transmission. These limitations have prompted research efforts to investigate the effects of combining biomaterial scaffolds with biochemical cues to augment bone repair. The goal of this study was to use a critically-sized rat femoral segmental defect model to investigate the efficacy of a delivery system consisting of an electrospun polycaprolactone (PCL) nanofiber mesh tube with a silk fibroin hydrogel for local recombinant bone morphogenetic protein 2 (BMP-2) delivery. Bilateral 8 mm segmental femoral defects were formed in 13-week-old Sprague Dawley rats. Perforated electrospun PCL nanofiber mesh tubes were fitted into the adjacent native bone such that the lumen of the tubes contained the defect (Kolambkar et al., 2011b). Silk hydrogels with or without BMP-2 were injected into the defect. Bone regeneration was longitudinally assessed using 2D X-ray radiography and 3D microcomputed topography (μCT). Following sacrifice at 12 weeks after surgery, the extracted femurs were either subjected to biomechanical testing or assigned for histology. The results demonstrated that silk was an effective carrier for BMP-2. Compared to the delivery system without BMP-2, the delivery system that contained BMP-2 resulted in more bone formation (p<0.05) at 4, 8, 12 weeks after surgery. Biomechanical properties were also significantly improved in the presence of BMP-2 (p<0.05) and were comparable to age-matched intact femurs. Histological evaluation of the defect region indicated that the silk hydrogel has been completely degraded by the end of the study. Based on these results, we conclude that a BMP-2 delivery system consisting of an electrospun PCL nanofiber mesh tube with a silk hydrogel presents an effective strategy for functional repair of large bone defects. PMID:22658161

  4. Expression of a Truncated ATHB17 Protein in Maize Increases Ear Weight at Silking

    PubMed Central

    Creelman, Robert A.; Griffith, Cara; Ahrens, Jeffrey E.; Taylor, J. Philip; Murphy, Lesley R.; Manjunath, Siva; Thompson, Rebecca L.; Lingard, Matthew J.; Back, Stephanie L.; Larue, Huachun; Brayton, Bonnie R.; Burek, Amanda J.; Tiwari, Shiv; Adam, Luc; Morrell, James A.; Caldo, Rico A.; Huai, Qing; Kouadio, Jean-Louis K.; Kuehn, Rosemarie; Sant, Anagha M.; Wingbermuehle, William J.; Sala, Rodrigo; Foster, Matt; Kinser, Josh D.; Mohanty, Radha; Jiang, Dongming; Ziegler, Todd E.; Huang, Mingya G.; Kuriakose, Saritha V.; Skottke, Kyle; Repetti, Peter P.; Reuber, T. Lynne; Ruff, Thomas G.; Petracek, Marie E.; Loida, Paul J.

    2014-01-01

    ATHB17 (AT2G01430) is an Arabidopsis gene encoding a member of the α-subclass of the homeodomain leucine zipper class II (HD-Zip II) family of transcription factors. The ATHB17 monomer contains four domains common to all class II HD-Zip proteins: a putative repression domain adjacent to a homeodomain, leucine zipper, and carboxy terminal domain. However, it also possesses a unique N-terminus not present in other members of the family. In this study we demonstrate that the unique 73 amino acid N-terminus is involved in regulation of cellular localization of ATHB17. The ATHB17 protein is shown to function as a transcriptional repressor and an EAR-like motif is identified within the putative repression domain of ATHB17. Transformation of maize with an ATHB17 expression construct leads to the expression of ATHB17Δ113, a truncated protein lacking the first 113 amino acids which encodes a significant portion of the repression domain. Because ATHB17Δ113 lacks the repression domain, the protein cannot directly affect the transcription of its target genes. ATHB17Δ113 can homodimerize, form heterodimers with maize endogenous HD-Zip II proteins, and bind to target DNA sequences; thus, ATHB17Δ113 may interfere with HD-Zip II mediated transcriptional activity via a dominant negative mechanism. We provide evidence that maize HD-Zip II proteins function as transcriptional repressors and that ATHB17Δ113 relieves this HD-Zip II mediated transcriptional repression activity. Expression of ATHB17Δ113 in maize leads to increased ear size at silking and, therefore, may enhance sink potential. We hypothesize that this phenotype could be a result of modulation of endogenous HD-Zip II pathways in maize. PMID:24736658

  5. LIM-homeodomain transcription factor Awh is a key component activating all three fibroin genes, fibH, fibL and fhx, in the silk gland of the silkworm, Bombyx mori.

    PubMed

    Kimoto, Mai; Tsubota, Takuya; Uchino, Keiro; Sezutsu, Hideki; Takiya, Shigeharu

    2015-01-01

    In the silkworm Bombyx mori, three fibroin genes, fibroin-heavy-chain (fibH), fibroin-light-chain (fibL) and fibrohexamerin (fhx), are coexpressed only in the posterior silk gland (PSG) cells, while the sericin genes encoding silk glue proteins are expressed in the middle silk gland (MSG) cells. Silk gland factor-2 (SGF-2) is a PSG-specific activator complex of fibH, composed of a LIM-homeodomain protein, Awh, and its cofactors, Ldb and Lcaf. We investigated whether SGF-2 can activate other fibroin genes using transgenic silkworms. The genes for Ldb and Lcaf were expressed ubiquitously in various tissues, while the gene for Awh was expressed strictly specific in PSG of the wild type silkworms. Misexpression of Awh in transgenic silkworms induced ectopic expression of fibL and fhx as well as fibH in MSG. Coincidently with the induction of fibL and fhx by Awh, binding of SGF-2 to the promoter of fibL and fhx was detected in vitro, and SGF-2 binds directly to the fhx core promoter. Ectopic expression of the fibroin genes was observed at high levels in the middle part of MSG. Moreover, fibL and fhx were induced in the anterior silk gland (ASG) of the transgenic silkworms, but fibH was not. These results indicate that Awh is a key activator of all three fibroin genes, and the activity is probably regulated in conjunction with additional factors. PMID:25449130

  6. Silk inverse opals

    NASA Astrophysics Data System (ADS)

    Kim, Sunghwan; Mitropoulos, Alexander N.; Spitzberg, Joshua D.; Tao, Hu; Kaplan, David L.; Omenetto, Fiorenzo G.

    2012-12-01

    Periodic nanostructures provide the facility to control and manipulate the flow of light through their lattices. Three-dimensional photonic crystals enable the controlled design of structural colour, which can be varied by infiltrating the structure with different (typically liquid) fillers. Here, we report three-dimensional photonic crystals composed entirely of a purified natural protein (silk fibroin). The biocompatibility of this protein, as well as its favourable material properties and ease of biological functionalization, present opportunities for otherwise unattainable device applications such as bioresorbable integration of structural colour within living tissue or lattice functionalization by means of organic and inorganic material doping. We present a silk inverse opal that demonstrates a pseudo-photonic bandgap in the visible spectrum and show its associated structural colour beneath biological tissue. We also leverage silk's facile dopability to manufacture a gold nanoparticle silk inverse opal and demonstrate patterned heating mediated by enhancement of nanoparticle absorption at the band-edge frequency of the photonic crystal.

  7. A juvenile hormone transcription factor Bmdimm-fibroin H chain pathway is involved in the synthesis of silk protein in silkworm, Bombyx mori.

