Sample records for silk sericin protein

  1. Applications of natural silk protein sericin in biomaterials

    Microsoft Academic Search

    Yu-Qing Zhang

    2002-01-01

    Silk sericin is a natural macromolecular protein derived from silkworm Bombyx mori. During the various stages of producing raw silk and textile, sericin can be recovered for other uses. Also, sericin recovery reduces the environmental impact of silk manufacture. Sericin protein is useful because of its properties. The protein resists oxidation, is antibacterial, UV resistant, and absorbs and releases moisture

  2. Silk gland sericin protein membranes: Fabrication and characterization for potential biotechnological applications

    Microsoft Academic Search

    Biraja C. Dash; Biman B. Mandal; S. C. Kundu

    2009-01-01

    This study describes the potential use of silk gland sericin protein as a biocompatible natural biopolymer in its native form. The membranes were fabricated using native silk sericin protein extracted from middle silk gland of Antheraea mylitta, a non-mulberry tropical tasar silkworm without using any cross-linking agent. The fabricated membranes were biophysically characterized and optimized for cell culture. Silk sericin

  3. Silk protein lithography as a route to fabricate sericin microarchitectures.

    PubMed

    Kurland, Nicholas E; Dey, Tuli; Wang, Congzhou; Kundu, Subhas C; Yadavalli, Vamsi K

    2014-07-01

    Photolithographic fabrication via a "silk sericin photoresist" is used to form precise protein microstructures directly and rapidly on a variety of substrates. High-resolution and fidelity architectures in two and three dimensions with line widths down to 1 ?m are formed. Photo-crosslinked protein structures provide structural iridescence and guide cell adhesion with precise spatial control. PMID:24737390

  4. Consumption of silk protein, sericin elevates intestinal absorption of zinc, iron, magnesium and calcium in rats

    Microsoft Academic Search

    Masahiro Sasaki; Hideyuki Yamada; Norihisa Kato

    2000-01-01

    Rats were examined for the effect of consumption of silk protein, sericin on the intestinal absorption of Zn, Fe, Mg and Ca. Male Wistar rats were fed on the diet containing either 23% egg albumin or 20% egg albumin plus 3% sericin for 12 d. Consumption of sericin elevated the apparent absorption of Zn, Fe, Mg and Ca (41%, 41%,

  5. Immobilization of l-asparaginase on the microparticles of the natural silk sericin protein and its characters

    Microsoft Academic Search

    Yu-Qing Zhang; Mei-Lin Tao; Wei-De Shen; Yu-Zhen Zhou; Yue Ding; Yan Ma; Wen-Lin Zhou

    2004-01-01

    The natural silk sericin recovered from Bombyx mori silk waste by the degumming processing in the high-temperature and high-pressure is a macromolecular protein. Amino acid composition and molecular weight range of the sericin protein as a vector for enzyme immobilization were investigated. The silk sericin protein with different molecular mass from 50 to 200kDa was poorly soluble microparticles with an

  6. Silk Protein Sericin Accelerates Proliferation of Various Mammalian Cells

    Microsoft Academic Search

    Satoshi Terada; Kana Yanagihara; Kozue Kaito; Masao Miki; Masahiro Sasaki; Kazuhisa Tsujimoto; Hideyuki Yamada

    Sericin protein derived from silkworm cocoon was added to the culture of various mammalian cell lines including human hepatoblastoma HepG2, human fibroblasts and so on. The proliferations of all cell lines tested were accelerated in the presence of sericin and its mitogenic activity was comparable to that of bovine serum albumin (BSA), one of the best supplements for the culture

  7. Silk sericin protein of tropical tasar silkworm inhibits UVB-induced apoptosis in human skin keratinocytes

    Microsoft Academic Search

    Rupesh Dash; Mahitosh Mandal; Sudip K. Ghosh; S. C. Kundu

    2008-01-01

    The silk protein sericin has been identified as a potent antioxidant and photoprotective agent against ultraviolet B (UVB)\\u000a irradiation in mouse skin model. In this study, we have investigated the anti-apoptotic effect of sericin in UVB (30 mJ\\/cm2)-irradiated human keratinocytes. Flow cytometry analysis has shown that pre-treatment with sericin inhibits UVB-induced apoptosis.\\u000a The pre-treatment with sericin suppresses bax expression, up-regulates the

  8. The promotion of osseointegration of titanium surfaces by coating with silk protein sericin.

    PubMed

    Nayak, Sunita; Dey, Tuli; Naskar, Deboki; Kundu, Subhas C

    2013-04-01

    A promising strategy to influence the osseointegration process around orthopaedic titanium implants is the immobilization of bioactive molecules. This recruits appropriate interaction between the surface and the tissue by directing cells adhesion, proliferation, differentiation and active matrix remodelling. In this study, we aimed to investigate the functionalization of metallic implant titanium with silk protein sericin. Titanium surface was immobilized with non-mulberry Antheraea mylitta sericin using glutaraldehyde as crosslinker. To analyse combinatorial effects the sericin immobilized titanium was further conjugated with integrin binding peptide sequence Arg-Gly-Asp (RGD) using ethyl (dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide as coupling agents. The surface of sericin immobilized titanium was characterized biophysically. Osteoblast-like cells were cultured on sericin and sericin/RGD functionalized titanium and found to be more viable than those on pristine titanium. The enhanced adhesion, proliferation, and differentiation of osteoblast cells were observed. RT-PCR analysis showed that mRNA expressions of bone sialoprotein, osteocalcin and alkaline phosphatase were upregulated in osteoblast cells cultured on sericin and sericin/RGD immobilized titanium substrates. Additionally, no significant amount of pro-inflammatory cytokines TNF-?, IL-1? and nitric oxide production were recorded when macrophages cells and osteoblast-macrophages co culture cells were grown on sericin immobilized titanium. The findings demonstrate that the sericin immobilized titanium surfaces are potentially useful bioactive coated materials for titanium-based medical implants. PMID:23357374

  9. Sericin separation from silk degumming wastewater

    Microsoft Academic Search

    Pilanee Vaithanomsat; Vichien Kitpreechavanich

    2008-01-01

    This study was conducted to the recovery of sericin protein produced during silk degumming process. Sericin waste solution from conventional degumming process in Thailand contained high BOD (4840mg\\/L), COD (8870mg\\/L) and nitrogen content (0.11%). This indicated protein contamination and high cost for wastewater treatment. To reduce the treatment costs as well as to recover valuable sericin protein, membrane filtration and

  10. Rat islet culture in serum-free medium containing silk protein sericin

    Microsoft Academic Search

    Mitsuhiro Morikawa; Toshihisa Kimura; Makoto Murakami; Kanji Katayama; Satoshi Terada; Akio Yamaguchi

    2009-01-01

    Background  The development of islet cultures is desirable for successful clinical islet transplantation. Fetal bovine serum (FBS) has\\u000a been used as a supplement in islet culture medium, but it may be an unsuitable supplement due recent animal health problems.\\u000a We have evaluated the use of the silk protein, sericin, derived from Bombyx mori as a replacement for FBS in islet culture

  11. Monitoring of inflammatory mediators induced by silk sericin

    Microsoft Academic Search

    Pornanong Aramwit; Sorada Kanokpanont; Wanchai De-Eknamkul; Teerapol Srichana

    2009-01-01

    Silk proteins have been shown to be good candidates for biomedical materials. However, there have been some reports regarding immunological and allergic responses to silk sericin. Our objective was to investigate the inflammatory mediators induced by sericin both in vitro and in vivo. Mouse monocyte and alveolar macrophage cell lines were used for monitoring levels of interleukin (IL)-1? and tumor

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

    Microsoft Academic Search

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

    2003-01-01

    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

  13. An optimized sericin-1 expression system for mass-producing recombinant proteins in the middle silk glands of transgenic silkworms.

    PubMed

    Wang, Feng; Xu, Hanfu; Yuan, Lin; Ma, Sanyuan; Wang, Yuancheng; Duan, Xiaoli; Duan, Jianping; Xiang, Zhonghuai; Xia, Qingyou

    2013-10-01

    The middle silk gland (MSG) of silkworm is thought to be a potential host for mass-producing valuable recombinant proteins. Transgenic MSG expression systems based on the usage of promoter of sericin1 gene (sericin-1 expression system) have been established to produce various recombinant proteins in MSG. However, further modifying the activity of the sericin-1 expression system to yield higher amounts of recombinant proteins is still necessary. In this study, we provide an alternative modification strategy to construct an efficient sericin-1 expression system by using the hr3 enhancer (hr3 CQ) from a Chongqing strain of the Bombyx mori nuclear polyhedrosis virus (BmNPV) and the 3'UTRs of the fibroin heavy chain (Fib-HPA), the fibroin light chain (Fib-LPA), and Sericin1 (Ser1PA) genes. We first analyzed the effects of these DNA elements on expression of luciferase, and found that the combination of hr3 CQ and Ser1PA was most effective to increase the activity of luciferase. Then, hr3 CQ and Ser1PA were used to modify the sericin1 expression system. Transgenic silkworms bearing these modified sericin1 expression vectors were generated by a piggyBac transposon mediated genetic transformation method. Our results showed that mRNA level of DsRed reporter gene in transgenic silkworms containing hr3 CQ and Ser1PA significantly increased by 9 fold to approximately 83 % of that of endogenous sericin1. As the results of that, the production of recombinant RFP increased by 16 fold to 9.5 % (w/w) of cocoon shell weight. We conclude that this modified sericin-1 expression system is efficient and will contribute to the MSG as host to mass produce valuable recombinant proteins. PMID:23435751

  14. 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

  15. Preparation and characterization of sericin powder extracted from silk industry wastewater

    Microsoft Academic Search

    Jin-Hong Wu; Zhang Wang; Shi-Ying Xu

    2007-01-01

    In this study, we developed a new effective technology for the extraction of sericin from silk wastewater. Sericin was extracted with 75% (v\\/v) ethanol to obtain crude powder. The chemical composition of sericin powder, including protein, sugar, ash, and amino acid, was assayed in detail. The molecular weight distribution of sericin was also investigated by gel filtration chromatography, sodium dodecyl

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

    Microsoft Academic Search

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

    2007-01-01

    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, 13C NMR, and XRD,

  17. Fabrication of silk sericin nanofibers from a silk sericin-hope cocoon with electrospinning method.

    PubMed

    Zhang, Xianhua; Khan, Md Majibur Rahman; Yamamoto, Toshio; Tsukada, Masuhiro; Morikawa, Hideaki

    2012-03-01

    In this study, silk sericin nanofibers from sericin hope-silkworm, whose cocoons consist almost exclusively of sericin were successfully prepared by electrospinning method. Scanning electron microscopy (SEM) was used to observe the morphology of the fibers. The effect of spinning conditions, including the concentration of sericin cocoon solution, acceleration voltage, spinning distance and flow rate on the fiber morphologies and the size distribution of sericin nanofibers were examined. The structure and physical properties were also observed by Fourier transform infrared (FT-IR), differential scanning calorimetry (DSC) and thermogravimetric analysis (TG). The optimum conditions for producing finely thinner fibrous sericin nanofibers without beads were the concentration of sericin solution above 6-8 wt%, acceleration voltage ranging from 25 to 32 kV, spinning distance above 9 cm, and flow rate above 0.06 cm min(-1). The mean diameter of as spun sericin fibers varied from 114 to 430 nm at the different spinning conditions. In the as-spun fibers, silk sericin was present in a random coil conformation, while after methanol treatment, the molecular structure of silk sericin was transformed into a ?-sheet containing structure. Sericin hope nanofiber demonstrated thermal degradation at lower temperature than the sericin hope cocoon, which probably due to the randomly coiled rich structure of the sericin hope nanofiber. PMID:22198656

  18. Identification and characterization of a novel sericin gene expressed in the anterior middle silk gland of the silkworm Bombyx mori

    Microsoft Academic Search

    Yoko Takasu; Hiromi Yamada; Toshiki Tamura; Hideki Sezutsu; Kazuei Mita; Kozo Tsubouchi

    2007-01-01

    Sericin is a group of proteins expressed in the middle silk gland that covers the surface of fibroin in the cocoon filament of Bombyx mori. Sericin consists of several serine-rich proteins with different molecular masses. Sericin A is one of the proteins and is produced in the anterior portion of the middle silk gland. To identify the gene coding for

  19. Treatment of silk production wastewaters by membrane processes for sericin recovery

    Microsoft Academic Search

    Goksen Capar; S. Seylan Aygun; M. Rusen Gecit

    2008-01-01

    Sericin protein, although a valuable resource for many industries including cosmetics, pharmaceutical and biomedical, has been discarded as a waste in silk industry, causing environmental pollution. This paper describes determination of a membrane-based process for sericin recovery from cocoon cooking wastewaters (CCW) that will enable value-added utilization of waste sericin. The iso-electric point (pI) of sericin was found as 5–6,

  20. Isolation and bioactivities of a non-sericin component from cocoon shell silk sericin of the silkworm Bombyx mori.

    PubMed

    Wang, Hai-Yan; Wang, Yuan-Jing; Zhou, Li-Xia; Zhu, Lin; Zhang, Yu-Qing

    2012-02-01

    The cocoon shell of the silkworm Bombyx mori consists of silk fibroin fiber (70%) surrounded by a sericin layer made up of sericin (25%) and non-sericin (5%) components. The non-sericin component which consists of carbohydrate, salt, wax, flavonoids and derivatives is often overlooked in applied research into sericin and its hydrolysate. Here, sericin and non-sericin compounds were obtained from the sericin layer of five types of cocoon shell by means of degumming in water followed by extraction and separation in ethanol. These ethanol extracts were found to mainly contain flavonoids and free amino acids possessing scavenging activities of the 2,2-diphenyl -1-picrylhydrazyl (DPPH) free radical and inhibiting activities of tyrosinase, which were much greater than the corresponding activities of the purified sericin proteins. The extracts also strongly inhibited ?-glucosidase while the sericins had no such activity. In particular, the inhibitory activities of the ethanol extract of Daizo cocoons were much greater than those of the other cocoons. The IC(50) values of the Daizo cocoons for DPPH free radicals, tyrosinase, and ?-glucosidase were 170, 27, and 110 ?g mL(-1), respectively. The bioactivities of the non-sericin component were much higher than the activity of sericin alone. In addition, the in vivo test showed preliminarily that the administration of the non-sericin component had effectively resistant activity against streptozocin (STZ) oxidation and that of the purified sericin could also evidently decrease the induction ratio of diabetic mice induced by STZ. Therefore, ethanol extract protocols of the sericin layer of cocoon shells provide a novel stock which, together with sericin protein, has potential uses in functional food, biotechnological and medical applications. PMID:22101964

  1. Anti-inflammatory potential of silk sericin.

    PubMed

    Aramwit, Pornanong; Towiwat, Pasarapa; Srichana, Teerapol

    2013-04-01

    Silk sericin was found to suppress the production of pro-inflammatory cytokines, which are related to the inflammatory reaction. The objectives of this study were to investigate the anti-inflammatory effect of sericin in vivo using the carrageenan-induced rat edema model and changes in the histology of tissues. The effects of sericin on the expression of COX-2 and iNOS were also evaluated. Sericin solutions at 0.004-0.080 mg/mL were applied topically to the top of the hind paw and carrageenan (1.0 mg) was injected subcutaneously to the plantar surface of the right hind paw. Our results indicated that sericin significantly reduced the inflammation in rats' paw compared with the negative control (water and acetone) and its effect at 0.080 mg/mL was only slightly lower than that of 1.0% w/v indomethacin. Similar numbers of polymorphonuclear and macrophage cells were found in rats' tissue treated with indomethacin and sericin solution, while the numbers were significantly higher in their absence. The gene expression results by RT-PCR showed that the COX-2 and iNOS genes were down-regulated in samples treated with sericin in a dose dependent manner. These data indicated that the anti-inflammatory properties of sericin may be partly attributable to the suppression of the COX-2 enzyme and nitric oxide production. PMID:23738464

  2. 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.

  3. 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

  4. Isolation, purification and characterization of silk protein sericin from cocoon peduncles of tropical tasar silkworm, Antheraea mylitta

    Microsoft Academic Search

    Rupesh Dash; Soumen Mukherjee; S. C. Kundu

    2006-01-01

    A high molecular weight water-soluble glue protein, sericin was identified in the cocoon peduncle (a strong thread connecting the cocoons to the branches of the tree with a ring) of the tropical tasar silkworm, Antheraea mylitta. The sericin was isolated by 8M urea containing 1% sodium dodecyl sulfate and ?-mercaptoethenol (2%) or by 1% sodium chloride. The protein was purified

  5. Functional conservation and structural diversification of silk sericins in two moth species.

    PubMed

    Zurovec, Michal; Kludkiewicz, Barbara; Fedic, Robert; Sulitkova, Jitka; Mach, Vaclav; Kucerova, Lucie; Sehnal, Frantisek

    2013-06-10

    Sericins are hydrophilic structural proteins produced by caterpillars in the middle section of silk glands and layered over fibroin proteins secreted in the posterior section. In the process of spinning, fibroins form strong solid filaments, while sericins seal the pair of filaments into a single fiber and glue the fiber into a cocoon. Galleria mellonella and the previously examined Bombyx mori harbor three sericin genes that encode proteins containing long repetitive regions. Galleria sericin genes are similar to each other and the protein repeats are built from short and extremely serine-rich motifs, while Bombyx sericin genes are diversified and encode proteins with long and complex repeats. Developmental changes in sericin properties are controlled at the level of gene expression and splicing. In Galleria , MG-1 sericin is produced throughout larval life until the wandering stage, while the production of MG-2 and MG-3 reaches a peak during cocoon spinning. PMID:23593923

  6. Effects of silk sericin on the proliferation and apoptosis of colon cancer cells.

    PubMed

    Kaewkorn, Waraporn; Limpeanchob, Nanteetip; Tiyaboonchai, Waree; Pongcharoen, Sutatip; Sutheerawattananonda, Manote

    2012-01-01

    Sericin is a silk protein woven from silkworm cocoons (Bombyx mori). In animal model, sericin has been reported to have anti-tumoral action against colon cancer. The mechanisms underlying the activity of sericin against cancer cells are not fully understood. The present study investigated the effects of sericin on human colorectal cancer SW480 cells compared to normal colonic mucosal FHC cells. Since the size of the sericin protein may be important for its activity, two ranges of molecular weight were tested. Sericin was found to decrease SW480 and FHC cell viability. The small sericin had higher anti-proliferative effects than that of the large sericin in both cell types. Increased apoptosis of SW480 cells is associated with increased caspase-3 activity and decreased Bcl-2 expression. The anti-proliferative effect of sericin was accompanied by cell cycle arrest at the S phase. Thus, sericin reduced SW480 cell viability by inducing cell apoptosis via caspase-3 activation and down-regulation of Bcl-2 expression. The present study provides scientific data that support the protective effect of silk sericin against cancer cells of the colon and suggests that this protein may have significant health benefits and could potentially be developed as a dietary supplement for colon cancer prevention. PMID:22688983

  7. Cryopreservation for bovine embryos in serum-free freezing medium containing silk protein sericin.

    PubMed

    Isobe, Tomohiro; Ikebata, Yoshihisa; Onitsuka, Takeshi; Do, Lanh Thi Kim; Sato, Yoko; Taniguchi, Masayasu; Otoi, Takeshige

    2013-10-01

    Because the use of serum in the embryo cryopreservation increases the probability of animal health problems such as bovine spongiform encephalopathy (BSE) and viral infections, this study was conducted to examine the effects of sericin supplementation for serum-free freezing medium on the survival and development of bovine embryos after freezing-thawing and direct transfer to recipients. When in vitro-produced bovine embryos were frozen conventionally in the freezing medium supplemented with various concentrations (0.1%, 0.5%, and 1.0%) of sericin, the percentages of damaged zona pellucida, survival, and development of frozen-thawed embryos were similar to those of embryos frozen in freezing medium supplemented with 0.4% bovine serum albumin (BSA) and 20% fetal bovine serum (FBS) (0.4BSA/20F; control). When in vivo-derived embryos were frozen with 0.4BSA/20F (control), 0.5% sericin +20% FBS (0.5S/20F) or 0.5% sericin (0.5S) and were subsequently transferred directly to recipients, the percentages of recipients with pregnancy and normal calving in the 0.5S/20F group were higher than those in the control group (47.3% vs. 40.1% and 94.6% vs. 87.3%, respectively). Moreover, the percentages of recipients with pregnancy and normal calving (42.2% and 92.4%, respectively) in the 0.5S group were similar with those of other groups. In conclusion, these results indicate that serum-free freezing medium supplemented with sericin is available for the cryopreservation of bovine embryos and that it is beneficial for the elimination of a potential source of biological contamination by serum or BSA. PMID:23850826

  8. Silk sericin–insulin bioconjugates: Synthesis, characterization and biological activity

    Microsoft Academic Search

    Yu-Qing Zhang; Yan Ma; Yun-Yue Xia; Wei-De Shen; Jian-Ping Mao; Ren-Yu Xue

    2006-01-01

    When silk fiber derived from Bombyx mori was subjected to degumming treatments twice in water and subsequent degraded processing in slightly alkaline aqueous solution under high-temperature and high-pressure, the water-soluble silk sericin peptides (SS) with different molecular mass from 10 to 70 kDa were obtained. The sericin peptides could be conjugated covalently with insulin alone with cross-linking reagent glutaraldehyde. The physicochemical

  9. Novel silk sericin\\/gelatin 3-D scaffolds and 2-D films: Fabrication and characterization for potential tissue engineering applications

    Microsoft Academic Search

    Biman B. Mandal; Anjana S. Priya; S. C. Kundu

    2009-01-01

    In this study, we report for the first time the fabrication of novel 3-D sericin\\/gelatin scaffolds and 2-D films using non-mulberry Antheraea mylitta silk cocoon sericin protein. The matrices were fabricated, biophysically characterized and optimized for cell culture applications. Blended sericin\\/gelatin 3-D scaffolds were highly porous with an optimum pore size of 170±20?m. The scaffolds were robust with enhanced mechanical

  10. Dietary silk protein, sericin, improves epidermal hydration with increased levels of filaggrins and free amino acids in NC/Nga mice.

    PubMed

    Kim, Hyunae; Lim, Yu-ji; Park, Ji-Ho; Cho, Yunhi

    2012-11-28

    Epidermal hydration is maintained primarily by natural moisturising factors (NMF), of which free amino acids (AA) are major constituents that are generated by filaggrin degradation. To identify dietary sources that may improve skin dryness of atopic dermatitis (AD), we investigated dietary effects of silk proteins, sericin and fibroin, on epidermal levels of hydration, filaggrins and free AA, as well as PPAR?, peptidylarginine deiminase-3 (PAD3) and caspase-14 proteins involved in filaggrin expression and degradation processes. NC/Nga mice, an animal model of AD, were fed a control diet (group CA: atopic control) or diets with 1 % sericin (group S) or fibroin (group F) for 10 weeks. In group S, epidermal levels of hydration, total filaggrins and total free AA, as well as PPAR?, PAD3 and caspase-14, which were reduced in group CA, were increased to higher or similar levels of a normal control group of BALB/c mice (group C). Furthermore, profilaggrin, a precursor with multiple filaggrin repeats, and three repeat intermediates were increased, while two repeat intermediates and filaggrin were decreased in parallel with increased levels of glutamate and serine, major AA of NMF in group S. Despite increased levels of total filaggrins, total free AA, PPAR? and PAD3, epidermal levels of hydration, glutamate, serine and caspase-14 were not increased, but other minor AA of NMF were highly detected in group F. Dietary sericin improves epidermal hydration in parallel with enhancing profilaggrin expression and degradation into free AA that is coupled with elevated levels of PPAR?, PAD3 and caspase-14 proteins. PMID:22244094

  11. Using FTIR spectroscopy to detect sericin on historic silk

    Microsoft Academic Search

    XiaoMei Zhang; Paul Wyeth

    2010-01-01

    Silks represent some of the most precious ancient and historic textile artefacts in collections worldwide. Their optimum preservation\\u000a demands an appreciation of their characteristics. One important concern, especially with regard to ancient Chinese silks,\\u000a is whether the fabrics have been degummed. Silks with remnant sericin gum coating the fibroin fibres would require different\\u000a conservation protocol. In previous research on aged

  12. 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

  13. Identification of a Novel Type of Silk Protein and Regulation of Its Expression*

    E-print Network

    ?urovec, Michal

    , which are produced in the posterior section of silk glands, and the sericins, which are secreted silk gland sections (MSG) secrete several sericins that provide this thread with sticky coating cord is spun (1). The analysis of silk proteins is difficult because only some of the sericins

  14. Non-mulberry silk sericin/poly (vinyl alcohol) hydrogel matrices for potential biotechnological applications.

    PubMed

    Mandal, Biman B; Ghosh, Borna; Kundu, S C

    2011-08-01

    This study reports a novel biopolymeric matrix fabricated by chemically cross-linking poly (vinyl alcohol) with silk sericin protein obtained from cocoons of the tropical tasar silkworm Antheraea mylitta. Glutaraldehyde was used as a cross-linking agent with hydrochloric acid acting as an initiator. The matrices were biophysically characterized and the cytocompatibility of the matrices was evaluated for their suitability as biomaterials. The surface morphology was assessed using atomic force microscopy while the changes taking place after cross-linking were confirmed by Fourier transform infrared spectroscopy. The enhanced thermal stability of the constructs was assessed by thermogravimetric and differential scanning calorimetry. Fourier transform infrared spectroscopy analysis showed that sericin was chemically cross-linked with poly (vinyl alcohol) using glutaraldehyde. Silk sericin protein demonstrated a favorable effect on animal cell culture by successfully improving the adhering and spreading of cells on the poorly adhering surface of poly (vinyl alcohol). Confocal microscopy revealed cell spreading and actin filament development in sericin/poly (vinyl alcohol) hydrogel matrices. These findings prove the potential of non-mulberry silk sericin/poly (vinyl alcohol) hydrogel matrices to be used as biocompatible and biopolymeric material for tissue-engineering and biotechnological applications. PMID:21549749

  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. Ca2+-induced self-assembly of Bombyx mori silk sericin into a nanofibrous network-like protein matrix for directing controlled nucleation of hydroxylapatite nano-needles

    PubMed Central

    Zhou, Guanshan; Shuai, Yajun; Wang, Jie; Zhu, Liangjun

    2015-01-01

    Bone biomineralization is a well-regulated protein-mediated process where hydroxylapatite (HAP) crystals are nucleated with preferred orientation within self-assembled protein matrix. Mimicking this process is a promising approach to the production of bone-like protein/mineral nanocomposites for bone repair and regeneration. Towards the goal of fabricating such nanocomposites from sericin, a protein spun by Bombyx mori (B.mori) silkworm, and bone mineral HAP, for the first time we investigated the chemical mechanism underpinning the synergistic processes of the conformational change/self-assembly of B.mori sericin (BS) as well as the nucleation of HAP on the resultant self-assembled BS matrix. We found that BS, rich in anionic amino acid residues, could bind Ca2+ ions from the HAP precursor solution through electrostatic attraction. The Ca2+binding drove the conformational change of BS from random coils into ?-sheets and its concomitant self-assembly into interconnected nanofibrous network-like protein matrix, which initiated the nucleation and growth of HAP crystals. HAP crystals directed by the resultant self-assembled BS matrix grew preferentially along their crystallographic c-axis, leading to the formation of HAP nano-needles. The HAP nano-needles in the self-assembled BS matrix were subsequently aggregated into globules, probably driven by the hydrogen bonding between C=O groups of BS and O-H groups of HAP nano-needles. The present work sheds light on the chemical mechanisms of BS self-assembly and the controlled mineralization directed by the self-assembled matrix. We also found that the resultant nanocomposites could promote the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells. Thus our work also generates a biomimetic approach to bone-like silk protein/mineral nanocomposite scaffolds that can find potential applications in bone repair and regeneration. PMID:26029374

  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 ?g/mL can promote the migration of fibroblast L929 cells similar to epidermal growth factor (positive control) at 100 ?g/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. Structure and expression of the silk adhesive protein Ser2 in Bombyx mori Barbara Kludkiewicz a,b

    E-print Network

    ?urovec, Michal

    in revised form 27 November 2009 Accepted 30 November 2009 Keywords: Silkworm Sericin Cocoon Silk gland Repetitive sequence a b s t r a c t Sericins are soluble silk components encoded in Bombyx mori by three later in the last larval instar as the major sericins of cocoon silk. These proteins are, however

  19. Tuning molecular weights of Bombyx mori (B. mori) silk sericin to modify its assembly structures and materials formation.

    PubMed

    Yang, Mingying; Shuai, Yajun; Zhou, Guanshan; Mandal, Namita; Zhu, Liangjun; Mao, Chuanbin

    2014-08-27

    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 ?-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 the scaffolds for tissue engineering. PMID:25050697

  20. Preparation of self-assembled silk sericin nanoparticles

    Microsoft Academic Search

    Kwang Yong Cho; Jae Yu Moon; Yong Woo Lee; Kwang Gill Lee; Joo Hong Yeo; Hae Yong Kweon; Ki Ho Kim; Chong Su Cho

    2003-01-01

    Silk sericin (SS) possessing moisture-retaining property was reacted with activated poly(ethylene glycol) (PEG) to obtain self-assembled SS nanoparticles. The aliphatic and aromatic hydroxyl groups of serine and tyrosine residues as the reaction sites in SS were clarified by amino acid analysis and 1H NMR spectroscopy, respectively. From IR and circular dichroism (CD) measurements, introduction of PEG into SS induced the

  1. Graft Copolymerization of Methyl Methacrylate onto Silk Sericin Initiated by Ceric Ammonium Nitrate

    Microsoft Academic Search

    Yan Song; Yong Jin; Deqing Wei; Jing Sun

    2006-01-01

    A novel redox system, ceric ammonium nitrate (CAN)?silk sericin (SS) was applied to initiate graft copolymerization of methyl methacrylate (MMA) onto silk sericin in an acidic aqueous solution medium. The effects of monomer concentration, initiator concentration, reaction temperature, and time on grafting parameters had been investigated in detail. The results showed that the graft copolymerization occurred even at as a

  2. 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 ?-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

  3. Biomimetic nucleation of hydroxyapatite crystals mediated by Antheraea pernyi silk sericin promotes osteogenic differentiation of human bone marrow derived mesenchymal stem cells.

    PubMed

    Yang, Mingying; Shuai, Yajun; Zhang, Can; Chen, Yuyin; Zhu, Liangjun; Mao, Chuanbin; OuYang, Hongwei

    2014-04-14

    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 ?-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

  4. A germline transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon

    Microsoft Academic Search

    Masahiro Tomita; Rika Hino; Shingo Ogawa; Masashi Iizuka; Takahiro Adachi; Katsuhiko Shimizu; Hisaya Sotoshiro; Katsutoshi Yoshizato

    2007-01-01

    A silk thread of the silkworm, Bombyx mori, is composed of the insoluble inner fibroin and the hydrophilic outer sericin layer, which are synthesized in the posterior\\u000a and middle silk gland (MSG), respectively. This study aimed to develop a novel sericin 1 gene (ser1) promoter-driven recombinant expression system using transgenic silkworms, in which recombinant proteins are synthesized\\u000a in MSG and

  5. 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.

  6. Accelerated healing of full-thickness wounds by genipin-crosslinked silk sericin/PVA scaffolds.

    PubMed

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

    2013-01-01

    Silk sericin has recently been studied for its advantageous biological properties, including its ability to promote wound healing. This study developed a delivery system to accelerate the healing of full-thickness wounds. Three-dimensional scaffolds were fabricated from poly(vinyl alcohol) (PVA), glycerin (as a plasticizer) and genipin (as a crosslinking agent), with or without sericin. The physical and biological properties of the genipin-crosslinked sericin/PVA scaffolds were investigated and compared with those of scaffolds without sericin. The genipin-crosslinked sericin/PVA scaffolds exhibited a higher compressive modulus and greater swelling in water than the scaffolds without sericin. Sericin also exhibited controlled release from the scaffolds. The genipin-crosslinked sericin/PVA scaffolds promoted the attachment and proliferation of L929 mouse fibroblasts. After application to full-thickness rat wounds, the wounds treated with genipin-crosslinked sericin/PVA scaffolds showed a significantly greater reduction in wound size, collagen formation and epithelialization compared with the control scaffolds without sericin but lower numbers of macrophages and multinucleated giant cells. These results indicate that the delivery of sericin from the novel genipin-crosslinked scaffolds efficiently healed the wound. Therefore, these genipin-crosslinked sericin/PVA scaffolds represent a promising candidate for the accelerated healing of full-thickness wounds. PMID:23307034

  7. Mineralization and biocompatibility of Antheraea pernyi (A. pernyi) silk sericin film for potential bone tissue engineering.

    PubMed

    Yang, Mingying; Mandal, Namita; Shuai, Yajun; Zhou, Guanshan; Min, Sijia; Zhu, Liangjun

    2014-01-01

    This study aimed to investigate the mineralization of Antheraea pernyi (A. pernyi) silk sericin. Mineralization of A. pernyi sericin was performed by alternative soaking in calcium and phosphate. The inhibition of precipitation of calcium carbonate and von Kossa staining on A. pernyi sericin were tested, and the corresponding results prove that A. pernyi sericin has Ca binding activity. Scanning electron microscope (SEM) observation shows that spherical crystals could be nucleated on the A. pernyi sericin film. These crystals were confirmed to be hydroxyapatite according to FT-IR and XRD spectra, indicating that A. pernyi sericin is capable of mineralization. In addition, cell adhesion and growth activity assay demonstrate that A. pernyi sericin shows excellent biocompatibility for the growth of MG-63 cells. PMID:24211968

  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. Inhibitory effects of silk protein, sericin on UVB-induced acute damage and tumor promotion by reducing oxidative stress in the skin of hairless mouse

    Microsoft Academic Search

    Siqin Zhaorigetu; Noriyuki Yanaka; Masahiro Sasaki; Hiromitsu Watanabe; Norihisa Kato

    2003-01-01

    This study was conducted to assess protective effect of an antioxidant protein, sericin, on UVB-induced acute damage and tumor promotion in mouse skin. In experiment 1, HR-1 hairless mice were treated with 180 mJ\\/cm2 of ultraviolet B light (UVB) once daily for 1 and 7 days. The treatment for 7 days caused red sunburn lesions of the skin. The intensity

  10. Natural protective glue protein, sericin bioengineered by silkworms: Potential for biomedical and biotechnological applications

    Microsoft Academic Search

    Subhas C. Kundu; Biraja C. Dash; Rupesh Dash; David L. Kaplan

    2008-01-01

    Silk proteins consist of a fibrous core protein, fibroin, and glue proteins called sericins, which envelop the fibroin fiber with successive sticky layers that help in the formation of a cocoon by cementing together the silk fibers. This system is essentially a protein–fiber composite system, with fibers of the high-molecular-weight fibroin proteins surrounded by the glue-like continuous phase of the

  11. Tyrosinase-catalyzed grafting of sericin peptides onto chitosan and production of protein–polysaccharide bioconjugates

    Microsoft Academic Search

    Anna Anghileri; Raija Lantto; Kristiina Kruus; Cristina Arosio; Giuliano Freddi

    2007-01-01

    The capability of Agaricus bisporus tyrosinase to catalyze the oxidation of tyrosine residues of silk sericin was studied under homogeneous reaction conditions, by using sericin peptides purified from industrial wastewater as the substrate. Tyrosinase was able to oxidize about 57% of sericin-bound tyrosine residues. The reaction rate was higher than with silk fibroin, but lower than with other silk-derived model

  12. 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

  13. 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

  14. pH responsive poly amino-acid hydrogels formed via silk sericin templating.

    PubMed

    Kurland, Nicholas E; Ragland, Robert B; Zhang, Aolin; Moustafa, Mahmoud E; Kundu, Subhas C; Yadavalli, Vamsi K

    2014-09-01

    Poly(amino acid) hydrogels have attracted a great deal of attention as biodegradable biomaterials that can limit products of synthetic polymer degradation. Here we report on a stimuli-responsive, porous, composite biomaterial based on the protein templating of the poly(amino acid) hydrogel from poly(aspartic acid) with the silk protein sericin. This low-cost, biocompatible and biodegradable hydrogel demonstrates a greatly increased porosity and improvement in volumetric swelling over networks formed from pure poly(aspartic acid). The swelling capacity measured over a range of pH values surrounding physiological pH 7.0 demonstrates a linear profile, in which hydrogel volume and mass increase to a maximum, with an increase as a function of higher sericin content. In comparison to pure poly(aspartic acid), this demonstrates a nearly 3-fold increase in retention volume at basic pH. The increase in swelling is also demonstrated by the increase in porosity and internal micro-architecture of the hydrogel networks. The biomaterial is then shown to perform well as a scaffold for cells with high mechanical strength and integrity. This protein- and homo poly(amino acid)-based super-swelling hydrogel has applications in drug delivery and tissue engineering as an economical and environmentally friendly biomaterial, in addition to ensuring the species incorporated maintain their biocompatibility during processing. PMID:25073107

  15. Hox transcription factor Antp regulates sericin-1 gene expression in the terminal differentiated silk gland of Bombyx mori.

    PubMed

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

    2014-02-01

    Hox genes are well-known master regulators in developmental morphogenesis along the anteroposterior axis of animals. However, the molecular mechanisms by which Hox proteins regulate their target genes and determine cell fates are not fully understood. The silk gland of Bombyx mori is a tubular tissue divided into several subparts along the anteroposterior axis, and the silk genes are expressed with specific patterns. The sericin-1 gene (ser1) is expressed in the middle silk gland (MSG) with sublocal specificity. Here we show that the Hox protein Antp is a component of the middle silk gland-specific complex, MIC (MSG-intermolt-specific complex), binds to the essential promoter element of ser1, and activates its expression. Ectopic expression of Antp in transgenic silkworms induced the expression of ser1 in the posterior silk gland (PSG), but not in the anterior part of MSG (MSG-A). Correspondingly, a MIC-like complex was formed by the addition of recombinant Antp in extracts from PSG with its cofactors Exd and Hth, but not in extracts from MSG-A. Splicing patterns of ser1 mRNA induced by the ectopic expression of Antp in PSG were almost the same as those in MSG at the fifth instar and altered depending on the induction timing of Antp. Other Hox genes were expressed with sublocal specificity in the silk gland. The Bombyx silk gland might provide a useful system for understanding how Hox proteins select and regulate their target genes. PMID:24333180

  16. 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

  17. 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.

  18. Effect of methyl alcohol on the morphology and conformational characteristics of silk sericin

    Microsoft Academic Search

    KwangGill Lee; HaeYong Kweon; Joo Hong Yeo; Soon Ok Woo; Yong Woo Lee; Chong-Su Cho; Ki Ho Kim; Young Hwan Park

    2003-01-01

    Effects of methyl alcohol on the morphology and conformational characteristics of silk sericin (SS) were studied. Scanning electron microscope showed that morphology of SS lyophilized was dramatically changed from sponge-like structure to spherical fine particle type. X-ray diffraction method, infrared spectroscopy, and differential scanning calorimetry showed that the conformation of SS was random coil structure regardless of the addition of

  19. 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 ?-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 ?asSO 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 ?OH 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.

  20. 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 ?L/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 ?-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 ?-sheet structure. PMID:22935694

  1. Development of ethyl alcohol-precipitated silk sericin/polyvinyl alcohol scaffolds for accelerated healing of full-thickness wounds.

    PubMed

    Siritienthong, Tippawan; Ratanavaraporn, Juthamas; Aramwit, Pornanong

    2012-12-15

    Silk sericin has been recently reported for its advantageous biological properties to promote wound healing. In this study, we established that the ethyl alcohol (EtOH) could be used to precipitate sericin and form the stable sericin/polyvinyl alcohol (PVA) scaffolds without the crosslinking. The sericin/PVA scaffolds were fabricated via freeze-drying and subsequently precipitating in various concentrations of EtOH. The EtOH-precipitated sericin/PVA scaffolds showed denser structure, higher compressive modulus, but lower water swelling ability than the non-precipitated scaffolds. Sericin could be released from the EtOH-precipitated sericin/PVA scaffolds in a sustained manner. After cultured with L929 mouse fibroblasts, the 70 vol% EtOH-precipitated sericin/PVA scaffolds showed the highest potential to promote cell proliferation. After applied to the full-thickness wounds of rats, the 70 vol% EtOH-precipitated sericin/PVA scaffolds showed significantly higher percentage of wound size reduction and higher extent of type III collagen formation and epithelialization, compared with the control scaffolds without sericin. The accelerated wound healing by the 70 vol% EtOH-precipitated sericin/PVA scaffolds was possibly due to (1) the bioactivity of sericin itself to promote wound healing, (2) the sustained release of precipitated sericin from the scaffolds, and (3) the activation and recruitment of wound healing-macrophages by sericin to the wounds. This finding suggested that the EtOH-precipitated sericin/PVA scaffolds were more effective for the wound healing, comparing with the EtOH-precipitated PVA scaffolds without sericin. PMID:23022662

  2. 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.