    PubMed

    Zhao, Xiao-Ming; Liu, Chun; Jiang, Li-Jun; Li, Qiong-Yan; Zhou, Meng-Ting; Cheng, Ting-Cai; Mita, Kazuei; Xia, Qing-You

    2015-01-01

    The genes responsible for silk biosynthesis are switched on and off at particular times in the silk glands of Bombyx mori. This switch appears to be under the control of endogenous and exogenous hormones. However, the molecular mechanisms by which silk protein synthesis is regulated by the juvenile hormone (JH) are largely unknown. Here, we report a basic helix-loop-helix transcription factor, Bmdimm, its silk gland-specific expression, and its direct involvement in the regulation of fibroin H-chain (fib-H) by binding to an E-box (CAAATG) element of the fib-H gene promoter. Far-Western blots, enzyme-linked immunosorbent assays, and co-immunoprecipitation assays revealed that Bmdimm protein interacted with another basic helix-loop-helix transcription factor, Bmsage. Immunostaining revealed that Bmdimm and Bmsage proteins are co-localized in nuclei. Bmdimm expression was induced in larval silk glands in vivo, in silk glands cultured in vitro, and in B. mori cell lines after treatment with a JH analog. The JH effect on Bmdimm was mediated by the JH-Met-Kr-h1 signaling pathway, and Bmdimm expression did not respond to JH by RNA interference with double-stranded BmKr-h1 RNA. These data suggest that the JH regulatory pathway, the transcription factor Bmdimm, and the targeted fib-H gene contribute to the synthesis of fibroin H-chain protein in B. mori. PMID:25371208

  8. Sericin-carboxymethyl cellulose porous matrices as cellular wound dressing material.

    PubMed

    Nayak, Sunita; Kundu, S C

    2014-06-01

    In this study, porous three-dimensional (3D) hydrogel matrices are fabricated composed of silk cocoon protein sericin of non-mulberry silkworm Antheraea mylitta and carboxymethyl cellulose. The matrices are prepared via freeze-drying technique followed by dual cross-linking with glutaraldehyde and aluminum chloride. The microstructure of the hydrogel matrices is assessed using scanning electron microscopy and biophysical characterization are carried out using Fourier transform infrared spectroscopy and X-ray diffraction. The transforming growth factor β1 release from the cross-linked matrices as a growth factor is evaluated by immunosorbent assay. Live dead assay and 3-[4,5-dimethylthiazolyl-2]-2,5-diphenyl tetrazolium bromide assay show no cytotoxicity of blended matrices toward human keratinocytes. The matrices support the cell attachment and proliferation of human keratinocytes as observed through scanning electron microscope and confocal images. Gelatin zymography demonstrates the low levels of matrix metalloproteinase 2 (MMP-2) and insignificant amount of MMP-9 in the culture media of cell seeded matrices. Low inflammatory response of the matrices is indicated through tumor necrosis factor alpha release assay. The results indicate that the fabricated matrices constitute 3D cell-interactive environment for tissue engineering applications and its potential use as a future cellular biological wound dressing material. PMID:23853114

  9. Effects of degumming conditions on electro-spinning rate of regenerated silk.

    PubMed

    Yoon, Kyunghwan; Lee, Ha Ni; Ki, Chang Seok; Fang, Dufei; Hsiao, Benjamin S; Chu, Benjamin; Um, In Chul

    2013-10-01

    Electro-spun silk webs are potentially good candidates as tissue engineering scaffolds owing to their good bio- and cyto-compatibility. However, the low fabrication rate of electro-spun silk mats has been one of the obstacles in the mass production of such nanofibrous silk mats in applications to the biomedical field. In this study, the effects of degumming ratio and silk concentration on the electro-spinning process were investigated by using regenerated silk with different residual sericin contents and different silk concentrations in terms of the morphology and structure of the electro-spun silk web. The rate of production of electro-spun silk mats could be increased by approximately 5 fold at a degumming ratio of 19.5%. The electro-spinning rate of silk was affected by two main factors: (1) dope solution viscosity and (2) degumming ratio of silk. The conductivity of the silk dope solution, however, had little effects on the electro-spinning of regenerated silk. A constant spun fiber morphology was observed within the electro-spinning rate range (0.3-1.4 ml/h). Fourier transform infrared spectroscopy showed that partial β-sheet crystallization occurred during electro-spinning. The molecular conformation was relatively unaffected by the electro-spinning rate of silk. PMID:23817099

  10. Silk-elastin-like protein polymer matrix for intraoperative delivery of an oncolytic vaccinia virus

    PubMed Central

    Price, Daniel L.; Li, Pingdong; Chen, Chun-Hao; Wong, Danni; Yu, Zhenkun; Chen, Nanhai G.; Yu, Yong A.; Szalay, Aladar A.; Cappello, Joseph; Fong, Yuman; Wong, Richard J.

    2016-01-01

    Background Oncolytic viral efficacy may be limited by the penetration of the virus into tumors. This may be enhanced by intraoperative application of virus immediately after surgical resection. Methods Oncolytic vaccinia virus GLV-1h68 was delivered in silk-elastin-like protein polymer (SELP) in vitro and in vivo in anaplastic thyroid carcinoma cell line 8505c in nude mice. Results GLV-1h68 in SELP infected and lysed anaplastic thyroid cancer cells in vitro equally as effectively as in phosphate-buffered saline (PBS), and at 1 week retains a thousand fold greater infectious plaque-forming units. In surgical resection models of residual tumor, GLV-1h68 in SELP improves tumor control and shows increased viral β-galactosidase expression as compared to PBS. Conclusion The use of SELP matrix for intraoperative oncolytic viral delivery protects infectious viral particles from degradation, facilitates sustained viral delivery and transgene expression, and improves tumor control. Such optimization of methods of oncolytic viral delivery may enhance therapeutic outcomes. PMID:25244076

  11. Silk-Elastinlike Protein Polymer Liquid Chemoembolic for Localized Release of Doxorubicin and Sorafenib.

    PubMed

    Poursaid, Azadeh; Jensen, Mark Martin; Nourbakhsh, Ida; Weisenberger, Mitchell; Hellgeth, John W; Sampath, Sujatha; Cappello, Joseph; Ghandehari, Hamidreza

    2016-08-01

    Locoregional therapies for cancer are minimally invasive procedures in which the treatment is administered directly into cancerous tissue. Transarterial chemoembolization (TACE) is used to treat intermediate stage hepatocellular carcinoma (HCC). TACE uses an embolic material to block blood flow while coadministering a chemotherapeutic to the neoplastic tissue. Liquid embolics capable of drug loading are at the forefront of development as they allow for deeper permeation of tumor vasculature, increase neoplasm exposure to therapeutics, and resist revascularization by occupying both large and small diameter vessels. In this work, two chemotherapeutics used in the treatment of HCC, doxorubicin and sorafenib, were incorporated into the in situ gelling liquid embolic composed of a silk-elastinlike protein polymer (SELP-815 K). The base forms of the drugs had no significant effect on the viscosity, the gelation kinetics, and the gel stiffness of the SELP: all properties essential for the successful performance of an injectable liquid embolic. In vitro release studies indicated that the SELP liquid embolic delivered doxorubicin and sorafenib, either alone or in combination, at therapeutically relevant concentrations for a minimum of 14 and 30 days, respectively. PMID:27295352

  12. Recent progress in development of transgenic silkworms overexpressing recombinant human proteins with therapeutic potential in silk glands.

    PubMed

    Itoh, Kohji; Kobayashi, Isao; Nishioka, So-Ichiro; Sezutsu, Hideki; Machii, Hiroaki; Tamura, Toshiki

    2016-02-01

    Since 2000, transgenic silkworms have been developed to produce recombinant proteins with therapeutic potential for future clinical use, including antibody preparations. Lysosomal storage diseases (LSDs) are inherited metabolic disorders caused by mutations of lysosomal enzymes associated with excessive accumulation of natural substrates and neurovisceral symptoms. Over the past few years, enzyme replacement therapy (ERT) with human lysosomal enzymes produced by genetically engineered mammalian cell lines has been used clinically to treat several patients with an LSD involving multi-organ symptoms. ERT is based on the incorporation of recombinant glycoenzymes by their binding to glycan receptors on the surface of target cells and their subsequent delivery to lysosomes. However, ERT has several disadvantages, including difficulty mass producing human enzymes, dangers of pathogen contamination, and high costs. Recently, the current authors have succeeded in producing transgenic silkworms overexpressing human lysosomal enzymes in the silk glands and the authors have purified catalytically active enzymes from the middle silk glands. Silk gland-derived human enzymes carrying high-mannose and pauci-mannose N-glycans were endocytosed by monocytes via the mannose receptor pathway and were then delivered to lysosomes. Conjugates with cell-penetrating peptides were also taken up by cultured fibroblasts derived from patients with enzyme deficiencies to restore intracellular catalytic activity and reduce the excessive accumulation of substrates in patient fibroblasts. Transgenic silkworms overexpressing human lysosomal enzymes in the silk glands could serve as future bioresources that provide safe therapeutic enzymes for the treatment of LSDs. Combining recent developments in transglycosylation technology with microbial endoglycosidases will promote the development of therapeutic glycoproteins as bio-medicines. PMID:26971553

  13. Silk-silica composites from genetically engineered chimeric proteins: materials properties correlate with silica condensation rate and colloidal stability of the proteins in aqueous solution

    PubMed Central

    Belton, David J.; Mieszawska, Aneta J.; Currie, Heather A.; Kaplan, David L.; Perry, Carole C.

    2012-01-01

    The aim of the study was to determine the extent and mechanism of influence on silica condensation that is presented by a range of known silicifying recombinant chimeras (R5- SSKKSGSYSGSKGSKRRIL; A1- SGSKGSKRRIL; and Si4-1- MSPHPHPRHHHT and repeats thereof) attached at the N-terminus end of a 15 mer repeat of the 32 amino acid consensus sequence of the major ampullate dragline Spindroin 1 (Masp1) Nephila clavipes spider silk sequence ([SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQG]15X). The influence of the silk/chimera ratio was explored through the adjustment of the type and number of silicifying domains, (denoted X above), and the results were compared with their non chimeric counterparts and the silk from Bombyx mori. The effect of pH (3–9) on reactivity was also explored. Optimum conditions for rate and control of silica deposition were determined and the solution properties of the silks were explored to determine their mode(s) of action. For the silica-silk-chimera materials formed there is a relationship between the solution properties of the chimeric proteins (ability to carry charge), the pH of reaction and the solid state materials that are generated. The region of colloidal instability correlates with the pH range observed for morphological control and coincides with the pH range for the highest silica condensation rates. With this information it should be possible to predict how chimeric or chemically modified proteins will affect structure and morphology of materials produced under controlled conditions and extend the range of composite materials for a wide spectrum of uses in the biomedical and technology fields. PMID:22313382

  14. Analysis of tissue-specific region in sericin 1 gene promoter of Bombyx mori

    SciTech Connect

    Liu Yan; Yu Lian; Guo Xiuyang; Guo Tingqing; Wang Shengpeng; Lu Changde . E-mail: cdlu@sibs.ac.cn

    2006-03-31

    The gene encoding sericin 1 (Ser1) of silkworm (Bombyx mori) is specifically expressed in the middle silk gland cells. To identify element involved in this transcription-dependent spatial restriction, truncation of the 5' terminal from the sericin 1 (Ser1) promoter is studied in vivo. A 209 bp DNA sequence upstream of the transcriptional start site (-586 to -378) is found to be responsible for promoting tissue-specific transcription. Analysis of this 209 bp region by overlapping deletion studies showed that a 25 bp region (-500 to -476) suppresses the ectopic expression of the Ser1 promoter. An unknown factor abundant in fat body nuclear extracts is shown to bind to this 25 bp fragment. These results suggest that this 25 bp region and the unknown factor are necessary for determining the tissue-specificity of the Ser1 promoter.

  15. Mechanical Improvements to Reinforced Porous Silk Scaffolds

    PubMed Central

    Gil, Eun Seok; Kluge, Jonathan A.; Rockwood, Danielle N.; Rajkhowa, Rangam; Wang, Lijing; Wang, Xungai; Kaplan, David L

    2012-01-01

    Load bearing porous biodegradable scaffolds are required to engineer functional tissues such as bone. Mechanical improvements to porogen leached scaffolds prepared from silk proteins were systematically studied through the addition of silk particles in combination with silk solution concentration, exploiting interfacial compatibility between the two components. Solvent solutions of silk up to 32 w/v% were successfully prepared in hexafluoroisopropanaol (HFIP) for the study. The mechanical properties of the reinforced silk scaffolds correlated to the material density and matched by a power law relationship, independent of the ratio of silk particles to matrix. These results were similar to the relationships previously shown for cancellous bone. The mechanism behind the increased mechanical properties was a densification effect, and not the effect of including stiffer silk particles into the softer silk continuous matrix. A continuous interface between the silk matrix and the silk particles, as well as homogeneous distribution of the silk particles within the matrix were observed. Furthermore, we note that the roughness of the pore walls was controllable by varying the ratio of particles matrix, providing a route to control topography. The rate of proteolytic hydrolysis of the scaffolds decreased with increase in mass of silk used in the matrix and with increasing silk particle content. PMID:21793193

  16. Modifying the Mechanical Properties of Silk Fiber by Genetically Disrupting the Ionic Environment for Silk Formation.

    PubMed

    Wang, Xin; Zhao, Ping; Li, Yi; Yi, Qiying; Ma, Sanyuan; Xie, Kang; Chen, Huifang; Xia, Qingyou

    2015-10-12

    Silks are widely used biomaterials, but there are still weaknesses in their mechanical properties. Here we report a method for improving the silk fiber mechanical properties by genetic disruption of the ionic environment for silk fiber formation. An anterior silk gland (ASG) specific promoter was identified and used for overexpressing ion-transporting protein in the ASG of silkworm. After isolation of the transgenic silkworms, we found that the metal ion content, conformation and mechanical properties of transgenic silk fibers changed accordingly. Notably, overexpressing endoplasmic reticulum Ca2+-ATPase in ASG decreased the calcium content of silks. As a consequence, silk fibers had more α-helix and β-sheet conformations, and their tenacity and extension increased significantly. These findings represent the in vivo demonstration of a correlation between metal ion content in the spinning duct and the mechanical properties of silk fibers, thus providing a novel method for modifying silk fiber properties. PMID:26302212

  17. Silkworms transformed with chimeric silkworm/spider silk genes spin composite silk fibers with improved mechanical properties

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The development of a spider silk manufacturing process is of great interest. piggyBac vectors were used to create transgenic silkworms encoding chimeric silkworm/spider silk proteins. The silk fibers produced by these animals were composite materials that included chimeric silkworm/spider silk prote...

  18. Spider wrapping silk fibre architecture arising from its modular soluble protein precursor.

    PubMed

    Tremblay, Marie-Laurence; Xu, Lingling; Lefèvre, Thierry; Sarker, Muzaddid; Orrell, Kathleen E; Leclerc, Jérémie; Meng, Qing; Pézolet, Michel; Auger, Michèle; Liu, Xiang-Qin; Rainey, Jan K

    2015-01-01

    Spiders store spidroins in their silk glands as high concentration aqueous solutions, spinning these dopes into fibres with outstanding mechanical properties. Aciniform (or wrapping) silk is the toughest spider silk and is devoid of the short amino acid sequence motifs characteristic of the other spidroins. Using solution-state NMR spectroscopy, we demonstrate that the 200 amino acid Argiope trifasciata AcSp1 repeat unit contrasts with previously characterized spidroins, adopting a globular 5-helix bundle flanked by intrinsically disordered N- and C-terminal tails. Split-intein-mediated segmental NMR-active isotope-enrichment allowed unambiguous demonstration of modular and malleable "beads-on-a-string" concatemeric behaviour. Concatemers form fibres upon manual drawing with silk-like morphology and mechanical properties, alongside secondary structuring and orientation consistent with native AcSp1 fibres. AcSp1 structural stability varies locally, with the fifth helix denaturing most readily. The structural transition of aciniform spidroin from a mostly α-helical dope to a mixed α-helix/β-sheet-containing fibre can be directly related to spidroin architecture and stability. PMID:26112753

  19. Spider wrapping silk fibre architecture arising from its modular soluble protein precursor

    PubMed Central

    Tremblay, Marie-Laurence; Xu, Lingling; Lefèvre, Thierry; Sarker, Muzaddid; Orrell, Kathleen E.; Leclerc, Jérémie; Meng, Qing; Pézolet, Michel; Auger, Michèle; Liu, Xiang-Qin; Rainey, Jan K.