  3. Isolation and processing of silk proteins for biomedical applications.

    PubMed

    Kundu, Banani; Kurland, Nicholas E; Yadavalli, Vamsi K; Kundu, Subhas C

    2014-09-01

    Silk proteins of silkworms are chiefly composed of core fibroin protein and glycoprotein sericin that glues fibroin. Unique mechanical properties, cyto-compatibility and controllable biodegradability facilitate the use of fibroin in biomedical applications. Sericin serves as additive in cosmetic and food industries, as mitotic factor in cell culture media, anti-cancerous drug, anticoagulant and as biocompatible coating. For all these uses; aqueous solutions of silk proteins are preferred. Therefore, an accurate understanding of extraction procedure of silk proteins from their sources is critical. A number of protocols exist, amongst which it is required to settle a precise and easy one with desired yield and least down-stream processing. Here, we report extraction of proteins employing methods mentioned in literature using cocoons of mulberry and nonmulberry silks. This study reveals sodium carbonate salt-boiling system is the most efficient sericin extraction procedure for all silk variants. Lithium bromide is observed as the effective fibroin dissolution system for mulberry silk cocoons; whereas heterogeneous species-dependent result is obtained in case of nonmulberry species. We further show the effect of common post processing on nanoscale morphology of mulberry silk fibroin films. This knowledge eases the adoption and fabrication of silk biomaterials in devices and therapeutic delivery systems. PMID:24971560

  4. Novel enhancer and promoter elements indispensable for the tissue-specific expression of the sericin-1 gene of the silkworm Bombyx mori

    Microsoft Academic Search

    Shigeharu Takiya; Hiroshige Inoue; Mai Kimoto

    2011-01-01

    Sericins are glue proteins produced specifically in the middle silk gland (MSG) of the silkworm Bombyx mori, while the silk fiber protein, fibroin, is produced in the posterior silk gland (PSG). These silk proteins are expected to be useful biomaterials in medical technology as well as biotechnology. In this study, we analyzed promoter elements of the sericin-1 gene (ser1) in

  5. Transgenic silkworms that weave recombinant proteins into silk cocoons.

    PubMed

    Tomita, Masahiro

    2011-04-01

    As a result of breeding for more than 4,000 years, the silkworm, Bombyx mori, has acquired the ability to synthesize bulk amounts of silk proteins in its silk glands. To utilize this capacity for mass production of useful proteins, transgenic silkworms were generated that synthesized recombinant proteins in the silk gland and secreted them into the silk cocoon. The silk gland is classified into two main regions: the posterior (PSG) and the middle silk gland (MSG). By controlling the expressed regions of the recombinant protein gene in the silk gland, we were able to control the localization of the synthesized protein in the silk thread. Expression in the PSG or MSG led to localization in the insoluble fibroin core or hydrophilic outer sericin layer, respectively. This review focuses on the expression of recombinant protein in the MSG of transgenic silkworms. The recombinant protein secreted in the sericin layer is extractable from the cocoon with only a small amount of endogenous silk protein contamination by soaking the cocoon in mild aqueous solutions. The possibility of utilizing transgenic silkworms as a valuable tool for the mass production of therapeutic and industrially relevant recombinant proteins is discussed. PMID:21184136

  6. Synthesis and properties of silk sericin- g -poly(acrylic acid-co-acrylamide) superabsorbent hydrogel

    Microsoft Academic Search

    Xiaobin Hu

    2011-01-01

    A novel superabsorbent hydrogel has been synthesized with the crosslinking graft copolymerization of acrylic acid (AA) and\\u000a acrylamide onto the chain of silk sericin. Potassium persulfate (KPS)–sodium sulfite (NaHSO3) as redox initiation system and N,N?-methylenebisacrylamide (MBA) as crosslinker were used. The structure of the product characterized by Fourier transform infrared\\u000a absorption spectroscopy and the surface morphology of the hydrogel were

  7. 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 30min 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

  8. 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 enzymatic degradability make these proteins interesting candidates for biomedical applications. PMID:23504416

  9. 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

  10. Enzymatic production of bioactive peptides from sericin recovered from silk industry wastewater

    Microsoft Academic Search

    Jin-Hong Wu; Zhang Wang; Shi-Ying Xu

    2008-01-01

    Bioactive peptides were produced from sericin, a by-product from silk industry wastewater, by using Protease P. The optimal hydrolysis conditions (pH of 8.40, temperature of 43.97°C and the enzyme\\/substrate ratio of 3:100) were estimated by using response surface methodology with a maximum degree of hydrolysis of 14.43% and the minimum IC50 value for the ferrous ion-chelating activity of 0.128mg\\/mL. The

  11. Comparative proteomics reveal diverse functions and dynamic changes of Bombyx mori silk proteins spun from different development stages.

    PubMed

    Dong, Zhaoming; Zhao, Ping; Wang, Chen; Zhang, Yan; Chen, Jianping; Wang, Xin; Lin, Ying; Xia, Qingyou

    2013-11-01

    Silkworms (Bombyx mori) produce massive amounts of silk proteins to make cocoons during the final stages of larval development. Although the major components, fibroin and sericin, have been the focus for a long time, few researchers have realized the complexity of the silk proteome. We collected seven kinds of silk fibers spun by silkworm larvae at different developmental stages: the silks spun by new hatched larvae, second instar day 0 larvae, third instar day 0 larvae, fourth instar day 0 larvae, and fourth instar molting larvae, the scaffold silk used to attach the cocoon to the substrate and the cocoon silk. Analysis by liquid chromatography-tandem mass spectrometry identified 500 proteins from the seven silks. In addition to the expected fibroins, sericins, and some known protease inhibitors, we also identified further protease inhibitors, enzymes, proteins of unknown function, and other proteins. Unsurprisingly, our quantitative results showed fibroins and sericins were the most abundant proteins in all seven silks. Except for fibroins and sericins, protease inhibitors, enzymes, and proteins of unknown function were more abundant than other proteins. We found significant change in silk protein compositions through development, being consistent with their different biological functions and complicated formation. PMID:24093152

  12. 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

  13. Proteomic analysis of sericin in Bombyx mori cocoons

    Microsoft Academic Search

    Xin Du; Jun Li; Yuyin Chen

    2011-01-01

    Cocoon sericin plays an important role in the reeling of silk and serves as a valuable biomaterial in the field of biomedicine,\\u000a skincare, and food industries; however, knowledge about cocoon sericin proteins has been limited. For a comprehensive study\\u000a on sericin, cocoons of eight varieties of silkworm of different geographic origin and with varied cocoon color were analyzed\\u000a utilizing proteomics

  14. Silk Protein Sericin Improves Mammalian Cell Culture

    Microsoft Academic Search

    Satoshi Terada; Naoki Takada; Kazuaki Itoh; Takuya Saitoh; Masahiro Sasaki; Hideyuki Yamada

    Mammalian cell cultures generally require supplementation with fetal bovine serum (FBS), or its replacement, into the culture\\u000a media. Sera contain various unidentified and unknown factors and the risk of infections, including bovine spongiform encephalopathy\\u000a (BSE), is of serious concern. Therefore, the supplementation of sera into culture media is a major obstacle for purification\\u000a to recover cell products and this limits

  15. Preliminary characterization of genipin-cross-linked silk sericin/poly(vinyl alcohol) films as two-dimensional wound dressings for the healing of superficial wounds.

    PubMed

    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

  16. 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

    2014-12-01

    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

  17. Application of sericin to modify textile supports

    Microsoft Academic Search

    I. Belhaj Khalifa; N. Ladhari; M. Touay

    2012-01-01

    In this study, we fixed silk sericin in fabrics as a finishing product. First of all, we have extracted sericin from silkworm cocoons in order to obtain the optimum yield of sericin that is not denatured. For this reason, we have searched the optimum parameters for a favorite sericin extraction. Then, the sericin extracted was fixed onto wool and cotton

  18. Application of sericin to modify textile supports

    Microsoft Academic Search

    I. Belhaj Khalifa; N. Ladhari; M. Touay

    2011-01-01

    In this study, we fixed silk sericin in fabrics as a finishing product. First of all, we have extracted sericin from silkworm cocoons in order to obtain the optimum yield of sericin that is not denatured. For this reason, we have searched the optimum parameters for a favorite sericin extraction. Then, the sericin extracted was fixed onto wool and cotton

  19. 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

  20. 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.

  1. Construction of Silk Fiber Core in Lepidoptera Frantisek Sehnal* and Michal Zurovec

    E-print Network

    ?urovec, Michal

    thread, it has long been known that the raw silk filament contains a sericin coating soluble in hot as a product of the posterior region of the gland, whereas sericin is produced in the middle region that also associated with two small proteins. The sericin coating, which functions as glue during cocoon construction

  2. 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

  3. 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.

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

    PubMed Central

    2014-01-01

    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. PMID:24533676

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

    PubMed

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

    2014-01-01

    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. PMID:24533676

  6. Silk-based biomaterials

    Microsoft Academic Search

    Gregory H. Altman; Frank Diaz; Caroline Jakuba; Tara Calabro; Rebecca L. Horan; Jingsong Chen; Helen Lu; John Richmond; David L. Kaplan

    2003-01-01

    Silk from the silkworm, Bombyx mori, has been used as biomedical suture material for centuries. The unique mechanical properties of these fibers provided important clinical repair options for many applications. During the past 20 years, some biocompatibility problems have been reported for silkworm silk; however, contamination from residual sericin (glue-like proteins) was the likely cause. More recent studies with well-defined

  7. Biomaterials 24 (2003) 401416 Silk-based biomaterials

    E-print Network

    Lu, Helen H.

    2003-01-01

    biocompatibility problems have been reported for silkworm silk; however, contamination from residual sericin (glue Science Ltd. All rights reserved. Keywords: Silk; Sericin; Fibroin; Foreign body response; Suture; Tissue

  8. Potential of 2D crosslinked sericin membranes with improved biostability for skin tissue engineering.

    PubMed

    Nayak, Sunita; Talukdar, Sarmistha; Kundu, Subhas C

    2012-03-01

    Silk sericin protein is a natural, hydrophilic, macromolecular glycoprotein mainly synthesized in the middle silk gland of the silkworm. It constitutes 25-30% of the silk cocoon. Sericin proteins have antioxidant, antimicrobial, UV-resistant properties, promote wound healing and support cell proliferation even in serum-free media. Most of the sericin is discarded as waste in silk processing industries. This study aims at improving the mechanical strength and stability of sericin extracted from the silk cocoons during processing and utilize it as a biocompatible natural biopolymer in biomedical applications. Crosslinked sericin membranes, from the cocoon of non-mulberry tropical silkworm, Antheraea mylitta, were prepared using gluteraldehyde as the crosslinking agent. Physical and structural characteristics of the membranes were analyzed using scanning electron microscopy, atomic force microscopy, Fourier transform infrared spectroscopy and X-ray diffraction along with swelling and degradation studies. The secondary structure of the membrane indicates that crosslinking provides a more integrated structure that significantly improves the stability and mechanical strength of the membranes. In vitro cytocompatibility of the membranes was evaluated by MTT assay and cell cycle analysis of feline fibroblast cells. The adherence, growth and proliferation patterns of cells on membranes were assessed by confocal microscopy, which demonstrated that the latter is non-toxic and supports cell growth. Cell cycle analyses indicate cytocompatibility with normal cell cycle pattern. This study reveals that silk sericin protein can be used as a biocompatible natural biopolymer for various applications in the biomedical field. PMID:22327482

  9. Stem cell-based tissue engineering with silk biomaterials

    Microsoft Academic Search

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

    2006-01-01

    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

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

    PubMed

    Takechi, Tayori; Wada, Ritsuko; Fukuda, Tsubasa; Harada, Kazuki; Takamura, Hitoshi

    2014-05-01

    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

  11. 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

  12. A novel poly(?-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(?-glutamic acid)/silk sericin (?-PGA/SS) hydrogel has been developed and used as wound dressing. The physical and chemical properties of the ?-PGA/SS gels were systemically investigated. Furthermore, these ?-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 ?-PGA/SS hydrogel might have a wide range of applications in biomedical and clinical areas. PMID:25579954

  13. Preparation of silk sericin beads using LiCl\\/DMSO solvent and their potential as a drug carrier for oral administration

    Microsoft Academic Search

    Hanjin Oh; Ji Young Lee; Arum Kim; Chang Seok Ki; Jong Wook Kim; Young Hwan Park; Ki Hoon Lee

    2007-01-01

    Silk sericin (SS) was fabricated into beads using LiCl\\/DMSO solution as a solvent. Up to 30 % (w\\/v) of SS could be dissolved\\u000a within 3 hours, and the shape of solidified SS depends on the concentration of SS. Ethanol was the best coagulant among alcohols,\\u000a making beads with suitable mechanical strength for further application. SS beads swell more at a

  14. 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

  15. 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

  16. Insect silk contains both a Kunitz-type and a unique Kazal-type proteinase inhibitor

    E-print Network

    ?urovec, Michal

    Republic Insect silk is made up of structural fibrous (fibroins) and sticky (sericins) proteinsInsect silk contains both a Kunitz-type and a unique Kazal-type proteinase inhibitor Xavier Nirmala inhibit bacterial and fungal proteinases (subtilisin, proteinase K and pronase). These `silk protei- nase

  17. 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. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 76-83, 2014. PMID:23606462

  18. Dietary sericin enhances epidermal levels of glucosylceramides and ceramides with up-regulating protein expressions of glucosylceramide synthase, ?-glucocerebrosidase and acidic sphingomyelinase in NC/Nga mice.

    PubMed

    Kim, Hyunae; Lee, Jongsun; Cho, Yunhi

    2012-12-01

    We have previously reported that dietary sericin improves epidermal dryness with the increased total Ceramide (Cer) in NC/Nga mice, an animal model of atopic dermatitis (AD). In this study, we hypothesized that the increased level of total Cer induced by dietary sericin would be related to the altered metabolism of glucosylceramide (GlcCer) and sphingomyelin (SM), major precursors of Cer generation. NC/Nga mice were fed a control diet (group CA: atopic control) or diets with 1% silk protein, either sericin (group S) or fibroin (group F) for 10 weeks. In the epidermis of group CA, total Cer (including Cer1, 2, 3/4 and 6) and all GlcCer species were reduced; these levels in group S were increased to levels similar to or higher than in the normal control group of BALB/c mice (group C). In addition, the protein expressions, but not mRNA expressions, of GlcCer synthase, ?-glucocerebrosidase, and acidic sphingomyelinase, enzymes for GlcCer synthesis, GlcCer and SM hydrolysis, respectively, were highly increased in group S. The epidermal levels of total Cer (including Cer2, 3/4, and 6) and all GlcCer species and of these enzyme proteins in group F were lower than in group S. Notably, alterations in total SM, SM1, SM3, and SM synthase 1, which were increased in group CA, were not significant between groups S and F. Cer5 and SM2 were not altered among groups. Dietary sericin enhanced the epidermal levels of all GlcCer and most Cer species with up-regulating protein expressions of GlcCer synthase, ?-glucocerebrosidase, and acidic sphingomyelinase. PMID:23244541

  19. Thermal crystallization mechanism of silk fibroin protein

    Microsoft Academic Search

    Xiao Hu

    2009-01-01

    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:

  20. A Sericin-Derived Peptide Protects Sf9 Insect Cells from Death Caused by Acute Serum Deprivation

    Microsoft Academic Search

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

    2005-01-01

    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. 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 stage before crystallization. (3) The beta-sheet crystallization kinetics in silk fibroin protein were measured using X-ray, FTIR and heat flow, and the structure reveals the formation mechanism of the silk crystal network. Avrami kinetics theories, which were established for studies of synthetic polymer crystal growth, were for the first time extended to investigate protein self-assembly in multiblock silk fibroin samples. The Avrami exponent, n, was close to two for all methods, indicating formation of beta sheet crystals in silk proteins is different from the 3-D spherulitic crystal growth found in most synthetic homopolymers. A microphase separation pattern after chymotrypsin enzyme biodegradation was shown in the protein structures using scanning electron microscopy. A model was then used to explain the crystallization of silk fibroin protein by analogy to block copolymers. (4) The effects of metal ions during the crystallization of silk fibroin was investigated using thermal analysis. Advanced thermal analysis methods were used to analyze the thermal protein-metallic ion interactions in silk fibroin proteins. Results show that K+ and Ca2+ metallic salts play different roles in silk fibroin proteins, which either reduce (K+) or increase (Ca2+ ) the glass transition (Tg) of pure silk protein and affect the thermal stability of this structure.

  2. Generation of a transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon: Production of recombinant human serum albumin

    Microsoft Academic Search

    Shingo Ogawa; Masahiro Tomita; Katsuhiko Shimizu; Katsutoshi Yoshizato

    2007-01-01

    In this study we produced germline transgenic silkworms that spin cocoons containing recombinant human serum albumin (rHSA) in the sericin layer. A piggyBac-based transformation vector was constructed that carried HSA cDNA driven by sericin-1 gene promoter, viral enhancer hr3, and gene encoding viral trans-activator IE1. Isolated silk glands were bombarded with the vector and transplanted into host larvae. Three days

  3. Inhibition of the binding of MSG-intermolt-specific complex, MIC, to the sericin-1 gene promoter and sericin-1 gene expression by POU-M1/SGF-3.

    PubMed

    Kimoto, Mai; Kitagawa, Tsuyuki; Kobayashi, Isao; Nakata, Tomohiro; Kuroiwa, Asato; Takiya, Shigeharu

    2012-11-01

    The sericin-1 gene encoding a glue protein is expressed in the middle silk gland (MSG) of the silkworm, Bombyx mori. A member of the class III POU domain transcription factors, POU-M1, was cloned as the factor bound to the SC site of the sericin-1 promoter and has been proposed to be a positive transcription factor. In this study, we analyzed the expression pattern of the POU-M1 gene in fourth and fifth instars in comparison with the pattern of the sericin-1 gene. The POU-M1 gene was expressed strongly in the region anterior to the sericin-1-expressing portion of the silk gland at both feeding stages. As the sericin-1-expressing region expands from the posterior to middle portions of the MSG in the fifth instar, the POU-M1-expressing region retreated from the middle to anterior portion. Introduction of the expression vector of POU-M1 into the silk glands by gene gun technology repressed promoter activity of the sericin-1 gene, suggesting that POU-M1 regulates the sericin-1 gene negatively. An in vitro binding assay showed that POU-M1 bound not only to the SC site but also to other promoter elements newly detected in vivo. Another spatiotemporal specific factor MIC binds to these elements, and POU-M1 competed with MIC to bind at the -70 site essential for promoter activity. These results suggest that POU-M1 is involved in restricting the anterior boundary of the sericin-1-expressing region in the silk gland by inhibiting the binding of the transcriptional activator to the promoter elements. PMID:23070540

  4. Transgenic silkworms that weave recombinant proteins into silk cocoons

    Microsoft Academic Search

    Masahiro Tomita

    2011-01-01

    As a result of breeding for more than 4,000 years, the silkworm, Bombyx mori, has acquired the ability to synthesize bulk amounts of silk proteins in its silk glands. To utilize this capacity for mass\\u000a production of useful proteins, transgenic silkworms were generated that synthesized recombinant proteins in the silk gland\\u000a and secreted them into the silk cocoon. The silk gland

  5. 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

  6. 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

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

    E-print Network

    Buehler, Markus J.

    A Materiomics Approach to Spider Silk: Protein Molecules to Webs ANNA TARAKANOVA1 and MARKUS J-assembling silk biopolymers have been extensively studied experimentally and in computa- tional investigations length scales in silk, ranging from atomistic models of protein constituents to the spider web

  8. Degradation of sericin (degumming) of Persian silk by ultrasound and enzymes as a cleaner and environmentally friendly process

    Microsoft Academic Search

    Niyaz Mohammad Mahmoodi; Mokhtar Arami; Firoozmehr Mazaheri; Shahram Rahimi

    2010-01-01

    Degumming is a surface modification process of natural fibers such as silk. In this paper, the feasibility of degumming with ultrasonic, ultrasonic–soap, and ultrasonic–enzyme (alcalase, savinase, and mixtures of these enzymes) processes as cleaner and environmentally friendly surface modification techniques of Persian silk were investigated. The effectiveness of parameters such as sonication time, soap, ultrasound–enzyme, enzyme concentration, degumming time and enzymes

  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. High-toughness silk produced by a transgenic silkworm expressing spider (Araneus ventricosus) dragline silk protein.

    PubMed

    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

  11. Silks produced by insect labial glands

    PubMed Central

    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 ?-sheets interrupted with other structures such as ?-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

  12. Transgenic silkworms (Bombyx mori) produce recombinant spider dragline silk in cocoons.

    PubMed

    Wen, Hongxiu; Lan, Xiqian; Zhang, Yuansong; Zhao, Tianfu; Wang, Yujun; Kajiura, Zenta; Nakagaki, Masao

    2010-04-01

    Spider dragline silk is a unique fibrous protein with a combination of tensile strength and elasticity, but the isolation of large amounts of silk from spiders is not feasible. In this study, we generated germline-transgenic silkworms (Bombyx mori) that spun cocoons containing recombinant spider silk. A piggyBac-based transformation vector was constructed that carried spider dragline silk (MaSp1) cDNA driven by the sericin 1 promoter. Silkworm eggs were injected with the vector, producing transgenic silkworms displaying DsRed fluorescence in their eyes. Genotyping analysis confirmed the integration of the MaSp1 gene into the genome of the transgenic silkworms, and silk protein analysis revealed its expression and secretion in the cocoon. Compared with wild-type silk, the recombinant silk displayed a higher tensile strength and elasticity. The results indicate the potential for producing recombinant spider silk in transgenic B. mori. PMID:19633923

  13. 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

  14. 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

  15. Functional expression of a Bombyx mori cocoonase: potential application for silk degumming.

    PubMed

    Rodbumrer, Prangprapai; Arthan, Dumrongkiet; Uyen, Utai; Yuvaniyama, Jirundon; Svasti, Jisnuson; Wongsaengchantra, Pramvadee Y

    2012-12-01

    Cocoon, a shelter for larva development to silk moth, contains the fibrous protein fibroin, which is coated by the globular protein sericin. Emergence of the silk moth requires the action of cocoonase, a protease secreted by the pupa. The full-length prococoonase cDNA, with 780 bp open reading frame encoding 260 amino acids, was cloned by reverse transcription from total RNA of the head of 6-day-old Thai-silk Bombyx mori pupa. Only the gene fragment lacking the propeptide encoding sequence was successfully expressed in Pichia pastoris, yielding an extracellularly active cocoonase. The recombinant cocoonase was purified to homogeneity by 80% ammonium-sulfate fractionation and CM-Sepharose chromatography, and its internal peptide sequences were analyzed by nano liquid chromatography-mass spectrometry/mass spectrometry. This monomeric protein has native molecular weight of 26 kDa by gel exclusion analysis and 25 kDa subunit size by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme hydrolyses sericin but does not hydrolyse fibroin, as shown by radial diffusion on thin-layer enzyme assay (RD-TEA). Scanning electron microscopy showed that purified recombinant cocoonase could remove sericin from natural silk completely in 24 h, without damaging fibroin, using only 1 immobilized sericin unit (ISU) of enzyme as determined by RD-TEA. Natural cocoonase isolated from B. mori pupa could also digest sericin effectively, but required more enzymes (2 ISU) and longer time (48 h). In comparison, a commercial enzyme, alcalase, with the same activity not only showed less complete digestion of sericin but also caused damage of fibroin. These results suggest that recombinant B. mori cocoonase is potentially useful for silk degumming. PMID:23169343

  16. Evidence for an immune function of lepidopteran silk proteins.

    PubMed

    Korayem, Ahmed M; Hauling, Thomas; Lesch, Christine; Fabbri, Marco; Lindgren, Malin; Loseva, Olga; Schmidt, Otto; Dushay, Mitchell S; Theopold, Ulrich

    2007-01-12

    Hemolymph coagulation stops bleeding and protects against infection. Clotting factors include both proteins that are conserved during evolution as well as more divergent proteins in different species. Here we show that several silk proteins also appear in the clot of the greater wax moth Galleria mellonella. RT-PCR analysis reveals that silk proteins are expressed in immune tissues and induced upon wounding in both Galleria and Ephestia kuehniella, a second pyralid moth. Our results support the idea that silk proteins were co-opted for immunity and coagulation during evolution. PMID:17126296

  17. Tubuliform silk protein: A protein with unique molecular characteristics and mechanical properties in the spider silk fibroin family

    Microsoft Academic Search

    M. Tian; R. V. Lewis

    2006-01-01

    Orb–web weavers can produce up to six different types of silk and a glue for various functions. Tubuliform silk is unique\\u000a among them due to its distinct amino acid composition, specific time of production, and atypical mechanical properties. To\\u000a study the protein composing this silk, tubuliform gland cDNA libraries were constructed from three orb–weaving spiders Argiope\\u000a aurantia, Araneus gemmoides, and

  18. Genetic variants of the Bombyx mori silkworm encoding sericin proteins of different lengths

    Microsoft Academic Search

    Takuma Gamo

    1982-01-01

    A variant sericin polypeptide originally found by acid gel electrophoresis in the Nd-s mutant strain of the silkworm, Bombyx mori, has been analyzed genetically. The vriant polypeptide (called S-2v) is encoded by a gene which behaves as a codominant allele of the gene encoding the standard S-2 sericin polypeptide. Linkage analysis locates these alleles at 0.0 map unit on chromosome

  19. Production of synthetic spider dragline silk protein in Pichia pastoris

    Microsoft Academic Search

    S. R. Fahnestock; L. A. Bedzyk

    1997-01-01

    The methylotrophic yeast Pichia pastoris was tested as a host for the production of long, repetitive protein polymers. Synthetic genes for a designed analog of a\\u000a spider dragline silk protein were readily expressed at high levels under control of the methanol-inducible AOX1 promoter. Transformants containing multiple gene copies produced elevated levels of silk protein, but of a variety of altered

  20. Laser Ablation of Silk Protein (Fibroin) Films

    NASA Astrophysics Data System (ADS)

    Tsuboi, Yasuyuki; Adachi, Hisanori; Yamada, Kazushi; Miyasaka, Hiroshi; Itaya, Akira

    2002-07-01

    Fibroin is the main protein component of silk and is expected to have functional applications in bioelectronics and medicine. We investigated nanosecond (ns) pulsed laser ablation of solid fibroin films with/without a dye as a photosensitizer. Laser lights at 248 nm and 532/355/351 nm excited the peptide bond of fibroin and the dye, respectively. The neat film irradiated at 248 nm was scarcely accessible to etching and swelling, and instead, a microscopic pattern (structure) was formed. In contrast, for ablation of the doped film at 532/355/351 nm, we found marked swelling (height ˜500 ?m) and deep etching (depth ˜10 ?m) on the irradiated surfaces. The dye-photosensitized ablation was brought about by a photothermal mechanism, whereas ablation of neat films may be induced by another process, such as a photochemical one. The ablation processes are discussed in terms of the properties of fibroin and the mode of excitation.

  1. Sericin accelerates the production of hyaluronan and decreases the incidence of polyspermy fertilization in bovine oocytes during in vitro maturation.

    PubMed

    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

  2. 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

  3. Engineered spider silk protein-based composites for drug delivery.

    PubMed

    Hardy, John G; Leal-Egaña, Aldo; Scheibel, Thomas R

    2013-10-01

    Silk protein-based materials are promising materials for the delivery of drugs and other active ingredients, due to their processability, biocompatibility, and biodegradability. The preparation of films composed of an engineered spider silk protein (eADF4(C16)) in combination with either a polyester (polycaprolactone) or a polyurethane (pellethane), and their physical properties are described. The release profiles are affected by both the film composition and the presence of enzymes, and release can be observed over a period of several weeks. Such silk-based composites have potential as drug eluting biocompatible coatings or implantable devices. PMID:23881554

  4. 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

  5. Coatings and films made of silk proteins.

    PubMed

    Borkner, Christian B; Elsner, Martina B; Scheibel, Thomas

    2014-09-24

    Silks are a class of proteinaceous materials produced by arthropods for various purposes. Spider dragline silk is known for its outstanding mechanical properties, and it shows high biocompatibility, good biodegradability, and a lack of immunogenicity and allergenicity. The silk produced by the mulberry silkworm B. mori has been used as a textile fiber and in medical devices for a long time. Here, recent progress in the processing of different silk materials into highly tailored isotropic and anisotropic coatings for biomedical applications such as tissue engineering, cell adhesion, and implant coatings as well as for optics and biosensors is reviewed. PMID:25004395

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

    Microsoft Academic Search

    Hajime Mori; Masuhiro Tsukada

    2000-01-01

    The interest in silk fibroin morphology and structure have increased due to its attractiveness for bio-related applications. Silk fibers have been used as sutures for a long time in the surgical field, due to the biocompatibility of silk fibroin fibers with human living tissue. In addition, it has been demonstrated that silk can be used as a substrate for enzyme

  7. Spider silks: recombinant synthesis, assembly, spinning, and engineering of synthetic proteins

    PubMed Central

    Scheibel, Thomas

    2004-01-01

    Since thousands of years humans have utilized insect silks for their own benefit and comfort. The most famous example is the use of reeled silkworm silk from Bombyx mori to produce textiles. In contrast, despite the more promising properties of their silk, spiders have not been domesticated for large-scale or even industrial applications, since farming the spiders is not commercially viable due to their highly territorial and cannibalistic nature. Before spider silks can be copied or mimicked, not only the sequence of the underlying proteins but also their functions have to be resolved. Several attempts to recombinantly produce spider silks or spider silk mimics in various expression hosts have been reported previously. A new protein engineering approach, which combines synthetic repetitive silk sequences with authentic silk domains, reveals proteins that closely resemble silk proteins and that can be produced at high yields, which provides a basis for cost-efficient large scale production of spider silk-like proteins. PMID:15546497

  8. Controlled hydrogel formation of a recombinant spider silk protein.

    PubMed

    Schacht, Kristin; Scheibel, Thomas

    2011-07-11

    Due to their biocompatibility, biodegradability, and low immunogenicity, recombinant spider silk proteins have a high potential for a variety of applications when processed into morphologies such as films, capsules, beads, or hydrogels. Here, hydrogels made of the engineered and recombinantly produced spider silk protein eADF4(C16) were analyzed in detail. It has previously been shown that eADF4(C16) nanofibrils self-assemble by a mechanism of nucleation-aggregation, providing the basis of silk hydrogels. We focused on establishing a reproducible gelation process by employing different protein concentrations, chemical crosslinking, and functionalization of eADF4(C16) with fluorescein. Fluorescein strongly influenced assembly as well as the properties of the hydrogels, such as pore sizes and mechanical behavior, possibly due to its interference with packing of silk nanofibrils during hydrogel formation. PMID:21612299

  9. Carbon dioxide induced silk protein gelation for biomedical applications.

    PubMed

    Floren, Michael L; Spilimbergo, Sara; Motta, Antonella; Migliaresi, Claudio

    2012-07-01

    We present a novel method to fabricate silk fibroin hydrogels using high pressure carbon dioxide (CO(2)) as a volatile acid without the need for chemical cross-linking agents or surfactants. The simple and efficient recovery of CO(2) post processing results in a remarkably clean production method offering tremendous benefit toward materials processing for biomedical applications. Further, with this novel technique we reveal that silk protein gelation can be considerably expedited under high pressure CO(2) with the formation of extensive ?-sheet structures and stable hydrogels at processing times less than 2 h. We report a significant influence of the high pressure CO(2) processing environment on silk hydrogel physical properties such as porosity, sample homogeneity, swelling behavior and compressive properties. Microstructural analysis revealed improved porosity and homogeneous composition among high pressure CO(2) specimens in comparison to the less porous and heterogeneous structures of the citric acid control gels. The swelling ratios of silk hydrogels prepared under high pressure CO(2) were significantly reduced compared to the citric acid control gels, which we attribute to enhanced physical cross-linking. Mechanical properties were found to increase significantly for the silk hydrogels prepared under high pressure CO(2), with a 2- and 3-fold increase in the compressive modulus of the 2 and 4 wt % silk hydrogels over the control gels, respectively. We adopted a semiempirical theoretical model to elucidate the mechanism of silk protein gelation demonstrated here. Mechanistically, the rate of silk protein gelation is believed to be a function of the kinetics of solution acidification from absorbed CO(2) and potentially accelerated by high pressure effects. The attractive features of the method described here include the acceleration of stable silk hydrogel formation, free of residual mineral acids or chemical cross-linkers, reducing processing complexity, and avoiding adverse biological responses, while providing direct manipulation of hydrogel physical properties for tailoring toward specific biomedical applications. PMID:22657735

  10. 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

  11. Tubuliform silk protein: A protein with unique molecular characteristics and mechanical properties in the spider silk fibroin family

    NASA Astrophysics Data System (ADS)

    Tian, M.; Lewis, R. V.

    2006-02-01

    Orb-web weavers can produce up to six different types of silk and a glue for various functions. Tubuliform silk is unique among them due to its distinct amino acid composition, specific time of production, and atypical mechanical properties. To study the protein composing this silk, tubuliform gland cDNA libraries were constructed from three orb-weaving spiders Argiope aurantia, Araneus gemmoides, and Nephila clavipes. Amino acid composition comparison between the predicted tubuliform silk protein sequence (TuSp1) and the corresponding gland protein confirms that TuSp1 is the major component in tubuliform gland in three spiders. Sequence analysis suggests that TuSp1 shares no significant similarity with its paralogues, while it has conserved sequence motifs with the most primitive spider, Euagrus chisoseus silk protein. The presence of large side-chain amino acids in TuSp1 sequence is consistent with the frustrated ?-sheet crystalline structure of tubuliform silk observed in transmission electron microscopy. Repeat unit comparison within species as well as among three spiders exhibits high sequence conservation. Parsimony analysis based on carboxy terminal sequence shows that Argiope and Araneus are more closely related than either is to Nephila which is consistent with phylogenetic analysis based on morphological evidence.

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

    PubMed

    Keten, Sinan; Buehler, Markus J

    2010-12-01

    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 3?-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. Production of spider silk proteins in tobacco and potato

    Microsoft Academic Search

    Jürgen Scheller; Karl-Heinz Gührs; Frank Grosse; Udo Conrad

    2001-01-01

    Spider dragline silk is a proteinaceous fiber with remarkable mechanical properties that make it attractive for technical applications. Unfortunately, the material cannot be obtained in large quantities from spiders. We have therefore generated transgenic tobacco and potato plants that express remarkable amounts of recombinant Nephila clavipes dragline proteins. Using a gene synthesis approach, the recombinant proteins exhibit homologies of >90%

  14. Silk-tropoelastin protein films for nerve guidance.

    PubMed

    White, James D; Wang, Siran; Weiss, Anthony S; Kaplan, David L

    2015-03-01

    Peripheral nerve regeneration may be enhanced through the use of biodegradable thin film biomaterials as highly tuned inner nerve conduit liners. Dorsal root ganglion neuron and Schwann cell responses were studied on protein films comprising silk fibroin blended with recombinant human tropoelastin protein. Tropoelastin significantly improved neurite extension and enhanced Schwann cell process length and cell area, while the silk provided a robust biomaterial template. Silk-tropoelastin blends afforded a 2.4-fold increase in neurite extension, when compared to silk films coated with poly-d-lysine. When patterned by drying on grooved polydimethylsiloxane (3.5 ?m groove width, 0.5 ?m groove depth), these protein blends induced both neurite and Schwann cell process alignment. Neurons were functional as assessed using patch-clamping, and displayed action potentials similar to those cultured on poly(lysine)-coated glass. Taken together, silk-tropoelastin films offer useful biomaterial interfacial platforms for nerve cell control, which can be considered for neurite guidance, disease models for neuropathies and surgical peripheral nerve repairs. PMID:25481743

  15. Sericin-fixed filk fiber as an immobilization support of enzyme

    Microsoft Academic Search

    Ki Hoon Lee; Gyung Don Kang; Bong Seob Shin; Young Hwan Park

    2005-01-01

    In this study, we attempted to evaluate a novel use of sericin-fixed silk fiber (SFx) as an immobilization support of enzyme.\\u000a Sericin was fixed on the silk fiber using glutaraldehyde as a fixation reagent. After 6 hours of fixation, the degree of fixation\\u000a increases linearly with linear decrease of the amount of bound ?-chymotrypsin (CT). This suggests that the increase

  16. Interfacial rheological properties of recombinant spider-silk proteins Cyrille Vzya

    E-print Network

    Bausch, Andreas

    Interfacial rheological properties of recombinant spider-silk proteins Cyrille Vézya E27 Lehrstuhl to characterize the adsorption kinetics and the rheology of spider-silk films formed at an oil water interface. The high surface activity of the engineered spider-silk proteins results in a fast formation of highly

  17. Novel enhancer and promoter elements indispensable for the tissue-specific expression of the sericin-1 gene of the silkworm Bombyx mori.

    PubMed

    Takiya, Shigeharu; Inoue, Hiroshige; Kimoto, Mai

    2011-08-01

    Sericins are glue proteins produced specifically in the middle silk gland (MSG) of the silkworm Bombyx mori, while the silk fiber protein, fibroin, is produced in the posterior silk gland (PSG). These silk proteins are expected to be useful biomaterials in medical technology as well as biotechnology. In this study, we analyzed promoter elements of the sericin-1 gene (ser1) in vivo by introducing reporter constructs into silk glands via gene gun technology. The region from -1602 to +47 was sufficient to induce MSG-specific expression. The 5' deletion mutants showed a three-step decrease in promoter activity with the key sequences located between -1362 and -1250, -201 and -116, and -115 and -37. We detected a tissue- and stage-specific factor complex (MSG-intermolt-specific complex: MIC) bound to the sequence elements around the -1350, -320, -180, and -70 regions. A mutation in the -70 region, which inhibits MIC-binding, diminished almost all promoter activity, while another mutation that did not inhibit MIC-binding showed no effect on promoter activity. The results suggest that the binding of MIC to the above elements is intrinsic for the spatiotemporal specificity of ser1 in vivo. PMID:21496486

  18. Protein families, natural history and biotechnological aspects of spider silk.

    PubMed

    Bittencourt, D; Oliveira, P F; Prosdocimi, F; Rech, E L

    2012-01-01

    Spiders are exceptionally diverse and abundant organisms in terrestrial ecosystems and their evolutionary success is certainly related to their capacity to produce different types of silks during their life cycle, making a specialized use on each of them. Presenting particularly tandemly arranged amino acid repeats, silk proteins (spidroins) have mechanical properties superior to most synthetic or natural high-performance fibers, which makes them very promising for biotechnology industry, with putative applications in the production of new biomaterials. During the evolution of spider species, complex behaviors of web production and usage have been coupled with anatomical specialization of spinning glands. Spiders retaining ancestral characters, such as the ones belonging to the Mygalomorph group, present simpler sorts of webs used mainly to build burrows and egg sacs, and their silks are produced by globular undifferentiated spinning glands. In contrast, Araneomorphae spiders have a complex spinning apparatus, presenting up to seven morphologically distinct glands, capable to produce a more complex set of silk polymers with different degrees of rigidness and elasticity associated with distinct behaviors. Aiming to provide a discussion involving a number of spider silks' biological aspects, in this review we present descriptions of members from each family of spidroin identified from five spider species of the Brazilian biodiversity, and an evolutionary study of them in correlation with the anatomical specialization of glands and spider's spinning behaviors. Due to the biotechnological importance of spider silks for the production of new biomaterials, we also discuss about the new possible technical and biomedical applications of spider silks and the current status of it. PMID:22911606

  19. Folding behavior of four silks of giant honey bee reflects the evolutionary conservation of aculeate silk proteins.

    PubMed

    Maitip, Jakkrawut; Trueman, Holly E; Kaehler, Benjamin D; Huttley, Gavin A; Chantawannakul, Panuwan; Sutherland, Tara D

    2015-04-01

    Multiple gene duplication events in the precursor of the Aculeata (bees, ants, hornets) gave rise to four silk genes. Whilst these homologs encode proteins with similar amino acid composition and coiled coil structure, the retention of all four homologs implies they each are important. In this study we identified, produced and characterized the four silk proteins from Apis dorsata, the giant Asian honeybee. The proteins were readily purified, allowing us to investigate the folding behavior of solutions of individual proteins in comparison to mixtures of all four proteins at concentrations where they assemble into their native coiled coil structure. In contrast to solutions of any one protein type, solutions of a mixture of the four proteins formed coiled coils that were stable against dilution and detergent denaturation. The results are consistent with the formation of a heteromeric coiled coil protein complex. The mechanism of silk protein coiled coil formation and evolution is discussed in light of these results. PMID:25712559

  20. 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

  1. Biosynthesis and Applications of Silk?like and Collagen?like Proteins

    Microsoft Academic Search

    Jia Huang; C. Wong Po Foo; David L. Kaplan

    2007-01-01

    Genetic engineering provides a useful strategy to generate native and designed fibrous proteins, such as silks and collagens, for fundamental and applied studies. Recent studies on biosynthesized spider and silkworm silks, and human collagens, are reviewed with a focus on genes, expression systems and biomaterial applications. Genetically engineered silks and collagens can be exploited for fundamental biophysical studies of the

  2. 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.

  3. Sericin protects against diabetes-induced injuries in sciatic nerve and related nerve cells.

    PubMed

    Song, Chengjun; Yang, Zhenjun; Zhong, Meirong; Chen, Zhihong

    2013-02-25

    Sericin from discarded silkworm cocoons of silk reeling has been used in different fields, such as cosmetology, skin care, nutrition, and oncology. The present study established a rat model of type 2 diabetes by consecutive intraperitoneal injections of low-dose (25 mg/kg) streptozotocin. After intragastrical perfusion of sericin for 35 days, blood glucose levels significantly declined, and the expression of neurofilament protein in the sciatic nerve and nerve growth factor in L4-6 spinal ganglion and anterior horn cells significantly increased. However, the expression of neuropeptide Y in spinal ganglion and anterior horn cells significantly decreased in model rats. These findings indicate that sericin protected the sciatic nerve and related nerve cells against injury in a rat type 2 diabetic model by upregulating the expression of neurofilament protein in the sciatic nerve and nerve growth factor in spinal ganglion and anterior horn cells, and downregulating the expression of neuropeptide Y in spinal ganglion and anterior horn cells. PMID:25206693

  4. 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.

  5. Structure of a protein superfiber: spider dragline silk.

    PubMed Central

    Xu, M; Lewis, R V

    1990-01-01

    Spider major ampullate (dragline) silk is an extracellular fibrous protein with unique characteristics of strength and elasticity. The silk fiber has been proposed to consist of pseudocrystalline regions of antiparallel beta-sheet interspersed with elastic amorphous segments. The repetitive sequence of a fibroin protein from major ampullate silk of the spider Nephila clavipes was determined from a partial cDNA clone. The repeating unit is a maximum of 34 amino acids long and is not rigidly conserved. The repeat unit is composed of three different segments: (i) a 6 amino acid segment that is conserved in sequence but has deletions of 3 or 6 amino acids in many of the repeats; (ii) a 13 amino acid segment dominated by a polyalanine sequence of 5-7 residues; (iii) a 15 amino acid, highly conserved segment. The latter is predominantly a Gly-Gly-Xaa repeat with Xaa being alanine, tyrosine, leucine, or glutamine. The codon usage for this DNA is highly selective, avoiding the use of cytosine or guanine in the third position. A model for the physical properties of fiber formation, strength, and elasticity, based on this repetitive protein sequence, is presented. PMID:2402494

  6. 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

  7. 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

  8. 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 ?m diameter and 10 ?m length) of silk biopolymer were observed upon irradiation with large fluences.