    2015-01-01

    Spiders store spidroins in their silk glands as high concentration aqueous solutions, spinning these dopes into fibres with outstanding mechanical properties. Aciniform (or wrapping) silk is the toughest spider silk and is devoid of the short amino acid sequence motifs characteristic of the other spidroins. Using solution-state NMR spectroscopy, we demonstrate that the 200 amino acid Argiope trifasciata AcSp1 repeat unit contrasts with previously characterized spidroins, adopting a globular 5-helix bundle flanked by intrinsically disordered N- and C-terminal tails. Split-intein-mediated segmental NMR-active isotope-enrichment allowed unambiguous demonstration of modular and malleable “beads-on-a-string” concatemeric behaviour. Concatemers form fibres upon manual drawing with silk-like morphology and mechanical properties, alongside secondary structuring and orientation consistent with native AcSp1 fibres. AcSp1 structural stability varies locally, with the fifth helix denaturing most readily. The structural transition of aciniform spidroin from a mostly α-helical dope to a mixed α-helix/β-sheet-containing fibre can be directly related to spidroin architecture and stability. PMID:26112753

  20. Resistive Switching: Physically Transient Resistive Switching Memory Based on Silk Protein (Small 20/2016).

    PubMed

    Wang, Hong; Zhu, Bowen; Ma, Xiaohua; Hao, Yue; Chen, Xiaodong

    2016-05-01

    On page 2715, physically transient resistive switching memory based on silk fibroin is demonstrated by Y. Hao, X. Chen, and co-workers. The memory devices can be absolutely dissolved in deionized water or phosphate-buffered saline in 2 hours. The transient devices have the potential for application in secure data storage systems and biocompatible electronics. PMID:27198963

  1. Spider wrapping silk fibre architecture arising from its modular soluble protein precursor

    NASA Astrophysics Data System (ADS)

    Tremblay, Marie-Laurence; Xu, Lingling; Lefèvre, Thierry; Sarker, Muzaddid; Orrell, Kathleen E.; Leclerc, Jérémie; Meng, Qing; Pézolet, Michel; Auger, Michèle; Liu, Xiang-Qin; Rainey, Jan K.

    2015-06-01

    Spiders store spidroins in their silk glands as high concentration aqueous solutions, spinning these dopes into fibres with outstanding mechanical properties. Aciniform (or wrapping) silk is the toughest spider silk and is devoid of the short amino acid sequence motifs characteristic of the other spidroins. Using solution-state NMR spectroscopy, we demonstrate that the 200 amino acid Argiope trifasciata AcSp1 repeat unit contrasts with previously characterized spidroins, adopting a globular 5-helix bundle flanked by intrinsically disordered N- and C-terminal tails. Split-intein-mediated segmental NMR-active isotope-enrichment allowed unambiguous demonstration of modular and malleable “beads-on-a-string” concatemeric behaviour. Concatemers form fibres upon manual drawing with silk-like morphology and mechanical properties, alongside secondary structuring and orientation consistent with native AcSp1 fibres. AcSp1 structural stability varies locally, with the fifth helix denaturing most readily. The structural transition of aciniform spidroin from a mostly α-helical dope to a mixed α-helix/β-sheet-containing fibre can be directly related to spidroin architecture and stability.

  2. Protein secondary structure of Green Lynx spider dragline silk investigated by solid-state NMR and X-ray diffraction

    PubMed Central

    Xu, Dian; Shi, Xiangyan; Thompson, Forrest; Weber, Warner S.; Mou, Qiushi; Yarger, Jeffery L

    2016-01-01

    In this study, the secondary structure of the major ampullate silk from Peucetia viridans (Green Lynx) spiders is characterized by X-ray diffraction and solid-state NMR spectroscopy. From X-ray diffraction measurement, β-sheet nanocrystallites were observed and found to be highly oriented along the fiber axis, with an orientational order, fc ≈ 0.98. The size of the nanocrystallites was determined to be on average 2.5 nm × 3.3 nm × 3.8 nm. Besides a prominent nanocrystalline region, a partially oriented amorphous region was also observed with an fa ≈ 0.89. Two-dimensional 13C–13C through-space and through-bond solid-state NMR experiments were employed to elucidate structure details of P. viridans silk proteins. It reveals that β-sheet nanocrystallites constitutes 40.0 ± 1.2% of the protein and are dominated by alanine-rich repetitive motifs. Furthermore, based upon the NMR data, 18 ± 1% of alanine, 60 ± 2% glycine and 54 ± 2% serine are incorporated into helical conformations. PMID:26226457

  3. Protein secondary structure of Green Lynx spider dragline silk investigated by solid-state NMR and X-ray diffraction.

    PubMed

    Xu, Dian; Shi, Xiangyan; Thompson, Forrest; Weber, Warner S; Mou, Qiushi; Yarger, Jeffery L

    2015-11-01

    In this study, the secondary structure of the major ampullate silk from Peucetia viridans (Green Lynx) spiders is characterized by X-ray diffraction and solid-state NMR spectroscopy. From X-ray diffraction measurement, β-sheet nanocrystallites were observed and found to be highly oriented along the fiber axis, with an orientational order, fc≈0.98. The size of the nanocrystallites was determined to be on average 2.5nm×3.3nm×3.8nm. Besides a prominent nanocrystalline region, a partially oriented amorphous region was also observed with an fa≈0.89. Two-dimensional (13)C-(13)C through-space and through-bond solid-state NMR experiments were employed to elucidate structure details of P. viridans silk proteins. It reveals that β-sheet nanocrystallites constitutes 40.0±1.2% of the protein and are dominated by alanine-rich repetitive motifs. Furthermore, based upon the NMR data, 18±1% of alanine, 60±2% glycine and 54±2% serine are incorporated into helical conformations. PMID:26226457

  4. Effect of degumming time on silkworm silk fibre for biodegradable polymer composites

    NASA Astrophysics Data System (ADS)

    Ho, Mei-po; Wang, Hao; Lau, Kin-tak

    2012-02-01

    Recently, many studies have been conducted on exploitation of natural materials for modern product development and bioengineering applications. Apart from plant-based materials (such as sisal, hemp, jute, bamboo and palm fibre), animal-based fibre is a kind of sustainable natural materials for making novel composites. Silkworm silk fibre extracted from cocoon has been well recognized as a promising material for bio-medical engineering applications because of its superior mechanical and bioresorbable properties. However, when producing silk fibre reinforced biodegradable/bioresorbable polymer composites, hydrophilic sericin has been found to cause poor interfacial bonding with most polymers and thus, it results in affecting the resultant properties of the composites. Besides, sericin layers on fibroin surface may also cause an adverse effect towards biocompatibility and hypersensitivity to silk for implant applications. Therefore, a proper pre-treatment should be done for sericin removal. Degumming is a surface modification process which allows a wide control of the silk fibre's properties, making the silk fibre possible to be used for the development and production of novel bio-composites with unique/specific mechanical and biodegradable properties. In this paper, a cleaner and environmentally friendly surface modification technique for tussah silk in polymer based composites is proposed. The effectiveness of different degumming parameters including degumming time and temperature on tussah silk is discussed through the analyses of their mechanical and morphological properties. Based on results obtained, it was found that the mechanical properties of tussah silk are affected by the degumming time due to the change of the fibre structure and fibroin alignment.

  5. From Micelles to Fibers: Balancing Self-Assembling and Random Coiling Domains in pH-Responsive Silk-Collagen-Like Protein-Based Polymers

    PubMed Central

    2014-01-01

    We study the self-assembly of genetically engineered protein-based triblock copolymers consisting of a central pH-responsive silk-like middle block (SHn, where SH is a silk-like octapeptide, (GA)3GH and n is the number of repeats) flanked by hydrophilic random coil outer blocks (C2). Our previous work has already shown that triblocks with very long midblocks (n = 48) self-assemble into long, stiff protein filaments at pH values where the middle blocks are uncharged. Here we investigate the self-assembly behavior of the triblock copolymers for a range of midblock lengths, n = 8, 16, 24, 48. Upon charge neutralization of SHn by adjusting the pH, we find that C2SH8C2 and C2SH16C2 form spherical micelles, whereas both C2SH24C2 and C2SH48C2 form protein filaments with a characteristic beta-roll secondary structure of the silk midblocks. Hydrogels formed by C2SH48C2 are much stronger and form much faster than those formed by C2SH24C2. Enzymatic digestion of much of the hydrophilic outer blocks is used to show that with much of the hydrophilic outer blocks removed, all silk-midblocks are capable of self-assembling into stiff protein filaments. In that case, reduction of the steric repulsion by the hydrophilic outer blocks also leads to extensive fiber bundling. Our results highlight the opposing roles of the hydrophilic outer blocks and central silk-like midblocks in driving protein filament formation. They provide crucial information for future designs of triblock protein-based polymers that form stiff filaments with controlled bundling, that could mimick properties of collagen in the extracellular matrix. PMID:25133990