  9. Molecular Evolution of Lepidopteran Silk Proteins: Insights from the Ghost Moth, Hepialus californicus

    Microsoft Academic Search

    Matthew A. Collin; Kazuei Mita; Frantisek Sehnal; Cheryl Y. Hayashi

    2010-01-01

    Silk production has independently evolved in numerous arthropod lineages, such as Lepidoptera, the moths and butterflies.\\u000a Lepidopteran larvae (caterpillars) synthesize silk proteins in modified salivary glands and spin silk fibers into protective\\u000a tunnels, escape lines, and pupation cocoons. Molecular sequence data for these proteins are necessary to determine critical\\u000a features of their function and evolution. To this end, we constructed

  10. Synthesis and characterization of cell-adhesive silk-like proteins constructed from the sequences of Anaphe silk fibroin and fibronectin.

    PubMed

    Tanaka, Chikako; Asakura, Tetsuo

    2009-04-13

    New silk-like recombinant proteins, [(AAG)(6)ASTGRGDSPAAS](n) and [(AG)(9)ASTGRGDSPAAS](n), with high cell adhesive activities were designed and produced from E. coli. These are recombinant proteins with characteristic sequences from the silk fibroin of a wild silkworm, Anaphe , and the cell adhesive region, including the sequence RGD derived from fibronectin. They showed higher cell adhesion activity than the parent protein, Anaphe silk fibroin without the RGD sequence. In addition, the activities were very similar to that of collagen, which acted as a positive control. Thus, it is demonstrated that the primary structure of Anaphe silk fibroin, which is composed largely of alanine and glycine residues, can be used as a platform for the basic structures of silk-like cell adhesive proteins. The structural characterization of the silk-like recombinant proteins was performed with (13)C CP/MAS NMR. PMID:19236090

  11. Sericin, a protein derived from silkworms, accelerates the proliferation of several mammalian cell lines including a hybridoma

    Microsoft Academic Search

    Satoshi Terada; Taeko Nishimura; Masahiro Sasaki; Hideyuki Yamada; Masao Miki

    2002-01-01

    Sericin, a constituent of the silkworm cocoon, was added to the culture of four mammalian cell lines: murine hybridoma 2E3-O,human\\u000a hepatoblastoma HepG2, human epithelial HeLa and human embryonal kidney 293 cells. The proliferation of all cell lineswas accelerated\\u000a in the presence of sericin. The hybridoma cellline was further studied. The 2E3-O cell line was so well adapted to serum-free\\u000a medium

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

    Microsoft Academic Search

    Yan Liu; Lian Yu; Xiuyang Guo; Tingqing Guo; Shengpeng Wang; Changde Lu

    2006-01-01

    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 209bp DNA sequence upstream of the transcriptional start site (?586 to ?378) is found to be

  13. A Novel Model System for Design of Biomaterials Based on Recombinant Analogs of Spider Silk Proteins

    Microsoft Academic Search

    Vladimir G. Bogush; Olga S. Sokolova; Lyubov I. Davydova; Dmitri V. Klinov; Konstantin V. Sidoruk; Natalya G. Esipova; Tatyana V. Neretina; Igor A. Orchanskyi; Vsevolod Yu Makeev; Vladimir G. Tumanyan; Konstantin V. Shaitan; Vladimir G. Debabov; Mikhail P. Kirpichnikov

    2009-01-01

    Spider dragline silk possesses impressive mechanical and biochemical properties. It is synthesized by a couple of major ampullate\\u000a glands in spiders and comprises of two major structural proteins—spidroins 1 and 2. The relationship between structure and\\u000a mechanical properties of spider silk is not well understood. Here, we modeled the complete process of the spider silk assembly\\u000a using two new recombinant

  14. Hierarchical structures made of proteins. The complex architecture of spider webs and their constituent silk proteins.

    PubMed

    Heim, Markus; Römer, Lin; Scheibel, Thomas

    2010-01-01

    Biopolymers fulfil a variety of different functions in nature. They conduct various processes inside and outside cells and organisms, with a functionality ranging from storage of information to stabilization, protection, shaping, transport, cellular division, or movement of whole organisms. Within the plethora of biopolymers, the most sophisticated group is of proteinaceous origin: the cytoskeleton of a cell is made of protein filaments that aid in pivotal processes like intracellular transport, movement, and cell division; geckos use a distinct arrangement of keratin-like filaments on their toes which enable them to walk up smooth surfaces, such as walls, and even upside down across ceilings; and spiders spin silks that are extra-corporally used for protection of offspring and construction of complex prey traps. The following tutorial review describes the hierarchical organization of protein fibers, using spider dragline silk as an example. The properties of a dragline silk thread originate from the strictly controlled assembly of the underlying protein chains. The assembly procedure leads to protein fibers showing a complex hierarchical organization comprising three different structural phases. This structural organization is responsible for the outstanding mechanical properties of individual fibers, which out-compete even those of high-performance artificial fibers like Kevlar. Web-weaving spiders produce, in addition to dragline silk, other silks with distinct properties, based on slightly variant constituent proteins--a feature that allows construction of highly sophisticated spider webs with well designed architectures and with optimal mechanical properties for catching prey. PMID:20023846

  15. 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

  16. Biological responses to spider silk-antibiotic fusion protein

    PubMed Central

    Gomes, Sílvia; Gallego-Llamas, Jabier; Leonor, Isabel B.; Mano, João F.; Reis, Rui L.; Kaplan, David L.

    2011-01-01

    The development of a new generation of multifunctional biomaterials is a continual goal for the field of materials science. The in vivo functional behaviour of a new fusion protein that combines the mechanical properties of spider silk with the antimicrobial properties of hepcidin was addressed in this study. This new chimeric protein, termed 6mer+hepcidin, fuses spider dragline consensus sequences (6mer) and the antimicrobial peptide hepcidin as we have recently described, with retention of bactericidal activity and low cytotoxicity. In the present study mice subcutaneous implants were studied to access the in vivo biological response to the 6mer+hepcidin, which were compared with controls of silk alone (6mer), poly-lactic-glycolic-acid (PLGA) films and empty defects. Along with visual observations, flow cytometry and histology analyses were used to determine the number and type of inflammatory cells at the implantation site. The results show a mild to low inflammatory reaction to the implanted materials and no apparent differences between the 6mer+hepcidin films and the other experimental controls, demonstrating that the new fusion protein has good in vivo biocompatibility, while maintaining antibiotic function. PMID:22514077

  17. Purification and cytotoxicity of tag-free bioengineered spider silk proteins.

    PubMed

    Dams-Kozlowska, Hanna; Majer, Agnieszka; Tomasiewicz, Paulina; Lozinska, Jolanta; Kaplan, David L; Mackiewicz, Andrzej

    2013-02-01

    Bioengineered spider silk-like proteins can serve as biomaterials for various biomedical applications. These proteins can be assembled in several morphological forms such as films, microcapsules, spheres, fibers, gels, and scaffolds. However, crucial points for recombinant spider silks for human use are toxicity and immunogenicity. To assess this issue, two bioengineered spider silk proteins composed of different numbers of repetitive motifs of the consensus repeats from spidroin-1 from Nephila clavipes (15X and 6X) were cloned and expressed in Escherichia coli. The proteins were free of tag sequence and were purified using two methods based on (1) thermal and (2) organic acid resistance of the spider silks. The soluble spider silk proteins were not cytotoxic and did not activate macrophages over a wide range of concentrations, except when present at the highest concentration. Films made of the different silk variants supported the growth of the cells. Based on these data, and as the biodegradation rate of silk is very slow, the bioengineered spider silks are presumed safe biomaterials for biomedical applications. PMID:22865581

  18. Purification and cytotoxcicity of tag-free bioengineered spider silk proteins3

    PubMed Central

    Dams-Kozlowska, Hanna; Majer, Agnieszka; Tomasiewicz, Paulina; Lozinska, Jolanta; Kaplan, David L.; Mackiewicz, Andrzej

    2012-01-01

    Bioengineered spider silk-like proteins can serve as biomaterials for various biomedical applications. These proteins can be assembled in several morphological forms such as films, microcapsules, spheres, fibers, gels and scaffolds. However, crucial points for recombinant spider silks for human use are toxicity and immunogenicity. To assess this issue two bioengineered spider silk proteins composed of different numbers of repetitive motifs of the consensus repeats from spidroin-1 from Nephila clavipes (15X and 6X) were cloned and expressed in E. coli. The proteins were free of tag-sequence and were purified using two methods based on (i) thermal and (ii) organic acid resistance of the spider silks. The soluble spider silk proteins were not cytotoxic and did not activate macrophages over a wide range of concentrations, except when present at the highest concentration. Films made of the different silk variants supported the growth of the cells. Based on these data, and since the biodegradation rate of silk is very slow, the bioengineered spider silks are presumed safe biomaterials for biomedical applications. PMID:22865581

  19. Silk-elastinlike protein polymers for matrix-mediated cancer gene therapy

    Microsoft Academic Search

    Joshua A. Gustafson; Hamidreza Ghandehari

    2010-01-01

    Silk-elastinlike protein polymers (SELPs) are recombinant polymers designed from silk fibroin and mammalian elastin amino acid repeats. These are versatile materials that have been examined as controlled release systems for intratumoral gene delivery. SELP hydrogels comprise monodisperse and tunable polymers that have the capability to control and localize the release and expression of plasmid DNA and viruses. This article reviews

  20. 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

  1. 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

  2. Enhancing analysis of cells and proteins by fluorescence imaging on silk-based biomaterials: modulating the autofluorescence of silk.

    PubMed

    Neo, Puay Yong; Tan, Daryl Jian-An; Shi, Pujiang; Toh, Siew Lok; Goh, James Cho-Hong

    2015-02-01

    Silk is a versatile and established biomaterial for various tissue engineering purposes. However, it also exhibits strong autofluorescence signals-thereby hindering fluorescence imaging analysis of cells and proteins on silk-derived biomaterials. Sudan Black B (SB) is a lysochrome dye commonly used to stain lipids in histology. It has also been reported to be able to quench autofluorescence of tissues in histology and has been tested on artificial biomedical polymers in recent years. It was hypothesized that SB would exert similar quenching effects on silk, modulating the autofluorescence signals, and thereby enabling improved imaging analysis of cells and molecules of interests. The quenching effect of SB on the intrinsic fluorescence properties of silk and on commercial fluorescent dyes were first investigated in this study. SB was then incorporated into typical fluorescence-based staining protocols to study its effectiveness in improving fluorescence-based imaging of the cells and proteins residing with the silk-based biomaterials. Silk processed into various forms of biomaterials (e.g., films, sponges, fibers, and electrospun mats) was seeded with cells and cultured in vitro. At sacrificial time points, specimens were harvested, fixed, and prepared for fluorescence staining. SB, available commercially as a powder, was dissolved in 70% ethanol (0.3% [w/v]) to form staining solutions. SB treatment was introduced at the last step of typical immunofluorescence staining protocols for 15-120?min. For actin staining protocols by phalloidin toxin, SB staining solutions were added before and after permeabilization with Triton-X for 15-30?min. Results showed that ideal SB treatment duration is about 15?min. Apart from being able to suppress the autofluorescence of silk, this treatment duration was also not too long to adversely affect the fluorescent labeling probes used. The relative improvement brought about by SB treatment was most evident in the blue and green emission wavelengths compared with the red emission wavelength. This study has showed that the use of SB is a cost and time effective approach to enhance fluorescence-based imaging analyses of cell-seeded silk biomaterials, which otherwise would have been hindered by the unmodulated autofluorescence signals. PMID:25050876

  3. Protein composition correlates with the mechanical properties of spider ( Argiope trifasciata ) dragline silk.

    PubMed

    Marhabaie, Mohammad; Leeper, Thomas C; Blackledge, Todd A

    2014-01-13

    We investigated the natural variation in silk composition and mechanical performance of the orb-weaving spider Argiope trifasciata at multiple spatial and temporal scales in order to assess how protein composition contributes to the remarkable material properties of spider dragline silk. Major ampullate silk in orb-weaving spiders consists predominantly of two proteins (MaSp1 and MaSp2) with divergent amino acid compositions and functionally different microstructures. Adjusting the expression of these two proteins therefore provides spiders with a simple mechanism to alter the material properties of their silk. We first assessed the reliability and precision of the Waters AccQ-Tag amino acid composition analysis kit for determining the amino acid composition of small quantities of spider silk. We then tested how protein composition varied within single draglines, across draglines spun by the same spider on different days, and finally between spiders. Then, we correlated chemical composition with the material properties of dragline silk. Overall, we found that the chemical composition of major ampullate silk was in general homogeneous among individuals of the same population. Variation in chemical composition was not detectable within silk spun by a single spider on a single day. However, we found that variation within a single spider's silk across different days could, in rare instances, be greater than variation among individual spiders. Most of the variation in silk composition in our investigation resulted from a small number of outliers (three out of sixteen individuals) with a recent history of stress, suggesting stress affects silk production process in orb web spiders. Based on reported sequences for MaSp genes, we developed a gene expression model showing the covariation of the most abundant amino acids in major ampullate silk. Our gene expression model supports that dragline silk composition was mostly determined by the relative abundance of MaSp1 and MaSp2. Finally, we showed that silk composition (especially proline content) strongly correlated with some measures of mechanical performance, particularly how much fibers shrunk during supercontraction as well as their breaking strains. Our findings suggest that spiders are able to change the relative expression rates of different MaSp genes to produce silk fibers with different chemical compositions, and hence, different material properties. PMID:24313814

  4. 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 (?) 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

  5. Quantitative Correlation Between the Protein Primary Sequences and Secondary Structures in Spider Dragline Silks

    PubMed Central

    Jenkins, Janelle E.; Creager, Melinda S.; Lewis, Randolph V.; Holland, Gregory P.; Yarger, Jeffery L.

    2009-01-01

    Synthetic spider silk holds great potential for use in various applications spanning medical uses to ultra lightweight armor, however producing synthetic fibers with mechanical properties comparable to natural spider silk has eluded the scientific community. Natural dragline spider silks are commonly made from proteins that contain highly repetitive amino acid motifs, adopting an array of secondary structures. Before further advances can be made in the production of synthetic fibers based on spider silk proteins, it is imperative to know the percentage of each amino acid in the protein that forms a specific secondary structure. Linking these percentages to the primary amino acid sequence of the protein will establish a structural foundation for synthetic silk. In this study, Nuclear Magnetic Resonance (NMR) techniques are used to quantify the percentage of Ala, Gly, and Ser that form both ?-sheet and helical secondary structures. The fraction of these three amino acids and their secondary structure are quantitatively correlated to the primary amino acid sequence for the proteins that comprise major and minor ampullate silk from the Nephila clavipes spider providing a blueprint for synthetic spider silks. PMID:20000730

  6. Insect Biochemistry and Molecular Biology 28 (1998) 121130 Evolution of repetitive proteins: spider silks from Nephila clavipes

    E-print Network

    Beckwitt, Richard

    1998-01-01

    have obtained two variants of the Spidroin 1 (NCF-1) silk gene sequence from Nephila clavipes. One Elsevier Science Ltd. All rights reserved. Keywords: Spider silk; Spidroin; Fibroin; Repetitive protein published partial cDNA sequences of two silk genes (Spidroin 1 and 2, also called NCF-1 and NCF-2) from

  7. 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

  8. Review the role of terminal domains during storage and assembly of spider silk proteins.

    PubMed

    Eisoldt, Lukas; Thamm, Christopher; Scheibel, Thomas

    2012-06-01

    Fibrous proteins in nature fulfill a wide variety of functions in different structures ranging from cellular scaffolds to very resilient structures like tendons and even extra-corporal fibers such as silks in spider webs or silkworm cocoons. Despite their different origins and sequence varieties many of these fibrous proteins share a common building principle: they consist of a large repetitive core domain flanked by relatively small non-repetitive terminal domains. Amongst protein fibers, spider dragline silk shows prominent mechanical properties that exceed those of man-made fibers like Kevlar. Spider silk fibers assemble in a spinning process allowing the transformation from an aqueous solution into a solid fiber within milliseconds. Here, we highlight the role of the non-repetitive terminal domains of spider dragline silk proteins during storage in the gland and initiation of the fiber assembly process. PMID:22057429

  9. Preparation of sericin microparticles by electrohydrodynamic spraying and their application in drug delivery

    Microsoft Academic Search

    Hanjin Oh; Moo Kon Kim; Ki Hoon Lee

    2011-01-01

    Sericin (SS) is a protein present in silkworm cocoons that is normally discarded in the degumming process. Recycling sericin\\u000a can expand the library of natural polymers used in the materials field. In this study, sericin microparticles were prepared\\u000a using an electrohydrodynamic (EHD) spraying technique. The sizes of the SS microparticles, which can be controlled by the\\u000a concentration of the dope

  10. 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

  11. Properties and antityrosinase activity of sericin from various extraction methods.

    PubMed

    Aramwit, Pornanong; Damrongsakkul, Siriporn; Kanokpanont, Sorada; Srichana, Teerapol

    2010-02-01

    The present study investigated the chemical properties and antityrosinase activities of SS (silk sericin) extracted from different Thai silk strains via various extraction methods. Different silk strains contain distinct SS with various amino acid compositions, which are significantly influenced by the extraction method used. Urea extraction of SS was the only method that provided clearly distinguishable bands and had the most significant impact on SS conformation as illustrated by FTIR (Fourier-transform infrared) spectra. The use of urea or either acidic or alkaline chemicals in the extraction process also influenced SS thermal behaviour. With regard to biological activity, SS extracted using urea exhibited the highest antityrosinase activity, whereas alkali-degraded SS showed no inhibition of mushroom tyrosinase. Pigments, primarily flavonoids and carotenoids from silk cocoons, were also found to enhance tyrosinase inhibition of SS. PMID:20055756

  12. Brown widow (Latrodectus geometricus) major ampullate silk protein and its material properties.

    PubMed

    Motriuk-Smith, Dagmara; Lewis, Randolph V

    2004-01-01

    Major ampullate (dragline) silk is the main web component as well as the silk that spiders use for a lifeline when they fall. This silk has a breaking stress of 4.6 GPa, which is similar to that of Kevlar. The majority of the previous mechanical testing studies involved the major ampullate silk from orb-weaving spiders. To date, there have been no reports on dragline silk mechanical properties from a cob-weaver, brown widow Latrodectus geometricus. L. geometricus dragline was found to be composed of MaSp1, MaSp2, and MaSp-like proteins all of which have highly conserved amino acid motifs, especially the GGX, GA and poly A for MaSp1 and GPGGX and poly A for MaSp2. These sequences are the same as those found in the silks of orb-weaving spiders. To determine if protein sequences influence the material properties of the silk, mechanical testing was performed on single strands of silk fibers from adult female L. geometricus spiders. The 3 cm long silk fibers were tested for breaking stress and strain with a MTS Synergie 100 mechanical testing system using a 50 g load cell with the cross-head speed set at 10 mm/min. The breaking stress and strain were measured for 20 replicate samples and averaged. The values of 0.83 +/- 0.19 GPa for stress and 0.14 +/- 0.06 for strain shows that brown widow dragline is weaker than the orb-weaving spiders. PMID:15133936

  13. 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 ?-helical in solution to increasingly ?-sheet in fibers. PMID:23209681

  14. 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

  15. Bio-inspired Silicification of Silica-binding Peptide-Silk Protein Chimeras: Comparison of Chemically and Genetically Produced Proteins

    PubMed Central

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

    2012-01-01

    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, 25 equivalents 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 to 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 which can be used to prepare a range of materials for biomedical and sensor based applications. PMID:22229696

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

    E-print Network

    Keten, Sinan

    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 ...

  17. Light can transform the secondary structure of silk protein

    NASA Astrophysics Data System (ADS)

    Tsuboi, Y.; Ikejiri, T.; Shiga, S.; Yamada, K.; Itaya, A.

    Fibroin is the main component of silk and is expected to be used as a novel functional material in medicine and bioelectronics. The main secondary structures of this protein are of the random-coil and the ?-sheet types. In this study, we carried out laser-induced transformation of the secondary structure, from the random-coil type to the ?-sheettype, in solid fibroin films. We prepared two types of fibroin films with the random-coil structure. One is a fibroin film doped with a dye as a photosensitizer with a small amount (1 wt%), and the other is a neat fibroin film. The former was excited at 532 nm and the latter was excited at 266 nm. Irradiations were carried out with fluences much lower than each ablation threshold. The excitation of the dye at 532 nm did not affect the secondary structure of the random-coil type. By contrast, 266-nm laser irradiation, which excites tryptophan (an amino-acid residue) involved in fibroin, created the ?-sheetdomain in the film. The structural transformation was revealed by infrared absorption spectroscopy and atomic force microscopy.

  18. 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.

  19. Resistance to Toxoplasma gondii infection in mice treated with silk protein by enhanced immune responses.

    PubMed

    Moon, Joung-Ho; Pyo, Kyoung-Ho; Jung, Bong-Kwang; Chun, Hyang Sook; Chai, Jong-Yil; Shin, Eun-Hee

    2011-09-01

    This study investigated whether elevated host immune capacity can inhibit T. gondii infection. For this purpose, we used silk protein extracted from Bombyx mori cocoons as a natural supplement to augment immune capacity. After silk protein administration to BALB/c mice for 6 weeks, ratios of T lymphocytes (CD4(+) and CD8(+) T-cells) and splenocyte proliferative capacities in response to Con A or T. gondii lysate antigen (TLA) were increased. Of various cytokines, which regulate immune systems, Th1 cytokines, such as IFN-?, IL-2, and IL-12, were obviously increased in splenocyte primary cell cultures. Furthermore, the survival of T. gondii (RH strain)-infected mice increased from 2 days to 5 or more days. In a state of immunosuppression induced by methylprednisolone acetate, silk protein-administered mice were resistant to reduction in T-lymphocyte (CD4(+) and CD8(+) T-cells) numbers and the splenocyte proliferative capacity induced by Con A or TLA with a statistical significance. Taken together, our results suggest that silk protein augments immune capacity in mice and the increased cellular immunity by silk protein administration increases host protection against acute T. gondii infection. PMID:22072834

  20. Production and characterization of a silk-like hybrid protein, based on the polyalanine region of Samia cynthia ricini silk fibroin and a cell adhesive region derived from fibronectin

    Microsoft Academic Search

    Tetsuo Asakura; Chikako Tanaka; Mingying Yang; Juming Yao; Masato Kurokawa

    2004-01-01

    There are a variety of silkworms and silk fibroins produced by them. Silks have many inherent suitable properties for biomaterials. In this paper, a novel silk-like hybrid protein, [DGG(A)12GGAASTGRGDSPAAS]5, which consists of polyalanine region of silk fibroin from a wild silkworm, Samia cynthia ricini, and cell adhesive region including Arg-Gly-Asp (RGD) sequence, derived from fibronectin, was designed and produced. The

  1. Mulberry non-engineered silk gland protein vis-à-vis silk cocoon protein engineered by silkworms as biomaterial matrices

    Microsoft Academic Search

    Joydip Kundu; Moumita Dewan; Sarani Ghoshal; S. C. Kundu

    2008-01-01

    Silk fibroin from silk gland of Bombyx mori 5th instar larvae was utilized to fabricate films, which may find possible applications as two-dimensional matrices for tissue\\u000a engineering. Bombyx mori cocoon fibroin is well characterized as potential biomaterial by virtue of its good mechanical strength, water stability,\\u000a thermal properties, surface roughness and biocompatibility. The present study aims to characterize the biophysical,

  2. 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 ?-sheet formation. A higher density of condensed silica formed on the films containing the lowest ?-sheet content while the films with the highest ?-sheet content precipitated the lowest density of silica, revealing an inverse correlation between the ?-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

  3. Convergently-evolved structural anomalies in the coiled coil domains of insect silk proteins.

    PubMed

    Sutherland, Tara D; Trueman, Holly E; Walker, Andrew A; Weisman, Sarah; Campbell, Peter M; Dong, Zhaoming; Huson, Mickey G; Woodhead, Andrea L; Church, Jeffrey S

    2014-06-01

    The use of coiled coil proteins as the basis of silk materials is an engineering solution that has evolved convergently in at least five insect lineages-the stinging hymenopterans (ants, bees, hornets), argid sawflies, fleas, lacewings, and praying mantises-and persisted throughout large radiations of these insect families. These coiled coil silk proteins share a characteristic distinct from other coiled coil proteins, in that they are fabricated into solid materials after accumulating as highly concentrated solutions within dedicated glands. Here, we relate the amino acid sequences of these proteins to the secondary and tertiary structural information available from biophysical methods such as X-ray scattering, nuclear magnetic resonance and Raman spectroscopy. We investigate conserved and convergently evolved features within these proteins and compare these to the features of classic coiled coil proteins including tropomyosin and leucine zippers. Our analysis finds that the coiled coil domains of insect silk proteins have several common structural anomalies including a high prevalence of alanine residues in core positions. These atypical features of the coiled coil fibrous proteins - which likely produce deviations from canonical coiled-coil structure - likely exist due to selection pressures related to the process of silk fabrication and the final function of the proteins. PMID:24434611

  4. A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning

    PubMed Central

    Teulé, Florence; Cooper, Alyssa R; Furin, William A; Bittencourt, Daniela; Rech, Elibio L; Brooks, Amanda; Lewis, Randolph V

    2009-01-01

    The extreme strength and elasticity of spider silks originate from the modular nature of their repetitive proteins. To exploit such materials and mimic spider silks, comprehensive strategies to produce and spin recombinant fibrous proteins are necessary. This protocol describes silk gene design and cloning, protein expression in bacteria, recombinant protein purification and fiber formation. With an improved gene construction and cloning scheme, this technique is adaptable for the production of any repetitive fibrous proteins, and ensures the exact reproduction of native repeat sequences, analogs or chimeric versions. The proteins are solubilized in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at 25–30% (wt/vol) for extrusion into fibers. This protocol, routinely used to spin single micrometer-size fibers from several recombinant silk-like proteins from different spider species, is a powerful tool to generate protein libraries with corresponding fibers for structure–function relationship investigations in protein-based biomaterials. This protocol may be completed in 40 d. PMID:19229199

  5. Transgenic protein production in silkworm silk glands requires cathepsin and chitinase of Autographa californica multicapsid nucleopolyhedrovirus.

    PubMed

    Wöltje, Michael; Böbel, Melanie; Rheinnecker, Michael; Tettamanti, Gianluca; Franzetti, Eleonora; Saviane, Alessio; Cappellozza, Silvia

    2014-05-01

    The silkworm Bombyx mori represents an established in vivo system for the production of recombinant proteins. Baculoviruses have been extensively investigated and optimised for the expression of high protein levels inside the haemolymph of larvae and pupae of this lepidopteran insect. Current technology includes deletion of genes responsible for the activity of virus-borne proteases, which in wild-type viruses, cause liquefaction of the host insect and enhance horizontal transmission of newly synthesised virus particles. Besides the haemolymph, the silk gland of B. mori provides an additional expression system for recombinant proteins. In this paper, we investigated how silk gland can be efficiently infected by a Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). We demonstrated that the viral chitinase and the cysteine protease cathepsin are necessary to permit viral entry into the silk gland cells of intrahaemocoelically infected B. mori larvae. Moreover, for the first time, we showed AcMNPV crossing the basal lamina of silk glands in B. mori larvae, and we assessed a new path of infection of silk gland cells that can be exploited for protein production. PMID:24477386

  6. 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

  7. Designing recombinant spider silk proteins to control assembly

    Microsoft Academic Search

    Stefan Winkler; Sandra Szela; Peter Avtges; Regina Valluzzi; Daniel A. Kirschner; David Kaplan

    1999-01-01

    The consensus repeat sequence found in the dragline silk from the spider, Nephila clavipes, was redesigned to incorporate a redox trigger flanking the beta-sheet forming polyalanine sequences. The methionine redox trigger, in the oxidized state, was incorporated to prevent the formation of the beta sheets, while in the reduced state would not result in sterical limitations to beta sheet formation.

  8. 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

  9. Snmp-1, a Novel Membrane Protein of Olfactory Neurons of the Silk Moth Antheraea polyphemus with Homology to the CD36 Family of

    E-print Network

    Vogt, Richard G.

    Snmp-1, a Novel Membrane Protein of Olfactory Neurons of the Silk Moth Antheraea polyphemus of the wild silk moth Antheraea polyphemus. We have purified and cloned a prominent 67-kDa protein which we of olfactory neuron receptor membranes of the wild silk moth Antheraea polyphemus. The morphology of the A

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

    PubMed Central

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

    2010-01-01

    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

  11. Silk and collagen scaffolds for tendon reconstruction.

    PubMed

    Kwon, Soon-Yong; Chung, Jin-Wha; Park, Hee-Jung; Jiang, Yuan-Yuan; Park, Jung-Keug; Seo, Young-Kwon

    2014-04-01

    In this study, silk thread (Bombyx mori) was braided to a tube-like shape and sericin was removed from the silk tube. Thereafter, collagen/chondroitin-6-sulfate solution was poured into the silk tube, and the lyophilization process was performed. To assess the inflammatory response in vivo, raw silk and sericin-free silk tubes were implanted in the subcutaneous layer of mice. After 10 days of in vivo implantation, mild inflammatory responses were observed around the sericin-free silk tubes, and severe inflammation with the presence of neutrophils and macrophages was observed around the raw silk tubes. At 24 weeks post implantation, the regenerated tendon had a thick, cylindrical, grayish fibrous structure and a shiny white appearance, similar to that of the native tendon in the rabbit model of tendon defect. The average tensile strength of the native tendons was 220 ± 20 N, whereas the average tensile strength of the regenerated tendons was 167 ± 30 N and the diameter of the regenerated tendon (3 ± 0.2 mm) was similar to that of the native tendons (4 ± 0.3 mm). Histologically, the regenerated tendon resembled the native tendon, and all the regenerated tissues showed organized bundles of crimped fibers. Masson trichrome staining was performed for detecting collagen synthesis, and it showed that the artificial tendon was replaced by new collagen fibers and extracellular matrix. However, the regenerated tendon showed fibrosis to a certain degree. In conclusion, the artificial tendon, comprising a braided silk tube and lyophilized collagen sponge, was optimal for tendon reconstruction. Thus, this study showed an improved regeneration of neo-tendon tissues, which have the structure and tensile strength of the native tendon, with the use of the combination of collagen and silk scaffold. PMID:24705339

  12. 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

  13. Snmp1, a Novel Membrane Protein of Olfactory Neurons of the Silk Moth Antheraea polyphemus with Homology to the CD36 Family of Membrane Proteins

    Microsoft Academic Search

    Matthew E. Rogers; Ming Sun; Michael R. Lerner; Richard G. Vogt

    1997-01-01

    While olfactory neurons of silk moths are well known for their exquisite sensitivity to sex pheromone odor- ants, molecular mechanisms underlying this sensitivity are poorly understood. In searching for proteins that might support olfactory mechanisms, we characterized the protein profile of olfactory neuron receptor mem- branes of the wild silk moth Antheraea polyphemus .W e have purified and cloned a

  14. Structure and organization of the Bombyx mori sericin 1 gene and of the sericins 1 deduced from the sequence of the Ser 1B cDNA

    Microsoft Academic Search

    Annie Garel; Gilbert Deleage; Jean-Claude Prudhomme

    1997-01-01

    The sericin 1 primary transcript of the silkworm Bombyx mori is differentially spliced via a tissue- and developmentally-regulated process. From a middle silk gland cDNA library, we have elucidated the sequence of one of the four mRNAs, the 4.0 kb Ser1B mRNA. Determination of alternative or constitutive exons and intron-exon boundaries allowed us to establish the nine exon-eight intron structure

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

    PubMed

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

    2013-07-01

    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

  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. Silk fibroin protein from mulberry and non-mulberry silkworms: cytotoxicity, biocompatibility and kinetics of L929 murine fibroblast adhesion

    Microsoft Academic Search

    Chitrangada Acharya; Sudip K. Ghosh; S. C. Kundu

    2008-01-01

    Silks fibers and films fabricated from fibroin protein of domesticated mulberry silkworm cocoon have been traditionally utilized\\u000a as sutures in surgery and recently as biomaterial films respectively. Here, we explore the possibility of application of silk\\u000a fibroin protein from non-mulberry silkworm cocoon as a potential biomaterial aid. In terms of direct inflammatory potential,\\u000a fibroin proteins from Antheraea mylitta and Bombyx

  18. Synthetic spider silk fibers spun from Pyriform Spidroin 2, a glue silk protein discovered in orb-weaving spider attachment discs.

    PubMed

    Geurts, Paul; Zhao, Liang; Hsia, Yang; Gnesa, Eric; Tang, Simon; Jeffery, Felicia; La Mattina, Coby; Franz, Andreas; Larkin, Leah; Vierra, Craig

    2010-12-13

    Spider attachment disc silk fibers are spun into a viscous liquid that rapidly solidifies, gluing dragline silk fibers to substrates for locomotion or web construction. Here we report the identification and artificial spinning of a novel attachment disc glue silk fibroin, Pyriform Spidroin 2 (PySp2), from the golden orb weaver Nephila clavipes . MS studies support PySp2 is a constituent of the pyriform gland that is spun into attachment discs. Analysis of the PySp2 protein architecture reveals sequence divergence relative to the other silk family members, including the cob weaver glue silk fibroin PySp1. PySp2 contains internal block repeats that consist of two subrepeat units: one dominated by Ser, Gln, and Ala and the other Pro-rich. Artificial spinning of recombinant PySp2 truncations shows that the Ser-Gln-Ala-rich subrepeat is sufficient for the assembly of polymeric subunits and subsequent fiber formation. These studies support that both orb- and cob-weaving spiders have evolved highly polar block-repeat sequences with the ability to self-assemble into fibers, suggesting a strategy to allow fiber fabrication in the liquid environment of the attachment discs. PMID:21053953

  19. 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

  20. [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

  1. Involvement of trypsin-digested silk peptides in the induction of RAW264.7 macrophage activation.

    PubMed

    Pyo, Kyoung-Ho; Kim, Min-Ki; Shin, Kwang-Soon; Chun, Hyang Sook; Shin, Eun-Hee

    2013-12-01

    The activation of macrophages by trypsin-digested silk peptides was investigated by considering CD1 lb and CD40 expression in the RAW264.7 cell, a murine macrophage. Silk protein hydrolysates were digested with trypsin, following by centrifugal purification using the Centriprep 30k concentrator. Trypsin-digested total silk peptides and its centrifugal fractions were tested for macrophage activation in vitro. The functional peptide of fractionated silk peptides was examined by LC/MS/MS analysis. Trypsin-digested and fractionated silk peptides of more than 30 kDa induced an increase in the activation markers CD1 lb and CD40 in RAW264.7 cells. These results are supported by morphological changes reflecting an increase in the number of dendrites in activated cells. The fractionated silk peptides examined by LC/MS/MS contained partial peptides of Bombyx mori fibroin. These results suggest that the activation of RAW264.7 macrophages may be induced not by sericin-derived peptides but by fibroin-derived ones. PMID:24555292

  2. Gene delivery mediated by recombinant silk proteins containing cationic and cell binding motifs.

    PubMed

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

    2010-08-17

    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

  3. 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. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 237-245, 2014. PMID:23784754

  4. 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 spidroin-1 from Nephila spiders provide the basis for mechanical-elastic property studies of silk for biotechnological and biomedical potential applications. This article is part of a Special Issue entitled: Proteomics of non-model organisms. PMID:24434585

  5. Use of Sericin in Feltproofing of Wool

    Microsoft Academic Search

    O. G. Allam; H. El-Sayed; A. Kantouch; K. Haggag

    2009-01-01

    A new process for feltproofing of wool fibers based on the biopolymer sericin is reported. The susceptibility of wool to sericin has been enhanced by creation of new active sites along the wool keratin molecules, such as cysteic acid and S-cystine sulphonate residues, by pretreatment with hydrogen peroxide and sodium sulphite. The sericin-combining capacity of wool has been even enhanced

  6. Carbonization of a stable ?-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 ?-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

  7. Carbonization of a stable ?-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 ?-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

  8. Non-bioengineered silk gland fibroin protein: characterization and evaluation of matrices for potential tissue engineering applications.

    PubMed

    Mandal, Biman B; Kundu, Subhas C

    2008-08-15

    The possibility of using wild non-mulberry silk protein as a biopolymer remains unexplored compared to domesticated mulberry silk protein. One of the main reasons for this was for not having any suitable method of extraction of silk protein fibroin from cocoons and silk glands. In this study non-bioengineered non-mulberry silk gland fibroin protein from tropical tasar silkworm Antheraea mylitta, is regenerated and characterized using 1% (w/v) sodium dodecyl sulfate (SDS). The new technique is important and unique because it uses a mild surfactant for fibroin dissolution and is advantageous over other previous reported techniques using chaotropic salts. Fabricated fibroin films are smooth as confirmed by atomic force microscopy. Circular dichroism spectrometry along with Fourier transformed infrared spectroscopy and X-ray diffraction reveal random coil/alpha-helix conformations in regenerated fibroin which transform to beta-sheets, resulting in crystalline structure and protein insolubility through ethanol treatment. Differential scanning calorimetry shows an increase in glass transition (Tg) temperature and enhanced degradation temperature on alcohol treatment. Enhanced cell attachment and viability of AH927 feline fibroblasts were observed on fibroin matrices. Higher mechanical strength along with controllable water stability of regenerated gland fibroin films make non-mulberry Indian tropical tasar silk gland fibroin protein a promising biomaterial for tissue engineering applications. PMID:18383269

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

    PubMed Central

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

    2010-01-01

    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

  10. Novel genes differentially expressed between posterior and median silk gland identified by SAGE-aided transcriptome analysis.

    PubMed

    Royer, Corinne; Briolay, Jérôme; Garel, Annie; Brouilly, Patrick; Sasanuma, Shun-ichi; Sasanuma, Motoe; Shimomura, Michihiko; Keime, Céline; Gandrillon, Olivier; Huang, Yongping; Chavancy, Gérard; Mita, Kazuei; Couble, Pierre

    2011-02-01

    Serial analysis of gene expression (SAGE) profiles, from posterior and median cells of the silk gland of Bombyx mori, were analyzed and compared, so as to identify their respective distinguishing functions. The annotation of the SAGE libraries was performed with a B. mori reference tag collection, which was extracted from a novel set of Bombyx ESTs, sequenced from the 3' side. Most of the tags appeared at similar relative concentration within the two libraries, and corresponded with region-specific and highly abundant silk proteins. Strikingly, in addition to tags from silk protein mRNAs, 19 abundant tags were found (? 0.1%), in the median cell library, which were absent in the posterior cell tag collection. With the exception of tags from SP1 mRNA, no PSG specific tags were found in this subset class. The analysis of some of the MSG-specific transcripts, suggested that middle silk gland cells have diversified functions, in addition to their well characterized role in silk sericins synthesis and secretion. PMID:21078388

  11. Unfolding the multi-length scale domain structure of silk fibroin protein

    Microsoft Academic Search

    Hennady Shulha; Cheryl Wong Po Foo; David L. Kaplan; Vladimir V. Tsukruk

    2006-01-01

    Multi-scale force spectroscopy was applied to measure unfolding properties and the internal domain structure of Bombyx mori silk fibroin. We demonstrated that the complex multi-domain sequence and block design in this protein can be directly related to multi-stage unfolding behavior of the specific regions through the use of force extension measurements. These new findings suggest relationships between polymer block chemistry

  12. [Biological synthesis and purification of spider dragline silk protein polymers containing RGD three peptide].

    PubMed

    Li, Min; Huang, Jiankun; Tu, Guiyun; Huang, Xi

    2004-12-01

    Spider dragline silk is one of most perfect fibrous proteins in nature. As biomaterials, it has a wide application in tissue engineering due to its unique mechanical properties, good biocompatibility, slow degradation. In this paper, based on the highly repetitive sequence of spider dragline silk and with the introduced RGD peptide codons which involve cell adhesion, the DNA monomer sequence encoding RGD-spider dragline silk was synthesized, and then was used to construct the multimers by the strategy of "head to tail"; the multimers were ligated into prokaryotic expression vector pET-30a, and then the B121 (DE3) pLyS, were transformed the expression of recombinant protein was induced by the addition of IPTG. SDS-PAGE analysis shows that the molecular weight of products expressed here are 35KD and 60KD respectively in agreement with the desired. Western assay was used for determining the specification of products. Further, the purification process was groped for the producing of large quantity of synthetic proteins through high density fermentation. PMID:15646353

  13. New and highly efficient expression systems for expressing selectively foreign protein in the silk glands of transgenic silkworm.

    PubMed

    Zhao, Aichun; Zhao, Tianfu; Zhang, Yuansong; Xia, Qingyou; Lu, Cheng; Zhou, Zeyang; Xiang, Zhonghuai; Nakagaki, M

    2010-02-01

    We constructed three different fibroin H-chain expression systems to estimate the efficacy of producing recombinant proteins in the cocoon of transgenic silkworms. The results showed that the three different EGFP/H-chain fusion genes were all expressed selectively in the posterior silk gland of the transgenic silkworm. The recombinant protein content of transgenic silkworm cocoons is up to 15% (w/w) when using the most highly efficient H-chain expression system. To our knowledge, in comparison with silkworm silk gland expression systems in the literature, the highly efficient expression system developed in this study is the most efficient silkworm silk gland expression system to date. This expression system is the best candidate for foreign gene production and for creation of novel functional silk material. The results suggested the N-terminal domain and the intron of the H-chain gene are important in the secretion of fibroin and its transcription, respectively. PMID:19533404

  14. Molecular mechanisms of spider silk

    Microsoft Academic Search

    X. Hu; K. Vasanthavada; K. Kohler; S. McNary; A. M. F. Moore; C. A. Vierra

    2006-01-01

    .  Spiders spin high-performance silks through the expression and assembly of tissue-restricted fibroin proteins. Spider silks\\u000a are composite protein biopolymers that have complex microstructures. Retrieval of cDNAs and genomic DNAs encoding silk fibroins\\u000a has revealed an association between the protein sequences and structure-property relationships. However, before spider silks\\u000a can be subject to genetic engineering for commercial applications, the complete protein sequences

  15. Complexation-triggerable liposome mixed with silk protein and chitosan.

    PubMed

    Hong, Yeon-Ji; Kim, Jin-Chul

    2015-08-01

    Complexation-triggerable liposomes were prepared by modifying the surface of egg phosphatidylcholine (EPC) liposomes with hydrophobicized silk fibroin (HmSF) and hydrophobicized chitosan (HmCh). Maximum complexation, determined by measuring the diameter of complexation, was found when the ratio of HmSF to HmCh was 14:1, so they were immobilized on the surface of liposomes at the same ratio. The degree of fluorescence quenching of calcein in liposomal suspension was as high as 68% when the ratio of surface modifier (HmSF + HmCh) to EPC was 1:15. When the ratio was increased to 1:5, the degree of quenching decreased to 32%, indicating the inefficient formation of liposome. Liposome mixed with the surface modifier was multi-lamellar vesicle on TEM photo. And, the mean diameter was larger than those of liposome mixed with either HmSF or HmCh, possibly due to insoluble complex on the liposomal surface. The liposome exhibited a pH-sensitive release and triggered the release at pH 5.5 and 6.0. It is believed that complexation is responsible for the promoted release at those pH values. PMID:26059216

  16. Fibrous proteins—silk as a model system

    Microsoft Academic Search

    David L. Kaplan

    1998-01-01

    The repetitive nature of fibrous proteins leads to regular secondary and higher order structures. This attribute suggests that these polymers are useful models to gain insight into structure-function relationships. Genetic variants of these proteins can be constructed from precisely defined synthetic oligonucleotides to encode the specific sized protein of interest. The biosynthesis and assembly of this type of protein is

  17. Dielectric breakdown strength of regenerated silk fibroin films as a function of protein conformation.

    PubMed

    Dickerson, Matthew B; Fillery, Scott P; Koerner, Hilmar; Singh, Kristi M; Martinick, Katie; Drummy, Lawrence F; Durstock, Michael F; Vaia, Richard A; Omenetto, Fiorenzo G; Kaplan, David L; Naik, Rajesh R

    2013-10-14

    Derived from Bombyx mori cocoons, regenerated silk fibroin (RSF) exhibits excellent biocompatibility, high toughness, and tailorable biodegradability. Additionally, RSF materials are flexible, optically clear, easily patterned with nanoscale features, and may be doped with a variety bioactive species. This unique combination of properties has led to increased interest in the use of RSF in sustainable and biocompatible electronic devices. In order to explore the applicability of this biopolymer to the development of future bioelectronics, the dielectric breakdown strength (Ebd) of RSF thin films was quantified as a function of protein conformation. The application of processing conditions that increased ?-sheet content (as determined by FTIR analysis) and produced films in the silk II structure resulted in RSF materials with improved Ebd with values reaching up to 400 V/?m. PMID:23987229

  18. Spider silk proteins: recent advances in recombinant production, structure-function relationships and biomedical applications.

    PubMed

    Rising, Anna; Widhe, Mona; Johansson, Jan; Hedhammar, My

    2011-01-01

    Spider dragline silk is an outstanding material made up of unique proteins-spidroins. Analysis of the amino acid sequences of full-length spidroins reveals a tripartite composition: an N-terminal non-repetitive domain, a highly repetitive central part composed of approximately 100 polyalanine/glycine rich co-segments and a C-terminal non-repetitive domain. Recent molecular data on the terminal domains suggest that these have different functions. The composite nature of spidroins allows for recombinant production of individual and combined regions. Miniaturized spidroins designed by linking the terminal domains with a limited number of repetitive segments recapitulate the properties of native spidroins to a surprisingly large extent, provided that they are produced and isolated in a manner that retains water solubility until fibre formation is triggered. Biocompatibility studies in cell culture or in vivo of native and recombinant spider silk indicate that they are surprisingly well tolerated, suggesting that recombinant spider silk has potential for biomedical applications. PMID:20668909

  19. Comparative architecture of silks, fibrous proteins and their encoding genes in insects and spiders

    Microsoft Academic Search

    Catherine L. Craig; Christian Riekel

    2002-01-01

    The known silk fibroins and fibrous glues are thought to be encoded by members of the same gene family. All silk fibroins sequenced to date contain regions of long-range order (crystalline regions) and\\/or short-range order (non-crystalline regions). All of the sequenced fibroin silks (Flag or silk from flagelliform gland in spiders; Fhc or heavy chain fibroin silks produced by Lepidoptera

  20. 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 ?g/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

  1. 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 ?g/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

  2. Physical Crosslinking Modulates Sustained Drug Release from Recombinant Silk-Elastinlike Protein Polymer for Ophthalmic Applications

    PubMed Central

    Teng, Weibing; Cappello, Joseph; Wu, Xiaoyi

    2011-01-01

    We evaluated the drug release capability of optically transparent recombinant silk-elastinlike protein polymer, SELP-47K, films to sustainably deliver the common ocular antibiotic, ciprofloxacin. The ciprofloxacin release kinetics from drug-loaded SELP-47K films treated with ethanol or methanol vapor to induce different densities of physical crosslinking was investigated. Additionally, the drug-loaded protein films were embedded in a protein polymer coating to further prolong the release of the drug. Drug-loaded SELP-47K films released ciprofloxacin for up to 132 hours with near first-order release kinetics. Polymer coating of drug-loaded films prolonged drug release for up to 220 hours. The antimicrobial activity of ciprofloxacin released from the drug delivery matrices was not impaired by the film casting process or the ethanol or methanol treatments. The mechanism of drug release was elucidated by analyzing the physical properties of the film specimens, including equilibrium swelling, soluble fraction, surface roughness and hydrophobicity. Additionally, the conformation of the SELP-47K and its physical crosslinks in the films was analyzed by FTIR and Raman spectroscopy. A three-parameter physics based model accurately described the release rates observed for the various film and coating treatments and attributed the effects to the degree of physical crosslinking of the films and to an increasing affinity of the drug with the polymer network. Together, these results indicate that optically transparent silk-elastinlike protein films may be attractive material candidates for novel ophthalmic drug delivery devices. PMID:21839125

  3. 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 ?1 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

  4. Spider silk proteins: recent advances in recombinant production, structure–function relationships and biomedical applications

    Microsoft Academic Search

    Anna Rising; Mona Widhe; Jan Johansson; My Hedhammar

    2011-01-01

    Spider dragline silk is an outstanding material made up of unique proteins—spidroins. Analysis of the amino acid sequences\\u000a of full-length spidroins reveals a tripartite composition: an N-terminal non-repetitive domain, a highly repetitive central\\u000a part composed of approximately 100 polyalanine\\/glycine rich co-segments and a C-terminal non-repetitive domain. Recent molecular\\u000a data on the terminal domains suggest that these have different functions. The

  5. Production and characterization of a silk-like hybrid protein, based on the polyalanine region of Samia cynthia ricini silk fibroin and a cell adhesive region derived from fibronectin.

    PubMed

    Asakura, Tetsuo; Tanaka, Chikako; Yang, Mingying; Yao, Juming; Kurokawa, Masato

    2004-02-01

    There are a variety of silkworms and silk fibroins produced by them. Silks have many inherent suitable properties for biomaterials. In this paper, a novel silk-like hybrid protein, [DGG(A)(12)GGAASTGRGDSPAAS](5), which consists of polyalanine region of silk fibroin from a wild silkworm, Samia cynthia ricini, and cell adhesive region including Arg-Gly-Asp (RGD) sequence, derived from fibronectin, was designed and produced. The genes encoding the hybrid protein were constructed and expressed in Escherichia coli. The main conformation of the polyalanine region, that is, either alpha-helix or beta-sheet, could be easily controlled by treatment with different acidic solvents, trifluoroacetic acid or formic acid, respectively. This structural change was monitored with 13C CP/MAS NMR. Higher cell adhesive and growth activities of the hybrid protein compared with those of collagen were obtained. PMID:14607499

  6. Characterization of silk fibroin modified surface: a proteomic view of cellular response proteins induced by biomaterials.

    PubMed

    Yang, Ming-Hui; Yuan, Shyng-Shiou; Chung, Tze-Wen; Jong, Shiang-Bin; Lu, Chi-Yu; Tsai, Wan-Chi; Chen, Wen-Cheng; Lin, Po-Chiao; Chiang, Pei-Wen; Tyan, Yu-Chang

    2014-01-01

    The purpose of this study was to develop the pathway of silk fibroin (SF) biopolymer surface induced cell membrane protein activation. Fibroblasts were used as an experimental model to evaluate the responses of cellular proteins induced by biopolymer material using a mass spectrometry-based profiling system. The surface was covered by multiwalled carbon nanotubes (CNTs) and SF to increase the surface area, enhance the adhesion of biopolymer, and promote the rate of cell proliferation. The amount of adhered fibroblasts on CNTs/SF electrodes of quartz crystal microbalance (QCM) greatly exceeded those on other surfaces. Moreover, analyzing differential protein expressions of adhered fibroblasts on the biopolymer surface by proteomic approaches indicated that CD44 may be a key protein. Through this study, utilization of mass spectrometry-based proteomics in evaluation of cell adhesion on biopolymer was proposed. PMID:24818131

  7. Characterization of Silk Fibroin Modified Surface: A Proteomic View of Cellular Response Proteins Induced by Biomaterials

    PubMed Central

    Yang, Ming-Hui; Yuan, Shyng-Shiou; Chung, Tze-Wen; Jong, Shiang-Bin; Lu, Chi-Yu; Tsai, Wan-Chi; Chen, Wen-Cheng; Lin, Po-Chiao; Chiang, Pei-Wen; Tyan, Yu-Chang

    2014-01-01

    The purpose of this study was to develop the pathway of silk fibroin (SF) biopolymer surface induced cell membrane protein activation. Fibroblasts were used as an experimental model to evaluate the responses of cellular proteins induced by biopolymer material using a mass spectrometry-based profiling system. The surface was covered by multiwalled carbon nanotubes (CNTs) and SF to increase the surface area, enhance the adhesion of biopolymer, and promote the rate of cell proliferation. The amount of adhered fibroblasts on CNTs/SF electrodes of quartz crystal microbalance (QCM) greatly exceeded those on other surfaces. Moreover, analyzing differential protein expressions of adhered fibroblasts on the biopolymer surface by proteomic approaches indicated that CD44 may be a key protein. Through this study, utilization of mass spectrometry-based proteomics in evaluation of cell adhesion on biopolymer was proposed. PMID:24818131

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

    SciTech Connect

    Liu Yan [College of Biomedical Engineering and Instrument Science, Zhejiang University, Hangzhou 310027 (China); Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Yu Lian [College of Biomedical Engineering and Instrument Science, Zhejiang University, Hangzhou 310027 (China); Zhejiang Province Key Laboratory of Preventive Veterinary Medicine, Institute of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310029 (China); Guo Xiuyang [Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Guo Tingqing [Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Wang Shengpeng [Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Lu Changde [Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China)]. 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.

  9. Spidroin N-terminal domain promotes a pH-dependent association of silk proteins during self-assembly.

    PubMed

    Gaines, William A; Sehorn, Michael G; Marcotte, William R

    2010-12-24

    Spider silks are spun from concentrated solutions of spidroin proteins. The appropriate timing of spidroin assembly into organized fibers must be highly regulated to avoid premature fiber formation. Chemical and physical signals presented to the silk proteins as they pass from the ampulle and through the tapered duct include changes in ionic environment and pH as well as the introduction of shear forces. Here, we show that the N-terminal domain of spidroins from the major ampullate gland (MaSp-NTDs) for both Nephila and Latrodectus spiders associate noncovalently as homodimers. The MaSp-NTDs are highly pH-responsive and undergo a structural transition in the physiological pH range of the spider duct. Tryptophan fluorescence of the MaSp-NTDs reveals a change in conformation when pH is decreased, and the pH at which the transition occurs is determined by the amount and type of salt present. Size exclusion chromatography and pulldown assays both indicate that the lower pH conformation is associated with a significantly increased MaSp-NTD homodimer stability. By transducing the duct pH signal into specific protein-protein interactions, this conserved spidroin domain likely contributes significantly to the silk-spinning process. Based on these results, we propose a model of spider silk assembly dynamics as mediated through the MaSp-NTD. PMID:20959449

  10. A pH-dependent dimer lock in spider silk protein.

    PubMed

    Landreh, Michael; Askarieh, Glareh; Nordling, Kerstin; Hedhammar, My; Rising, Anna; Casals, Cristina; Astorga-Wells, Juan; Alvelius, Gunvor; Knight, Stefan D; Johansson, Jan; Jörnvall, Hans; Bergman, Tomas

    2010-11-26

    Spider dragline silk, one of the strongest polymers in nature, is composed of proteins termed major ampullate spidroin (MaSp) 1 and MaSp2. The N-terminal (NT) domain of MaSp1 produced by the nursery web spider Euprosthenops australis acts as a pH-sensitive relay, mediating spidroin assembly at around pH 6.3. Using amide hydrogen/deuterium exchange combined with mass spectrometry (MS), we detected pH-dependent changes in deuterium incorporation into the core of the NT domain, indicating global structural stabilization at low pH. The stabilizing effects were diminished or abolished at high ionic strength, or when the surface-exposed residues Asp40 and Glu84 had been exchanged with the corresponding amides. Nondenaturing electrospray ionization MS revealed the presence of dimers in the gas phase at pH values below--but not above--6.4, indicating a tight electrostatic association that is dependent on Asp40 and Glu84 at low pH. Results from analytical ultracentrifugation support these findings. Together, the data suggest a mechanism whereby lowering the pH to <6.4 results in structural changes and alteration of charge-mediated interactions between subunits, thereby locking the spidroin NT dimer into a tight entity important for aggregation and silk formation. PMID:20887730

  11. 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 ?s and in a pH-dependent manner. Equilibrium denaturation and temperature-jump relaxation experiments revealed cooperative and ultrafast folding in only 60 ?s. 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

  12. Swelling behavior of a genetically engineered silk-elastinlike protein polymer hydrogel.

    PubMed

    Dinerman, Adam A; Cappello, Joseph; Ghandehari, Hamidreza; Hoag, Stephen W

    2002-11-01

    The influence of environmental conditions such as pH, temperature, and ionic strength on the equilibrium swelling ratio of physically crosslinked networks of a genetically engineered silk-elastinlike protein-based copolymer (SELP) with an amino acid repeat sequence of [(GVGVP)4GKGVP(GVGVP)3(GAGAGS)4]12 was investigated. The effects of gelation cure time and initial polymer concentration on the equilibrium swelling ratio and soluble fraction of the hydrogels were also studied. It was found that the soluble fraction linearly correlated with the initial polymer concentration at higher gelation times. Soluble fraction results suggest that final hydrogel water content may be controlled by both initial polymer concentration and gelation time. Equilibrium swelling studies demonstrated that these hydrogels are relatively insensitive to environmental changes such as pH, temperature, and ionic strength. Over the concentration range studied, it was found that an increase in gelation time at 37 degrees C resulted in lower hydrogel weight equilibrium swelling ratios, which corresponds to less soluble polymer released post-gelation. Together, these results have implications for the controlled delivery of bioactive agents from silk-elastinlike hydrogels. PMID:12194523

  13. Influence of polymer structure and biodegradation on DNA release from silk-elastinlike protein polymer hydrogels.

    PubMed

    Hwang, David; Moolchandani, Vikas; Dandu, Ramesh; Haider, Mohamed; Cappello, Joseph; Ghandehari, Hamidreza

    2009-02-23

    Silk-elastinlike protein polymers (SELPs) of varying ratios and lengths of silk and elastin blocks capable of hydrogel formation were evaluated as matrices for controlled delivery of plasmid DNA. Influence of polymer structure, ionic strength of the media and gelation time on DNA release from two structurally related hydrogels, SELP-47K and SELP-415K, was evaluated. The influence of elastase-induced degradation on the swelling behavior and DNA release from these hydrogels was investigated. Results indicate that release is a function of polymer structure, concentration and cure time. SELP-415K which has twice the number of elastin units as that of SELP-47K demonstrated higher release than that of SELP-47K. DNA release from these hydrogels is an inverse function of polymer concentration and cure time, with higher release observed at lower polymer concentration and shorter cure time. Results indicate that ionic strength of the media governs the rate of release. An increase in swelling ratio was observed in the presence of elastase at 12 wt.% composition for both SELP analogs. Release in the presence of elastase was enhanced due to increased swelling ratio and loss of hydrogel integrity. These studies allude to the utility of recombinant techniques to control plasmid DNA release and biodegradation in SELP hydrogels. PMID:19027056

  14. AFM study of morphology and mechanical properties of a chimeric spider silk and bone sialoprotein protein for bone regeneration

    PubMed Central

    Gomes, Sílvia; Numata, Keiji; Leonor, Isabel B.; Mano, João F.; Reis, Rui L.; Kaplan, David L.

    2011-01-01

    Atomic force microscopy (AFM) was used to assess a new chimeric protein consisting of a fusion protein of the consensus repeat for Nephila clavipes spider dragline protein and bone sialoprotein (6mer+BSP). The elastic modulus of this protein in film form was assessed through force curves, and film surface roughness was also determined. The results showed a significant difference between the elastic modulus of the chimeric silk protein, 6mer+BSP, and control films consisting of only the silk component (6mer). The behaviour of the 6mer+BSP and 6mer proteins in aqueous solution in the presence of calcium (Ca) ions was also assessed to determine interactions between the inorganic and organic components related to bone interactions, anchoring and biomaterial network formation. The results demonstrated the formation of protein networks in the presence of Ca2+ ions, characteristics that may be important in the context of controlling materials assembly and properties related to bone-formation with this new chimeric silk-BSP protein. PMID:21370930

  15. Ancient properties of spider silks revealed by the complete gene sequence of the prey-wrapping silk protein (AcSp1).

    PubMed

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

    2013-03-01

    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

  16. 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).

  17. Cre-mediated targeted gene activation in the middle silk glands of transgenic silkworms (Bombyx mori).

    PubMed

    Duan, Jianping; Xu, Hanfu; Ma, Sanyuan; Guo, Huizhen; Wang, Feng; Zhao, Ping; Xia, Qingyou

    2013-06-01

    Cre-mediated recombination is widely used to manipulate defined genes spatiotemporally in vivo. The present study evaluated the Cre/loxP system in Bombyx mori by establishing two transgenic lines. One line contained a Cre recombinase gene controlled by a sericin-1 gene (Ser1) promoter. The other line contained a loxP-Stop-loxP-DsRed cassette driven by the same Ser1 promoter. The precise deletion of the Stop fragment was found to be triggered by Cre-mediated site-specific excision, and led to the expression of DsRed fluorescence protein in the middle silk glands of all double-transgenic hybrids. This result was also confirmed by phenotypical analysis. Hence, the current study demonstrated the feasibility of Cre-mediated site-specific recombination in B. mori, and opened a new window for further refining genetic tools in silkworms. PMID:23264031

  18. 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

  19. Engineering of recombinant spider silk proteins allows defined uptake and release of substances.

    PubMed

    Doblhofer, Elena; Scheibel, Thomas

    2015-03-01

    Drug delivery carriers stabilize drugs and control their release, expanding the therapeutic window, and avoiding side effects of otherwise freely diffusing drugs in the human body. Materials used as carrier vehicles have to be biocompatible, biodegradable, nontoxic, and nonimmunogenic. Previously, particles made of the recombinant spider silk protein eADF4(C16) could be effectively loaded with positively and neutrally charged model substances. Here, a new positively charged variant thereof, named eADF4(?16), has been engineered. Its particle formation is indistinguishable to that of polyanionic eADF4(C16), but in contrast polycationic eADF4(?16) allows incorporation of negatively charged substances. Both high-molecular-weight substances, such as nucleic acids, and low-molecular-weight substances could be efficiently loaded onto eADF4(?16) particles, and release of nucleic acids was shown to be well controlled. PMID:25546241

  20. New and highly efficient expression systems for expressing selectively foreign protein in the silk glands of transgenic silkworm

    Microsoft Academic Search

    Aichun Zhao; Tianfu Zhao; Yuansong Zhang; Qingyou Xia; Cheng Lu; Zeyang Zhou; Zhonghuai Xiang; M. Nakagaki

    2010-01-01

    We constructed three different fibroin H-chain expression systems to estimate the efficacy of producing recombinant proteins\\u000a in the cocoon of transgenic silkworms. The results showed that the three different EGFP\\/H-chain fusion genes were all expressed\\u000a selectively in the posterior silk gland of the transgenic silkworm. The recombinant protein content of transgenic silkworm\\u000a cocoons is up to 15% (w\\/w) when using

  1. Surface induced nanofiber growth by self-assembly of a silk-elastinlike protein polymer

    PubMed Central

    Hwang, Wonseok; Kim, Bo-Hyun; Dandu, Ramesh; Cappello, Joseph; Ghandehari, Hamidreza; Seog, Joonil

    2009-01-01

    Many synthetic and natural peptides are known to self-assemble to form various nanostructures such as nanofibers, hollow tubes, or ring-like structures. Some of the synthetic peptide molecules are specifically designed to produce well-defined nanostructures by controlling intermolecular interactions. Many environmental conditions such as salt concentration, pH, temperature, and surface characteristics influence intermolecular interactions, hence the process of the self-assembly. Here we studied self-assembly of a genetically engineered protein polymer composed of silk-like and elastin-like repeats on a mica surface. Silk-elastinlike protein polymers (SELPs) consist of tandem repeats of Gly-Ala-Gly-Ala-Gly-Ser from Bombyx mori (silkworm) and Gly-Val-Gly-Val-Pro from mammalian elastin. At a very low polymer concentration of 1 ?g/ml, SELPs self-assembled into nanofibrous structures on a mica surface. Examination using atomic force microscopy (AFM) and dynamic light scattering techniques showed that SELPs self-assembled into nanofibers in the presence of the mica surface but not in the bulk state. Ionic strength had a significant influence on nanofiber growth, indicating the importance of electrostatic interactions between the polymer and the mica surface. At low ionic strength, the kinetics of nanofiber growth indicates that the mica surface effectively removed a lag phase by providing nucleating sites, facilitating nanofiber self-assembly of SELPs. Further examination of self-assembly on various surfaces such as silicon, positively charged surface, and hydrophobic surface revealed that negatively charged hydrophilic surface provides optimal surface to facilitate self-assembly of SELPs. PMID:19803470

  2. Free radical scavenging and tyrosinase inhibition activity of oils and sericin extracted from Thai native silkworms (Bombyx mori).

    PubMed

    Manosroi, Aranya; Boonpisuttinant, Korawinwich; Winitchai, Supanida; Manosroi, Worapaka; Manosroi, Jiradej

    2010-08-01

    Oils and sericin were extracted from pupae and silk cocoons, respectively, of the five Thai native silkworms (Bombyx mori, Linnaeus (Bombycidae)), namely, Keawsakol, Nangnoi, Somrong, Nangleung, and Noneruesee, which are variations of the same species. The oils were extracted by a hot process using Soxhlet apparatus and a cold process using petroleum ether, while sericin was extracted by basic hydrolysis and autoclaving. Sericin from the five Thai native silkworms showed free radical scavenging activity lower than the standard antioxidants (vitamin C, vitamin E, and BHT) by about 20-100-fold, but all oils gave higher activity than that of the standard linoleic acid by 11-22-fold. Oil extracted from Noneruesee by the cold process gave the highest DPPH scavenging activity, compared with other oil samples. All sericin samples showed tyrosinase inhibition activity with IC(50) values in the range of 1.2-18.76 mg/mL, but only oils from Noneruesee extracted by the hot process, and Nangleung, Somrong, and Noneruesee extracted by the cold process, showed this activity. Oil extracted by the hot process and sericin by basic hydrolysis from Noneruesee gave the highest tyrosinase inhibition activity, but lower than that of the standards vitamin C and kojic acid by 20-49 and 3-8 times, respectively. This study has suggested that sericin and oil from Noneruesee extracted by basic hydrolysis and the cold process, which gave the highest tyrosinase inhibition and free radical scavenging activity, respectively, can be applied in antiaging and whitening cosmetic products. PMID:20673171

  3. Regenerated silk fibers: Structural studies and solid state NMR techniques for efficient multiple distance determinations in proteins

    NASA Astrophysics Data System (ADS)

    Liivak, Oskar

    2000-09-01

    Material Science is the science of understanding the relationship between the molecular level structure of a material and its macroscopic properties. Such research requires both the ability to determine molecular structure and the ability to control and modify the molecular structure. The present research into silks, especially the dragline silk from the spider Nephila clavipes , is occurring at a time when these two criteria are beginning to be met for proteins like spider silk. Genetic engineering has evolved to the point where material scientists have full control over the primary sequence of amino acids that comprise proteins. In addition, solid state nuclear magnetic resonance (NMR) techniques exist which allow us to probe molecular structure. This work applies solid state NMR to the study of the structure of silk fibers. In particular, we focus on techniques of fiber regeneration from solution. The purpose is not only to develop the techniques by which genetically engineered fibers could be spun into fibers for mass production but also as a tool into fundamental silk research. Results on these regenerated fibers show a correlation between the fraction of the silk's alanine residues which are in the ?-sheet conformation and the ultimate tensile strength of the fibers. In addition, in a clever mating of the fiber regeneration technique and the solid state NMR distance measurement experiment, rotational echo double resonance (REDOR), we investigate the supramolecular topology of the alanine ?-sheet crystals. Even though the REDOR technique has failings for the complicated ISn spin systems found in the silk samples, a qualitative analysis does indicate that the ?-sheet crystals are intermolecular. Finally, we investigate a new class of REDOR-like experiments which are designed to overcome the failings of REDOR in ISn spin systems. Experimental data is shown to validate these ideas. An alternate pulse sequence is also introduced and verified with experimental data. This pulse sequence highlights the similarities between multiple quantum NMR and REDOR. From this connection, we name this new class of experiments Multiple Quantum-REDOR. These experiments should allow for efficient simultaneous multiple distance determinations in proteins.

  4. 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

  5. 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

  6. TNF-? blocker effect of naringenin-loaded sericin microparticles that are potentially useful in the treatment of psoriasis.

    PubMed

    Chlapanidas, Theodora; Perteghella, Sara; Leoni, Flavio; Faragò, Silvio; Marazzi, Mario; Rossi, Daniela; Martino, Emanuela; Gaggeri, Raffaella; Collina, Simona

    2014-01-01

    This study aims to evaluate the effect of combined use of the racemic flavanone Naringenin (NRG) and the protein sericin as TNF-? blockers. Sericin (SMs) and (R/S) NRG-loaded Sericin (SNRGMs) microparticles were prepared by spray-drying, characterized in terms of morphology and particle size distribution, and encapsulation efficiency was determined. Concerning morphology and particle size distribution of microparticles, results indicated that they were not affected by the presence of NRG. The encapsulation efficiency was almost quantitative (93%), thus proving that sericin can be advantageously loaded with (R/S) NRG. Biological evaluation of (R/S) NRG, SMs and SNRGMs was then performed in lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (hPBMC). SNRGMs resulted cytotoxic at the higher dose used (200 ?g/mL) and the effect was greater than (R/S) NRG alone. Moreover, even if sericin alone was not effective in suppressing LPS-induced serum TNF-? levels, SNRGMs loaded with 9.3% of (R/S) NRG were significantly more potent than (R/S) NRG alone. In summary, this study provides the proof of concept that sericin-based microspheres loaded with TNF-?-blockers could contribute to the down regulation of the cytokine and represents the starting point for the development of new topical formulations for the treatment of middle-stage psoriasis. PMID:25101847

  7. TNF-? Blocker Effect of Naringenin-Loaded Sericin Microparticles that Are Potentially Useful in the Treatment of Psoriasis

    PubMed Central

    Chlapanidas, Theodora; Perteghella, Sara; Leoni, Flavio; Faragò, Silvio; Marazzi, Mario; Rossi, Daniela; Martino, Emanuela; Gaggeri, Raffaella; Collina, Simona

    2014-01-01

    This study aims to evaluate the effect of combined use of the racemic flavanone Naringenin (NRG) and the protein sericin as TNF-? blockers. Sericin (SMs) and (R/S) NRG-loaded Sericin (SNRGMs) microparticles were prepared by spray-drying, characterized in terms of morphology and particle size distribution, and encapsulation efficiency was determined. Concerning morphology and particle size distribution of microparticles, results indicated that they were not affected by the presence of NRG. The encapsulation efficiency was almost quantitative (93%), thus proving that sericin can be advantageously loaded with (R/S) NRG. Biological evaluation of (R/S) NRG, SMs and SNRGMs was then performed in lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (hPBMC). SNRGMs resulted cytotoxic at the higher dose used (200 ?g/mL) and the effect was greater than (R/S) NRG alone. Moreover, even if sericin alone was not effective in suppressing LPS-induced serum TNF-? levels, SNRGMs loaded with 9.3% of (R/S) NRG were significantly more potent than (R/S) NRG alone. In summary, this study provides the proof of concept that sericin-based microspheres loaded with TNF-?-blockers could contribute to the down regulation of the cytokine and represents the starting point for the development of new topical formulations for the treatment of middle-stage psoriasis. PMID:25101847

  8. Study of Protein Conformation and Orientation in Silkworm and Spider Silk Fibers Using Raman Microspectroscopy

    Microsoft Academic Search

    Marie-Eve Rousseau; Thierry Lefèvre; Lilyane Beaulieu; Tetsuo Asakura; Michel Pézolet

    2004-01-01

    Raman microspectroscopy has been used for the first time to determine quantitatively the orientation of the ‚-sheets in silk monofilaments from Bombyx moriand Samia cynthia ricini silkworms, and from the spider Nephila edulis. It is shown that, for systems with uniaxial symmetry such as silk, it is possible to determine the order parameters ?P2? and ?P4? of the orientation distribution

  9. Influence of sericin/TiO? nanocomposite on cotton fabric: part 1. Enhanced antibacterial effect.

    PubMed

    Doakhan, S; Montazer, M; Rashidi, A; Moniri, R; Moghadam, M B

    2013-05-15

    Sericin as a biological material was extracted from raw silk by boiling in hot water and nano-TiO2 was dispersed in its solution. The prepared finishing agents with and without polycarboxylic acid cross-linking agents were treated on cotton fabric using pad-dry-cure. Presence of sericin, nano-TiO2, and cross-linking agents on cotton fabric was confirmed by at least one of the following experimental FTIR, SEM, EDX, and XRD. The antibacterial activity and the durability of modified cotton fabrics were investigated against one Gram-positive bacterium (Staphylococcus aureus) and one Gram-negative bacterium (Escherichia coli). The finishing treatment on the cotton fabric was more effective against S. aureus than E. coli. The fabrics treated with nano-TiO2 were possessed more activity against bacteria as compared to sericin and also considerably improved with given nanocomposite. The antibacterial activity of treated fabrics with cross-linking agents has not been considerably changed after 20 and 40 launderings. The fabrics treated with given nanocomposites did not dramatically affect the breaking strength. PMID:23544628

  10. Effect of an artificial silk elastin-like protein on the migration and collagen production of mouse fibroblasts.

    PubMed

    Ozaki, Chisa; Somamoto, Satoshi; Kawabata, Shingo; Tabata, Yasuhiko

    2014-01-01

    A silk elastin-like protein (SELP) is an artificial compound composing silk fibroin-like and elastin-like tandem repeats. The objective of this study is to evaluate the SELP effect on the migration, proliferation, and proteins production of L929 mouse fibroblasts. Upon culturing with different concentrations of SELP, the cells migration and their collagen production significantly enhanced in the SELP concentrations from 10(-3) to 10 ?g/ml. However, irrespective of the SELP concentration, no difference in the production of fibronectin, basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and stromal cell-derived factor 1? (SDF-1?) was observed. When the migration of mouse peritoneal macrophages by SELP was evaluated, significant enhancement of macrophages migration was observed in any concentration. It is concluded that the SELP has a potential to promote the migration of fibroblasts and macrophages, and the fibroblast collagen production. PMID:24941248

  11. Expression of a truncated ATHB17 protein in maize increases ear weight at silking.

    PubMed

    Rice, Elena A; Khandelwal, Abha; 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 ?-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 ATHB17?113, a truncated protein lacking the first 113 amino acids which encodes a significant portion of the repression domain. Because ATHB17?113 lacks the repression domain, the protein cannot directly affect the transcription of its target genes. ATHB17?113 can homodimerize, form heterodimers with maize endogenous HD-Zip II proteins, and bind to target DNA sequences; thus, ATHB17?113 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 ATHB17?113 relieves this HD-Zip II mediated transcriptional repression activity. Expression of ATHB17?113 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

  12. 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 ?-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 ATHB17?113, a truncated protein lacking the first 113 amino acids which encodes a significant portion of the repression domain. Because ATHB17?113 lacks the repression domain, the protein cannot directly affect the transcription of its target genes. ATHB17?113 can homodimerize, form heterodimers with maize endogenous HD-Zip II proteins, and bind to target DNA sequences; thus, ATHB17?113 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 ATHB17?113 relieves this HD-Zip II mediated transcriptional repression activity. Expression of ATHB17?113 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

  13. 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

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

    PubMed Central

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

    2011-01-01

    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 have completely been 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

  15. Cell Research | Vol 21 No 6 | June 2011 RESEARCH HIGHLIGHT

    E-print Network

    Belles, Xavier

    and is synthesized by the posterior silk gland. This fibroin core is covered with a layer of protein called sericin, which is produced in the middle silk gland [3, 5]. The weight contents of fibroin and sericin in total

  16. 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 ?-sheet crystallization occurred during electro-spinning. The molecular conformation was relatively unaffected by the electro-spinning rate of silk. PMID:23817099

  17. 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

  18. Spider silks and their applications.

    PubMed

    Kluge, Jonathan A; Rabotyagova, Olena; Leisk, Gary G; Kaplan, David L

    2008-05-01

    Spider silks are characterized by remarkable diversity in their chemistry, structure and functions, ranging from orb web construction to adhesives and cocoons. These unique materials have prompted efforts to explore potential applications of spider silk equivalent to those of silkworm silks, which have undergone 5,000 years of domestication and have a variety of uses, from textiles to biomedical materials. Recent progress in genetic engineering of spider silks and the development of new chimeric spider silks with enhanced functions and specific characteristics have advanced spider silk technologies. Further progress in yields of expressed spider-silk proteins, in the control of self-assembly processes and in the selective exploration of material applications is anticipated in the future. The unique features of spider silks, the progress and challenges in the cloning and expression of these silks, environmentally triggered silk assembly and disassembly and the formation of fibers, films and novel chimeric composite materials from genetically engineered spider silks will be reviewed. PMID:18367277

  19. Development of silk-like materials based on Bombyx mori and Nephila clavipes dragline silk fibroins

    Microsoft Academic Search

    Mingying Yang; Junji Kawamura; Zhenghua Zhu; Kazuo Yamauchi; Tetsuo Asakura

    2009-01-01

    In order to develop new silk-like materials in the form of fiber and non-woven nano-fiber, this study synthesized a new silk-like protein by selecting the sequence from the crystalline region of Bombyx mori silk fibroin, (GAGSGA)6, to imitate the processing condition of the silk fibroin with the combination of the sequence YGGLGSQGAGRG, the hydrophilic motif of spider dragline silk which

  20. Science Nation: Got Silk?

    NSDL National Science Digital Library

    Spider silk fibers are stronger than almost any other man-made fiber and they're also elastic. There are a lot of potential applications for the fiber, but how do you come up with enough raw material? With help from the National Science Foundation (NSF), researchers have figured out a way to put the spider's silk-making genes into goats in a way that they only make the protein in their milk.

  1. Nanostructure and molecular mechanics of spider dragline silk

    E-print Network

    Buehler, Markus J.

    Nanostructure and molecular mechanics of spider dragline silk protein assemblies Sinan Keten1, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139, USA Spider silk is a self structure of silk proteins has a direct influence on the stiffness, toughness and failure strength of silk

  2. Solute diffusion in genetically engineered silk-elastinlike protein polymer hydrogels.

    PubMed

    Dinerman, Adam A; Cappello, Joseph; Ghandehari, Hamidreza; Hoag, Stephen W

    2002-08-21

    The partitioning and diffusion behavior of theophylline, vitamin B(12), and cytochrome c in physically crosslinked networks of a genetically engineered silk-elastinlike protein-based (SELP) copolymer with an amino acid sequence of [(GVGVP)(4)GKGVP(GVGVP)(3)(GAGAGS)(4)](12) was investigated. The effect of gelation kinetics on the equilibrium swelling ratio and normalized dimensions of loaded SELP hydrogel disks before and after release studies was also examined. Size dependent release behavior was quantified by diffusion studies with equilibrium loaded SELP hydrogels. Direct loading diffusion studies confirmed that hydrogels produced by direct incorporation of cytochrome c with the aqueous SELP solution did not significantly influence the release behavior compared to equilibrium loaded hydrogels. An overall increase in the equilibrium swelling ratio after the release studies was observed. Analysis of the hydrogel disk dimensions after the release studies revealed no expansion of the disk dimensions. The apparent increase in the equilibrium swelling ratio was most likely due to a decrease in the hydrogel crosslinking density following the removal of the polymer soluble fraction over the course of the release study. PMID:12175743

  3. 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

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

    2012-03-01

    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](15)X). 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

  4. 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

  5. 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.

  6. Electrostatic charges instigate 'concertina-like' mechanisms of molecular toughening in MaSp1 (spider silk) proteins.

    PubMed

    Pahlevan, Mahdi; Toivakka, Martti; Alam, Parvez

    2014-08-01

    According to a recent article authored by Ortega-Jimenez and Dudley [1], the capture success of spiders is in part due to electrostatic charges on the surfaces of insects that macroscopically deform the spider web and increase the chances of insect-web contact. In this brief communication, we further show that electrostatic charges instigate a molecular 'concertina-like' mechanism of deformation in MaSp1 protein, which effectively begins the toughening-up of dragline silk threads prior to insect-web contact. PMID:24907767

  7. Silk Electrogel Rheology

    NASA Astrophysics Data System (ADS)

    Tabatabai, A. P.; Urbach, J. S.; Blair, D. L.; Kaplan, D. L.

    2014-03-01

    We present experimental results on the rheology on electrogels derived from aqueous solutions of reconstituted Bombyx Mori silk fibroin protein. Through electrochemistry, the silk protein solution develops local pH changes resulting in the assembly of protein into a weak gel. We determine the physical properties of the electrogels by performing rheology and observe that they exhibit the characteristics of a crosslinked biopolymer network. Interestingly, we find that these silk gels exhibit linear elasticity over a range of up to two orders of magnitude larger than most crosslinked biopolymer networks. Moreover, the nonlinear rheology exhibits a strain-stiffening behavior that is fundamentally different than the strain-stiffening observed in crosslinked biopolymers. Through rheological techniques we aim to understand this distinctive material that cannot be explained by current polymeric models. This work is supported by a grant from the AFOSR FA9550-07-1-0130.

  8. Sericin Enhances the Bioperformance of Collagen-Based Matrices Preseeded with Human-Adipose Derived Stem Cells (hADSCs).

    PubMed

    Dinescu, Sorina; Galateanu, Bianca; Albu, Madalina; Cimpean, Anisoara; Dinischiotu, Anca; Costache, Marieta

    2013-01-01

    Current clinical strategies for adipose tissue engineering (ATE), including autologous fat implants or the use of synthetic surrogates, not only are failing in the long term, but also can't face the latest requirements regarding the aesthetic restoration of the resulted imperfections. In this context, modern strategies in current ATE applications are based on the implantation of 3D cell-scaffold bioconstructs, designed for prospective achievement of in situ functional de novo tissue. Thus, in this paper, we reported for the first time the evaluation of a spongious 60% collagen and 40% sericin scaffold preseeded with human adipose-derived stem cells (hADSCs) in terms of biocompatibility and adipogenic potential in vitro. We showed that the addition of the sticky protein sericin in the composition of a classical collagen sponge enhanced the adhesion and also the proliferation rate of the seeded cells, thus improving the biocompatibility of the novel scaffold. In addition, sericin stimulated PPAR?2 overexpression, triggering a subsequent upregulated expression profile of FAS, aP2 and perilipin adipogenic markers. These features, together with the already known sericin stimulatory potential on cellular collagen production, promote collagen-sericin biomatrix as a good candidate for soft tissue reconstruction and wound healing applications. PMID:23325052

  9. 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...

  10. 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

  11. Snmp-1, a novel membrane protein of olfactory neurons of the silk moth Antheraea polyphemus with homology to the CD36 family of membrane proteins.

    PubMed

    Rogers, M E; Sun, M; Lerner, M R; Vogt, R G

    1997-06-01

    While olfactory neurons of silk moths are well known for their exquisite sensitivity to sex pheromone odorants, molecular mechanisms underlying this sensitivity are poorly understood. In searching for proteins that might support olfactory mechanisms, we characterized the protein profile of olfactory neuron receptor membranes of the wild silk moth Antheraea polyphemus. We have purified and cloned a prominent 67-kDa protein which we have named Snmp-1 (sensory neuron membrane protein-1). Northern blot analysis suggests that Snmp-1 is uniquely expressed in antennal tissue; in situ hybridization and immunocytochemical analyses show that Snmp-1 is expressed in olfactory neurons and that the protein is localized to the cilia, dendrites, and somata but not the axons. Snmp-1 mRNA expression increases significantly 1-2 days before the end of adult development, coincident with the functional maturation of the olfactory system. Sequence analysis suggests Snmp-1 is homologous with the CD36 protein family, a phylogenetically diverse family of receptor-like membrane proteins. CD36 family proteins are characterized as having two transmembrane domains and interacting with proteinaceous ligands; Snmp-1 is the first member of this family identified in nervous tissue. These findings argue that Snmp-1 has an important role in olfaction; possible roles of Snmp-1 in odorant detection are discussed. PMID:9169446

  12. 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 ?-helical dope to a mixed ?-helix/?-sheet-containing fibre can be directly related to spidroin architecture and stability. PMID:26112753

  13. 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 ?-helical dope to a mixed ?-helix/?-sheet-containing fibre can be directly related to spidroin architecture and stability. PMID:26112753

  14. NMR analysis of the fibronectin cell-adhesive sequence, Arg-Gly-Asp, in a recombinant silk-like protein and a model peptide.