  6. Silk-elastinlike protein polymers improve the efficacy of adenovirus thymidine kinase enzyme prodrug therapy of head and neck tumors

    PubMed Central

    Greish, Khaled; Frandsen, Jordan; Scharff, Stephanie; Gustafson, Joshua; Cappello, Joseph; Li, Daqing; O’Malley, Bert W.; Ghandehari, Hamidreza

    2010-01-01

    Background Adenoviral directed enzyme prodrug therapy is a promising approach for head and neck cancer gene therapy. Challenges with this approach however are transient gene expression and dissemination of viruses to distant organs. Methods We used recombinant silk-elastinlike protein copolymer (SELP) matrices for intratumoral delivery of adenoviruses containing both thymidine kinase-1, and luciferase genes in a nude mice model of JHU-022 head and neck tumor. Hydrogels made from two SELP analogues (47K and 815K) with similar silk to elastinlike block ratios but different block lengths were studied for intratumoral viral delivery. Tumor bearing mice were followed up for tumor progression and luciferase gene expression concomitantly for five weeks. Polymer’s safety was evaluated through body weight change, blood count, liver and kidney functions in addition to gross and microscopic histological examination. Results SELP 815K analogues efficiently controlled the duration and extent of transfection in tumors for up to 5 weeks with no detectable spread to the liver. About five-fold greater reduction in tumor volume was obtained with matrix-mediated delivery compared to intra-tumoral injection of adenoviruses in saline. SELP matrix proved safe in all injected mice compared to control group. Conclusion SELP- controlled gene delivery approach could potentially improve the anticancer activity of virus-mediated gene therapy while limiting viral spread to normal organs. PMID:20603862

  7. Immobilization of sericin molecules via amorphous carbon plasma modified-polystyrene dish for serum-free culture

    NASA Astrophysics Data System (ADS)

    Tunma, Somruthai; Song, Doo-Hoon; Kim, Si-Eun; Kim, Kyoung-Nam; Han, Jeon-Geon; Boonyawan, Dheerawan

    2013-10-01

    In this study, we focused on sericin hydrolysates, originating from silkworm used in serum-free human bone marrow-derived mesenchymal stem cells (hBM-MSCs) culture. We reported the effect of a covalent linkage between a bioactive protein molecule and polystyrene dish surface via a carbon intermediate layer which can slow down the release rate of protein compounds into the phosphate buffer saline (PBS) solution. Films of amorphous carbon (a-C) and functionalized-carbon were deposited on PS culture dish surfaces by using a DC magnetron sputtering system and RF PECVD system. We found that a-C based-films can increase the hydrophilicity and biocompatibility of polystyrene (PS) dishes, especially a-C films and a-C:N2 films showed good attachment of hBM-MSCs at 24 h. However, in the case of silica surface (a-C:SiOx films), the cells showed a ragged and unattached boundary resulting from the presence of surface silanol groups. For the UV-vis absorbance, all carbon modified-PS dishes showed a lower release rate of sericin molecules into PBS solution than PS control. This revealed that the functionalized carbon could be enhanced by specific binding properties with given molecules. The carbon-coated PS dishes grafting with sericin protein were used in a serum-free condition. We also found that hBM-MSCs have higher percentage of proliferated cells at day 7 for the modified dishes with carbon films and coated with sericin than the PS control coated with sericin. The physical film properties were measured by atomic force microscopy (AFM), scanning electron microscope (SEM) and contact angle measurement. The presence of sbnd NH2 groups of sericin compounds on the PS dish was revealed by Fourier transform infrared spectroscopy (FTIR). The stability of covalent bonds of sericin molecules after washing out ungrafted sericin was confirmed by X-ray photoelectron spectroscopy (XPS).

  8. Interaction of recombinant analogs of spider silk proteins 1F9 and 2E12 with phospholipid membranes.

    PubMed

    Antonenko, Yuri N; Perevoshchikova, Irina V; Davydova, Lyubov I; Agapov, Igor A; Bogush, Vladimir G

    2010-06-01

    Recombinant analogs of spider dragline silk proteins 1F9 and 2E12 are characterized by numerous repeats consisting of hydrophobic poly-Ala blocks and Gly-rich sequences with a substantial number of positively charged amino acid residues which suggest a pronounced ability to interact with negatively charged phospholipid membranes. Actually both proteins displayed substantial binding affinity towards lipid vesicles formed of acidic lipids as measured by fluorescence correlation spectroscopy (FCS) using rhodamine-labeled conjugates of the proteins. Both proteins did not induce liposome leakage, fusion or breakdown, but were able to bring about liposome aggregation. 1F9 was more active in the induction of liposome aggregation compared to 2E12. Interestingly, 2E12 markedly decreased the rate of calcium-induced liposome fusion. Circular dichroism data showed that binding of the proteins to negatively charged phosphatidylserine liposomes provoked transition from the left-handed helix of polyproline II (PPII) type to beta-structures and alpha-helices. The data suggested predominantly surface location of membrane bound proteins without significant perturbation of their hydrophobic core. PMID:20214876

  9. Effect of BBX-B8 overexpression on development, body weight, silk protein synthesis and egg diapause of Bombyx mori.

    PubMed

    Zheng, Xiaojian; Gong, Yongchang; Kumar, Dhiraj; Chen, Fei; Kuan, Sulan; Liang, Zi; Hu, Xiaolong; Cao, Guangli; Xue, Renyu; Gong, Chengliang

    2016-08-01

    Bombyxin (BBX) is an insulin-like peptide exists in the silkworm Bombyx mori. Our previous studies on the effects of inhibiting BBX-B8 expression found that BBX-B8 is important for the development of organ, reproduction and trehalose metabolism in the silkworms. In this paper, we investigated the expression profile of the BBX-B8 gene and effect of BBX-B8 overexpression on the development, body weight, silk protein synthesis and egg diapause of B. mori to further understand BBX-B8 functions. BBX-B8 gene expression could be detected in the brains, midguts, anterior silkglands, ovaries, testes, fat bodies, hemolymph, malpighian tubules and embryos by RT-PCR, however it was mainly expressed in the brain. Western blots showed that the change in BBX-B8 expression was not obvious in the brain of 1- to 4-day-old larvae of fifth instar silkworms, but expression increased substantially at 5- to 6-day-old larvae of fifth instar silkworms. Transgenic silkworms overexpressing BBX-B8 were obtained by introducing non-transposon transgenic vector pIZT-B8 containing a BBX-B8 gene driven by Orgyia pseudotsugata nucleopolyhedrovirus IE2 promoter into the genome. Development duration of the transgenic silkworms was delayed by 2.5-3.5 days. Cocoon shell weight of transgenic silkworms was reduced by 4.79 % in females and 7.44 % in males, pupal weight of transgenic silkworms was reduced 6.75 % in females and 13.83 % in males compared to non-transgenic silkworms, and 5.56-14.29 % of transgenic moths laid nondiapausing eggs. All results indicated that BBX-B8 plays an important role in the development, silk protein synthesis and egg diapause of silkworm. PMID:26951193

  10. Mechanical properties of regenerated Bombyx mori silk fibers and recombinant silk fibers produced by transgenic silkworms.

    PubMed

    Zhu, Zhenghua; Kikuchi, Yuka; Kojima, Katsura; Tamura, Toshiki; Kuwabara, Nobuo; Nakamura, Takashi; Asakura, Tetsuo

    2010-01-01

    Regenerated silk fibroin fibers from the cocoons of silkworm, Bombyx mori, were prepared with hexafluoro solvents, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) or hexafluoroacetone-trihydrate (HFA), as dope solvents and methanol as coagulation solvent. The regenerated fiber prepared from the HFIP solution showed slightly larger tensile strength when the draw ratio is 1:3 than that of native silk fiber, but the strength of the regenerated fiber with draw ratio 1:3 from the HFA solution is much lower than that of native silk fiber. This difference in the tensile strength of the regenerated silk fibers between two dope solvents comes from the difference in the long-range orientation of the crystalline region rather than that of short-range structural environment such as the fraction of beta-sheet structure. The increase in the biodegradation was observed for the regenerated silk fiber compared with native silk fiber. Preparations of regenerated silk fibroin fibers containing spider silk sequences were obtained by mixing silk fibroins and silk-like proteins with characteristic sequences from a spider, Naphila clavipes, to produce drag-line silk in E. coli in the fluoro solvents. A small increase in the tensile strength was obtained by adding 5% (w/w) of the silk-like protein to the silk fibroin. The production of silk fibroin fibers with these spider silk sequences was also performed with transgenic silkworms. Small increase in the tensile strength of the fibers was obtained without significant change in the elongation-at-break. PMID:20178693

  11. Diverse formulas for spider dragline fibers demonstrated by molecular and mechanical characterization of spitting spider silk.

    PubMed

    Correa-Garhwal, Sandra M; Garb, Jessica E

    2014-12-01

    Spider silks have outstanding mechanical properties. Most research has focused on dragline silk proteins (major ampullate spidroins, MaSps) from orb-weaving spiders. Using silk gland expression libraries from the haplogyne spider Scytodes thoracica, we discovered two novel spidroins (S. thoracica fibroin 1 and 2). The amino acid composition of S. thoracica silk glands and dragline fibers suggest that fibroin 1 is the major component of S. thoracica dragline silk. Fibroin 1 is dominated by glycine-alanine motifs, and lacks sequence motifs associated with orb-weaver MaSps. We hypothesize fibroin 2 is a piriform or aciniform silk protein, based on amino acid composition, spigot morphology, and phylogenetic analyses. S. thoracica's dragline silk is less tough than previously reported, but is still comparable to other dragline silks. Our analyses suggest that dragline silk proteins evolved multiple times. This demonstrates that spider dragline silk is more diverse than previously understood, providing alternative high performance silk designs. PMID:25340514

  12. Reproducing Natural Spider Silks' Copolymer Behavior in Synthetic Silk Mimics

    SciTech Connect

    An, Bo; Jenkins, Janelle E; Sampath, Sujatha; Holland, Gregory P; Hinman, Mike; Yarger, Jeffery L; Lewis, Randolph

    2012-10-30

    Dragline silk from orb-weaving spiders is a copolymer of two large proteins, major ampullate spidroin 1 (MaSp1) and 2 (MaSp2). The ratio of these proteins is known to have a large variation across different species of orb-weaving spiders. NMR results from gland material of two different species of spiders, N. clavipes and A. aurantia, indicates that MaSp1 proteins are more easily formed into β-sheet nanostructures, while MaSp2 proteins form random coil and helical structures. To test if this behavior of natural silk proteins could be reproduced by recombinantly produced spider silk mimic protein, recombinant MaSp1/MaSp2 mixed fibers as well as chimeric silk fibers from MaSp1 and MaSp2 sequences in a single protein were produced based on the variable ratio and conserved motifs of MaSp1 and MaSp2 in native silk fiber. Mechanical properties, solid-state NMR, and XRD results of tested synthetic fibers indicate the differing roles of MaSp1 and MaSp2 in the fiber and verify the importance of postspin stretching treatment in helping the fiber to form the proper spatial structure.

  13. Silk Gland Factor-2, Involved in Fibroin Gene Transcription, Consists of LIM Homeodomain, LIM-interacting, and Single-stranded DNA-binding Proteins*

    PubMed Central

    Ohno, Kaoru; Sawada, Jun-ichi; Takiya, Shigeharu; Kimoto, Mai; Matsumoto, Akiko; Tsubota, Takuya; Uchino, Keiro; Hui, Chi-chung; Sezutsu, Hideki; Handa, Hiroshi; Suzuki, Yoshiaki

    2013-01-01

    SGF-2 binds to promoter elements governing posterior silk gland-specific expression of the fibroin gene in Bombyx mori. We purified SGF-2 and showed that SGF-2 contains at least four gene products: the silkworm orthologues of LIM homeodomain protein Awh, LIM domain-binding protein (Ldb), a sequence-specific single-stranded DNA-binding protein (Lcaf), and the silk protein P25/fibrohexamerin (fhx). Using co-expression of these factors in Sf9 cells, Awh, Ldb, and Lcaf proteins were co-purified as a ternary complex that bound to the enhancer sequence in vitro. Lcaf interacts with Ldb as well as Awh through the conserved regions to mediate transcriptional activation in yeast. Misexpression of Awh in transgenic silkworms induces ectopic expression of the fibroin gene in the middle silk glands, where Ldb and Lcaf are expressed. Taken together, this study demonstrates that SGF-2 is a multisubunit activator complex containing Awh. Moreover, our results suggest that the Ldb·Lcaf protein complex serves as a scaffold to facilitate communication between transcriptional control elements. PMID:24022586

  14. Silk from Crickets: A New Twist on Spinning

    PubMed Central

    Walker, Andrew A.; Weisman, Sarah; Church, Jeffrey S.; Merritt, David J.; Mudie, Stephen T.; Sutherland, Tara D.

    2012-01-01

    Raspy crickets (Orthoptera: Gryllacrididae) are unique among the orthopterans in producing silk, which is used to build shelters. This work studied the material composition and the fabrication of cricket silk for the first time. We examined silk-webs produced in captivity, which comprised cylindrical fibers and flat films. Spectra obtained from micro-Raman experiments indicated that the silk is composed of protein, primarily in a beta-sheet conformation, and that fibers and films are almost identical in terms of amino acid composition and secondary structure. The primary sequences of four silk proteins were identified through a mass spectrometry/cDNA library approach. The most abundant silk protein was large in size (300 and 220 kDa variants), rich in alanine, glycine and serine, and contained repetitive sequence motifs; these are features which are shared with several known beta-sheet forming silk proteins. Convergent evolution at the molecular level contrasts with development by crickets of a novel mechanism for silk fabrication. After secretion of cricket silk proteins by the labial glands they are fabricated into mature silk by the labium-hypopharynx, which is modified to allow the controlled formation of either fibers or films. Protein folding into beta-sheet structure during silk fabrication is not driven by shear forces, as is reported for other silks. PMID:22355311

  15. Transmission Electron Microscopy of Bombyx Mori Silk Fibers

    NASA Astrophysics Data System (ADS)

    Shen, Y.; Martin, D. C.

    1997-03-01

    The microstructure of B. Mori silk fibers before and after degumming was examined by TEM, selected area electron diffraction (SAED), WAXS and low voltage SEM. SEM micrographs of the neat cocoon revealed a network of pairs of twisting filaments. After degumming, there were only individual filaments showing a surface texture consistent with an oriented fibrillar structure in the fiber interior. WAXS patterns confirmed the oriented beta-sheet crystal structure common to silkworm and spider silks. Low dose SAED results were fully consistent with the WAXS data, and revealed that the crystallographic texture did not vary significantly across the fiber diameter. TEM observations of microtomed fiber cross sections indicated a somewhat irregular shape, and also revealed a 0.5-2 micron sericin coating which was removed by the degumming process. TEM observations of the degummed silk fiber showed banded features with a characteristic spacing of nominally 600 nm along the fiber axis. These bands were oriented in a roughly parabolic or V-shape pointing along one axis within a given fiber. We hypothesize that this orientation is induced by the extrusion during the spinning process. Equatorial DF images revealed that axial and lateral sizes of the β-sheet crystallites in silk fibroin ranged from 20 to 170 nm and from 1 to 24 nm, respectively. Crazes developed in the degummed silk fiber parallel to the fiber direction. The formation of these crazes suggests that there are significant lateral interactions between fibrils in silk fibers.