    PubMed

    Asakura, Tetsuo; Nishi, Hirohito; Nagano, Aya; Yoshida, Ai; Nakazawa, Yasumoto; Kamiya, Masakatsu; Demura, Makoto

    2011-11-14

    It is well established that by introducing the cell-adhesive sequence Arg-Gly-Asp (RGD) from fibronectin into Bombyx mori silk fibroin by covalent coupling or bioengineering techniques, excellent biomaterials have been developed with the modified silk fibroin. However, there is no report about the structure and dynamics of the RGD moiety in the silk fibroin. To clarify the origin of such a high cell adhesion character and to design new recombinant silk protein with higher cell adhesion ability, it is necessary to characterize the structure and dynamics of the RGD moiety introduced into silk fibroin. In this study, the structure and dynamics of the RGD moiety in a recombinant silk-like protein, SLPF(10), consisting of the repeated silk fibroin sequence (AGSGAG)(3) and the sequence ASTGRGDSPA including the RGD moiety, were studied using solution NMR. The (1)H, (15)N, and (13)C chemical shifts indicate that the RGD moiety, as well as the silk fibroin sequence, takes a random coil form with high mobility in aqueous solution. Next, a (13)C solid-state NMR study was performed on a (13)C selectively labeled model peptide, AGSGAG[3-(13)C]A(7)GSGAGAGSGGT[2-(13)C]G(19)R[1-(13)C]G(21)DSPAGGGAGAGSGAG. After formic acid treatment, an increase in the ?-sheet fraction for the AGSGAG sequence and peak narrowing of the residues around the RGD moiety were observed in the dry state. The latter indicates a decrease in the chemical shift distribution although the RGD moiety is still in random coil. A decrease in the peak intensities of the RGD moiety in the swollen state after immersing it in distilled water was observed, indicating high mobility of the RGD sequence in the peptide in the swollen state. Thus, the random coil state of the RGD moiety in the recombinant silk-like protein is maintained in aqueous solution and also in both dry and swollen state. This is similar to the case of the RGD moiety in fibronectin. The presence of the linker ASTG at the N-terminus and SPAGG at the C-terminus seems important to maintain the random coil form and the flexible state of the RGD sequence in order to permit access for binding to various integrins. PMID:21955288

  15. Skin equivalent tissue-engineered construct: co-cultured fibroblasts/ keratinocytes on 3D matrices of sericin hope cocoons.

    PubMed

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

    2013-01-01

    The development of effective and alternative tissue-engineered skin replacements to autografts, allografts and xenografts has became a clinical requirement due to the problems related to source of donor tissue and the perceived risk of disease transmission. In the present study 3D tissue engineered construct of sericin is developed using co-culture of keratinocytes on the upper surface of the fabricated matrices and with fibroblasts on lower surface. Sericin is obtained from "Sericin Hope" silkworm of Bombyx mori mutant and is extracted from cocoons by autoclave. Porous sericin matrices are prepared by freeze dried method using genipin as crosslinker. The matrices are characterized biochemically and biophysically. The cell proliferation and viability of co-cultured fibroblasts and keratinocytes on matrices for at least 28 days are observed by live/dead assay, Alamar blue assay, and by dual fluorescent staining. The growth of the fibroblasts and keratinocytes in co-culture is correlated with the expression level of TGF-?, b-FGF and IL-8 in the cultured supernatants by enzyme-linked immunosorbent assay. The histological analysis further demonstrates a multi-layered stratified epidermal layer of uninhibited keratinocytes in co-cultured constructs. Presence of involucrin, collagen IV and the fibroblast surface protein in immuno-histochemical stained sections of co-cultured matrices indicates the significance of paracrine signaling between keratinocytes and fibroblasts in the expression of extracellular matrix protein for dermal repair. No significant amount of pro inflammatory cytokines (TNF-?, IL-1? and nitric oxide) production are evidenced when macrophages grown on the sericin matrices. The results all together depict the potentiality of sericin 3D matrices as skin equivalent tissue engineered construct in wound repair. PMID:24058626

  16. Skin Equivalent Tissue-Engineered Construct: Co-Cultured Fibroblasts/ Keratinocytes on 3D Matrices of Sericin Hope Cocoons

    PubMed Central

    Nayak, Sunita; Dey, Sancharika; Kundu, Subhas C.

    2013-01-01

    The development of effective and alternative tissue-engineered skin replacements to autografts, allografts and xenografts has became a clinical requirement due to the problems related to source of donor tissue and the perceived risk of disease transmission. In the present study 3D tissue engineered construct of sericin is developed using co-culture of keratinocytes on the upper surface of the fabricated matrices and with fibroblasts on lower surface. Sericin is obtained from “Sericin Hope” silkworm of Bombyx mori mutant and is extracted from cocoons by autoclave. Porous sericin matrices are prepared by freeze dried method using genipin as crosslinker. The matrices are characterized biochemically and biophysically. The cell proliferation and viability of co-cultured fibroblasts and keratinocytes on matrices for at least 28 days are observed by live/dead assay, Alamar blue assay, and by dual fluorescent staining. The growth of the fibroblasts and keratinocytes in co-culture is correlated with the expression level of TGF-?, b-FGF and IL-8 in the cultured supernatants by enzyme-linked immunosorbent assay. The histological analysis further demonstrates a multi-layered stratified epidermal layer of uninhibited keratinocytes in co-cultured constructs. Presence of involucrin, collagen IV and the fibroblast surface protein in immuno-histochemical stained sections of co-cultured matrices indicates the significance of paracrine signaling between keratinocytes and fibroblasts in the expression of extracellular matrix protein for dermal repair. No significant amount of pro inflammatory cytokines (TNF-?, IL-1? and nitric oxide) production are evidenced when macrophages grown on the sericin matrices. The results all together depict the potentiality of sericin 3D matrices as skin equivalent tissue engineered construct in wound repair. PMID:24058626

  17. Conservation of Essential Design Features in Coiled Coil Silks

    Microsoft Academic Search

    Tara D. Sutherland; Sarah Weisman; Holly E. Trueman; Alagacone Sriskantha; John W. H. Trueman; Victoria S. Haritos

    2007-01-01

    Silks are strong protein fibers produced by a broad array of spiders and insects. The vast majority of known silks are large, repetitive proteins assembled into extended b-sheet structures. Honeybees, however, have found a radically different evolutionary solution to the need for a building material. The 4 fibrous proteins of honeybee silk are small (;30 kDa each) and nonrepetitive and

  18. Prediction of the structure of a silk-like protein in oligomeric states using explicit and implicit solvent models.

    PubMed

    Razzokov, Jamoliddin; Naderi, Saber; van der Schoot, Paul

    2014-08-01

    We perform Replica Exchange Molecular Dynamics (REMD) simulations on a silk-like protein design with amino-acid sequence [(Gly-Ala)3-Gly-Glu]5 to investigate the stability of a single protein, a dimer, a trimer and a tetramer made up of these proteins starting from ?-roll and ?-sheet structures in both explicit (TIP3P) and implicit (GBSA) solvent models. Our simulation results for the implicit solvent model agree with those for the explicit solvent model for simulation times up to the longest tested, being 30 ns per replica. From this we infer that the implicit solvent model that we use is reliable, allowing us to reach much longer time scales (up to 200 ns per replica). We find that the self-assembly of fibers of these proteins in solution must be a nucleated process, involving nuclei made up of at least three monomers. We also find that the conformation of the protein changes upon assembly, i.e., there is a transition from a disordered globular state to an ordered ?-sheet structure in the self-assembled state of aggregates containing more than two monomers. This indicates that autosteric effects must be important in the polymerization of this protein, reminiscent of what is observed for ?-amyloids. Our findings are consistent with recent experimental results on a protein with an amino acid sequence similar to that of the protein we study. PMID:24937549

  19. The molecular structure of spider dragline silk: Folding and orientation of the protein backbone

    PubMed Central

    van Beek, J. D.; Hess, S.; Vollrath, F.; Meier, B. H.

    2002-01-01

    The design principles of spider dragline silk, nature's high-performance fiber, are still largely unknown, in particular for the noncrystalline glycine-rich domains, which form the bulk of the material. Here we apply two-dimensional solid-state NMR to determine the distribution of the backbone torsion angles (?,?) as well as the orientation of the polypeptide backbone toward the fiber at both the glycine and alanine residues. Instead of an “amorphous matrix,” suggested earlier for the glycine-rich domains, these new data indicate that all domains in dragline silk have a preferred secondary structure and are strongly oriented, with the chains predominantly parallel to the fiber. As proposed previously, the alanine residues are predominantly found in a ? sheet conformation. The glycine residues are partly incorporated into the ? sheets and otherwise form helical structures with an approximate 3-fold symmetry. PMID:12149440

  20. Biomimetic magnetic silk scaffolds.

    PubMed

    Samal, Sangram K; Dash, Mamoni; Shelyakova, Tatiana; Declercq, Heidi A; Uhlarz, Marc; Bañobre-López, Manuel; Dubruel, Peter; Cornelissen, Maria; Herrmannsdörfer, Thomas; Rivas, Jose; Padeletti, Giuseppina; De Smedt, Stefaan; Braeckmans, Kevin; Kaplan, David L; Dediu, V Alek

    2015-03-25

    Magnetic silk fibroin protein (SFP) scaffolds integrating magnetic materials and featuring magnetic gradients were prepared for potential utility in magnetic-field assisted tissue engineering. Magnetic nanoparticles (MNPs) were introduced into SFP scaffolds via dip-coating methods, resulting in magnetic SFP scaffolds with different strengths of magnetization. Magnetic SFP scaffolds showed excellent hyperthermia properties achieving temperature increases up to 8 °C in about 100 s. The scaffolds were not toxic to osteogenic cells and improved cell adhesion and proliferation. These findings suggest that tailored magnetized silk-based biomaterials can be engineered with interesting features for biomaterials and tissue-engineering applications. PMID:25734962

  1. Silk Fibroin Electrogelation Mechanisms

    PubMed Central

    Lu, Qiang; Huang, Yongli; Li, Mingzhong; Zuo, Baoqi; Lu, Shenzhou; Wang, Jiannan; Zhu, Hesun; Kaplan, David L.

    2012-01-01

    A silk fibroin gel system (e-gel), formed with weak electric fields has potential utility in medical materials and devices. The mechanism of silk e-gel formation was studied to gain additional insight into the process and control of the material properties. Silk fibroin nanoparticles with sizes of several ten nanometers, composed of metastable conformations, were involved in e-gel formation. Under electric fields, the nanoparticles rapidly assembled into larger nano- or microspheres with size ranges from tens nanometers to several microns. Repulsive forces from the negative surface charge of the acidic groups on the protein were screened by the local decrease in solution pH in the vicinity of the positive electrode. By controlling the formation and content of silk fibroin nanoparticles e-gel could be formed even from low concentration silk fibroin solutions (1%). When e-gel was reversed to the solution state, the aggregated nano- and microspheres dispersed into solution, a significant observation related to future applications for this process, such as for drug delivery. PMID:21345387

  2. 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 ?-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.

  3. Molecular biology of spider silk

    Microsoft Academic Search

    Stefan Winkler; David L Kaplan

    2000-01-01

    Spider silks are an intriguing family of fibrous proteins due to their highly repetitive primary sequence, their solution properties and their assembly and processing into fibers with remarkable mechanical properties. Current research efforts aimed at understanding and manipulating genes encoding these proteins are helping to gain insight into the relationships between protein sequence, protein assembly and macromolecular properties.

  4. Extreme Diversity, Conservation, and Convergence of Spider Silk Fibroin Sequences

    Microsoft Academic Search

    John Gatesy; Cheryl Hayashi; Dagmara Motriuk; Justin Woods; Randolph Lewis

    2001-01-01

    Spiders (Araneae) spin high-performance silks from liquid fibroin proteins. Fibroin sequences from basal spider lineages reveal mosaics of amino acid motifs that differ radically from previously described spider silk sequences. The silk fibers of Araneae are constructed from many protein designs. Yet, the repetitive sequences of fibroins from orb-weaving spiders have been maintained, presumably by stabilizing selection, over 125 million

  5. Silk gland factor-2, involved in fibroin gene transcription, consists of LIM homeodomain, LIM-interacting, and single-stranded DNA-binding proteins.

    PubMed

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

    2013-11-01

    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

  6. Silk tape nanostructure and silk gland anatomy of trichoptera.

    PubMed

    Ashton, Nicholas N; Taggart, Daniel S; Stewart, Russell J

    2012-06-01

    Caddisflys (order Trichoptera) construct elaborate protective shelters and food harvesting nets with underwater adhesive silk. The silk fiber resembles a nanostructured tape composed of thousands of nanofibrils (? 120 nm) oriented with the major axis of the fiber, which in turn are composed of spherical subunits. Weaker lateral interactions between nanofibrils allow the fiber to conform to surface topography and increase contact area. Highly phosphorylated (pSX)(4) motifs in H-fibroin blocks of positively charged basic residues are conserved across all three suborders of Trichoptera. Electrostatic interactions between the oppositely charged motifs could drive liquid-liquid phase separation of silk fiber precursors into a complex coacervates mesophase. Accessibility of phosphoserine to an anti-phosphoserine antibody is lower in the lumen of the silk gland storage region compared to the nascent fiber formed in the anterior conducting channel. The phosphorylated motifs may serve as a marker for the structural reorganization of the silk precursor mesophase into strongly refringent fibers. The structural change occurring at the transition into the conducting channel makes this region of special interest. Fiber formation from polyampholytic silk proteins in Trichoptera may suggest a new approach to create synthetic silk analogs from water-soluble precursors. PMID:21953029

  7. Influence of repeat numbers on self-assembly rates of repetitive recombinant spider silk proteins.

    PubMed

    Humenik, Martin; Magdeburg, Michael; Scheibel, Thomas

    2014-06-01

    Assembly of recombinant spider silk variants eADF4(Cn) comprising different numbers (n) of the consensus sequence motif C, derived from the natural Araneus diadematus dragline silk ADF4, yielded indistinguishable nanofibrils in cases of n?2. The C-module comprises 35 amino acids rich in glycine and proline residues (in GPGXY repeats) and one polyalanine stretch (Ala)8. All variants were found to be intrinsically disordered in solution, and upon fibril formation they converted into a cross-? structure. Heterologous seeding indicated high structural compatibility between the different eADF4(Cn) variants, however, their assembly kinetics differed in dependence of the number of repeats. Kinetic analysis revealed a nucleation-growth mechanism typical for the formation of cross-?-fibrils, with nucleation rates as well as growth rates increasing with increasing numbers of repeats. Strikingly, the single C-module did not self-assemble into fibrils, but upon addition of heterologous seeds fibril growth could be observed. Apparently, interconnecting of at least two C-modules significantly facilitates the structural transformation from a disordered state into ?-sheet structures, which is necessary for nucleation and beneficial for fibril growth. PMID:24657229

  8. Reproducing Natural Spider Silks’ Copolymer Behavior in Synthetic Silk Mimics

    PubMed Central

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

    2012-01-01

    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 ?-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. PMID:23110450

  9. Reproducing natural spider silks' copolymer behavior in synthetic silk mimics.

    PubMed

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

    2012-12-10

    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 ?-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. PMID:23110450

  10. Conformational Transitions in Model Silk Peptides

    Microsoft Academic Search

    Donna Wilson; Regina Valluzzi; David Kaplan

    2000-01-01

    Protein structural transitions and ?-sheet formation are a common problem both in vivo and in vitro and are of critical relevance in disparate areas such as protein processing and ?-amyloid and prion behavior. Silks provide a “databank” of well-characterized polymorphic sequences, acting as a window onto structural transitions. Peptides with conformationally polymorphic silk-like sequences, expected to exhibit an intractable ?-sheet

  11. Characterization and optimization of RGD-containing silk blends to support osteoblastic differentiation

    Microsoft Academic Search

    Abby W. Morgan; Kristen E. Roskov; Sheng Lin-Gibson; David L. Kaplan; Matthew L. Becker; Carl G. Simon Jr.

    2008-01-01

    The effect of blending two silk proteins, regenerated Bombyx mori fibroin and synthetic spidroin containing RGD, on silk film material structure (?-sheet content) and properties (solubility), as well as on biological response (osteoblast adhesion, proliferation and differentiation) was investigated. Although the elasticity and strength of silks make them attractive candidates for bone, ligament, and cartilage tissue engineering applications, silk proteins

  12. 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

  13. 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

  14. Silk from crickets: a new twist on spinning.

    PubMed

    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. Composition and Hierarchical Organisation of a Spider Silk

    PubMed Central

    Sponner, Alexander; Vater, Wolfram; Monajembashi, Shamci; Unger, Eberhard; Grosse, Frank; Weisshart, Klaus

    2007-01-01

    Albeit silks are fairly well understood on a molecular level, their hierarchical organisation and the full complexity of constituents in the spun fibre remain poorly defined. Here we link morphological defined structural elements in dragline silk of Nephila clavipes to their biochemical composition and physicochemical properties. Five layers of different make-ups could be distinguished. Of these only the two core layers contained the known silk proteins, but all can vitally contribute to the mechanical performance or properties of the silk fibre. Understanding the composite nature of silk and its supra-molecular organisation will open avenues in the production of high performance fibres based on artificially spun silk material. PMID:17912375

  16. 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

  17. Pulsed laser deposition of silk protein: Effect of photosensitized-ablation on the secondary structure in thin deposited films

    NASA Astrophysics Data System (ADS)

    Tsuboi, Yasuyuki; Goto, Masaharu; Itaya, Akira

    2001-06-01

    Silk fibroin is a simple protein expected to have functional applications in medicine and bioelectronics. The primary structure of this protein is quite simple, and the main secondary structures are ?-sheet crystals and amorphous random coils. In the present study, we investigated pulsed laser deposition (PLD) of fibroin with the ?-sheet structures as targets. The primary and secondary structures in films deposited were analyzed using infrared spectroscopy. Normal laser deposition at 351 nm using neat fibroin targets produced thin films of fibroin with a random coiled structure. Ablation was triggered by two-photonic excitation of the peptide chains, which resulted in the destruction of ?-sheet structure in PLD. In order to avoid the two-photonic excitation, we adopted a PLD method utilizing anthracene (5-0.1 wt %) in a photosensitized reaction involving doped fibroin targets. Laser light (351 or 355 nm) was absorbed only by anthracene, which plays an important role converting photon energy to thermal energy with great ablation efficiency. Thin fibroin films deposited by this method had both random coil and ?-sheet structures. As the dopant concentration and laser fluence decreased, the ratio of ?-sheet domain to random coil increased in thin deposited films.

  18. Pulsed laser deposition of silk protein: Effect of photosensitized-ablation on the secondary structure in thin deposited films

    SciTech Connect

    Tsuboi, Yasuyuki; Goto, Masaharu; Itaya, Akira

    2001-06-15

    Silk fibroin is a simple protein expected to have functional applications in medicine and bioelectronics. The primary structure of this protein is quite simple, and the main secondary structures are {beta}-sheet crystals and amorphous random coils. In the present study, we investigated pulsed laser deposition (PLD) of fibroin with the {beta}-sheet structures as targets. The primary and secondary structures in films deposited were analyzed using infrared spectroscopy. Normal laser deposition at 351 nm using neat fibroin targets produced thin films of fibroin with a random coiled structure. Ablation was triggered by two-photonic excitation of the peptide chains, which resulted in the destruction of {beta}-sheet structure in PLD. In order to avoid the two-photonic excitation, we adopted a PLD method utilizing anthracene (5{endash}0.1 wt%) in a photosensitized reaction involving doped fibroin targets. Laser light (351 or 355 nm) was absorbed only by anthracene, which plays an important role converting photon energy to thermal energy with great ablation efficiency. Thin fibroin films deposited by this method had both random coil and {beta}-sheet structures. As the dopant concentration and laser fluence decreased, the ratio of {beta}-sheet domain to random coil increased in thin deposited films. {copyright} 2001 American Institute of Physics.

  19. Molecular nanosprings in spider capture-silk threads

    Microsoft Academic Search

    Nathan Becker; Emin Oroudjev; Stephanie Mutz; Jason P. Cleveland; Paul K. Hansma; Cheryl Y. Hayashi; Dmitrii E. Makarov; Helen G. Hansma

    2003-01-01

    Spider capture silk is a natural material that outperforms almost any synthetic material in its combination of strength and elasticity. The structure of this remarkable material is still largely unknown, because spider-silk proteins have not been crystallized. Capture silk is the sticky spiral in the webs of orb-weaving spiders. Here we are investigating specifically the capture spiral threads from Araneus,

  20. Molecular nanosprings in spider capture-silk threads

    NASA Astrophysics Data System (ADS)

    Becker, Nathan; Oroudjev, Emin; Mutz, Stephanie; Cleveland, Jason P.; Hansma, Paul K.; Hayashi, Cheryl Y.; Makarov, Dmitrii E.; Hansma, Helen G.

    2003-04-01

    Spider capture silk is a natural material that outperforms almost any synthetic material in its combination of strength and elasticity. The structure of this remarkable material is still largely unknown, because spider-silk proteins have not been crystallized. Capture silk is the sticky spiral in the webs of orb-weaving spiders. Here we are investigating specifically the capture spiral threads from Araneus, an ecribellate orb-weaving spider. The major protein of these threads is flagelliform protein, a variety of silk fibroin. We present models for molecular and supramolecular structures of flagelliform protein, derived from amino acid sequences, force spectroscopy (molecular pulling) and stretching of bulk capture web. Pulling on molecules in capture-silk fibres from Araneus has revealed rupture peaks due to sacrificial bonds, characteristic of other self-healing biomaterials. The overall force changes are exponential for both capture-silk molecules and intact strands of capture silk.

  1. Materials fabrication from Bombyx mori silk fibroin

    Microsoft Academic Search

    Danielle N Rockwood; Rucsanda C Preda; Tuna Yücel; Xiaoqin Wang; Michael L Lovett; David L Kaplan

    2011-01-01

    Silk fibroin, derived from Bombyx mori cocoons, is a widely used and studied protein polymer for biomaterial applications. Silk fibroin has remarkable mechanical properties when formed into different materials, demonstrates biocompatibility, has controllable degradation rates from hours to years and can be chemically modified to alter surface properties or to immobilize growth factors. A variety of aqueous or organic solvent-processing

  2. Vortex-Induced Injectable Silk Fibroin Hydrogels

    Microsoft Academic Search

    Tuna Yucel; Peggy Cebe; David L. Kaplan

    2009-01-01

    A novel, to our knowledge, technique was developed to control the rate of ?-sheet formation and resulting hydrogelation kinetics of aqueous, native silk solutions. Circular dichroism spectroscopy indicated that vortexing aqueous solutions of silkworm silk lead to a transition from an overall protein structure that is initially rich in random coil to one that is rich in ?-sheet content. Dynamic

  3. Mechanical Response of Silk Crystalline Units from Force-Distribution Analysis

    E-print Network

    Gräter, Frauke

    crystals, opening up the road to predict full fiber mechanics. INTRODUCTION Silk proteins build upMechanical Response of Silk Crystalline Units from Force-Distribution Analysis Senbo Xiao, Wolfram of silk fibers is thought to be caused by embedded crystalline units acting as cross links of silk

  4. Mixed protein blends composed of gelatin and Bombyx mori silk fibroin: effects of solvent-induced crystallization and composition.

    PubMed

    Gil, Eun S; Frankowski, David J; Bowman, Michelle K; Gozen, Arif O; Hudson, Samuel M; Spontak, Richard J

    2006-03-01

    Novel protein blends have been prepared by mixing gelatin (G) with Bombyx mori silk fibroin (SF) and using aqueous methanol (MeOH) to post-induce SF crystallization. When co-cast from solution, amorphous blends of these polymers appear homogeneous, as discerned from visual observation, microscopy, and Fourier-transform infrared (FTIR) spectroscopy. Upon subsequent exposure to aqueous MeOH, SF undergoes a conformational change from random coil to beta sheet. This transformation occurs in pure SF, as well as in each of the G/SF blends, according to X-ray diffractometry and thermal calorimetry. The influence of MeOH-induced SF crystallization on structure and property development has been ascertained in terms of preparation history and blend composition. Thermal gravimetric analysis reveals that the presence of beta sheets in SF and G/SF blends improves thermal stability, while extensional rheometry confirms that SF crystallization enhances the tensile properties of the blends. By preserving a support scaffold above the G helix-to-coil transition temperature, the formation of crystalline SF networks in G/SF blends can be used to stabilize G-based hydrogels for biomaterial and pharmaceutical purposes. The present study not only examines the properties of G/SF blends before and after SF crystallization, but also establishes the foundation for future research into thermally responsive G/SF bioconjugates. PMID:16529407

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

    Microsoft Academic Search

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

    2007-01-01

    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 ?-sheet. Amino acid analysis shows that the protein is enriched in glycine and serine while the mole percentages of

  6. 13C NMR of Nephila clavipes major ampullate silk gland.

    PubMed

    Hijirida, D H; Do, K G; Michal, C; Wong, S; Zax, D; Jelinski, L W

    1996-12-01

    The major ampullate glands of the spider Nephila clavipes contain approximately 0.2 microliter each of a highly concentrated (approximately 50%) solution of silk fibroin. Therefore, the reservoir of silk in these glands presents an ideal opportunity to observe prefolded conformations of a protein in its native state. To this end, the structure and conformation of major ampullate gland silk fibroin within the glands of the spider N. clavipes were examined by 13C NMR spectroscopy. These results were compared to those from silk protein first drawn from the spinneret and then denatured. The 13C NMR chemical shifts, along with infrared and circular dichroism data, suggest that the silk fibroin in the glands exists in dynamically averaged helical conformations. Furthermore, there is no evidence of proline residues in U-(13)C-D-glucose-labeled silk. This transient prefolded "molten fibril" state may correspond to the silk I form found in Bombyx mori silk. There is no evidence of the final beta-sheet structure in the ampullate gland silk fibroin before final silk processing. However, the conformation of silk in the glands appears to be in a highly metastable state, as plasticization with water produces the beta-sheet structure. Therefore, the ducts connecting the ampullate glands to the spinnerets play a larger role in silk processing than previously thought. PMID:8968613

  7. 13C NMR of Nephila clavipes major ampullate silk gland.

    PubMed Central

    Hijirida, D H; Do, K G; Michal, C; Wong, S; Zax, D; Jelinski, L W

    1996-01-01

    The major ampullate glands of the spider Nephila clavipes contain approximately 0.2 microliter each of a highly concentrated (approximately 50%) solution of silk fibroin. Therefore, the reservoir of silk in these glands presents an ideal opportunity to observe prefolded conformations of a protein in its native state. To this end, the structure and conformation of major ampullate gland silk fibroin within the glands of the spider N. clavipes were examined by 13C NMR spectroscopy. These results were compared to those from silk protein first drawn from the spinneret and then denatured. The 13C NMR chemical shifts, along with infrared and circular dichroism data, suggest that the silk fibroin in the glands exists in dynamically averaged helical conformations. Furthermore, there is no evidence of proline residues in U-(13)C-D-glucose-labeled silk. This transient prefolded "molten fibril" state may correspond to the silk I form found in Bombyx mori silk. There is no evidence of the final beta-sheet structure in the ampullate gland silk fibroin before final silk processing. However, the conformation of silk in the glands appears to be in a highly metastable state, as plasticization with water produces the beta-sheet structure. Therefore, the ducts connecting the ampullate glands to the spinnerets play a larger role in silk processing than previously thought. PMID:8968613

  8. Silk film biomaterials for ocular surface repair

    NASA Astrophysics Data System (ADS)

    Lawrence, Brian David

    Current biomaterial approaches for repairing the cornea's ocular surface upon injury are partially effective due to inherent material limitations. As a result there is a need to expand the biomaterial options available for use in the eye, which in turn will help to expand new clinical innovations and technology development. The studies illustrated here are a collection of work to further characterize silk film biomaterials for use on the ocular surface. Silk films were produced from regenerated fibroin protein solution derived from the Bombyx mori silkworm cocoon. Methods of silk film processing and production were developed to produce consistent biomaterials for in vitro and in vivo evaluation. A wide range of experiments was undertaken that spanned from in vitro silk film material characterization to in vivo evaluation. It was found that a variety of silk film properties could be controlled through a water-annealing process. Silk films were then generated that could be use in vitro to produce stratified corneal epithelial cell sheets comparable to tissue grown on the clinical standard substrate of amniotic membrane. This understanding was translated to produce a silk film design that enhanced corneal healing in vivo on a rabbit injury model. Further work produced silk films with varying surface topographies that were used as a simplified analog to the corneal basement membrane surface in vitro. These studies demonstrated that silk film surface topography is capable of directing corneal epithelial cell attachment, growth, and migration response. Most notably epithelial tissue development was controllably directed by the presence of the silk surface topography through increasing cell sheet migration efficiency at the individual cellular level. Taken together, the presented findings represent a comprehensive characterization of silk film biomaterials for use in ocular surface reconstruction, and indicate their utility as a potential material choice in the development of innovative procedures and technologies for corneal repair.

  9. Enhancement of anti-inflammatory activity of PEP-1-FK506 binding protein by silk fibroin peptide.

    PubMed

    Kim, Dae Won; Hwang, Hyun Sook; Kim, Duk-Soo; Sheen, Seung Hoon; Heo, Dong Hwa; Hwang, Gyojun; Kang, Suk Hyung; Kweon, Haeyong; Jo, You-Young; Kang, Seok Woo; Lee, Kwang-Gill; Park, Jinseu; Eum, Won Sik; Cho, Yong-Jun; Choi, Soo Young

    2012-04-01

    Silk fibroin (SF) peptide has been traditionally used as a treatment for flatulence, spasms, and phlegm. In this study, we examined whether SF peptide enhanced the antiinflammatory effect of PEP-1-FK506 binding protein (PEP-1-FK506BP) through comparing the anti-inflammatory activities of SF peptide and/or PEP-1-FK506BP. In the presence or absence of SF peptide, transduction levels of PEP-1-FK506BP into HaCaT cells and mice skin and anti-inflammatory activities of PEP-1-FK506BP were identified by Western blot and histological analyses. SF peptide alone effectively reduced both mice ear edema and the elevated levels of cyclooxygenase-2, interleukin-6 and -1beta, and tumor necrosis factor-alpha, showing similar anti-inflammatory effect to that of PEP-1-FK506BP. Furthermore, co-treatment with SF peptide and PEP-1- FK506BP exhibited more enhanced anti-inflammatory effects than the samples treated with SF peptides or PEP- 1-FK506BP alone, suggesting the possibility that SF peptide and PEP-1-FK506BP might interact with each other. Moreover, the transduction data demonstrated that SF peptide did not affect the transduction of PEP-1- FK506BP into HaCaT cells and mice skin, indicating that the improvement of anti-inflammatory effect of PEP-1- FK506BP was not caused by enhanced transduction of PEP-1-FK506BP. Thus, these results suggest the possibility that co-treatment with SF peptide and PEP-1-FK506BP may be exploited as a useful therapy for various inflammationrelated diseases. PMID:22534296

  10. Structural characterization of nanofiber silk produced by embiopterans (webspinners)†

    PubMed Central

    Addison, J. Bennett; Popp, Thomas M. Osborn; Weber, Warner S.; Edgerly, Janice S.; Holland, Gregory P.; Yarger, Jeffery L.

    2014-01-01

    Embiopterans produce silken galleries and sheets using exceptionally fine silk fibers in which they live and breed. In this study, we use electron microscopy (EM), Fourier-transform infrared (FT-IR) spectroscopy, wide angle X-ray diffraction (WAXD) and solid-state nuclear magnetic resonance (ssNMR) techniques to elucidate the molecular level protein structure of webspinner (embiid) silks. Silks from two species Antipaluria urichi and Aposthonia ceylonica are studied in this work. Electron microscopy images show that the fibers are about 90–100 nm in diameter, making webspinner silks among the finest of all known animal silks. Structural studies reveal that the silk protein core is dominated by ?-sheet structures, and that the protein core is coated with a hydrophobic alkane-rich surface coating. FTIR spectra of native embiid silk shows characteristic alkane CH2 stretchings near 2800–2900 cm?1, which decrease approximately 50% after washing the silk with 2 : 1 CHCl3 : MeOH. Furthermore, 13C ssNMR data shows a significant CH2 resonance that is strongly affected by the presence of water, supporting the idea that the silk fibers are coated with a hydrocarbon-rich layer. Such a layer is likely used to protect the colonies from rain. FTIR data also suggests that embiid silks are dominated by ?-sheet secondary structures similar to spider and silkworm silk fibers. NMR data confirms the presence of ?-sheet nanostructures dominated by serine-rich repetitive regions. A deconvolution of the serine C? NMR resonance reveals that approximately 70% of all seryl residues exist in a ?-sheet structure. This is consistent with WAXD results that suggest webspinner silks are 70% crystalline, which is the highest crystalline fraction reported for any animal silks. The work presented here provides a molecular level structural picture of silk fibers produced by webspinners. PMID:25383190

  11. Molecular and Mechanical Characterization of Aciniform Silk: Uniformity of Iterated Sequence Modules in a Novel Member of the

    E-print Network

    Blackledge, Todd

    to seven different protein-based silks/glues that have diverse physical properties. The fibroin sequences constructed, and unique silk transcripts were sequenced. There was evidence for a single silk fibroin gene divergence at the sequence level among fibroin paralogs, suggest a possible linkage between silk fibroin

  12. Evaluation of Sericin as a Fetal Bovine Serum-Replacing Cryoprotectant During Freezing of Human Mesenchymal Stromal Cells and Human Osteoblast-Like Cells

    PubMed Central

    Verdanova, Martina; Pytlik, Robert

    2014-01-01

    A reliable, cryoprotective, xeno-free medium suitable for different cell types is highly desirable in regenerative medicine. There is danger of infection or allergic reaction with the use of fetal bovine serum (FBS), making it problematic for medical applications. The aim of the present study was to develop an FBS-free cryoprotective medium for human mesenchymal stromal cells (hMSCs; primary cells) and immortalized human osteoblasts (SAOS-2 cell line). Furthermore, we endeavored to eliminate or reduce the presence of dimethyl sulfoxide (DMSO) in the medium. Sericin, a sticky protein derived from the silkworm cocoon, was investigated as a substitute for FBS and DMSO in the freezing medium. Cell viability (24 hours after thawing, both hMSC and SAOS-2) and colony-forming ability (2 weeks after thawing, only for hMSCs) were both determined. The FBS-free medium with 1% sericin in 10% DMSO was found to be a suitable freezing medium for primary hMSCs, in contrast to immortalized human osteoblasts. Surprisingly, the storage of hMSCs in a cultivation medium with only 10% DMSO also provided satisfactory results. Any drop in DMSO concentration led to significantly worse survival of cells, with little improvement in hMSC survival in the presence of sericin. Thus, sericin may substitute for FBS in the freezing medium for primary hMSCs, but cannot substitute for DMSO. PMID:24749876

  13. Evaluation of sericin as a fetal bovine serum-replacing cryoprotectant during freezing of human mesenchymal stromal cells and human osteoblast-like cells.

    PubMed

    Verdanova, Martina; Pytlik, Robert; Kalbacova, Marie Hubalek

    2014-04-01

    A reliable, cryoprotective, xeno-free medium suitable for different cell types is highly desirable in regenerative medicine. There is danger of infection or allergic reaction with the use of fetal bovine serum (FBS), making it problematic for medical applications. The aim of the present study was to develop an FBS-free cryoprotective medium for human mesenchymal stromal cells (hMSCs; primary cells) and immortalized human osteoblasts (SAOS-2 cell line). Furthermore, we endeavored to eliminate or reduce the presence of dimethyl sulfoxide (DMSO) in the medium. Sericin, a sticky protein derived from the silkworm cocoon, was investigated as a substitute for FBS and DMSO in the freezing medium. Cell viability (24 hours after thawing, both hMSC and SAOS-2) and colony-forming ability (2 weeks after thawing, only for hMSCs) were both determined. The FBS-free medium with 1% sericin in 10% DMSO was found to be a suitable freezing medium for primary hMSCs, in contrast to immortalized human osteoblasts. Surprisingly, the storage of hMSCs in a cultivation medium with only 10% DMSO also provided satisfactory results. Any drop in DMSO concentration led to significantly worse survival of cells, with little improvement in hMSC survival in the presence of sericin. Thus, sericin may substitute for FBS in the freezing medium for primary hMSCs, but cannot substitute for DMSO. PMID:24749876

  14. Effect of pH on the structure of the recombinant C-terminal domain of Nephila clavipes dragline silk protein.

    PubMed

    Gauthier, Martin; Leclerc, Jérémie; Lefèvre, Thierry; Gagné, Stéphane M; Auger, Michèle

    2014-12-01

    Spider silk proteins undergo a complex series of molecular events before being converted into an outstanding hierarchically organized fiber. Recent literature has underlined the crucial role of the C-terminal domain in silk protein stability and fiber formation. However, the effect of pH remains to be clarified. We have thus developed an efficient purification protocol to obtain stable native-like recombinant MaSp1 C-terminal domain of Nephila clavipes (NCCTD). Its structure was investigated as a function of pH using circular dichroism, fluorescence and solution NMR spectroscopy. The results show that the NCCTD structure is very sensitive to pH and suggest that a molten globule state occurs at pH 5.0 and below. Electronic microscopy images also indicate fiber formation at low pH and coarser globular particles at more basic pH. The results are consistent with a spinning process model where the NCCTD acts as an aggregation nucleus favoring the ?-aggregation of the hydrophobic polyalanine repeats upon spinning. PMID:25337802

  15. History of Silk

    NSDL National Science Digital Library

    0000-00-00

    Nicely illustrated history of silk and explanation of silk production. A fine discussion of the moths, life history, and feeding are included, as well as the extensive cultural history of this outstanding insect product.

  16. Characterization and expression of a cDNA encoding a tubuliform silk protein of the golden web spider Nephila antipodiana

    Microsoft Academic Search

    W. Huang; Z. Lin; Y. M. Sin; D. Li; Z. Gong; D. Yang

    2006-01-01

    Spider silks are renowned for their excellent mechanical properties. Although several spider fibroin genes, mainly from dragline and capture silks, have been identified, there are still many members in the spider fibroin gene family remain uncharacterized. In this study, a novel silk cDNA clone from the golden web spider Nephila antipodiana was isolated. It is serine rich and contains two

  17. Study on the microstructure of African wild silk cocoon shells and fibers.

    PubMed

    Teshome, Addis; Vollrath, Fritz; Raina, Suresh K; Kabaru, J M; Onyari, J

    2012-01-01

    Silk fibers and cocoon shells from four African wild silkmoths Gonometa postica, Anaphe panda, Argema mimosae and Epiphora bauhiniae-were studied to gain insight into the structure-property-function relations and potential commercial application. The surface and cross-section of cocoon shells and fibers revealed the presence of prominent structural variations. Cocoon shells were multilayered and porous structures constructed from highly cross-linked fibers that are densely packed within the sericin/gum. Fibers had fibrillar sub-structures running along the fiber axis and with greater number and size of voids. The ecological significance and implication of these structures for further application are discussed. PMID:21986544

  18. Stylized Silk Painting.

    ERIC Educational Resources Information Center

    Skophammer, Karen

    1998-01-01

    Presents an art activity inspired by a workshop "Surrounded by Silk" given by Susan Skvoe in which the students create silk paintings. Explains that the students first sketch their floral design on paper, trace the design on the silk's surface, and apply liquid dye for color. Provides an easier activity for younger students. (CMK)

  19. Extreme Diversity, Conservation, and Convergence of Spider Silk Fibroin Sequences

    NASA Astrophysics Data System (ADS)

    Gatesy, John; Hayashi, Cheryl; Motriuk, Dagmara; Woods, Justin; Lewis, Randolph

    2001-03-01

    Spiders (Araneae) spin high-performance silks from liquid fibroin proteins. Fibroin sequences from basal spider lineages reveal mosaics of amino acid motifs that differ radically from previously described spider silk sequences. The silk fibers of Araneae are constructed from many protein designs. Yet, the repetitive sequences of fibroins from orb-weaving spiders have been maintained, presumably by stabilizing selection, over 125 million years of evolutionary history. The retention of these conserved motifs since the Mesozoic and their convergent evolution in other structural superproteins imply that these sequences are central to understanding the exceptional mechanical properties of orb weaver silks.

  20. Proteomic profiling of the photo-oxidation of silk fibroin: implications for historic tin-weighted silk.

    PubMed

    Solazzo, Caroline; Dyer, Jolon M; Deb-Choudhury, Santanu; Clerens, Stefan; Wyeth, Paul

    2012-01-01

    The stability of silk proteins to ultraviolet light is an issue of significant concern in both the appearance retention of silk-derived products and the preservation of historic silk textiles. Until now, evaluation of silk degradation has only been performed at the holistic, rather than molecular level. This article describes the first proteomic profiling of silk photo-oxidation, characterizing protein primary level modification leading to coloration changes, and evaluating the effects of tin weighting on photodegradation. Heavy-chain fibroin, the main proteinaceous component of the silk thread, is a repetitive, highly crystalline protein with a content rich in tyrosine. Photoproducts of tyrosine were characterized and the levels of oxidative modification at the protein primary structural level correlated with changes in coloration and tensile strength. The effect of tin as a weighting agent used on historical fabrics was examined. Tin-weighted fabrics were evaluated following two treatments (pink and dynamite) and proteomic analysis revealed a significant increase in oxidatively modified amino acid residues within the pink-treated silk. These findings offer new insight into the molecular-level oxidation of silk proteins under UV exposure, and the effects of silk treatments in either exacerbating or ameliorating this degradation. PMID:22554154

  1. The effect of sterilization on silk fibroin biomaterial properties.

    PubMed

    Rnjak-Kovacina, Jelena; DesRochers, Teresa M; Burke, Kelly A; Kaplan, David L

    2015-06-01

    The effects of common sterilization techniques on the physical and biological properties of lyophilized silk fibroin sponges are described. Sterile silk fibroin sponges were cast using a pre-sterilized silk fibroin solution under aseptic conditions or post-sterilized via autoclaving, ? radiation, dry heat, exposure to ethylene oxide, or hydrogen peroxide gas plasma. Low average molecular weight and low concentration silk fibroin solutions could be sterilized via autoclaving or filtration without significant loses of protein. However, autoclaving reduced the molecular weight distribution of the silk fibroin protein solution, and silk fibroin sponges cast from autoclaved silk fibroin were significantly stiffer compared to sponges cast from unsterilized or filtered silk fibroin. When silk fibroin sponges were sterilized post-casting, autoclaving increased scaffold stiffness, while decreasing scaffold degradation rate in vitro. In contrast, ? irradiation accelerated scaffold degradation rate. Exposure to ethylene oxide significantly decreased cell proliferation rate on silk fibroin sponges, which was rescued by leaching ethylene oxide into PBS prior to cell seeding. PMID:25761231

  2. Effects of RGDS sequence genetically interfused in the silk fibroin light chain protein on chondrocyte adhesion and cartilage synthesis

    Microsoft Academic Search

    Yusuke Kambe; Koji Yamamoto; Katsura Kojima; Yasushi Tamada; Naohide Tomita

    2010-01-01

    Initial chondrocyte–silk fibroin interactions are implicated in chondrogenesis when using fibroin as a scaffold for chondrocytes. Here, we focused on integrin-mediated cell–scaffold adhesion and prepared cell adhesive fibroin in which a tandem repeat of the Arg-Gly-Asp-Ser (RGDS) sequence was genetically interfused in the fibroin light chain (L-chain) (L-RGDS×2 fibroin). We investigated the effects of the sequence on chondrocyte adhesion and

  3. Characteristics of platelet gels combined with silk

    PubMed Central

    Pallotta, Isabella; Kluge, Jonathan A.; Moreau, Jodie; Calabrese, Rossella

    2014-01-01

    Platelet gel, a fibrin network containing activated platelets, is widely used in regenerative medicine due the capacity of platelet-derived growth factors to accelerate and direct healing processes. However, limitations to this approach include poor mechanical properties, relatively rapid degradation, and the lack of control of release of growth factors at the site of injection. These issues compromise the ability of platelet gels for sustained function in regenerative medicine. In the present study, a combination of platelet gels with silk fibroin gel was studied to address the above limitations. Mixing sonicated silk gels with platelet gels extended the release of growth factors without inhibiting gel forming ability. The released growth factors were biologically active and their delivery was modified further by manipulation of the charge of the silk protein. Moreover, the silk gel augmented both the rheological properties and compressive stiffness of the platelet gel, tuned by the silk concentration and/or silk/platelet gel ratio. Silk-platelet gel injections in nude rats supported enhanced cell infiltration and blood vessel formation representing a step towards new platelet gel formulations with enhanced therapeutic impact. PMID:24480538

  4. Silk fibroin biomaterials for tissue regenerations.

    PubMed

    Kundu, Banani; Rajkhowa, Rangam; Kundu, Subhas C; Wang, Xungai

    2013-04-01

    Regeneration of tissues using cells, scaffolds and appropriate growth factors is a key approach in the treatments of tissue or organ failure. Silk protein fibroin can be effectively used as a scaffolding material in these treatments. Silk fibers are obtained from diverse sources such as spiders, silkworms, scorpions, mites and flies. Among them, silk of silkworms is a good source for the development of biomedical device. It possesses good biocompatibility, suitable mechanical properties and is produced in bulk in the textile sector. The unique combination of elasticity and strength along with mammalian cell compatibility makes silk fibroin an attractive material for tissue engineering. The present article discusses the processing of silk fibroin into different forms of biomaterials followed by their uses in regeneration of different tissues. Applications of silk for engineering of bone, vascular, neural, skin, cartilage, ligaments, tendons, cardiac, ocular, and bladder tissues are discussed. The advantages and limitations of silk systems as scaffolding materials in the context of biocompatibility, biodegradability and tissue specific requirements are also critically reviewed. PMID:23137786

  5. The influence of specific binding of collagen-silk chimeras to silk biomaterials on hMSC behavior

    PubMed Central

    An, Bo; DesRochers, Teresa M.; Qin, Guokui; Xia, Xiaoxia; Thiagarajan, Geetha; Brodsky, Barbara; Kaplan, David

    2012-01-01

    Collagen-like proteins in the bacteria Streptococcus pyogenes adopt a triple-helix structure with a thermal stability similar to that of animal collagens, can be expressed in high yield in E. coli and can be easily modified through molecular biology techniques. However, potential applications for such recombinant collagens are limited by their lack of higher order structure to achieve the physical properties needed for most biomaterials. To overcome this problem, the S. pyrogenes collagen domain was fused to a repetitive Bombyx mori silk consensus sequence, as a strategy to direct specific non-covalent binding onto solid silk materials whose superior stability, mechanical and material properties have been previously established. This approach resulted in the successful binding of these new collagen-silk chimeric proteins to silk films and porous scaffolds, and the binding affinity could be controlled by varying the number of repeats in the silk sequence. To explore the potential of collagen-silk chimera for regulating biological activity, integrin (Int) and fibronectin (Fn) binding sequences from mammalian collagens were introduced into the bacterial collagen domain. The attachment of bioactive collagen-silk chimeras to solid silk biomaterials promoted hMSC spreading and proliferation substantially in comparison to the controls. The ability to combine the biomaterial features of silk with the biological activities of collagen allowed more rapid cell interactions with silk-based biomaterials, improved regulation of stem cell growth and differentiation, as well as the formation of artificial extracellular matrices useful for tissue engineering applications. PMID:23088839

  6. The influence of specific binding of collagen-silk chimeras to silk biomaterials on hMSC behavior.

    PubMed

    An, Bo; DesRochers, Teresa M; Qin, Guokui; Xia, Xiaoxia; Thiagarajan, Geetha; Brodsky, Barbara; Kaplan, David L

    2013-01-01

    Collagen-like proteins in the bacteria Streptococcus pyogenes adopt a triple-helix structure with a thermal stability similar to that of animal collagens, can be expressed in high yield in Escherichia coli and can be easily modified through molecular biology techniques. However, potential applications for such recombinant collagens are limited by their lack of higher order structure to achieve the physical properties needed for most biomaterials. To overcome this problem, the S. pyogenes collagen domain was fused to a repetitive Bombyx mori silk consensus sequence, as a strategy to direct specific non-covalent binding onto solid silk materials whose superior stability, mechanical and material properties have been previously established. This approach resulted in the successful binding of these new collagen-silk chimeric proteins to silk films and porous scaffolds, and the binding affinity could be controlled by varying the number of repeats in the silk sequence. To explore the potential of collagen-silk chimera for regulating biological activity, integrin (Int) and fibronectin (Fn) binding sequences from mammalian collagens were introduced into the bacterial collagen domain. The attachment of bioactive collagen-silk chimeras to solid silk biomaterials promoted hMSC spreading and proliferation substantially in comparison to the controls. The ability to combine the biomaterial features of silk with the biological activities of collagen allowed more rapid cell interactions with silk-based biomaterials, improved regulation of stem cell growth and differentiation, as well as the formation of artificial extracellular matrices useful for tissue engineering applications. PMID:23088839

  7. Sericins exhibit ROS-scavenging, anti-tyrosinase, anti-elastase, and in vitro immunomodulatory activities.

    PubMed

    Chlapanidas, Theodora; Faragò, Silvio; Lucconi, Giulia; Perteghella, Sara; Galuzzi, Marta; Mantelli, Melissa; Avanzini, Maria Antonietta; Tosca, Marta Cecilia; Marazzi, Mario; Vigo, Daniele; Torre, Maria Luisa; Faustini, Massimo

    2013-07-01

    Some biological properties of Bombyx mori sericins from twenty strains were investigated, fourteen fed with artificial diet, two with fresh mulberry leaves and four with both diets. Sericin exhibited ROS-scavenging, anti-tyrosinase and anti-elastase properties, the strain significantly influenced these properties, while diet only influenced the anti-tyrosinase activity. Sericins were clustered into 5 groups and one sericin from each group was further studied: sericins showed anti-proliferative activity on in vitro stimulated peripheral blood mononuclear cells; some strains decreased in vitro secretion of IFN?, while no effects were observed on TNF? and IL10 release. Therefore, a mixture of sericins extracted from the most promising strains may be useful for dermatological and cosmetic use. PMID:23541552

  8. Structure and properties of silk hydrogels.

    PubMed

    Kim, Ung-Jin; Park, Jaehyung; Li, Chunmei; Jin, Hyoung-Joon; Valluzzi, Regina; Kaplan, David L

    2004-01-01

    Control of silk fibroin concentration in aqueous solutions via osmotic stress was studied to assess relationships to gel formation and structural, morphological, and functional (mechanical) changes associated with this process. Environmental factors potentially important in the in vivo processing of aqueous silk fibroin were also studied to determine their contributions to this process. Gelation of silk fibroin aqueous solutions was affected by temperature, Ca(2+), pH, and poly(ethylene oxide) (PEO). Gelation time decreased with increase in protein concentration, decrease in pH, increase in temperature, addition of Ca(2+), and addition of PEO. No change of gelation time was observed with the addition of K(+). Upon gelation, a random coil structure of the silk fibroin was transformed into a beta-sheet structure. Hydrogels with fibroin concentrations >4 wt % exhibited network and spongelike structures on the basis of scanning electron microscopy. Pore sizes of the freeze-dried hydrogels were smaller as the silk fibroin concentration or gelation temperature was increased. Freeze-dried hydrogels formed in the presence of Ca(2+) exhibited larger pores as the concentration of this ion was increased. Mechanical compressive strength and modulus of the hydrogels increased with increase in protein concentration and gelation temperature. The results of these studies provide insight into the sol-gel transitions that silk fibroin undergoes in glands during aqueous processing while also providing important insight in the in vitro processing of these proteins into useful new materials. PMID:15132662

  9. Sericin\\/poly(vinyl alcohol) blend membranes for pervaporation separation of ethanol\\/water mixtures

    Microsoft Academic Search

    Marcelino L. Gimenes; Li Liu; Xianshe Feng

    2007-01-01

    Hydrophilic membranes were prepared from sericin and poly(vinyl alcohol) (PVA) for applications in alcohol dehydration by pervaporation. Sericin\\/PVA blend membranes were prepared by blending sericin and PVA, followed by chemical crosslinking with dimethylolurea. The permeation of water\\/ethanol mixtures through the membranes was investigated. It was shown that the blend membranes are preferentially permeable to water. In the temperature range of

  10. Mechanism of silk processing in insects and spiders

    NASA Astrophysics Data System (ADS)

    Jin, Hyoung-Joon; Kaplan, David L.

    2003-08-01

    Silk spinning by insects and spiders leads to the formation of fibres that exhibit high strength and toughness. The lack of understanding of the protein processing in silk glands has prevented the recapitulation of these properties in vitro from reconstituted or genetically engineered silks. Here we report the identification of emulsion formation and micellar structures from aqueous solutions of reconstituted silkworm silk fibroin as a first step in the process to control water and protein-protein interactions. The sizes (100-200nm diameter) of these structures could be predicted from hydrophobicity plots of silk protein primary sequence. These micelles subsequently aggregated into larger `globules' and gel-like states as the concentration of silk fibroin increased, while maintaining solubility owing to the hydrophilic regions of the protein interspersed among the larger hydrophobic regions. Upon physical shearing or stretching structural transitions, increased birefringence and morphological alignment were demonstrated, indicating that this process mimics the behaviour of similar native silk proteins in vivo. Final morphological features of these silk materials are similar to those observed in native silkworm fibres.

  11. Materials Fabrication from Bombyx mori Silk Fibroin

    PubMed Central

    Rockwood, Danielle N.; Preda, Rucsanda C.; Yücel, Tuna; Wang, Xiaoqin; Lovett, Michael L.; Kaplan, David L.

    2013-01-01

    Silk fibroin, derived from Bombyx mori cocoons, is a widely used and studied protein polymer for biomaterial applications. Silk fibroin has remarkable mechanical properties when formed into different materials, demonstrates biocompatibility, has controllable degradation rates from hours to years, and it can be chemically modified to alter surface properties or to immobilize growth factors. A variety of aqueous or organic solvent processing methods can be used to generate silk biomaterials for a range of applications. In this protocol we include methods to extract silk from B. mori cocoons in order to fabricate hydrogels, tubes, sponges, composites, fibers, microspheres and thin films. These materials can be used directly as biomaterials for implants, as scaffolding in tissue engineering and in vitro disease models, and for drug delivery. PMID:21959241

  12. Materials fabrication from Bombyx mori silk fibroin.

    PubMed

    Rockwood, Danielle N; Preda, Rucsanda C; Yücel, Tuna; Wang, Xiaoqin; Lovett, Michael L; Kaplan, David L

    2011-10-01

    Silk fibroin, derived from Bombyx mori cocoons, is a widely used and studied protein polymer for biomaterial applications. Silk fibroin has remarkable mechanical properties when formed into different materials, demonstrates biocompatibility, has controllable degradation rates from hours to years and can be chemically modified to alter surface properties or to immobilize growth factors. A variety of aqueous or organic solvent-processing methods can be used to generate silk biomaterials for a range of applications. In this protocol, we include methods to extract silk from B. mori cocoons to fabricate hydrogels, tubes, sponges, composites, fibers, microspheres and thin films. These materials can be used directly as biomaterials for implants, as scaffolding in tissue engineering and in vitro disease models, as well as for drug delivery. PMID:21959241

  13. Production of Bombyx mori silk fibroin incorporated with unnatural amino acids.

    PubMed

    Teramoto, Hidetoshi; Kojima, Katsura

    2014-07-14

    Silk fibroin incorporated with unnatural amino acids was produced by in vivo feeding of p-chloro-, p-bromo-, and p-azido-substituted analogues of L-phenylalanine (Phe) to transgenic silkworms (Bombyx mori) that expressed a mutant of phenylalanyl-tRNA synthetase with expanded substrate recognition capabilities in silk glands. Cutting down the content of Phe in the diet was effective for increasing the incorporation of Phe analogues but simultaneously caused a decrease of fibroin production. The azide groups incorporated in fibroin were active as chemical handles for click chemistry in both the solubilized and the solid (fibrous) states. The azides survived degumming in the boiling alkaline solution that is required for complete removal of the sericin layer, demonstrating that AzPhe-incorporated silk fibroin could be a versatile platform to produce "clickable" silk materials in various forms. This study indicates the huge potential of UAA mutagenesis as a novel methodology to alter the characteristics of B. mori silk. PMID:24884258

  14. Silk Nanospheres and Microspheres from Silk/PVA Blend Films for Drug Delivery

    PubMed Central

    Wang, Xiaoqin; Yucel, Tuna; Lu, Qiang; Hu, Xiao; Kaplan, David L.

    2009-01-01

    Silk fibroin protein-based micro- and nanospheres provide new options for drug delivery due to their biocompatibility, biodegradability and their tunable drug loading and release properties. In the present study, we report a new aqueous-based preparation method for silk spheres with controllable sphere size and shape. The preparation was based on phase separation between silk fibroin and polyvinyl alcohol (PVA) at a weight ratio of 1/1 and 1/4. Water-insoluble silk spheres were easily obtained from the blend in a three step process: (1) air-drying the blend solution into a film, (2) film dissolution in water and (3) removal of residual PVA by subsequent centrifugation. In both cases, the spheres had approximately 30% beta-sheet content and less than 5% residual PVA. Spindle-shaped silk particles, as opposed to the spherical particles formed above, were obtained by stretching the blend films before dissolving in water. Compared to the 1/1 ratio sample, the silk spheres prepared from the 1/4 ratio sample showed a more homogeneous size distribution ranging from 300 nm up to 20 ?m. Further studies showed that sphere size and polydispersity could be controlled either by changing the concentration of silk and PVA or by applying ultrasonication on the blend solution. Drug loading was achieved by mixing model drugs in the original silk solution. The distribution and loading efficiency of the drug molecules in silk spheres depended on their hydrophobicity and charge, resulting in different drug release profiles. The entire fabrication procedure could be completed within one day. The only chemical used in the preparation except water was PVA, an FDA-approved ingredient in drug formulations. Silk micro- and nanospheres reported have potential as drug delivery carriers in a variety of biomedical applications. PMID:19945157

  15. Structure-Function-Property-Design Interplay in Biopolymers: Spider Silk

    PubMed Central

    Tokareva, Olena; Jacobsen, Matthew; Buehler, Markus; Wong, Joyce; Kaplan, David L.

    2013-01-01

    Spider silks have been a focus of research for almost two decades due to their outstanding mechanical and biophysical properties. Recent advances in genetic engineering have led to the synthesis of recombinant spider silks, thus helping to unravel a fundamental understanding of structure-function-property relationships. The relationships between molecular composition, secondary structures, and mechanical properties found in different types of spider silks are described, along with a discussion of artificial spinning of these proteins and their bioapplications, including the role of silks in biomineralization and fabrication of biomaterials with controlled properties. PMID:23962644

  16. A Novel Growth Process of Calcium Carbonate Crystals in Silk Fibroin Hydrogel System

    E-print Network

    Paris-Sud XI, Université de

    to silk-fibroin-like protein in nacre in amino acid sequence and secondary structure [14] compared1 A Novel Growth Process of Calcium Carbonate Crystals in Silk Fibroin Hydrogel System Yufei Ma carbonate (CaCO3) crystal growth in the silk fibroin (SF) hydrogel with different concentrations by a simple

  17. Comparison of fibroin cDNAs from webspinning insects: insight into silk formation and function

    Microsoft Academic Search

    Matthew A. Collin; Janice S. Edgerly; Cheryl Y. Hayashi

    2011-01-01

    Embiopterans (webspinning insects) are renowned for their prolific use of silk. These organisms spin silk to construct elaborate networks of tubes in which they live, forage, and reproduce. The silken galleries are essential for protecting these soft-bodied insects from predators and other environmental hazards. Despite the ecological importance of embiopteran silk, very little is known about its constituent proteins. Here,

  18. Spectral analysis of induced color change on periodically nanopatterned silk films

    E-print Network

    Spectral analysis of induced color change on periodically nanopatterned silk films Jason J. Amsden1 on periodic nanopatterned 2D lattices in pure protein films of silk fibroin. We show here periodic lattices in silk fibroin films with feature sizes of hundreds of nanometers that exhibit different colors

  19. Silicon electronics on silk as a path to bioresorbable, implantable devices Dae-Hyeong Kim,1

    E-print Network

    Rogers, John A.

    Silicon electronics on silk as a path to bioresorbable, implantable devices Dae-Hyeong Kim,1 Yun water soluble and biocompatible silk substrates. Electrical, bending, water dissolution, and animal on nanomembranes of silicon, with biodegrad- able thin film substrates of silk protein, to yield a flexible system

  20. Transgenic silkworms ( Bombyx mori ) produce recombinant spider dragline silk in cocoons

    Microsoft Academic Search

    Hongxiu Wen; Xiqian Lan; Yuansong Zhang; Tianfu Zhao; Yujun Wang; Zenta Kajiura; Masao Nakagaki

    2010-01-01

    Spider dragline silk is a unique fibrous protein with a combination of tensile strength and elasticity, but the isolation\\u000a of large amounts of silk from spiders is not feasible. In this study, we generated germline-transgenic silkworms (Bombyx mori) that spun cocoons containing recombinant spider silk. A piggyBac-based transformation vector was constructed that carried spider dragline silk (MaSp1) cDNA driven by

  1. Sequential origin in the high performance properties of orb spider dragline silk

    PubMed Central

    Blackledge, Todd A.; Pérez-Rigueiro, José; Plaza, Gustavo R.; Perea, Belén; Navarro, Andrés; Guinea, Gustavo V.; Elices, Manuel

    2012-01-01

    Major ampullate (MA) dragline silk supports spider orb webs, combining strength and extensibility in the toughest biomaterial. MA silk evolved ~376?MYA and identifying how evolutionary changes in proteins influenced silk mechanics is crucial for biomimetics, but is hindered by high spinning plasticity. We use supercontraction to remove that variation and characterize MA silk across the spider phylogeny. We show that mechanical performance is conserved within, but divergent among, major lineages, evolving in correlation with discrete changes in proteins. Early MA silk tensile strength improved rapidly with the origin of GGX amino acid motifs and increased repetitiveness. Tensile strength then maximized in basal entelegyne spiders, ~230?MYA. Toughness subsequently improved through increased extensibility within orb spiders, coupled with the origin of a novel protein (MaSp2). Key changes in MA silk proteins therefore correlate with the sequential evolution high performance orb spider silk and could aid design of biomimetic fibers. PMID:23110251

  2. Effect of Strongly Alkaline Electrolyzed Water on Silk Degumming and the Physical Properties of the Fibroin Fiber.

    PubMed

    Cao, Ting-Ting; Wang, Yuan-Jing; Zhang, Yu-Qing

    2013-01-01

    Strongly alkaline electrolyzed water (SAEW) was prepared by electrolysis of tap water in a laboratory-made water electrolyzer. The pH of stored SAEW was stable for more than one month. The hardness of the electrolyzed water was 30% lower and the Na(+) concentration was 18% higher than those of the tap water. Silkworm cocoon shells were boiled in pH 11.50 SAEW at a ratio of 1?40?80 (W/V) for 20 min and the sericin layers around the silk fibroin fibers were removed completely. The tensile properties and thermal decomposition temperature of a single filament of silk fibroin obtained by the SAEW method were almost the same as those for the fiber obtained by the neutral soap, and much higher than those for the fiber obtained by Na2CO3 degumming. The results demonstrate that SAEW is an environmentally friendly and pollution-free silk degumming agent that allows highly efficient, low cost recovery of sericin. PMID:23824061

  3. Production of scFv-conjugated affinity silk film and its application to a novel enzyme-linked immunosorbent assay

    PubMed Central

    Sato, Mitsuru; Kojima, Katsura; Sakuma, Chisato; Murakami, Maria; Tamada, Yasushi; Kitani, Hiroshi

    2014-01-01

    Bombyx mori (silkworm) silk proteins have been utilized as unique biomaterials for various medical applications. To develop a novel affinity silk material, we generated a transgenic silkworm that spins silk protein containing the fibroin L-chain linked with the single-chain variable fragment (scFv) as a fusion protein. Previously, the scFv-conjugated “affinity” silk powder specifically immunoprecipitated its target protein, Wiskott-Aldrich syndrome protein. To expand the applicability of affinity silk materials, we processed the scFv-conjugated silk protein into a thin film by dissolving it in lithium bromide, then drying it in the wells of 96-well plates. Enzyme-linked immunosorbent assay demonstrated specific detection of Wiskott-Aldrich syndrome protein, both as a recombinant protein and in its native form extracted from mouse macrophages. These findings suggest that this scFv-conjugated silk film serves as the basis for an alternative immunodetection system. PMID:24518284

  4. Production of scFv-conjugated affinity silk film and its application to a novel enzyme-linked immunosorbent assay.

    PubMed

    Sato, Mitsuru; Kojima, Katsura; Sakuma, Chisato; Murakami, Maria; Tamada, Yasushi; Kitani, Hiroshi

    2014-01-01

    Bombyx mori (silkworm) silk proteins have been utilized as unique biomaterials for various medical applications. To develop a novel affinity silk material, we generated a transgenic silkworm that spins silk protein containing the fibroin L-chain linked with the single-chain variable fragment (scFv) as a fusion protein. Previously, the scFv-conjugated "affinity" silk powder specifically immunoprecipitated its target protein, Wiskott-Aldrich syndrome protein. To expand the applicability of affinity silk materials, we processed the scFv-conjugated silk protein into a thin film by dissolving it in lithium bromide, then drying it in the wells of 96-well plates. Enzyme-linked immunosorbent assay demonstrated specific detection of Wiskott-Aldrich syndrome protein, both as a recombinant protein and in its native form extracted from mouse macrophages. These findings suggest that this scFv-conjugated silk film serves as the basis for an alternative immunodetection system. PMID:24518284

  5. Early events in the evolution of spider silk genes.

    PubMed

    Starrett, James; Garb, Jessica E; Kuelbs, Amanda; Azubuike, Ugochi O; Hayashi, Cheryl Y

    2012-01-01

    Silk spinning is essential to spider ecology and has had a key role in the expansive diversification of spiders. Silk is composed primarily of proteins called spidroins, which are encoded by a multi-gene family. Spidroins have been studied extensively in the derived clade, Orbiculariae (orb-weavers), from the suborder Araneomorphae ('true spiders'). Orbicularians produce a suite of different silks, and underlying this repertoire is a history of duplication and spidroin gene divergence. A second class of silk proteins, Egg Case Proteins (ECPs), is known only from the orbicularian species, Lactrodectus hesperus (Western black widow). In L. hesperus, ECPs bond with tubuliform spidroins to form egg case silk fibers. Because most of the phylogenetic diversity of spiders has not been sampled for their silk genes, there is limited understanding of spidroin gene family history and the prevalence of ECPs. Silk genes have not been reported from the suborder Mesothelae (segmented spiders), which diverged from all other spiders >380 million years ago, and sampling from Mygalomorphae (tarantulas, trapdoor spiders) and basal araneomorph lineages is sparse. In comparison to orbicularians, mesotheles and mygalomorphs have a simpler silk biology and thus are hypothesized to have less diversity of silk genes. Here, we present cDNAs synthesized from the silk glands of six mygalomorph species, a mesothele, and a non-orbicularian araneomorph, and uncover a surprisingly rich silk gene diversity. In particular, we find ECP homologs in the mesothele, suggesting that ECPs were present in the common ancestor of extant spiders, and originally were not specialized to complex with tubuliform spidroins. Furthermore, gene-tree/species-tree reconciliation analysis reveals that numerous spidroin gene duplications occurred after the split between Mesothelae and Opisthothelae (Mygalomorphae plus Araneomorphae). We use the spidroin gene tree to reconstruct the evolution of amino acid compositions of spidroins that perform different ecological functions. PMID:22761664

  6. Early Events in the Evolution of Spider Silk Genes

    PubMed Central

    Starrett, James; Garb, Jessica E.; Kuelbs, Amanda; Azubuike, Ugochi O.; Hayashi, Cheryl Y.

    2012-01-01

    Silk spinning is essential to spider ecology and has had a key role in the expansive diversification of spiders. Silk is composed primarily of proteins called spidroins, which are encoded by a multi-gene family. Spidroins have been studied extensively in the derived clade, Orbiculariae (orb-weavers), from the suborder Araneomorphae (‘true spiders’). Orbicularians produce a suite of different silks, and underlying this repertoire is a history of duplication and spidroin gene divergence. A second class of silk proteins, Egg Case Proteins (ECPs), is known only from the orbicularian species, Lactrodectus hesperus (Western black widow). In L. hesperus, ECPs bond with tubuliform spidroins to form egg case silk fibers. Because most of the phylogenetic diversity of spiders has not been sampled for their silk genes, there is limited understanding of spidroin gene family history and the prevalence of ECPs. Silk genes have not been reported from the suborder Mesothelae (segmented spiders), which diverged from all other spiders >380 million years ago, and sampling from Mygalomorphae (tarantulas, trapdoor spiders) and basal araneomorph lineages is sparse. In comparison to orbicularians, mesotheles and mygalomorphs have a simpler silk biology and thus are hypothesized to have less diversity of silk genes. Here, we present cDNAs synthesized from the silk glands of six mygalomorph species, a mesothele, and a non-orbicularian araneomorph, and uncover a surprisingly rich silk gene diversity. In particular, we find ECP homologs in the mesothele, suggesting that ECPs were present in the common ancestor of extant spiders, and originally were not specialized to complex with tubuliform spidroins. Furthermore, gene-tree/species-tree reconciliation analysis reveals that numerous spidroin gene duplications occurred after the split between Mesothelae and Opisthothelae (Mygalomorphae plus Araneomorphae). We use the spidroin gene tree to reconstruct the evolution of amino acid compositions of spidroins that perform different ecological functions. PMID:22761664

  7. Vortex-induced injectable silk fibroin hydrogels.

    PubMed

    Yucel, Tuna; Cebe, Peggy; Kaplan, David L

    2009-10-01

    A novel, to our knowledge, technique was developed to control the rate of beta-sheet formation and resulting hydrogelation kinetics of aqueous, native silk solutions. Circular dichroism spectroscopy indicated that vortexing aqueous solutions of silkworm silk lead to a transition from an overall protein structure that is initially rich in random coil to one that is rich in beta-sheet content. Dynamic oscillatory rheology experiments collected under the same assembly conditions as the circular dichroism experiments indicated that the increase in beta-sheet content due to intramolecular conformational changes and intermolecular self-assembly of the silk fibroin was directly correlated with the subsequent changes in viscoelastic properties due to hydrogelation. Vortexing low-viscosity silk solutions lead to orders-of-magnitude increase in the complex shear modulus, G*, and formation of rigid hydrogels (G* approximately 70 kPa for 5.2 wt % protein concentration). Vortex-induced, beta-sheet-rich silk hydrogels consisted of permanent, physical, intermolecular crosslinks. The hydrogelation kinetics could be controlled easily (from minutes to hours) by changing the vortex time, assembly temperature and/or protein concentration, providing a useful timeframe for cell encapsulation. The stiffness of preformed hydrogels recovered quickly, immediately after injection through a needle, enabling the potential use of these systems for injectable cell delivery scaffolds. PMID:19804736

  8. Water-insoluble Silk Films with Silk I Structure

    SciTech Connect

    Lu, Q.; Hu, X; Wang, X; Kluge, J; Lu, S; Cebe, P; Kaplan, D

    2010-01-01

    Water-insoluble regenerated silk materials are normally produced by increasing the {beta}-sheet content (silk II). In the present study water-insoluble silk films were prepared by controlling the very slow drying of Bombyx mori silk solutions, resulting in the formation of stable films with a predominant silk I instead of silk II structure. Wide angle X-ray scattering indicated that the silk films stabilized by slow drying were mainly composed of silk I rather than silk II, while water- and methanol-annealed silk films had a higher silk II content. The silk films prepared by slow drying had a globule-like structure at the core surrounded by nano-filaments. The core region was composed of silk I and silk II, surrounded by hydrophilic nano-filaments containing random turns and {alpha}-helix secondary structures. The insoluble silk films prepared by slow drying had unique thermal, mechanical and degradative properties. Differential scanning calorimetry results revealed that silk I crystals had stable thermal properties up to 250 C, without crystallization above the T{sub g}, but degraded at lower temperatures than silk II structure. Compared with water- and methanol-annealed films the films prepared by slow drying had better mechanical ductility and were more rapidly enzymatically degraded, reflecting the differences in secondary structure achieved via differences in post processing of the cast silk films. Importantly, the silk I structure, a key intermediate secondary structure for the formation of mechanically robust natural silk fibers, was successfully generated by the present approach of very slow drying, mimicking the natural process. The results also point to a new mode of generating new types of silk biomaterials with enhanced mechanical properties and increased degradation rates, while maintaining water insolubility, along with a low {beta}-sheet content.

  9. Water-Insoluble Silk Films with Silk I Structure

    PubMed Central

    Lu, Qiang; Hu, Xiao; Wang, Xiaoqin; Kluge, Jonathan A.; Lu, Shenzhou; Cebe, Peggy; Kaplan, David L.

    2009-01-01

    Water-insoluble regenerated silk materials are normally achieved by increasing ?-sheet content (silk II). In the present study, water-insoluble silk films were prepared by controlling very slow drying of B. mori silk solutions, resulting in the formation of stable films with dominating silk I instead of silk II structure. Wide angle x-ray scattering (WAXS) indicated that the silk films stabilized by slow drying were mainly composed of silk I rather than silk II, while water- and methanol-annealed silk films had a higher silk II content. The silk films prepared through slow drying had a globule-like structure in the core with nano-filaments. The core region was composed of silk I and silk II, and these regions are surrounded by hydrophilic nano-filaments containing random, turns, and ?-helix secondary structures. The insoluble silk films prepared by slow drying had unique thermal, mechanical and degradative properties. DSC results revealed that silk I crystals had stable thermal properties up to 250°C, without crystallization above the Tg, but degraded in lower temperature than silk II structure. Compared with water- and methanol-annealed films, the films prepared through slow drying achieved better mechanical ductility and more rapid enzymatic degradation, reflective of the differences in secondary structure achieved via differences in post processing of the cast silk films. Importantly, the silk I structure, a key intermediate secondary structure for the formation of mechanically robust natural silk fibers, was successfully generated in the present approach of very slow drying, mimicking the natural process. The results also point to a new mode to generate new types of silk biomaterials, where mechanical properties can be enhanced, and degradation rates increased, yet water insolubility is maintained along with low beta sheet content. PMID:19874919

  10. Rapid communication: Computational simulation and analysis of a candidate for the design of a novel silk-based biopolymer.

    PubMed

    Golas, Ewa I; Czaplewski, Cezary

    2014-09-01

    This work theoretically investigates the mechanical properties of a novel silk-derived biopolymer as polymerized in silico from sericin and elastin-like monomers. Molecular Dynamics simulations and Steered Molecular Dynamics were the principal computational methods used, the latter of which applies an external force onto the system and thereby enables an observation of its response to stress. The models explored herein are single-molecule approximations, and primarily serve as tools in a rational design process for the preliminary assessment of properties in a new material candidate. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 915-923, 2014. PMID:24723330

  11. Peroxinectin catalyzed dityrosine crosslinking in the adhesive underwater silk of a casemaker caddisfly larvae, Hysperophylax occidentalis.

    PubMed

    Wang, Ching-Shuen; Ashton, Nicholas N; Weiss, Robert B; Stewart, Russell J

    2014-11-01

    Aquatic caddisfly larvae use sticky silk fibers as an adhesive tape to construct protective composite structures under water. Three new silk fiber components were identified by transcriptome and proteome analysis of the silk gland: a heme-peroxidase in the peroxinectin (Pxt) sub-family, a superoxide dismutase 3 (SOD3) that generates the H2O2 substrate of the silk fiber Pxt from environmental reactive oxygen species (eROS), and a novel structural component with sequence similarity to the elastic PEVK region of the muscle protein, titin. All three proteins are co-drawn with fibroins to form silk fibers. The Pxt and SOD3 enzymes retain activity in drawn fibers. In native fibers, Pxt activity and dityrosine crosslinks are co-localized at the boundary of a peripheral layer and the silk fiber core. To our knowledge, dityrosine crosslinks, heme peroxidase, and SOD3 activities have not been previously reported in an insect silk. The PEVK-like protein is homogeneously distributed throughout the fiber core. The results are consolidated into a model in which caddisfly silk Pxt-catalyzed dityrosine crosslinking occurs post-draw using H2O2 generated within the silk fibers by SOD3. The ROS substrate of caddisfly silk SOD3 occurs naturally in aquatic environments, from biotic and abiotic sources. The radially inhomogeneous dityrosine crosslinking and a potential titin-like PEVK protein network have important implications for the mechanical properties of caddifly silk fibers. PMID:25220661

  12. Silk Road Seattle

    NSDL National Science Digital Library

    Silk Road Seattle is a "collaborative public education project using the 'Silk Road' theme to explore cultural interaction across Eurasia from the beginning of the Common Era (A.D.) to the Sixteenth Century." The Silk Road is a term understood to have been the overland trade route from China to the Mediterranean, opened first in the 2nd century and coming to an end between the 15th and 17th centuries. Traditional discussions of the Silk Road, however, recognize that there were branches that went into South Asia, or extended from Central Asia north of the Caspian Sea to the Black Sea. Sponsored primarily by the Simpson Center for the Humanities at the University of Washington and directed by Professors Daniel C. Waugh, Joel Walker, and Cynthea Bogel, this Web site contains a vast range of Silk Road materials. These materials include a list of texts that may be used in teaching and learning about the Silk Road, a section on cities and architecture along the Silk Road, information on traditional culture in Central Asia, a page of teaching and learning guides that list and annotate Silk Road materials, and a virtual art exhibit. This site is still in a constructive stage; therefore, every link is not yet accessible. In short, this site may be of value for a range of audiences, including school children, teachers, college and graduate students, and independent adult learners.

  13. Hypotensive and vasorelaxant effects of sericin-derived oligopeptides in rats.

    PubMed

    Onsa-Ard, Amnart; Shimbhu, Dawan; Tocharus, Jiraporn; Sutheerawattananonda, Manote; Pantan, Rungusa; Tocharus, Chainarong

    2013-01-01

    Sericin-derived oligopeptides obtained from silk cocoons were investigated for the in vivo hypotensive effect and investigated for the underlying mechanism involved in vasodilation in isolated rat thoracic aorta. In normotensive anesthetized rats, oligopeptides induced an immediate and transient hypotensive activity. In rat aortic rings, oligopeptides induced a concentration-dependent vasorelaxation in vessels precontracted with both KCl and phenylephrine (PE) with endothelium-intact or endothelium-denuded rings. In endothelium-intact rings, pretreatment with N ? -Nitro-L-arginine methyl ester hydrochloride (L-NAME, 100?µM), an inhibitor of the NO synthase (NOS) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1?µM), a selective inhibitor of the guanylyl cyclase enzyme, significantly reduced the relaxant effect of oligopeptides. However, indomethacin, an inhibitor of the cyclooxygenase, had no effect on oligopeptides-induced relaxation. In addition, pretreatment with tetraethylammonium (TEA, 5?mM) reduced the maximal relaxant effect induced by oligopeptides. By contrast, relaxation was not affected by 4-aminopyridine (4-AP, 1?mM), glibenclamide (10?µM), or barium chloride (BaCl2, 1?mM). In depolarization Ca(2+)-free solution, oligopeptides inhibited calcium chloride- (CaCl2-) induced contraction in endothelium-denuded rings in a concentration-dependent manner. Nevertheless, oligopeptides attenuated transient contractions in Ca(2+)-free medium containing EGTA (1?mM) induced by 1?µM PE, but they were not affected by 20?mM caffeine. It is obvious that potent vasodilation effect of oligopeptides is mediated through both the endothelium and the vascular smooth muscle. PMID:24312733

  14. Hypotensive and Vasorelaxant Effects of Sericin-Derived Oligopeptides in Rats

    PubMed Central

    Pantan, Rungusa; Tocharus, Chainarong

    2013-01-01

    Sericin-derived oligopeptides obtained from silk cocoons were investigated for the in vivo hypotensive effect and investigated for the underlying mechanism involved in vasodilation in isolated rat thoracic aorta. In normotensive anesthetized rats, oligopeptides induced an immediate and transient hypotensive activity. In rat aortic rings, oligopeptides induced a concentration-dependent vasorelaxation in vessels precontracted with both KCl and phenylephrine (PE) with endothelium-intact or endothelium-denuded rings. In endothelium-intact rings, pretreatment with N?-Nitro-L-arginine methyl ester hydrochloride (L-NAME, 100?µM), an inhibitor of the NO synthase (NOS) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1?µM), a selective inhibitor of the guanylyl cyclase enzyme, significantly reduced the relaxant effect of oligopeptides. However, indomethacin, an inhibitor of the cyclooxygenase, had no effect on oligopeptides-induced relaxation. In addition, pretreatment with tetraethylammonium (TEA, 5?mM) reduced the maximal relaxant effect induced by oligopeptides. By contrast, relaxation was not affected by 4-aminopyridine (4-AP, 1?mM), glibenclamide (10?µM), or barium chloride (BaCl2, 1?mM). In depolarization Ca2+-free solution, oligopeptides inhibited calcium chloride- (CaCl2-) induced contraction in endothelium-denuded rings in a concentration-dependent manner. Nevertheless, oligopeptides attenuated transient contractions in Ca2+-free medium containing EGTA (1?mM) induced by 1?µM PE, but they were not affected by 20?mM caffeine. It is obvious that potent vasodilation effect of oligopeptides is mediated through both the endothelium and the vascular smooth muscle. PMID:24312733

  15. Silk Batik using Cochineal Dye

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The history of silk, including sericulture (the production of raw silk, which requires the raising of silkworms on their natural diet, mulberry leaves) and silk manufacturing, is rich and extensive. It encompasses several famous “silk roads” (trade routes), various cultures and technologies, ideas,...

  16. GC/MS-based metabolomic studies reveal key roles of glycine in regulating silk synthesis in silkworm, Bombyx mori.

    PubMed

    Chen, Quanmei; Liu, Xinyu; Zhao, Ping; Sun, Yanhui; Zhao, Xinjie; Xiong, Ying; Xu, Guowang; Xia, Qingyou

    2015-02-01

    Metabolic profiling of silkworm, especially the factors that affect silk synthesis at the metabolic level, is little known. Herein, metabolomic method based on gas chromatography-mass spectrometry was applied to identify key metabolic changes in silk synthesis deficient silkworms. Forty-six differential metabolites were identified in Nd group with the defect of silk synthesis. Significant changes in the levels of glycine and uric acid (up-regulation), carbohydrates and free fatty acids (down-regulation) were observed. The further metabolomics of silk synthesis deficient silkworms by decreasing silk proteins synthesis using knocking out fibroin heavy chain gene or extirpating silk glands operation showed that the changes of the metabolites were almost consistent with those of the Nd group. Furthermore, the increased silk yields by supplying more glycine or its related metabolite confirmed that glycine is a key metabolite to regulate silk synthesis. These findings provide important insights into the regulation between metabolic profiling and silk synthesis. PMID:25533535

  17. Carbondioxide gating in silk cocoon.

    PubMed

    Roy, Manas; Meena, Sunil Kumar; Kusurkar, Tejas Sanjeev; Singh, Sushil Kumar; Sethy, Niroj Kumar; Bhargava, Kalpana; Sarkar, Sabyasachi; Das, Mainak

    2012-12-01

    Silk is the generic name given to the fibrous proteins spun by a number of arthropods. During metamorphosis, the larva of the silk producing arthropods excrete silk-fiber from its mouth and spun it around the body to form a protective structure called cocoon. An adult moth emerges out from the cocoon after the dormant phase (pupal phase) varying from 2 weeks to 9 months. It is intriguing how CO(2)/O(2) and ambient temperature are regulated inside the cocoon during the development of the pupa. Here we show that the cocoon membrane is asymmetric, it allows preferential gating of CO(2) from inside to outside and it regulates a physiological temperature inside the cocoon irrespective of the surrounding environment temperature. We demonstrate that under simulating CO(2) rich external environment, the CO(2) does not diffuse inside the cocoon. Whereas, when CO(2) was injected inside the cocoon, it diffuses out in 20 s, indicating gating of CO(2) from inside to outside the membrane. Removal of the calcium oxalate hydrate crystals which are naturally present on the outer surface of the cocoon affected the complete blockade of CO(2) flow from outside to inside suggesting its role to trap most of the CO(2) as hydrogen bonded bicarbonate on the surface. The weaved silk of the cocoon worked as the second barrier to prevent residual CO(2) passage. Furthermore, we show that under two extreme natural temperature regime of 5 and 50 °C, a temperature of 25 and 34 °C respectively were maintained inside the cocoons. Our results demonstrate, how CO(2) gating and thermoregulation helps in maintaining an ambient atmosphere inside the cocoon for the growth of pupa. Such natural architectural control of gas and temperature regulation could be helpful in developing energy saving structures and gas filters. PMID:22791361

  18. Spider silks from plants - a challenge to create native-sized spidroins.

    PubMed

    Hauptmann, Valeska; Weichert, Nicola; Rakhimova, Marziya; Conrad, Udo

    2013-10-01

    Silk threads from spiders exhibit extraordinary mechanical properties, such as superior toughness and elasticity. Spider silks consist of several different large repetitive proteins that act as the basic materials responsible for these outstanding features. The production of spider silk protein variants in plants opens up new horizons in the production and functional investigation that enable the use of spider silks in innovative material development, nanotechnology and biomedicine in the future. This review summarizes and discusses production of spider silk protein variants in plants, especially with regards to plant expression systems, purification strategies, and characteristics of spider silk variants. Furthermore, the challenge of producing native-sized recombinant spidroins in planta is outlined, presenting three different strategies for achieving these high repetitive proteins with the help of non-repetitive C-terminal domains, crosslinking transglutaminase, and self-linking inteins. The potential of these fascinating proteins in medicine is also highlighted. PMID:24092675

  19. Effects of RGDS sequence genetically interfused in the silk fibroin light chain protein on chondrocyte adhesion and cartilage synthesis.