  16. The elaborate structure of spider silk

    PubMed Central

    Römer, Lin

    2008-01-01

    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. PMID:19221522

  17. Silk as an innovative biomaterial for cancer therapy.

    PubMed

    Jastrzebska, Katarzyna; Kucharczyk, Kamil; Florczak, Anna; Dondajewska, Ewelina; Mackiewicz, Andrzej; Dams-Kozlowska, Hanna

    2015-01-01

    Silk has been used for centuries in the textile industry and as surgical sutures. In addition to its unique mechanical properties, silk possesses other properties, such as biocompatibility, biodegradability and ability to self-assemble, which make it an interesting material for biomedical applications. Although silk forms only fibers in nature, synthetic techniques can be used to control the processing of silk into different morphologies, such as scaffolds, films, hydrogels, microcapsules, and micro- and nanospheres. Moreover, the biotechnological production of silk proteins broadens the potential applications of silk. Synthetic silk genes have been designed. Genetic engineering enables modification of silk properties or the construction of a hybrid silk. Bioengineered hybrid silks consist of a silk sequence that self-assembles into the desired morphological structure and the sequence of a polypeptide that confers a function to the silk biomaterial. The functional domains can comprise binding sites for receptors, enzymes, drugs, metals or sugars, among others. Here, we review the current status of potential applications of silk biomaterials in the field of oncology with a focus on the generation of implantable, injectable and targeted drug delivery systems and the three-dimensional cancer models based on silk scaffolds for cancer research. However, the systems described could be applied in many biomedical fields. PMID:25859397

  18. Silk as an innovative biomaterial for cancer therapy

    PubMed Central

    Jastrzebska, Katarzyna; Kucharczyk, Kamil; Florczak, Anna; Dondajewska, Ewelina; Mackiewicz, Andrzej; Dams-Kozlowska, Hanna

    2014-01-01

    Silk has been used for centuries in the textile industry and as surgical sutures. In addition to its unique mechanical properties, silk possesses other properties, such as biocompatibility, biodegradability and ability to self-assemble, which make it an interesting material for biomedical applications. Although silk forms only fibers in nature, synthetic techniques can be used to control the processing of silk into different morphologies, such as scaffolds, films, hydrogels, microcapsules, and micro- and nanospheres. Moreover, the biotechnological production of silk proteins broadens the potential applications of silk. Synthetic silk genes have been designed. Genetic engineering enables modification of silk properties or the construction of a hybrid silk. Bioengineered hybrid silks consist of a silk sequence that self-assembles into the desired morphological structure and the sequence of a polypeptide that confers a function to the silk biomaterial. The functional domains can comprise binding sites for receptors, enzymes, drugs, metals or sugars, among others. Here, we review the current status of potential applications of silk biomaterials in the field of oncology with a focus on the generation of implantable, injectable and targeted drug delivery systems and the three-dimensional cancer models based on silk scaffolds for cancer research. However, the systems described could be applied in many biomedical fields. PMID:25859397

  19. Protein unfolding versus β-sheet separation in spider silk nanocrystals

    NASA Astrophysics Data System (ADS)

    Alam, Parvez

    2014-03-01

    In this communication a mechanism for spider silk strain hardening is proposed. Shear failure of β-sheet nanocrystals is the first failure mode that gives rise to the creation of smaller nanocrystals, which are of higher strength and stiffness. β-sheet unfolding requires more energy than nanocrystal separation in a shear mode of failure. As a result, unfolding occurs after the nanocrystals separate in shear. β-sheet unfolding yields a secondary strain hardening effect once the β-sheet conformation is geometrically stable and acts like a unidirectional fibre in a fibre reinforced composite. The mechanism suggested herein is based on molecular dynamics calculations of residual inter-β-sheet separation strengths against residual intra-β-sheet unfolding strengths.

  20. Effects of the amino acid sequence on thermal conduction through β-sheet crystals of natural silk protein.

    PubMed

    Zhang, Lin; Bai, Zhitong; Ban, Heng; Liu, Ling

    2015-11-21

    Recent experiments have discovered very different thermal conductivities between the spider silk and the silkworm silk. Decoding the molecular mechanisms underpinning the distinct thermal properties may guide the rational design of synthetic silk materials and other biomaterials for multifunctionality and tunable properties. However, such an understanding is lacking, mainly due to the complex structure and phonon physics associated with the silk materials. Here, using non-equilibrium molecular dynamics, we demonstrate that the amino acid sequence plays a key role in the thermal conduction process through β-sheets, essential building blocks of natural silks and a variety of other biomaterials. Three representative β-sheet types, i.e. poly-A, poly-(GA), and poly-G, are shown to have distinct structural features and phonon dynamics leading to different thermal conductivities. A fundamental understanding of the sequence effects may stimulate the design and engineering of polymers and biopolymers for desired thermal properties. PMID:26455593

  1. Lithium-free processing of silk fibroin.

    PubMed

    Zheng, Zhaozhu; Guo, Shaozhe; Liu, Yawen; Wu, Jianbing; Li, Gang; Liu, Meng; Wang, Xiaoqin; Kaplan, David

    2016-09-01

    Silk fibroin protein was purified from Bombyx mori silkworm cocoons using a novel dialysis strategy to avoid fibroin aggregation and pre-mature formation of β-sheets. The degummed silk fibers were dissolved in Ajisawa's reagent, a mixture of CaCl2-EtOH-H2O, that is less expensive than lithium bromide. The dissolved solutions were dialyzed against either water or urea solution with a stepwise decrease in concentration. When the steps of 4 M-2 M-1 M-0 M urea (referred to as silk-TS-4210) were adopted, the purified silk fibroin had smaller aggregates (<10 nm), similar average molecular weight (225 kDa) and a lower content of β-sheet (∼15%) compared to the sample processing methods (silk-TS-210, 10, 0) studied here. This outcome was close to the fibroin purified by the lithium bromide (silk-Li-0) method. Polyvinyl alcohol-emulsified silk microspheres generated using the purified solution had a similar size distribution and morphology when compared to lithium bromide dissolved solutions, while glycerol-blended silk films showed different mechanical properties. The silk-Li-0 generated films with the highest breaking strength (5.7 MPa ± 0.3) while the silk-TS-4210 had the highest extension at break (215.1% ± 12.5). The films prepared from silk-TS-4210 were cytocompatible to support the adhesion and proliferation of human mesenchymal stem cells, with improvements compared to the other samples likely due to the porous morphology of these films. PMID:27298185

  2. Luminescent golden silk and fabric through in situ chemically coating pristine-silk with gold nanoclusters.

    PubMed

    Zhang, Pu; Lan, Jing; Wang, Yi; Xiong, Zu Hong; Huang, Cheng Zhi

    2015-01-01

    Silk is an excellent natural material and has been used for a variety of applications. Modification of the pristine silk is usually needed depending on the intended purpose. The technical treatments involved in the modification not only should be easy, rapid, environmentally friendly, and cheap but should also retain the features of the pristine silk. Herein, we demonstrate that luminescent silk and fabric can be produced through nanotechnology. The surface of the natural silk fiber is chemically coated with luminescent gold nanoclusters (AuNCs) composed of tens to hundreds of Au atoms through a redox reaction between the protein-based silk and an Au salt precursor. The luminescent silk coated with AuNCs (called golden silk) possesses good optical properties, including a relatively long wavelength emission, high quantum yields, a long fluorescent lifetime, and photostability. Moreover, golden silk prepared this way has better mechanical properties than pristine silk, is better able to inhibit UV, and has lower toxicity in vitro. This work not only provides an effective strategy for in situ preparation of luminescent metal nanoclusters on a solid substrate but also paves the way for large-scale and industrialized production of novel silk-based materials or fabrics through nanotechnology. PMID:25308521

  3. Microdissection of Black Widow Spider Silk-producing Glands

    PubMed Central

    Hsia, Yang; Gnesa, Eric; Zhao, Liang; Franz, Andreas; Vierra, Craig

    2011-01-01

    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

  4. Anti-EGFR-iRGD recombinant protein conjugated silk fibroin nanoparticles for enhanced tumor targeting and antitumor efficiency

    PubMed Central

    Bian, Xinyu; Wu, Puyuan; Sha, Huizi; Qian, Hanqing; Wang, Qing; Cheng, Lei; Yang, Yang; Yang, Mi; Liu, Baorui