    PubMed

    Kambe, Yusuke; Yamamoto, Koji; Kojima, Katsura; Tamada, Yasushi; Tomita, Naohide

    2010-10-01

    Initial chondrocyte-silk fibroin interactions are implicated in chondrogenesis when using fibroin as a scaffold for chondrocytes. Here, we focused on integrin-mediated cell-scaffold adhesion and prepared cell adhesive fibroin in which a tandem repeat of the Arg-Gly-Asp-Ser (RGDS) sequence was genetically interfused in the fibroin light chain (L-chain) (L-RGDSx2 fibroin). We investigated the effects of the sequence on chondrocyte adhesion and cartilage synthesis, in comparison to the effects of fibronectin. As the physicochemical surface properties (e.g., wettability and zeta potential) of the fibroin substrate were not affected by the modification, specific cell adhesion to the RGDS predominately changed the chondrocyte adhesive state. This suggestion was also supported by the competitive inhibition of chondrocyte attachment to the L-RGDSx2 fibroin substrate with soluble RGD peptides in the medium. Unlike fibronectin, the expression of RGDS in the fibroin L-chain had no effect on chondrocyte spreading area but enhanced mRNA expression levels of integrins alpha5 and beta1, and aggrecan at 12 h after seeding. Although both the sequence and fibronectin increased cell adhesive force, chondrocytes grown on the fibroin substrate exhibited a peak in the force with time in culture. These results suggested that moderate chondrocyte adhesion to fibroin induced by the RGDS sequence was able to maintain the chondrogenic phenotype and, from the histology findings, the sequence could facilitate chondrogenesis. PMID:20643479

  20. Silk material modification and microfabrication strategies for applications in biosensors and drug delivery

    NASA Astrophysics Data System (ADS)

    Tsioris, Konstantinos

    Silk has proven to be a promising biomaterial for the development of a novel generation of biomedical devices due to the material's intrinsic properties of biocompatibility and biodegradability. To further the development of silk-based devices, augmented functionality can be provided to silk by means of microfabrication and material functionalization. In this dissertation, we set out to explore possibilities of silk-based biomedical device development with particular attention to different fabrication strategies that can be leveraged for this purpose, taking inspiration from conventional lithography, contact imprinting and chemical modification of the silk biomaterial. In particular, we have produced a novel silk-based drug delivery device, in the form of microneedles. For this purpose we have developed micromolding strategies which allow the fabrication of high aspect ratio silk structures. Furthermore, we have produced a THz split ring resonator based sensor device on silk films. To realize this device, we have developed a convenient fabrication method, allowing transfer of previously microfabricated metal and oxide microstructures to the silk film surface. This method has proven useful for hard masking and patterning silk films with reactive ion etching. Moreover, we have explored patterning semiconductor and glass substrates with silk films. For this purpose we have modified a standard microfabrication method -lift off- to be amendable to silk. Furthermore, combining silk based fabrication and material functionalization has lead to microfluidics pH sensing applications. Finally we have explored the possibility of utilizing silk for injectable and biodegradable glucose sensors. For this purpose, we have doped silk hydrogels with a fluorescence based protein biosensor. The advanced silk fabrication and material synthesis strategies and the resulting novel devices presented here could potentially lead to a new class of biomedical applications.

  1. Molecular dynamics analysis of supercontraction in spider dragline silk

    E-print Network

    Batty, Laura

    2013-01-01

    Spider dragline silk is a material that has evolved over millions of years to develop finely tuned mechanical properties. It is a protein-based fiber, used as the main structural component in spider webs and as a lifeline ...

  2. Unraveled mechanism in silk engineering: Fast reeling induced silk toughening

    NASA Astrophysics Data System (ADS)

    Wu, Xiang; Liu, Xiang-Yang; Du, Ning; Xu, Gangqin; Li, Baowen

    2009-08-01

    We theoretically and experimentally study the mechanical response of silkworm and spider silks against stretching and the relationship with the underlying structural factors. It is found that the typical stress-strain profiles are predicted in good agreement with experimental measurements by implementing the "?-sheet splitting" mechanism we discovered and verified, primarily varying the secondary structure of protein macromolecules. The functions of experimentally observed structural factors responding to the external stress have been clearly addressed, and optimization of the microscopic structures to enhance the mechanical strength will be pointed out, beneficial to their biomedical and textile applications.

  3. Effect of sericin supplementation during in vitro maturation on the maturation, fertilization and development of porcine oocytes.

    PubMed

    Do, L T K; Namula, Z; Luu, V V; Sato, Y; Taniguchi, M; Isobe, T; Kikuchi, K; Otoi, T

    2014-04-01

    This study aimed to examine the effects of sericin supplementation during in vitro oocyte maturation on the nuclear maturation, fertilization and development of porcine oocytes. Cumulus-oocyte complexes (COCs) were cultured in maturation medium supplemented with 0 (control), 0.1, 0.5, 1.0, 2.5 or 5.0% sericin and were then subjected to in vitro fertilization and embryo culture. More COCs matured with 1.0% sericin underwent germinal vesicle breakdown and reached metaphase II compared with the control COCs matured without sericin (p < 0.01). The proportions of oocytes with DNA-fragmented nuclei did not differ between the groups, regardless of the sericin level. The total fertilization rate of oocytes matured with 1.0% sericin was higher (p < 0.05) than that of oocytes matured with 0.1%, 2.5% and 5.0% sericin. Supplementation with more than 1.0% sericin decreased the DNA fragmentation index of the blastocysts compared with the control group (p < 0.05). However, the supplementation of the maturation medium with sericin had no beneficial effects on the cleavage, development to the blastocyst stage and the total cell number of the embryos. Our findings indicate that supplementation with 1.0% sericin during maturation culture may improve the nuclear maturation and the quality of the embryos but does not affect blastocyst formation. PMID:24467637

  4. Fabrication and Characterization of Silk-Fibroin-Coated Quantum Dots

    Microsoft Academic Search

    Bhavik B. Nathwani; Mona Jaffari; Ameet R. Juriani; Anshu B. Mathur; Kenith E. Meissner

    2009-01-01

    We report a novel technique of directly coating colloidal CdSe\\/ZnS core\\/shell quantum dots (QDs) with silk fibroin (SF), a protein derived from the Bombyx mori silk worm. The approach results in protein-modified QDs with little or no particle aggregation, and mitigates the issue of biocompatibility. QDs have desirable optical properties, such as narrow-band emission, broadband absorption, high quantum yield, and

  5. Clay-Enriched Silk Biomaterials for Bone Formation

    PubMed Central

    Mieszawska, Aneta J.; Llamas, Jabier Gallego; Vaiana, Christopher A.; Kadakia, Madhavi P.; Naik, Rajesh R.; Kaplan, David L.

    2011-01-01

    The formation of silk protein/clay composite biomaterials for bone tissue formation is described. Silk fibroin serves as an organic scaffolding material offering mechanical stability suitable for bone specific uses. Clay montmorillonite (Cloisite ® Na+) and sodium silicate are sources of osteoinductive silica-rich inorganic species, analogous to bioactive bioglass-like bone repair biomaterial systems. Different clay particle-silk composite biomaterial films were compared to silk films doped with sodium silicate as controls for support of human bone marrow derived mesenchymal stem cells (hMSCs) in osteogenic culture. The cells adhered and proliferated on the silk/clay composites over two weeks. Quantitative real-time RT-PCR analysis revealed increased transcript levels for alkaline phosphatase (ALP), bone sialoprotein (BSP), and collagen type 1 (Col I) osteogenic markers in the cells cultured on the silk/clay films in comparison to the controls. Early evidence for bone formation based on collagen deposition at the cell-biomaterial interface was also found, with more collagen observed for the silk films with higher contents of clay particles. The data suggest that the silk/clay composite systems may be useful for further study toward bone regenerative needs. PMID:21549864

  6. Silk film biomaterials for cornea tissue engineering

    PubMed Central

    Lawrence, Brian D.; Marchant, Jeffrey K.; Pindrus, Mariya; Omenetto, Fiorenzo; Kaplan, David L.

    2009-01-01

    Biomaterials for corneal tissue engineering must demonstrate several critical features for potential utility in vivo, including transparency, mechanical integrity, biocompatibility and slow biodegradation. Silk film biomaterials were designed and characterized to meet these functional requirements. Silk protein films were used in a biomimetic approach to replicate corneal stromal tissue architecture. The films were 2 ?m thick to emulate corneal collagen lamellae dimensions, and were surface patterned to guide cell alignment. To enhance trans-lamellar diffusion of nutrients and to promote cell-cell interaction, pores with 0.5 to 5.0 ?m diameters were introduced into the silk films. Human and rabbit corneal fibroblast proliferation, alignment and corneal extracellular matrix expression on these films in both 2D and 3D cultures was demonstrated. The mechanical properties, optical clarity and surface patterned features of these films, combined with their ability to support corneal cell functions suggest this new biomaterial system offers important potential benefits for corneal tissue regeneration. PMID:19059642

  7. Lyophilized silk fibroin hydrogels for the sustained local delivery of therapeutic monoclonal antibodies.

    PubMed

    Guziewicz, Nicholas; Best, Annie; Perez-Ramirez, Bernardo; Kaplan, David L

    2011-04-01

    The development of sustained delivery systems compatible with protein therapeutics continues to be a significant unmet need. A lyophilized silk fibroin hydrogel matrix (lyogel) for the sustained release of pharmaceutically relevant monoclonal antibodies is described. Sonication of silk fibroin prior to antibody incorporation avoids exposing the antibody to the sol-gel transition inducing shear stress. Fourier Transform Infrared (FTIR) analysis showed no change in silk structural composition between hydrogel and lyogel or with increasing silk fibroin concentration. Antibody release from hydrogels occurred rapidly over 10 days regardless of silk concentration. Upon lyophilization, sustained antibody release was observed over 38 days from lyogels containing 6.2% (w/w) silk fibroin and above. In 3.2% (w/w) silk lyogels, antibody release was comparable to hydrogels. Swelling properties of lyogels followed a similar threshold behavior. Lyogels at 3.2% (w/w) silk recovered approximately 90% of their fluid mass upon rehydration, while approximately 50% fluid recovery was observed at 6.2% (w/w) silk and above. Antibody release was primarily governed by hydrophobic/hydrophilic silk-antibody interactions and secondarily altered by the hydration resistance of the lyogel. Hydration resistance was controlled by altering ?-sheet (crystalline) density of the matrix. The antibody released from lyogels maintained biological activity. Silk lyogels offer an advantage as a delivery matrix over other hydrogel materials for the slow release of the loaded protein, making lyogels suitable for long-term sustained release applications. PMID:21216004

  8. Effect of beta-sheet crystals on the thermal and rheological behavior of protein-based hydrogels derived from gelatin and silk fibroin.

    PubMed

    Gil, Eun S; Spontak, Richard J; Hudson, Samuel M

    2005-08-12

    Novel protein-based hydrogels have been prepared by blending gelatin (G) with amorphous Bombyx mori silk fibroin (SF) and subsequently promoting the formation of beta-sheet crystals in SF upon exposure to methanol or methanol/water solutions. Differential scanning calorimetry of the resultant hydrogels confirms the presence and thermoreversibility of the G helix-coil transition between ambient and body temperature at high G concentrations. At low G concentrations, this transition is shifted to higher temperatures and becomes progressively less pronounced. Complementary dynamic rheological measurements reveal solid-liquid cross-over at the G helix-coil transition temperature typically between 30 and 36 degrees C in blends prior to the formation of beta-sheet crystals. Introducing the beta-sheet conformation in SF stabilizes the hydrogel network and extends the solid-like behavior of the hydrogels to elevated temperatures beyond body temperature with as little as 10 wt.-% SF. The temperature-dependent elastic modulus across the G helix-coil transition is reversible, indicating that the conformational change in G can be used in stabilized G/SF hydrogels to induce thermally triggered encapsulant release. PMID:16080165

  9. Blueprint for a High-Performance Biomaterial: Full-Length Spider Dragline Silk Genes

    PubMed Central

    Ayoub, Nadia A.; Garb, Jessica E.; Tinghitella, Robin M.; Collin, Matthew A.; Hayashi, Cheryl Y.

    2007-01-01

    Spider dragline (major ampullate) silk outperforms virtually all other natural and manmade materials in terms of tensile strength and toughness. For this reason, the mass-production of artificial spider silks through transgenic technologies has been a major goal of biomimetics research. Although all known arthropod silk proteins are extremely large (>200 kiloDaltons), recombinant spider silks have been designed from short and incomplete cDNAs, the only available sequences. Here we describe the first full-length spider silk gene sequences and their flanking regions. These genes encode the MaSp1 and MaSp2 proteins that compose the black widow's high-performance dragline silk. Each gene includes a single enormous exon (>9000 base pairs) that translates into a highly repetitive polypeptide. Patterns of variation among sequence repeats at the amino acid and nucleotide levels indicate that the interaction of selection, intergenic recombination, and intragenic recombination governs the evolution of these highly unusual, modular proteins. Phylogenetic footprinting revealed putative regulatory elements in non-coding flanking sequences. Conservation of both upstream and downstream flanking sequences was especially striking between the two paralogous black widow major ampullate silk genes. Because these genes are co-expressed within the same silk gland, there may have been selection for similarity in regulatory regions. Our new data provide complete templates for synthesis of recombinant silk proteins that significantly improve the degree to which artificial silks mimic natural spider dragline fibers. PMID:17565367

  10. Segmented nanofibers of spider dragline silk: Atomic force microscopy and single-molecule force spectroscopy

    PubMed Central

    Oroudjev, E.; Soares, J.; Arcidiacono, S.; Thompson, J. B.; Fossey, S. A.; Hansma, H. G.

    2002-01-01

    Despite its remarkable materials properties, the structure of spider dragline silk has remained unsolved. Results from two probe microscopy techniques provide new insights into the structure of spider dragline silk. A soluble synthetic protein from dragline silk spontaneously forms nanofibers, as observed by atomic force microscopy. These nanofibers have a segmented substructure. The segment length and amino acid sequence are consistent with a slab-like shape for individual silk protein molecules. The height and width of nanofiber segments suggest a stacking pattern of slab-like molecules in each nanofiber segment. This stacking pattern produces nano-crystals in an amorphous matrix, as observed previously by NMR and x-ray diffraction of spider dragline silk. The possible importance of nanofiber formation to native silk production is discussed. Force spectra for single molecules of the silk protein demonstrate that this protein unfolds through a number of rupture events, indicating a modular substructure within single silk protein molecules. A minimal unfolding module size is estimated to be around 14 nm, which corresponds to the extended length of a single repeated module, 38 amino acids long. The structure of this spider silk protein is distinctly different from the structures of other proteins that have been analyzed by single-molecule force spectroscopy, and the force spectra show correspondingly novel features. PMID:11959907

  11. Effect of Sequence Features on Assembly of Spider Silk Block Copolymers

    PubMed Central

    Tokareva, Olena S.; Lin, Shangchao; Jacobsen, Matthew M.; Huang, Wenwen; Rizzo, Daniel; Li, David; Simon, Marc; Staii, Cristian; Cebe, Peggy; Wong, Joyce Y.; Buehler, Markus J.; Kaplan, David L.

    2014-01-01

    Bioengineered spider silk block copolymers were studied to understand the effect of protein chain length and sequence chemistry on the formation of secondary structure and materials assembly. Using a combination of in vitro protein design and assembly studies, we demonstrate that silk block copolymers possessing multiple repetitive units self-assemble into lamellar microstructures. Additionally, the study provides insights into the assembly behavior of spider silk block copolymers in concentrated salt solutions. PMID:24613991

  12. Effect of sequence features on assembly of spider silk block copolymers.

    PubMed

    Tokareva, Olena S; Lin, Shangchao; Jacobsen, Matthew M; Huang, Wenwen; Rizzo, Daniel; Li, David; Simon, Marc; Staii, Cristian; Cebe, Peggy; Wong, Joyce Y; Buehler, Markus J; Kaplan, David L

    2014-06-01

    Bioengineered spider silk block copolymers were studied to understand the effect of protein chain length and sequence chemistry on the formation of secondary structure and materials assembly. Using a combination of in vitro protein design and assembly studies, we demonstrate that silk block copolymers possessing multiple repetitive units self-assemble into lamellar microstructures. Additionally, the study provides insights into the assembly behavior of spider silk block copolymers in concentrated salt solutions. PMID:24613991

  13. Molecular and Mechanical Characterization of Aciniform Silk: Uniformity of Iterated Sequence Modules in a Novel Member of the Spider Silk Fibroin Gene Family

    Microsoft Academic Search

    Cheryl Y. Hayashi; Todd A. Blackledge; Randolph V. Lewis

    2004-01-01

    Araneoid spiders use specialized abdominal glands to produce up to seven different protein-based silks\\/glues that have diverse physical properties. The fibroin sequences that encode aciniform fibers (wrapping silk) and the mechanical properties of these fibers have not been characterized previously. To gain a better understanding of the molecular radiation of spider silk fibroin genes, cDNA libraries derived from aciniform glands

  14. Electrospun chitosan/sericin composite nanofibers with antibacterial property as potential wound dressings.

    PubMed

    Zhao, Rui; Li, Xiang; Sun, Bolun; Zhang, Ying; Zhang, Dawei; Tang, Zhaohui; Chen, Xuesi; Wang, Ce

    2014-07-01

    Chitosan and sericin are natural and low cost biomaterials. Both biomaterials displayed good compatibility to human tissues and antibacterial properties for biomedical application. In this study, we have successfully fabricated chitosan/sericin composite nanofibers by electrospinning. The obtained composite nanofibers were characterized using scanning electron microscopy (SEM), Fourier transform infrared spectrometer (FT-IR), X-ray diffraction (XRD), and thermogravimetric analysis (TGA) studies. The composite nanofibers had good morphology with diameter between 240nm and 380nm. In vitro methyl thiazolyl tetrazolium (MTT) assays demonstrated that the chitosan/sericin composite nanofibers were biocompatible and could promote the cell proliferation. Furthermore, the composite nanofibers showed good bactericidal activity against both of Gram-positive and Gram-negative bacteria. Thus, the chitosan/sericin composite nanofibers are promising for wound dressing applications. PMID:24769088

  15. Structure and properties of regenerated Antheraea pernyi silk fibroin in aqueous solution.

    PubMed

    Tao, Wei; Li, Mingzhong; Zhao, Chunxia

    2007-04-10

    Antheraea pernyi silk fibroin fibers were dissolved by aqueous lithium thiocyanate to obtain regenerated A. pernyi silk fibroin solution. By means of circular dichroism, (13)C NMR and Raman spectroscopy, the molecular conformation of regenerated A. pernyi silk fibroin in aqueous solution was investigated. The relationship of environmental factors and sol-gel transformation behavior of regenerated A. pernyi silk fibroin was also studied. The molecular conformations of regenerated A. pernyi silk fibroin mainly were alpha-helix and random coil in solution. There also existed a little beta-sheet conformation. It was obviously different with Bombyx mori silk fibroin, whose molecular conformation in solution was only random coil but no alpha-helix existence. With the increase of temperature and solution concentration and with the decrease of solution pH value, the gelation velocity of regenerated A. pernyi silk fibroin solution increased. Especially, it showed that A. pernyi silk fibroin was more sensitive to temperature than B. mori silk fibroin during the sol-gel transformation. The velocity increased obviously when the temperature was above 30 degrees C. During the sol-gel transformation, the molecular conformation of regenerated A. pernyi silk fibroin changed from random coil to beta-sheet structure. The results of these studies provided important insight into the preparation of new biomaterials by silk fibroin protein. PMID:17173967

  16. Development of an enzyme-linked-immunosorbent-assay technique for accurate identification of poorly preserved silks unearthed in ancient tombs.

    PubMed

    Zheng, Qin; Wu, Xiaofeng; Zheng, Hailing; Zhou, Yang

    2015-05-01

    We report the preparation of a specific fibroin antibody and its use for the identification of unearthed ancient silk relics. Based on the 12-amino-acid repeat sequence "GAGAGSGAGAGS", which is found in fibroin of the silkworm Bombyx mori, a specific antibody against fibroin was prepared in rabbits through peptide synthesis and carrier-protein coupling. This antibody was highly specific for fibroin found in silk. Using this antibody we have successfully identified four silk samples from different time periods. Our results reveal, for the first time, a method capable of detecting silk from a few milligrams of archaeological fabric that has been buried for thousands of years, confirming that the ancient practice of wearing silk products while praying for rebirth dated back to at least 400 BCE. This method also complements current approaches in silk detection, especially for the characterization of poorly preserved silks, promoting the investigation of silk origins and of ancient clothing cultures. PMID:25804731

  17. [Effect of salt on minor ampullate silk spidroin modules].

    PubMed

    Wang, Jia; Chen, Gefei; Meng, Qing

    2014-08-01

    To study the effect of physiological conditions on spidroins, we analyzed NTR1SR2CT module secondary structure, aggregation and silk-formation influenced by different salts (in different concentration intervals). According to the full-length Araneus ventricosus MiSp sequence, NTR1SR2CT module was constructed and expressed in Escherichia coli BL21 (DE3), and the recombinant proteins were purified by denaturation method mediated by 8 mol/L urea. Random coil and helix are the main secondary structures of NTR1SR2CT and could be induced into beta-sheet by drying natively and lyophilization, where methanol can be used as a promoter. Furthermore, potassium and phosphate cations can cause significant NTR1SR2CT protein aggregation and silk-formation. The results could be a basis for the determination of silk-formation mechanism, and also useful for industrialized generation of high performance spider silk-like fibers. PMID:25423761

  18. Electrospun silk-BMP-2 scaffolds for bone tissue engineering

    Microsoft Academic Search

    Chunmei Li; Charu Vepari; Hyoung-Joon Jin; Hyeon Joo Kim; David L. Kaplan

    2006-01-01

    Silk fibroin fiber scaffolds containing bone morphogenetic protein 2 (BMP-2) and\\/or nanoparticles of hydroxyapatite (nHAP) prepared via electrospinning were used for in vitro bone formation from human bone marrow-derived mesenchymal stem cells (hMSCs). BMP-2 survived the aqueous-based electrospinnig process in bioactive form. hMSCs were cultured for up to 31 days under static conditions in osteogenic media on the scaffolds (silk\\/PEO\\/BMP-2,

  19. Sonication-induced gelation of silk fibroin for cell encapsulation

    Microsoft Academic Search

    Xiaoqin Wang; Jonathan A. Kluge; Gary G. Leisk; David L. Kaplan

    2008-01-01

    Purified native silk fibroin forms ?-sheet-rich, physically cross-linked, hydrogels from aqueous solution, in a process influenced by environmental parameters. Previously we reported gelation times of days to weeks for aqueous native silk protein solutions, with high ionic strength and temperature and low pH responsible for increasing gelation kinetics. Here we report a novel method to accelerate the process and control

  20. Innovative multifunctional silk fibroin and hydrotalcite nanocomposites: a synergic effect of the components.

    PubMed

    Posati, Tamara; Benfenati, Valentina; Sagnella, Anna; Pistone, Assunta; Nocchetti, Morena; Donnadio, Anna; Ruani, Giampiero; Zamboni, Roberto; Muccini, Michele

    2014-01-13

    Novel hybrid functional materials are formed by combining hydrotalcite-like compounds and silk fibroin (SF-HTlc) via an environmental friendly aqueous process. The nanocomposites can be prepared with different weight ratio of the constituting components and preserve the conformational properties of the silk protein and the lamellar structure of hydrotalcites. Optical microscopy, scanning electron microscopy, and atomic force microscopy analyses show a good dispersion degree of the inorganic nanoparticles into the organic silk matrix. A mutual benefit on the stability of both organic and inorganic components was observed in the nanocomposites. SF-HTlc displayed limited dissolution of hydrotalcite in acidic medium, enhanced mechanical properties, and higher protease resistance of silk protein. The transparency, flexibility, and acidic environment resistance of silk fibroin combined to the protective and reinforcing properties of hydrotalcites generate a hybrid material, which is very attractive for applications in recently reported silk based opto-electronic and photonics technologies. PMID:24313841

  1. The expression pattern of four odorant-binding proteins in male and female silk moths, Bombyx mori.

    PubMed

    Maida, R; Mameli, M; Müller, B; Krieger, J; Steinbrecht, R A

    2005-03-01

    Four recombinant odorant-binding proteins of Bombyx mori, pheromone-binding protein (PBP), general odorant-binding protein 1 (GOBP1), general odorant-binding protein 2 (GOBP2) and antennal binding protein X (ABPX), were expressed in E. coli and used to raise polyclonal antisera. Immunoblots of antennal homogenates showed that these antisera were specific. In Western blot analysis and immunocytochemical labelling experiments, the sera against recombinant PBP and GOBP2 of B. mori gave identical results as sera against native PBP and GOBP2 of Antheraea polyphemus, respectively, thus confirming earlier results obtained with the latter. Labelling consecutive cross sections of various sensillum types with all four antisera revealed different labelling patterns in male and female sensilla (s.) trichodea and s. basiconica. Long s. trichodea in males and females represented uniform labelling types, whereas for short s. trichodea, s. intermedia, and s. basiconica a great variety of labelling patterns was observed, some being more common than others. Long s. trichodea, which in males are uniformly tuned to the pheromone components bombykol and bombykal, all strongly expressed PBP; labelling with antisera against the other three odorant-binding proteins hardly was above background, only in some hairs GOBP1 was expressed somewhat more strongly. Long s. trichodea of females, which respond specifically to linalool and benzoic acid, showed a different labelling pattern. Here, we observed strong labelling with antibodies against GOBP2 and medium labelling with anti-GOBP1, sometimes with anti-ABPX. S. basiconica in both sexes most commonly co-expressed GOBP1 and GOBP2, but other patterns were occasionally found, with some of them showing PBP expression, also in females. The great variety of labelling types in short s. trichodea, s. intermedia, and s. basiconica suggests a similar variety of functional subtypes as observed in plant odour-sensitive sensilla of other moth species. PMID:16374716

  2. Preparation of Carbon Nanotube-Composite

    E-print Network

    Sharma, Sundeep

    2011-08-08

    is derived in the pupal stage of bombyx mori. Bombyx mori cocoon consists of two proteins fibroin and sericin. Fibroin protein is the central structure of the silk and the sericin protein is the sticky material surrounding the fibroin. The scientists...

  3. Anterior cruciate ligament reconstruction in a rabbit model using silk-collagen scaffold and comparison with autograft.

    PubMed

    Bi, Fanggang; Shi, Zhongli; Liu, An; Guo, Peng; Yan, Shigui

    2015-01-01

    The objective of the present study was to perform an in vivo assessment of a novel silk-collagen scaffold for anterior cruciate ligament (ACL) reconstruction. First, a silk-collagen scaffold was fabricated by combining sericin-extracted knitted silk fibroin mesh and type I collagen to mimic the components of the ligament. Scaffolds were electron-beam sterilized and rolled up to replace the ACL in 20 rabbits in the scaffold group, and autologous semitendinosus tendons were used to reconstruct the ACL in the autograft control group. At 4 and 16 weeks after surgery, grafts were retrieved and analyzed for neoligament regeneration and tendon-bone healing. To evaluate neoligament regeneration, H&E and immunohistochemical staining was performed, and to assess tendon-bone healing, micro-CT, biomechanical test, H&E and Russell-Movat pentachrome staining were performed. Cell infiltration increased over time in the scaffold group, and abundant fibroblast-like cells were found in the core of the scaffold graft at 16 weeks postoperatively. Tenascin-C was strongly positive in newly regenerated tissue at 4 and 16 weeks postoperatively in the scaffold group, similar to observations in the autograft group. Compared with the autograft group, tendon-bone healing was better in the scaffold group with trabecular bone growth into the scaffold. The results indicate that the silk-collagen scaffold has considerable potential for clinical application. PMID:25938408

  4. The elaborate structure of spider silk: structure and function of a natural high performance fiber.

    PubMed

    Römer, Lin; Scheibel, Thomas

    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

  5. Stabilization of Enzymes in Silk Films

    PubMed Central

    Lu, Shenzhou; Wang, Xiaoqin; Lv, Qiang; Hu, Xiao; Uppal, Neha; Omenetto, Fiorenzo

    2009-01-01

    Material systems are needed that promote stabilization of entrained molecules, such as enzymes or therapeutic proteins, without destroying their activity. We demonstrate that the unique structure of silk fibroin protein, when assembled into the solid state, establishes an environment that is conducive to the stabilization of entrained proteins. Enzymes (glucose oxidase, lipase and horseradish peroxidase) entrapped in these films over ten months retained significant activity, even when stored at 37°C, and in the case of glucose oxidase did not lose any activity. Further, the mode of processing of the silk protein into the films could be correlated to the stability of the enzymes. The relationship between processing and stability offers a large suite of conditions within which to optimize such stabilization processes. Overall, the techniques reported here result in materials that stabilize enzymes to a remarkable extent, without the need for cryoprotectants, emulsifiers, covalent immobilization or other treatments. Further, these systems are amenable to optical characterization, environmental distribution without refrigeration, are ingestible, and offer potential use in vivo, since silk materials are biocompatible and FDA approved, degradable with proteases and currently used in biomedical devices. PMID:19323497

  6. Hierarchical Chain Model of Spider Capture Silk Elasticity

    E-print Network

    Haijun Zhou; Yang Zhang

    2005-01-24

    Spider capture silk is a biomaterial with both high strength and high elasticity, but the structural design principle underlying these remarkable properties is still unknown. It was revealed recently by atomic force microscopy that, an exponential force--extension relationship holds both for capture silk mesostructures and for intact capture silk fibers [N. Becker et al., Nature Materials 2, 278 (2003)]. In this Letter a simple hierarchical chain model was proposed to understand and reproduce this striking observation. In the hierarchical chain model, a polymer is composed of many structural motifs which organize into structural modules and supra-modules in a hierarchical manner. Each module in this hierarchy has its own characteristic force. The repetitive patterns in the amino acid sequence of the major flagelliform protein of spider capture silk is in support of this model.

  7. Cross-linking in the silks of bees, ants and hornets.

    PubMed

    Campbell, Peter M; Trueman, Holly E; Zhang, Qiang; Kojima, Katsura; Kameda, Tsunenori; Sutherland, Tara D

    2014-05-01

    Silk production is integral to the construction of nests or cocoons for many Aculeata, stinging Hymenopterans such as ants, bees and wasps. Here we report the sequences of new aculeate silk proteins and compare cross-linking among nine native silks from three bee species (Apis mellifera, Bombus terrestris and Megachile rotundata), three ant species (Myrmecia forficata, Oecophylla smaragdina and Harpegnathos saltator) and three hornets (Vespa analis, Vespa simillima and Vespa mandarinia). The well studied silks of spiders and silkworms are comprised of large proteins that are cross-linked and stabilized predominantly by intra and intermolecular beta sheet structure. In contrast, the aculeate silks are comprised of relatively small proteins that contain central coiled coil domains and comparatively reduced amounts of beta sheet structure. The hornet silks, which have the most beta sheet structure and the greatest amount of amino acid sequence outside the coiled-coil domains, dissolve in concentrated LiBr solution and appear to be stabilized predominantly by beta sheet structure like the classic silks. In contrast, the ant and bee silks, which have less beta sheet and less sequence outside the coiled-coil domains, could not be dissolved in LiBr and appear to be predominantly stabilized by covalent cross-linking. The iso-peptide cross-linker, ?-(?-glutamyl)-lysine that is produced by transglutaminase enzymes, was demonstrated to be present in all silks by mass spectrometry, but at greater levels in silks of ants and bees. The bee silks and ant cocoons, but not the Oecophylla nest silks, appeared to be further stabilized by tanning reactions. PMID:24607851

  8. Electrospun Silk Biomaterial Scaffolds for Regenerative Medicine

    PubMed Central

    Zhang, Xiaohui; Reagan, Michaela R; Kaplan, David L.

    2009-01-01

    Electrospinning is a versatile technique that enables the development of nanofiber-based biomaterial scaffolds. Scaffolds can be generated that are useful for tissue engineering and regenerative medicine since they mimic the nanoscale properties of certain fibrous components of the native extracellular matrix in tissues. Silk is a natural protein with excellent biocompatibility, remarkable mechanical properties as well as tailorable degradability. Integrating these protein polymer advantages with electrospinning results in scaffolds with combined biochemical, topographical and mechanical cues with versatility for a range of biomaterial, cell and tissue studies and applications. This review covers research related to electrospinning of silk, including process parameters, post treatment of the spun fibers, functionalization of nanofibers, and the potential applications for these material systems in regenerative medicine. Research challenges and future trends are also discussed. PMID:19643154

  9. Optically switchable natural silk

    NASA Astrophysics Data System (ADS)

    Krasnov, Igor; Krekiehn, Nicolai R.; Krywka, Christina; Jung, Ulrich; Zillohu, Ahnaf U.; Strunskus, Thomas; Elbahri, Mady; Magnussen, Olaf M.; Müller, Martin

    2015-03-01

    An optically active bio-material is created by blending natural silk fibers with photoisomerizable chromophore molecules—azobenzenebromide (AzBr). The material converts the energy of unpolarized light directly into mechanical work with a well-defined direction of action. The feasibility of the idea to produce optically driven microsized actuators on the basis of bio-material (silk) is proven. The switching behavior of the embedded AzBr molecules was studied in terms of UV/Vis spectroscopy. To test the opto-mechanical properties of the modified fibers and the structural changes they undergo upon optically induced switching, single fiber X-ray diffraction with a micron-sized synchrotron radiation beam was combined in situ with optical switching as well as with mechanical testing and monitoring. The crystalline regions of silk are not modified by the presence of the guest molecules, hence occupy only the amorphous part of the fibers. It is shown that chromophore molecules embedded into fibers can be reversibly switched between the trans and cis conformation by illumination with light of defined wavelengths. The host fibers respond to this switching with a variation of the internal stress. The amplitude of the mechanical response is independent of the applied external stress and its characteristic time is shorter than the relaxation time of the usual mechanical response of silk.

  10. Invited review the coiled coil silk of bees, ants, and hornets.

    PubMed

    Sutherland, Tara D; Weisman, Sarah; Walker, Andrew A; Mudie, Stephen T

    2012-06-01

    In this article, we review current knowledge about the silk produced by the larvae of bees, ants, and hornets [Apoidea and Vespoidea: Hymenoptera]. Different species use the silk either alone or in composites for a variety of purposes including mechanical reinforcement, thermal regulation, or humidification. The characteristic molecular structure of this silk is ?-helical proteins assembled into tetrameric coiled coils. Gene sequences from seven species are available, and each species possesses a copy of each of four related silk genes that encode proteins predicted to form coiled coils. The proteins are ordered at multiple length scales within the labial gland of the final larval instar before spinning. The insects control the morphology of the silk during spinning to produce either fibers or sheets. The silk proteins are small and non repetitive and have been produced artificially at high levels by fermentation in E. coli. The artificial silk proteins can be fabricated into materials with structural and mechanical properties similar to those of native silks. PMID:21830200

  11. Electrodeposition of Silk Fibroin on Metal Substrates

    Microsoft Academic Search

    Devid Maniglio; Walter Bonani; Gabrio Bortoluzzi; Eva Servoli; Antonella Motta; Claudio Migliaresi

    2010-01-01

    Silk fibroin, one of the most promising natural materials for tissue engineering, has positive interactions with the biological environment, particularly in the field of bone and cartilage regeneration. A new approach was developed to create hydrogels from water-based fibroin solutions by applying an electric field to effect protein migration and coagulation at the anode (Aluminium or Ti6Al4V alloy) of an

  12. Electrodeposited silk coatings for functionalized implant applications

    NASA Astrophysics Data System (ADS)

    Elia, Roberto

    The mechanical and morphological properties of titanium as well as its biocompatibility and osteoinductive characteristics have made it the material of choice for dental implant systems. Although the success rate of titanium implants exceeds 90% in healthy individuals, a large subset of the population has one or more risk factors that inhibit implant integration. Treatments and coatings have been developed to improve clinical outcomes via introduction of appropriate surface topography, texture and roughness or incorporation of bioactive molecules. It is essential that the coatings and associated deposition techniques are controllable and reproducible. Currently, methods of depositing functional coatings are dictated by numerous parameters (temperature, particle size distribution, pH and voltage), which result in variable coating thickness, strength, porosity and weight, and hinder or preclude biomolecule incorporation. Silk is a highly versatile protein with a unique combination of mechanical and physical properties, including tunable degradation, biocompatibility, drug stabilizing capabilities and mechanical properties. Most recently an electrogelation technique was developed which allows for the deposition of gels which dry seamlessly over the contoured topography of the conductive substrate. In this work we examine the potential use of silk electrogels as mechanically robust implant coatings capable of sequestering and releasing therapeutic agents. Electrodeposition of silk electrogels formed in uniform electric fields was characterized with respect to field intensity and deposition time. Gel formation kinetics were used to derive functions which allowed for the prediction of coating deposition over a range of process and solution parameters. Silk electrogel growth orientation was shown to be influenced by the applied electric field. Coatings were reproducible and tunable via intrinsic silk solution properties and extrinsic process parameters. Adhesion was modulated over a 10-fold range and implant insertion into bone mimics demonstrated that the coatings were able to withstand delamination forces experienced during these mock implantations. Antibiotic release from coated implant studs inhibited bacterial growth and dexamethasone release was shown to stimulate calcium deposition in mesenchymal stem cells.

  13. updated 1:45 p.m. CT,Sun., April 18, 2010 WASHINGTON -A brain implant made partly of silk

    E-print Network

    Rogers, John A.

    updated 1:45 p.m. CT,Sun., April 18, 2010 WASHINGTON - A brain implant made partly of silk can melt of Pennsylvania and Tufts University in Boston made the electrode arrays using protein from silk and thin metal electrodes. The silk is biocompatible and water-soluble, dissolving into the brain and leaving the electrodes

  14. 1H, 13C and 15N NMR assignments of the aciniform spidroin (AcSp1) repetitive domain of Argiope trifasciata wrapping silk.

    PubMed

    Xu, Lingling; Tremblay, Marie-Laurence; Meng, Qing; Liu, Xiang-Qin; Rainey, Jan K

    2012-10-01

    Spider silk is one of nature's most remarkable biomaterials due to extraordinary strength and toughness not found in today's synthetic materials. Of the seven types of silk, wrapping silk (AcSp1) is the most extensible of the types of silks and has no sequence similarity to the other types. Here we report the chemical shifts for the AcSp1 199 amino acid protein repeat unit and its anticipated secondary structure based on secondary chemical shifts. PMID:21989955

  15. Production of scFv-conjugated affinity silk powder by transgenic silkworm technology.

    PubMed

    Sato, Mitsuru; Kojima, Katsura; Sakuma, Chisato; Murakami, Maria; Aratani, Eriko; Takenouchi, Takato; Tamada, Yasushi; Kitani, Hiroshi

    2012-01-01

    Bombyx mori (silkworm) silk proteins are being utilized as unique biomaterials for medical applications. Chemical modification or post-conjugation of bioactive ligands expand the applicability of silk proteins; however, the processes are elaborate and costly. In this study, we used transgenic silkworm technology to develop single-chain variable fragment (scFv)-conjugated silk fibroin. The cocoons of the transgenic silkworm contain fibroin L-chain linked with scFv as a fusion protein. After dissolving the cocoons in lithium bromide, the silk solution was dialyzed, concentrated, freeze-dried, and crushed into powder. Immunoprecipitation analyses demonstrate that the scFv domain retains its specific binding activity to the target molecule after multiple processing steps. These results strongly suggest the promise of scFv-conjugated silk fibroin as an alternative affinity reagent, which can be manufactured using transgenic silkworm technology at lower cost than traditional affinity carriers. PMID:22496833

  16. Lyophilized silk fibroin hydrogels for the sustained local delivery of therapeutic monoclonal antibodies

    Microsoft Academic Search

    Nicholas Guziewicz; Annie Best; Bernardo Perez-Ramirez; David L. Kaplan

    2011-01-01

    The development of sustained delivery systems compatible with protein therapeutics continues to be a significant unmet need. A lyophilized silk fibroin hydrogel matrix (lyogel) for the sustained release of pharmaceutically relevant monoclonal antibodies is described. Sonication of silk fibroin prior to antibody incorporation avoids exposing the antibody to the sol–gel transition inducing shear stress. Fourier Transform Infrared (FTIR) analysis showed

  17. Ultrasonication and Genipin Cross-Linking to Prepare Novel Silk Fibroin–Gelatin Composite Hydrogel

    Microsoft Academic Search

    Wenqian Xiao; Wenlong Liu; Jing Sun; Xiuli Dan; Dan Wei; Hongsong Fan

    2012-01-01

    Hydrogels are highly desirable tissue engineering scaffolds due to their high water content and structural similarity to a natural extra cellular matrix. However, the extensive use of hydrogels is limited by their low strength and facile degradation. By combining mechanical integrity and slow degradation of silk fibroin with excellent bioactivity of gelatin, a novel biocompatible protein-based composite hydrogel of silk

  18. Relationships between supercontraction and mechanical properties of spider silk

    NASA Astrophysics Data System (ADS)

    Liu, Yi; Shao, Zhengzhong; Vollrath, Fritz

    2005-12-01

    Typical spider dragline silk tends to outperform other natural fibres and most man-made filaments. However, even small changes in spinning conditions can have large effects on the mechanical properties of a silk fibre as well as on its water uptake. Absorbed water leads to significant shrinkage in an unrestrained dragline fibre and reversibly converts the material into a rubber. This process is known as supercontraction and may be a functional adaptation for the silk's role in the spider's web. Supercontraction is thought to be controlled by specific motifs in the silk proteins and to be induced by the entropy-driven recoiling of molecular chains. In analogy, in man-made fibres thermal shrinkage induces changes in mechanical properties attributable to the entropy-driven disorientation of `unfrozen' molecular chains (as in polyethylene terephthalate) or the `broken' intermolecular hydrogen bonds (as in nylons). Here we show for Nephila major-ampullate silk how in a biological fibre the spinning conditions affect the interplay between shrinkage and mechanical characteristics. This interaction reveals design principles linking the exceptional properties of silk to its molecular orientation.