    2016-01-01

    In this study, we report a novel kind of targeting with paclitaxel (PTX)-loaded silk fibroin nanoparticles conjugated with iRGD–EGFR nanobody recombinant protein (anti-EGFR-iRGD). The new nanoparticles (called A-PTX-SF-NPs) were prepared using the carbodiimide-mediated coupling procedure and their characteristics were evaluated. The cellular cytotoxicity and cellular uptake of A-PTX-SF-NPs were also investigated. The results in vivo suggested that NPs conjugated with the recombinant protein exhibited more targeting and anti-neoplastic property in cells with high EGFR expression. In the in vivo antitumor efficacy assay, the A-PTX-SF-NPs group showed slower tumor growth and smaller tumor volumes than PTX-SF-NPs in a HeLa xenograft mouse model. A real-time near-infrared fluorescence imaging study showed that A-PTX-SF-NPs could target the tumor more effectively. These results suggest that the anticancer activity and tumor targeting of A-PTX-SF-NPs were superior to those of PTX-SF-NPs and may have the potential to be used for targeted delivery for tumor therapies. PMID:27313461

  5. Post-secretion processing influences spider silk performance.

    PubMed

    Blamires, Sean J; Wu, Chung-Lin; Blackledge, Todd A; Tso, I-Min

    2012-10-01

    Phenotypic variation facilitates adaptations to novel environments. Silk is an example of a highly variable biomaterial. The two-spidroin (MaSp) model suggests that spider major ampullate (MA) silk is composed of two proteins-MaSp1 predominately contains alanine and glycine and forms strength enhancing β-sheet crystals, while MaSp2 contains proline and forms elastic spirals. Nonetheless, mechanical properties can vary in spider silks without congruent amino acid compositional changes. We predicted that post-secretion processing causes variation in the mechanical performance of wild MA silk independent of protein composition or spinning speed across 10 species of spider. We used supercontraction to remove post-secretion effects and compared the mechanics of silk in this 'ground state' with wild native silks. Native silk mechanics varied less among species compared with 'ground state' silks. Variability in the mechanics of 'ground state' silks was associated with proline composition. However, variability in native silks did not. We attribute interspecific similarities in the mechanical properties of native silks, regardless of amino acid compositions, to glandular processes altering molecular alignment of the proteins prior to extrusion. Such post-secretion processing may enable MA silk to maintain functionality across environments, facilitating its function as a component of an insect-catching web. PMID:22628213

  6. Modeling the Strength of β-sheet Structures in Silk Crystals and Protein Molecules

    NASA Astrophysics Data System (ADS)

    Grubb, David

    2012-02-01

    The mechanical response of β-sheet structures to a tensile force directed along the axis of one chain can be modeled as an array of elastic springs. The 3-D potential of H-bonds in β-sheets gives a shear stiffness of 4.5Nm-1 and the chain repeat stiffness is 60Nm-1. Nanocrystals >3.5nm long with >=20 H-bonds/chain are the strong component of spider silk. They behave much like macro-scale objects, and two conditions must be met for pull-out failure: (1) the load on the most stressed H-bond exceeds the bond strength. (2) the energy of the system is lower after failure. (1) is the critical condition, and the predicted pull-out load is 3-4 times the H-bond strength. An energetically favorable `stick-slip' process is kinetically forbidden. Arrays within a single molecule such as titin have fewer bonds and can fail at low loads by the `stick-slip' process. The logarithmic rate dependence of failure load observed in AFM is 50pN/decade and the stick-slip prediction is 30pN/decade. Simulations at short times and high loads give slopes >10x higher, matching the prediction for failure at a single bond.

  7. Spider silk fibers spun from soluble recombinant silk produced in mammalian cells.

    PubMed

    Lazaris, Anthoula; Arcidiacono, Steven; Huang, Yue; Zhou, Jiang-Feng; Duguay, Francois; Chretien, Nathalie; Welsh, Elizabeth A; Soares, Jason W; Karatzas, Costas N

    2002-01-18

    Spider silks are protein-based "biopolymer" filaments or threads secreted by specialized epithelial cells as concentrated soluble precursors of highly repetitive primary sequences. Spider dragline silk is a flexible, lightweight fiber of extraordinary strength and toughness comparable to that of synthetic high-performance fibers. We sought to "biomimic" the process of spider silk production by expressing in mammalian cells the dragline silk genes (ADF-3/MaSpII and MaSpI) of two spider species. We produced soluble recombinant (rc)-dragline silk proteins with molecular masses of 60 to 140 kilodaltons. We demonstrated the wet spinning of silk monofilaments spun from a concentrated aqueous solution of soluble rc-spider silk protein (ADF-3; 60 kilodaltons) under modest shear and coagulation conditions. The spun fibers were water insoluble with a fine diameter (10 to 40 micrometers) and exhibited toughness and modulus values comparable to those of native dragline silks but with lower tenacity. Dope solutions with rc-silk protein concentrations >20% and postspinning draw were necessary to achieve improved mechanical properties of the spun fibers. Fiber properties correlated with finer fiber diameter and increased birefringence. PMID:11799236

  8. Functionalized silk biomaterials for wound healing.

    PubMed

    Gil, Eun Seok; Panilaitis, Bruce; Bellas, Evangelia; Kaplan, David L

    2013-01-01

    Silk protein-biomaterial wound dressings with epidermal growth factor (EGF) and silver sulfadiazine were studied with a cutaneous excisional mouse wound model. Three different material designs and two different drug incorporation techniques were studied to compare wound healing responses. Material formats included silk films, lamellar porous silk films and electrospun silk nanofibers, each studied with the silk matrix alone and with drug loading or drug coatings on the silk matrices. Changes in wound size and histological assessments of wound tissues showed that the functionalized silk biomaterial wound dressings increased wound healing rate, including reepithelialization, dermis proliferation, collagen synthesis and reduced scar formation, when compared to air-permeable Tegaderm tape (3M) (- control) and a commercial wound dressing, Tegaderm Hydrocolloid dressing (3M) (+ control). All silk biomaterials were effective for wound healing, while the lamellar porous films and electrospun nanofibers and the incorporation of EGF/silver sulfadiazine, via drug loading or coating, provided the most rapid wound healing responses. This systematic approach to evaluating functionalized silk biomaterial wound dressings demonstrates a useful strategy to select formulations for further study towards new treatment options for chronic wounds. PMID:23184644

  9. Self-assembled semi-crystallinity at parallel β-sheet nanocrystal interfaces in clustered MaSp1 (spider silk) proteins.

    PubMed

    Sintya, Erly; Alam, Parvez

    2016-01-01

    In this communication, we use molecular dynamics methods to model the self-assembly of semi-crystalline domains at β-sheet nanocrystal interfaces in clusters of spider silk (MaSp1) proteins. Our research elucidates that the energetics at interfaces between crystalline and amorphous domains control effectively, the extent to which semi-crystalline domains can form at interfaces. Stability at nanocrystal interfaces is not linearly related to the internal (bulk) stability of the β-sheet nanocrystal. Rather, interfacial stability is found to be highly sensitive to the number of alanine repeat units that make up each sheet. Intriguingly, the most stable interface for the development of semi-crystallinity is built up of polyalanine β-sheets of a length similar to that which is spun naturally in spider dragline silk. PMID:26478322

  10. Post-secretion processing influences spider silk performance

    PubMed Central

    Blamires, Sean J.; Wu, Chung-Lin; Blackledge, Todd A.; Tso, I-Min

    2012-01-01

    Phenotypic variation facilitates adaptations to novel environments. Silk is an example of a highly variable biomaterial. The two-spidroin (MaSp) model suggests that spider major ampullate (MA) silk is composed of two proteins—MaSp1 predominately contains alanine and glycine and forms strength enhancing β-sheet crystals, while MaSp2 contains proline and forms elastic spirals. Nonetheless, mechanical properties can vary in spider silks without congruent amino acid compositional changes. We predicted that post-secretion processing causes variation in the mechanical performance of wild MA silk independent of protein composition or spinning speed across 10 species of spider. We used supercontraction to remove post-secretion effects and compared the mechanics of silk in this ‘ground state’ with wild native silks. Native silk mechanics varied less among species compared with ‘ground state’ silks. Variability in the mechanics of ‘ground state’ silks was associated with proline composition. However, variability in native silks did not. We attribute interspecific similarities in the mechanical properties of native silks, regardless of amino acid compositions, to glandular processes altering molecular alignment of the proteins prior to extrusion. Such post-secretion processing may enable MA silk to maintain functionality across environments, facilitating its function as a component of an insect-catching web. PMID:22628213