  19. Immunolocalization of pheromone-binding protein and general odorant-binding protein in olfactory sensilla of the silk moths Antheraea and Bombyx

    Microsoft Academic Search

    R. A. Steinbrecht; M. Laue; G. Ziegelberger

    1995-01-01

    The distribution of odorant-binding proteins among olfactory sensilla of three moth species was studied by immuno-electron microscopy. Two polyclonal antisera were used in a post-embedding labelling protocol on sections of cryo-substituted antennae. The first was directed against the pheromone-binding protein (PBP) of Antheraea polyphemus, the second against the general odorant-binding protein (GOBP) of the same species. Immunoblots showed that these

  20. Photoresponsive retinal-modified silk-elastin copolymer

    PubMed Central

    Sun, Zhongyuan; Qin, Guokui; Xia, Xiaoxia; Cronin-Golomb, Mark; Omenetto, Fiorenzo; Kaplan, David L.

    2013-01-01

    The chimeric proteins, silk-elastin-like protein polymers (SELPs), consist of repeating units of silk and elastin to retain the mechanical strength of silk, while incorporating the dynamic environmental sensitivity of elastin. A retinal-modified silk-elastin-like protein polymer was prepared, modified and studied for photodynamic responses. The protein was designed, cloned, expressed and purified with lysine present in the elastin repeats. The purified protein was then chemically modified with the bio-compatible moiety retinal via the lysine side chains. Structural changes with the polymer were assessed before and after retinal modification using Fourier Transform Infrared Spectroscopy and Circular Dichroism Spectroscopy. Optical studies and spectral analysis were performed before and after retinal-modification. The random coil fraction of the protein increased after retinal modification while the ?-sheet fraction significantly decreased. Birefringence of the modified protein was induced when irradiated with a linearly polarized 488 nm laser light. Retinal modification of this protein offers a useful strategy for potential use in biosensors, controlled drug delivery and other areas of biomedical engineering. PMID:23383965

  1. Molecular mechanics of silk nanostructures under varied mechanical loading.

    PubMed

    Bratzel, Graham; Buehler, Markus J

    2012-06-01

    Spider dragline silk is a self-assembling tunable protein composite fiber that rivals many engineering fibers in tensile strength, extensibility, and toughness, making it one of the most versatile biocompatible materials and most inviting for synthetic mimicry. While experimental studies have shown that the peptide sequence and molecular structure of silk have a direct influence on the stiffness, toughness, and failure strength of silk, few molecular-level analyses of the nanostructure of silk assemblies, in particular, under variations of genetic sequences have been reported. In this study, atomistic-level structures of wildtype as well as modified MaSp1 protein from the Nephila clavipes spider dragline silk sequences, obtained using an in silico approach based on replica exchange molecular dynamics and explicit water molecular dynamics, are subjected to simulated nanomechanical testing using different force-control loading conditions including stretch, pull-out, and peel. The authors have explored the effects of the poly-alanine length of the N. clavipes MaSp1 peptide sequence and identify differences in nanomechanical loading conditions on the behavior of a unit cell of 15 strands with 840-990 total residues used to represent a cross-linking ?-sheet crystal node in the network within a fibril of the dragline silk thread. The specific loading condition used, representing concepts derived from the protein network connectivity at larger scales, have a significant effect on the mechanical behavior. Our analysis incorporates stretching, pull-out, and peel testing to connect biochemical features to mechanical behavior. The method used in this study could find broad applications in de novo design of silk-like tunable materials for an array of applications. PMID:22020792

  2. Silk constructs for delivery of musculoskeletal therapeutics.

    PubMed

    Meinel, Lorenz; Kaplan, David L

    2012-09-01

    Silk fibroin (SF) is a biopolymer with distinguishing features from many other bio- as well as synthetic polymers. From a biomechanical and drug delivery perspective, SF combines remarkable versatility for scaffolding (solid implants, hydrogels, threads, solutions), with advanced mechanical properties and good stabilization and controlled delivery of entrapped protein and small molecule drugs, respectively. It is this combination of mechanical and pharmaceutical features which renders SF so exciting for biomedical applications. This pattern along with the versatility of this biopolymer has been translated into progress for musculoskeletal applications. We review the use and potential of silk fibroin for systemic and localized delivery of therapeutics in diseases affecting the musculoskeletal system. We also present future directions for this biopolymer as well as the necessary research and development steps for their achievement. PMID:22522139

  3. Nanoconfinement of spider silk fibrils begets superior strength, extensibility, and toughness.

    PubMed

    Giesa, Tristan; Arslan, Melis; Pugno, Nicola M; Buehler, Markus J

    2011-11-01

    Silk is an exceptionally strong, extensible, and tough material made from simple protein building blocks. The molecular structure of dragline spider silk repeat units consists of semiamorphous and nanocrystalline ?-sheet protein domains. Here we show by a series of computational experiments how the nanoscale properties of silk repeat units are scaled up to create macroscopic silk fibers with outstanding mechanical properties despite the presence of cavities, tears, and cracks. We demonstrate that the geometric confinement of silk fibrils to diameters of 50 ± 30 nm is critical to facilitate a powerful mechanism by which hundreds of thousands of protein domains synergistically resist deformation and failure to provide enhanced strength, extensibility, and toughness at the macroscale, closely matching experimentally measured mechanical properties. Through this mechanism silk fibers exploit the full potential of the nanoscale building blocks, regardless of the details of microscopic loading conditions and despite the presence of large defects. Experimental results confirm that silk fibers are composed of silk fibril bundles with diameters in the range of 20-150 nm, in agreement with our predicted length scale. Our study reveals a general mechanism to map nanoscale properties to the macroscale and provides a potent design strategy toward novel fiber and bulk nanomaterials through hierarchical structures. PMID:21967633

  4. Properties and potential medical applications of silk fibers produced by Rothischildia lebeau.

    PubMed

    Reddy, Narendra; Jiang, Qiuran; Yang, Yiqi

    2013-01-01

    Rothischildia lebeau which belongs to the Saturniidae family of silk-producing insects secretes protein fibers with properties between that of the Bombyx mori and the common wild silks. Traditionally, wild silks produced by insects such as Antheraea mylitta are considerably coarser and have inferior tensile properties than the domesticated and most commonly used silk produced by B. mori. Recently, it has been demonstrated that some of the wild silks have unique properties and preferable for medical applications. Wild silks are comparatively easier to rear, produce larger cocoons, and could have unique properties. In this research, the structure and properties of the silk fibers produced by R. lebeau were studied and the potential of using the fibers for medical applications was investigated. Fibers produced by R. lebeau had average tensile strength of 3.3?g/den, similar to that of wild silks but lower than that of the B. mori silk. R. lebeau fibers were biocompatible and showed potential to be useful for tissue engineering and other medical applications. PMID:23594071

  5. Tuning chemical and physical cross-links in silk electrogels for morphological analysis and mechanical reinforcement.

    PubMed

    Lin, Yinan; Xia, Xiaoxia; Shang, Ke; Elia, Roberto; Huang, Wenwen; Cebe, Peggy; Leisk, Gary; Omenetto, Fiorenzo; Kaplan, David L

    2013-08-12

    Electrochemically controlled, reversible assembly of biopolymers into hydrogel structures is a promising technique for on-demand cell or drug encapsulation and release systems. An electrochemically sol-gel transition has been demonstrated in regenerated Bombyx mori silk fibroin, offering a controllable way to generate biocompatible and reversible adhesives and other biomedical materials. Despite the involvement of an electrochemically triggered electrophoretic migration of the silk molecules, the mechanism of the reversible electrogelation remains unclear. It is, however, known that the freshly prepared silk electrogels (e-gels) adopt a predominantly random coil conformation, indicating a lack of cross-linking as well as thermal, mechanical, and morphological stabilities. In the present work, the tuning of covalent and physical ?-sheet cross-links in silk hydrogels was studied for programming the structural properties. Scanning electron microscopy (SEM) revealed delicate morphology, including locally aligned fibrillar structures, in silk e-gels, preserved by combining glutaraldehyde-cross-linking and ethanol dehydration. Fourier transform infrared (FTIR) spectroscopic analysis of either electrogelled, vortex-induced or spontaneously formed silk hydrogels showed that the secondary structure of silk e-gels was tunable between non-?-sheet-dominated and ?-sheet-dominated states. Dynamic oscillatory rheology confirmed the mechanical reinforcement of silk e-gels provided by controlled chemical and physical cross-links. The selective incorporation of either chemical or physical or both cross-links into the electrochemically responsive, originally unstructured silk e-gel should help in the design for electrochemically responsive protein polymers. PMID:23859710

  6. Tuning Chemical and Physical Crosslinks in Silk Electrogels for Morphological Analysis and Mechanical Reinforcement

    PubMed Central

    Lin, Yinan; Xia, Xiaoxia; Shang, Ke; Elia, Roberto; Huang, Wenwen; Cebe, Peggy; Leisk, Gary; Omenetto, Fiorenzo; Kaplan, David L.

    2013-01-01

    Electrochemically controlled, reversible assembly of biopolymers into hydrogel structures is a promising technique for on-demand cell or drug encapsulation and release systems. An electrochemically sol-gel transition has been demonstrated in regenerated Bombyx mori silk fibroin, offering a controllable way to generate biocompatible and reversible adhesives and other biomedical materials. Despite the involvement of an electrochemically triggered electrophoretic migration of the silk molecules, the mechanism of the reversible electrogelation remains unclear. It is, however, known that the freshly prepared silk electrogels (e-gels) adopt a predominantly random coil conformation, indicating a lack of crosslinking as well as thermal, mechanical and morphological stabilities. In the present work, the tuning of covalent and physical ?-sheet crosslinks in silk hydrogels was studied for programming the structural properties. Scanning electron microscopy (SEM) revealed delicate morphology, including locally aligned fibrillar structures, in silk e-gels, preserved by combining glutaraldehyde-crosslinking and ethanol dehydration. Fourier transform infrared (FTIR) spectroscopic analysis of either electrogelled, vortex-induced or spontaneously formed silk hydrogels showed that the secondary structure of silk e-gels was tunable between non ?-sheet dominated and ?-sheet dominated states. Dynamic oscillatory rheology confirmed the mechanical reinforcement of silk e-gels provided by controlled chemical and physical crosslinks. The selective incorporation of either chemical or physical or both crosslinks into the electrochemically-responsive, originally unstructured silk e-gel should help in the design for electrochemically-responsive protein polymers. PMID:23859710

  7. Decrease in corneal damage due to benzalkonium chloride by the addition of sericin into timolol maleate eye drops.

    PubMed

    Nagai, Noriaki; Ito, Yoshimasa; Okamoto, Norio; Shimomura, Yoshikazu

    2013-01-01

    We investigated the protective effects of sericin on corneal damage due to benzalkonium chloride (BAC) used as a preservative in commercially available timolol maleate eye drops using rat debrided corneal epithelium and a human cornea epithelial cell line (HCE-T). Corneal wounds were monitored using a fundus camera TRC-50X equipped with a digital camera; eye drops were instilled into the rat eyes five times a day after corneal epithelial abrasion. The viability of HCE-T cells was calculated by TetraColor One; and Escherichia coli (ATCC 8739) were used to measure antimicrobial activity. The reducing effects on transcorneal penetration and intraocular pressure (IOP) of the eye drops were determined using rabbits. The corneal wound healing rate and rate constants (kH) as well as cell viability were higher following treatment with 0.005% BAC solution containing 0.1% sericin than in the case of treatment with BAC solution alone; the antimicrobial activity was approximately the same for BAC solutions with and without sericin. In addition, the kH for rat eyes instilled with commercially available timolol maleate eye drops containing 0.1% sericin was significantly higher than that of eyes instilled with timolol maleate eye drops without sericin, and the addition of sericin did not affect the corneal penetration or IOP reducing effect of commercially available timolol maleate eye drops. A preservative system comprising BAC and sericin may provide effective therapy for glaucoma patients requiring long-term anti-glaucoma agents. PMID:23470443

  8. Shear-induced rigidity in spider silk glands

    NASA Astrophysics Data System (ADS)

    Koski, Kristie J.; McKiernan, Keri; Akhenblit, Paul; Yarger, Jeffery L.

    2012-09-01

    We measure the elastic stiffnesses of the concentrated viscous protein solution of the dehydrated Nephila clavipes major ampullate gland with Brillouin light scattering. The glandular material shows no rigidity but possesses a tensile stiffness similar to that of spider silk. We show, however, that with application of a simple static shear, the mechanical properties of the spider gland protein mixture can be altered irreversibly, lowering symmetry and enabling shear waves to be supported, thus, giving rise to rigidity and yielding elastic properties similar to those of the naturally spun (i.e., dynamically sheared) silk.

  9. Nutrient Deprivation Induces Property Variations in Spider Gluey Silk

    PubMed Central

    Blamires, Sean J.; Sahni, Vasav; Dhinojwala, Ali; Blackledge, Todd A.; Tso, I-Min

    2014-01-01

    Understanding the mechanisms facilitating property variability in biological adhesives may promote biomimetic innovations. Spider gluey silks such as the spiral threads in orb webs and the gumfoot threads in cobwebs, both of which comprise of an axial thread coated by glue, are biological adhesives that have variable physical and chemical properties. Studies show that the physical and chemical properties of orb web gluey threads change when spiders are deprived of food. It is, however, unknown whether gumfoot threads undergo similar property variations when under nutritional stress. Here we tested whether protein deprivation induces similar variations in spiral and gumfoot thread morphology and stickiness. We manipulated protein intake for the orb web spider Nephila clavipes and the cobweb spider Latrodectus hesperus and measured the diameter, glue droplet volume, number of droplets per mm, axial thread width, thread stickiness and adhesive energy of their gluey silks. We found that the gluey silks of both species were stickier when the spiders were deprived of protein than when the spiders were fed protein. In N. clavipes a concomitant increase in glue droplet volume was found. Load-extension curves showed that protein deprivation induced glue property variations independent of the axial thread extensions in both species. We predicted that changes in salt composition of the glues were primarily responsible for the changes in stickiness of the silks, although changes in axial thread properties might also contribute. We, additionally, showed that N. clavipes' glue changes color under protein deprivation, probably as a consequence of changes to its biochemical composition. PMID:24523902

  10. Nutrient deprivation induces property variations in spider gluey silk.

    PubMed

    Blamires, Sean J; Sahni, Vasav; Dhinojwala, Ali; Blackledge, Todd A; Tso, I-Min

    2014-01-01

    Understanding the mechanisms facilitating property variability in biological adhesives may promote biomimetic innovations. Spider gluey silks such as the spiral threads in orb webs and the gumfoot threads in cobwebs, both of which comprise of an axial thread coated by glue, are biological adhesives that have variable physical and chemical properties. Studies show that the physical and chemical properties of orb web gluey threads change when spiders are deprived of food. It is, however, unknown whether gumfoot threads undergo similar property variations when under nutritional stress. Here we tested whether protein deprivation induces similar variations in spiral and gumfoot thread morphology and stickiness. We manipulated protein intake for the orb web spider Nephila clavipes and the cobweb spider Latrodectus hesperus and measured the diameter, glue droplet volume, number of droplets per mm, axial thread width, thread stickiness and adhesive energy of their gluey silks. We found that the gluey silks of both species were stickier when the spiders were deprived of protein than when the spiders were fed protein. In N. clavipes a concomitant increase in glue droplet volume was found. Load-extension curves showed that protein deprivation induced glue property variations independent of the axial thread extensions in both species. We predicted that changes in salt composition of the glues were primarily responsible for the changes in stickiness of the silks, although changes in axial thread properties might also contribute. We, additionally, showed that N. clavipes' glue changes color under protein deprivation, probably as a consequence of changes to its biochemical composition. PMID:24523902

  11. Changes in growth and cell wall extensibility of maize silks following pollination

    PubMed Central

    Sella Kapu, Nuwan U.; Cosgrove, Daniel J.

    2010-01-01

    In response to pollination maize silks undergo an accelerated process of senescence which involves an inhibition of elongation. To gain insight into the mechanism underlying this growth response, the relationships among silk elongation kinetics, cell wall biophysical properties, pollen tube growth, and expansin protein abundance were investigated. The inhibition of silk elongation became apparent beyond 12?h after pollination. Pollinated walls were less responsive in assays of extension induced by pollen ?-expansin. Expansin protein abundance and endogenous expansin activity were not considerably reduced after pollination. Silk wall plastic compliance was significantly reduced 6?h post-pollination and beyond, suggesting that the wall undergoes structural modifications leading to its rigidification in response to pollination. The reduction in the plastic compliance occurred locally and progressively, shortly after pollen tubes traversed through a region of silk. Though numerous pollen grains germinated and initiated pollen tubes at the silk tip, the density of pollen tubes gradually declined along the length of the silk and only 1–2 reached the ovary even 24?h after pollination. These results support the notion that pollination-induced cell wall rigidification plays multiple roles in maize reproduction, including inhibition of silk growth and prevention of polyspermy. PMID:20656797

  12. Silk electrogel coatings for titanium dental implants.

    PubMed

    Elia, Roberto; Michelson, Courtney D; Perera, Austin L; Harsono, Masly; Leisk, Gray G; Kugel, Gerard; Kaplan, David L

    2015-04-01

    The aim of this study was to develop biocompatible, biodegradable dental implant coatings capable of withstanding the mechanical stresses imparted during implant placement. Two techniques were developed to deposit uniform silk fibroin protein coatings onto dental implants. Two novel coating techniques were implemented to coat titanium shims, studs, and implants. One technique involved electrodeposition of the silk directly onto the titanium substrates. The second technique consisted of melting electrogels and dispensing the melted gels onto the titanium to form the coatings. Both techniques were tested for coating reproducibility using a stylus profilometer and a dial thickness gauge. The mechanical strength of adhered titanium studs was assessed using a universal mechanical testing machine. Uniform, controllable coatings were obtained from both the electrodeposition and melted electrogel coating techniques, tunable from 35 to 1654?µm thick under the conditions studied, and able to withstand delamination during implantation into implant socket mimics. Mechanical testing revealed that the adhesive strength of electrogel coatings, 0.369?±?0.09?MPa, rivaled other biologically derived coating systems such as collagen, hydroxyapatite, and chitosan (0.07-4.83?MPa). These novel silk-based techniques offer a unique approach to the deposition of safe, simple, mechanically robust, biocompatible, and degradable implant coatings. PMID:25425563

  13. In vitro study on silk fibroin textile structure for anterior cruciate ligament regeneration.

    PubMed

    Farè, Silvia; Torricelli, Paola; Giavaresi, Gianluca; Bertoldi, Serena; Alessandrino, Antonio; Villa, Tomaso; Fini, Milena; Tanzi, Maria Cristina; Freddi, Giuliano

    2013-10-01

    A novel hierarchical textile structure made of silk fibroin from Bombyx mori capable of matching the mechanical performance requirements of anterior cruciate ligament (ACL) and in vitro cell ingrowth is described. This sericin-free, Silk Fibroin Knitted Sheath with Braided Core (SF-KSBC) structure was fabricated using available textile technologies. Micro-CT analysis confirmed that the core was highly porous and had a higher degree of interconnectivity than that observed for the sheath. The in vivo cell colonization of the scaffolds is thus expected to penetrate even the internal parts of the structure. Tensile mechanical tests demonstrated a maximum load of 1212.4±56.4 N (under hydrated conditions), confirming the scaffold's suitability for ACL reconstruction. The absence of cytotoxic substances in the extracts of the SF-KSBC structure in culture medium was verified by in vitro tests with L929 fibroblasts. In terms of extracellular matrix production, Human Periodontal Ligament Fibroblasts (HPdLFs) cultured in direct contact with SF-KSBC, compared to control samples, demonstrated an increased secretion of aggrecan (PG) and fibronectin (FBN) at 3 and 7 days of culture, and no change in IL-6 and TNF-? secretion. Altogether, the outcomes of this investigation confirm the significant utility of this novel scaffold for ACL tissue regeneration. PMID:23910255

  14. Therapeutic effects of sericin on diabetic keratopathy in Otsuka Long-Evans Tokushima Fatty rats

    PubMed Central

    Nagai, Noriaki; Ito, Yoshimasa

    2013-01-01

    An Otsuka Long-Evans Tokushima Fatty (OLETF) rat provides a useful model for studies to develop corneal wound healing drugs for use in diabetic keratopathy resulting from type 2 diabetes mellitus. We investigated the effects of sericin on corneal wound healing in OLETF rats. Corneal wounds were prepared by removal of the corneal epithelium and documented using a TRC-50X. Sericin was instilled into the eyes of rats five times a day following corneal abrasion. The plasma levels of glucose, triglycerides, cholesterol and insulin in 38 wk old OLETF rats were significantly higher than in normal control rats (LETO rats), and the rate of corneal wound healing in OLETF rats was slower than in normal rat, probably due to the suppression of cell migration and proliferation caused by high plasma glucose levels. The corneal wounds of OLETF rats instilled with saline showed almost complete healing 72 h after corneal epithelial abrasion. On the other hand, the instillation of sericin has a potent effect in promoting wound healing and wound size reduction in OLETF rats and the wounds showed almost complete healing at 48 h after abrasion. The sericin may be an effective and safe drug to promote corneal wound healing in diabetic keratopathy. PMID:24379918

  15. Extraction conditions of Antheraea mylitta sericin with high yields and minimum molecular weight degradation.

    PubMed

    Yun, Haesung; Oh, Hanjin; Kim, Moo Kon; Kwak, Hyo Won; Lee, Jeong Yun; Um, In Chul; Vootla, Shyam Kumar; Lee, Ki Hoon

    2013-01-01

    Although the technique for extracting the Bombyx mori sericin has been extensively known, the extraction of sericin from wild-silkworm cocoons is not yet standardized. The aim of this study was to find the optimal conditions for the extraction of sericin from Antheraea mylitta cocoons, with high yields and minimum degradation. We attempted to apply various protocols for the extraction of the A. mylitta sericin (AmS). Among these, we found that the extraction of AmS with a sodium carbonate solution exhibited the highest yield except the conventional soap-alkali extraction. To find the optimal conditions for the AmS extraction with the sodium carbonate, we changed the concentration of sodium carbonate and the treatment time. With an increase in the sodium carbonate concentration and the extraction time, the yield of AmS increased, but the molecular weight (MW) of AmS decreased. Considering the yield, molecular weight distribution (MWD) and amino acid composition of AmS, we suggest that the optimal conditions for the AmS extraction require treatment with 0.02 M sodium carbonate and boiling for 60 min. PMID:23026092

  16. Therapeutic effects of sericin on diabetic keratopathy in Otsuka Long-Evans Tokushima Fatty rats.

    PubMed

    Nagai, Noriaki; Ito, Yoshimasa

    2013-12-15

    An Otsuka Long-Evans Tokushima Fatty (OLETF) rat provides a useful model for studies to develop corneal wound healing drugs for use in diabetic keratopathy resulting from type 2 diabetes mellitus. We investigated the effects of sericin on corneal wound healing in OLETF rats. Corneal wounds were prepared by removal of the corneal epithelium and documented using a TRC-50X. Sericin was instilled into the eyes of rats five times a day following corneal abrasion. The plasma levels of glucose, triglycerides, cholesterol and insulin in 38 wk old OLETF rats were significantly higher than in normal control rats (LETO rats), and the rate of corneal wound healing in OLETF rats was slower than in normal rat, probably due to the suppression of cell migration and proliferation caused by high plasma glucose levels. The corneal wounds of OLETF rats instilled with saline showed almost complete healing 72 h after corneal epithelial abrasion. On the other hand, the instillation of sericin has a potent effect in promoting wound healing and wound size reduction in OLETF rats and the wounds showed almost complete healing at 48 h after abrasion. The sericin may be an effective and safe drug to promote corneal wound healing in diabetic keratopathy. PMID:24379918

  17. Increasing silk fibre strength through heterogeneity of bundled fibrils

    PubMed Central

    Cranford, Steven W.

    2013-01-01

    Can naturally arising disorder in biological materials be beneficial? Materials scientists are continuously attempting to replicate the exemplary performance of materials such as spider silk, with detailed techniques and assembly procedures. At the same time, a spider does not precisely machine silk—imaging indicates that its fibrils are heterogeneous and irregular in cross section. While past investigations either focused on the building material (e.g. the molecular scale protein sequence and behaviour) or on the ultimate structural component (e.g. silk threads and spider webs), the bundled structure of fibrils that compose spider threads has been frequently overlooked. Herein, I exploit a molecular dynamics-based coarse-grain model to construct a fully three-dimensional fibril bundle, with a length on the order of micrometres. I probe the mechanical behaviour of bundled silk fibrils with variable density of heterogenic protrusions or globules, ranging from ideally homogeneous to a saturated distribution. Subject to stretching, the model indicates that cooperativity is enhanced by contact through low-force deformation and shear ‘locking’ between globules, increasing shear stress transfer by up to 200 per cent. In effect, introduction of a random and disordered structure can serve to improve mechanical performance. Moreover, addition of globules allows a tuning of free volume, and thus the wettability of silk (with implications for supercontraction). These findings support the ability of silk to maintain near-molecular-level strength at the scale of silk threads, and the mechanism could be easily adopted as a strategy for synthetic fibres. PMID:23486175

  18. Silk properties determined by gland-specific expression of a spider fibroin gene family.

    PubMed

    Guerette, P A; Ginzinger, D G; Weber, B H; Gosline, J M

    1996-04-01

    Spiders produce a variety of silks that range from Lycra-like elastic fibers to Kevlar-like superfibers. A gene family from the spider Araneus diadematus was found to encode silk-forming proteins (fibroins) with different proportions of amorphous glycine-rich domains and crystal domains built from poly(alanine) and poly(glycine-alanine) repeat motifs. Spiders produce silks of different composition by gland-specific expression of this gene family, which allows for a range of mechanical properties according to the crystal-forming potential of the constituent fibroins. These principles of fiber property control may be important in the development of genetically engineered structural proteins. PMID:8600519

  19. Analysis of a new type of major ampullate spider silk gene, MaSp1s.

    PubMed

    Han, Leng; Zhang, Lei; Zhao, Tianfu; Wang, Yujun; Nakagaki, Masao

    2013-05-01

    Spider dragline silk, which is secreted from the major ampullate silk glands, is a unique fibrous protein with a combination of tensile strength and elasticity. Here, we describe a new short type of dragline silk gene, Cyrtophora moluccensis MaSp1s. The full-length gene is only 1320 base pairs (bp), which encodes 439 amino acids that includes the intact non-repetitive N-terminal (149 residues), C-terminal (98 residues) and so-called repetitive regions (192 residues); the deduced molecular weight is approximately 40 kDa. The sequence analysis demonstrated that the two termini are highly homologous to the other characterized dragline silk genes but that the so-called repetitive region is different. Our results suggest that MaSp1s is a possible new characteristic dragline gene; the discovery of this gene should enhance our understanding of the major ampullate spider silk genes. PMID:23403024

  20. An emerging functional natural silk biomaterial from the only domesticated non-mulberry silkworm Samia ricini.

    PubMed

    Pal, Shilpa; Kundu, Joydip; Talukdar, Sarmistha; Thomas, Tintu; Kundu, Subhas C

    2013-08-01

    Mulberry silk fibroin is a widely used biomaterial and recent work on non-mulberry silk fibroin also suggests it may have similar uses. We expect silk fibroin from the only domesticated non-mulberry eri silkworm, Samia ricini, to possess useful properties as a biomaterial. Eri silk gland fibroin is a heterodimeric protein of approximately 450?kDa. Cytocompatibility evaluation with fibroblasts and osteoblast-like cells shows good cell attachment, viability and proliferation. The matrices, which have high thermal stability and good swellability, are also haemocompatible. Eri silk production is cost effective as no agronomic practices are required for their host plant cultivation. This fibroin provide new opportunities as an alternative natural functional biomaterial in various biomedical applications. PMID:23733347

  1. In situ Raman spectroscopic study of Al-infiltrated spider dragline silk under tensile deformation.

    PubMed

    Lee, Seung-Mo; Pippel, Eckhard; Moutanabbir, Oussama; Kim, Jae-Hyun; Lee, Hak-Joo; Knez, Mato

    2014-10-01

    Natural materials consisting of protein structures impregnated with a tiny amount of metals often exhibit impressive mechanical behavior, which represents a new design paradigm for the development of biomimetic materials. Here, we produced Al-infiltrated silks by applying a modified Al2O3 atomic layer deposition process to the dragline silk of the Nephila pilipes spider, which showed unusual mechanical properties. The deformation behavior of the molecular structure of the Al-infiltrated silk was investigated by performing in situ Raman spectroscopy, where Raman shifts were measured concurrently with macroscopic mechanical deformations. For identifying the role of the infiltrated Al atoms, the study was performed in parallel with untreated silk, and the results were compared. Our experimental results revealed that superior mechanical properties of the Al-infiltrated silk are likely to be caused by the alterations of the sizes of the ?-sheet crystals and their distribution. PMID:25203848

  2. Silk matrix for tissue engineered anterior cruciate ligaments

    Microsoft Academic Search

    Gregory H Altman; Rebecca L Horan; Helen H Lu; Jodie Moreau; Ivan Martin; John C Richmond; David L Kaplan

    2002-01-01

    A silk-fiber matrix was studied as a suitable material for tissue engineering anterior cruciate ligaments (ACL). The matrix was successfully designed to match the complex and demanding mechanical requirements of a native human ACL, including adequate fatigue performance. This protein matrix supported the attachment, expansion and differentiation of adult human progenitor bone marrow stromal cells based on scanning electron microscopy,

  3. Silk materials--a road to sustainable high technology.

    PubMed

    Tao, Hu; Kaplan, David L; Omenetto, Fiorenzo G

    2012-06-01

    This review addresses the use of silk protein as a sustainable material in optics and photonics, electronics and optoelectronic applications. These options represent additional developments for this technology platform that compound the broad utility and impact of this material for medical needs that have been recently described in the literature. The favorable properties of the material certainly make a favorable case for the use of silk, yet serve as a broad inspiration to further develop biological foundries for both the synthesis and processing of Nature's materials for technological applications. PMID:22553118

  4. All-water-based electron-beam lithography using silk as a resist

    NASA Astrophysics Data System (ADS)

    Kim, Sunghwan; Marelli, Benedetto; Brenckle, Mark A.; Mitropoulos, Alexander N.; Gil, Eun-Seok; Tsioris, Konstantinos; Tao, Hu; Kaplan, David L.; Omenetto, Fiorenzo G.

    2014-04-01

    Traditional nanofabrication techniques often require complex lithographic steps and the use of toxic chemicals. To move from the laboratory scale to large scales, nanofabrication should be carried out using alternative procedures that are simple, inexpensive and use non-toxic solvents. Recent efforts have focused on nanoimprinting and the use of organic resists (such as quantum dot-polymer hybrids, DNA and poly(ethylene glycol)), which still require, for the most part, noxious chemicals for processing. Significant advances have been achieved using `green' resists that can be developed with water, but so far these approaches have suffered from low electron sensitivity, line edge roughness and scalability constraints. Here, we present the use of silk as a natural and biofunctional resist for electron-beam lithography. The process is entirely water-based, starting with the silk aqueous solution and ending with simple development of the exposed silk film in water. Because of its polymorphic crystalline structure, silk can be used either as a positive or negative resist through interactions with an electron beam. Moreover, silk can be easily modified, thereby enabling a variety of `functional resists', including biologically active versions. As a proof of principle of the viability of all-water-based silk electron-beam lithography (EBL), we fabricate nanoscale photonic lattices using both neat silk and silk doped with quantum dots, green fluorescent proteins (GFPs) or horseradish peroxidase (HRP).

  5. Comparison of fibroin cDNAs from webspinning insects: insight into silk formation and function.

    PubMed

    Collin, Matthew A; Edgerly, Janice S; Hayashi, Cheryl Y

    2011-09-01

    Embiopterans (webspinning insects) are renowned for their prolific use of silk. These organisms spin silk to construct elaborate networks of tubes in which they live, forage, and reproduce. The silken galleries are essential for protecting these soft-bodied insects from predators and other environmental hazards. Despite the ecological importance of embiopteran silk, very little is known about its constituent proteins. Here, we characterize the silk protein cDNAs from four embiopteran species to better understand the function and evolution of these adaptive molecules. We show that webspinner fibroins (silk proteins) are highly repetitive in sequence and possess several conserved characteristics, despite differences in habitat preferences across species. The most striking similarities are in the codon usage biases of the fibroin genes, particularly in the repetitive regions, as well as sequence conservation of the carboxyl-terminal regions of the fibroins. Based on analyses of the silk genes, we propose hypotheses regarding codon bias and its effect on the translation and replication of these unusual genes. Furthermore, we discuss the significance of specific fibroin motifs to the mechanical and structural characteristics of silk fibers. Lastly, we report that the conservation of webspinner fibroin carboxyl-terminal regions suggests that fiber formation may occur through a mechanism analogous to that found in Lepidoptera. From these results, insight is gained into the tempo and mode of evolution that has shaped embiopteran fibroins. PMID:21741226

  6. Different Types of Peptide Detected by Mass Spectrometry among Fresh Silk and Archaeological Silk Remains for Distinguishing Modern Contamination

    PubMed Central

    Li, Li; Gong, Yuxuan; Yin, Hao; Gong, Decai

    2015-01-01

    Archaeological silk provides abundant information for studying ancient technologies and cultures. However, due to the spontaneous degradation and the damages from burial conditions, most ancient silk fibers which suffered the damages for thousands of years were turned into invisible molecular residues. For the obtained rare samples, extra care needs to be taken to accurately identify the genuine archaeological silk remains from modern contaminations. Although mass spectrometry (MS) is a powerful tool for identifying and analyzing the ancient protein residues, the traditional approach could not directly determine the dating and contamination of each sample. In this paper, a series of samples with a broad range of ages were tested by MS to find an effective and innovative approach to determine whether modern contamination exists, in order to verify the authenticity and reliability of the ancient samples. The new findings highlighted that the detected peptide types of the fibroin light chain can indicate the degradation levels of silk samples and help to distinguish contamination from ancient silk remains. PMID:26186676

  7. Structure to function: Spider silk and human collagen

    NASA Astrophysics Data System (ADS)

    Rabotyagova, Olena S.

    Nature has the ability to assemble a variety of simple molecules into complex functional structures with diverse properties. Collagens, silks and muscles fibers are some examples of fibrous proteins with self-assembling properties. One of the great challenges facing Science is to mimic these designs in Nature to find a way to construct molecules that are capable of organizing into functional supra-structures by self-assembly. In order to do so, a construction kit consisting of molecular building blocks along with a complete understanding on how to form functional materials is required. In this current research, the focus is on spider silk and collagen as fibrous protein-based biopolymers that can shed light on how to generate nanostructures through the complex process of self-assembly. Spider silk in fiber form offers a unique combination of high elasticity, toughness, and mechanical strength, along with biological compatibility and biodegrability. Spider silk is an example of a natural block copolymer, in which hydrophobic and hydrophilic blocks are linked together generating polymers that organize into functional materials with extraordinary properties. Since silks resemble synthetic block copolymer systems, we adopted the principles of block copolymer design from the synthetic polymer literature to build block copolymers based on spider silk sequences. Moreover, we consider spider silk to be an important model with which to study the relationships between structure and properties in our system. Thus, the first part of this work was dedicated to a novel family of spider silk block copolymers, where we generated a new family of functional spider silk-like block copolymers through recombinant DNA technology. To provide fundamental insight into relationships between peptide primary sequence, block composition, and block length and observed morphological and structural features, we used these bioengineered spider silk block copolymers to study secondary structure, morphological features and assembly. Aside from fundamental perspectives, we anticipate that these results will provide a blueprint for the design of precise materials for a range of potential applications such as controlled release devices, functional coatings, components of tissue regeneration materials and environmentally friendly polymers in future studies. In the second part of this work, human collagen type I was studied as another representative of the family of fibrous proteins. Collagen type I is the most abundant extracellular matrix protein in the human body, providing the basis for tissue structure and directing cellular functions. Collagen has a complex structural hierarchy, organized at different length scales, including the characteristic triple helical feature. In the present study we assessed the relationship between collagen structure (native vs. denatured) and sensitivity to UV radiation with a focus on changes in the primary structure, conformation, microstructure and material properties. Free radical reactions are involved in collagen degradation and a mechanism for UV-induced collagen degradation related to structure was proposed. The results from this study demonstrated the role of collagen supramolecular organization (triple helix) in the context of the effects of electromagnetic radiation on extracellular matrices. Owing to the fact that both silks and collagens are proteins that have found widespread interest for biomaterial related needs, we anticipate that the current studies will serve as a foundation for future biomaterial designs with controlled properties. Furthermore, fundamental insight into self-assembly and environmentally-2mediated degradation, will build a foundation for fundamental understanding of the remodeling and functions of these types of fibrous proteins in vivo and in vitro. This type of insight is essential for many areas of scientific inquiry, from drug delivery, to scaffolds for tissue engineering, and to the stability of materials in space.

  8. Sonication-induced gelation of silk fibroin for cell encapsulation.

    PubMed

    Wang, Xiaoqin; Kluge, Jonathan A; Leisk, Gary G; Kaplan, David L

    2008-03-01

    Purified native silk fibroin forms beta-sheet-rich, physically cross-linked, hydrogels from aqueous solution, in a process influenced by environmental parameters. Previously we reported gelation times of days to weeks for aqueous native silk protein solutions, with high ionic strength and temperature and low pH responsible for increasing gelation kinetics. Here we report a novel method to accelerate the process and control silk fibroin gelation through ultrasonication. Depending on the sonication parameters, including power output and time, along with silk fibroin concentration, gelation could be controlled from minutes to hours, allowing the post-sonication addition of cells prior to final gel setting. Mechanistically, ultrasonication initiated the formation of beta-sheets by alteration in hydrophobic hydration, thus accelerating the formation of physical cross-links responsible for gel stabilization. K(+) at physiological concentrations and low pH promoted gelation, which was not observed in the presence of Ca(2+). The hydrogels were assessed for mechanical properties and proteolytic degradation; reported values matched or exceeded other cell-encapsulating gel material systems. Human bone marrow derived mesenchymal stem cells (hMSCs) were successfully incorporated into these silk fibroin hydrogels after sonication, followed by rapid gelation and sustained cell function. Sonicated silk fibroin solutions at 4%, 8%, and 12% (w/v), followed by mixing in hMSCs, gelled within 0.5-2 h. The cells grew and proliferated in the 4% gels over 21 days, while survival was lower in the gels with higher protein content. Thus, sonication provides a useful new tool with which to initiate rapid sol-gel transitions, such as for cell encapsulation. PMID:18031805

  9. Emergent spacetimes By Silke Weinfurtner

    E-print Network

    Visser, Matt

    Jain, Anglea White, Crispin Gardiner and Bill Unruh #12;Emergent spacetimes By Silke Weinfurtner and collaborators: Matt Visser, Stefano Liberati, Piyush Jain, Anglea White, Crispin Gardiner and Bill Unruh #12

  10. Cell culture's spider silk road.

    PubMed

    Perkel, Jeffrey

    2014-06-01

    A number of synthetic and natural materials have been tried in cell culture and tissue engineering applications in recent years. Now Jeffrey Perkel takes a look at one new culture component that might surprise you-spider silk. PMID:24924388

  11. Carbon nanotubes on a spider silk scaffold

    PubMed Central

    Steven, Eden; Saleh, Wasan R.; Lebedev, Victor; Acquah, Steve F. A.; Laukhin, Vladimir; Alamo, Rufina G.; Brooks, James S.

    2013-01-01

    Understanding the compatibility between spider silk and conducting materials is essential to advance the use of spider silk in electronic applications. Spider silk is tough, but becomes soft when exposed to water. Here we report a strong affinity of amine-functionalised multi-walled carbon nanotubes for spider silk, with coating assisted by a water and mechanical shear method. The nanotubes adhere uniformly and bond to the silk fibre surface to produce tough, custom-shaped, flexible and electrically conducting fibres after drying and contraction. The conductivity of coated silk fibres is reversibly sensitive to strain and humidity, leading to proof-of-concept sensor and actuator demonstrations. PMID:24022336

  12. Nephila clavipes Flagelliform Silk-like GGX Motifs Contribute to Extensibility and Spacer Motifs Contribute to Strength in Synthetic Spider Silk Fibers

    PubMed Central

    Adrianos, Sherry L.; Teulé, Florence; Hinman, Michael B.; Jones, Justin A.; Weber, Warner S.; Yarger, Jeffery L.; Lewis, Randolph V.

    2013-01-01

    Flagelliform spider silk is the most extensible silk fiber produced by orb weaver spiders, though not as strong as the dragline silk of the spider. The motifs found in the core of the Nephila clavipes flagelliform Flag protein are: GGX, spacer, and GPGGX. Flag does not contain the polyalanine motif known to provide the strength of dragline silk. To investigate the source of flagelliform fiber strength, four recombinant proteins were produced containing variations of the three core motifs of the Nephila clavipes flagelliform Flag protein that produces this type of fiber. The as-spun fibers were processed in 80% aqueous isopropanol using a standardized process for all four fiber types, which produced improved mechanical properties. Mechanical testing of the recombinant proteins determined that the GGX motif contributes extensibility and the spacer motif contributes strength to the recombinant fibers. Recombinant protein fibers containing the spacer motif were stronger than the proteins constructed without the spacer that contained only the GGX motif or the combination of the GGX and GPGGX motifs. The mechanical and structural X-ray diffraction analysis of the recombinant fibers provide data that suggests a functional role of the spacer motif that produces tensile strength though the spacer motif is not clearly defined structurally. These results indicate that the spacer is likely a primary contributor of strength with the GGX motif supplying mobility to the protein network of native N. clavipes flagelliform silk fibers. PMID:23646825

  13. Nanolayer biomaterial coatings of silk fibroin for controlled release.

    PubMed

    Wang, Xianyan; Hu, Xiao; Daley, Andrea; Rabotyagova, Olena; Cebe, Peggy; Kaplan, David L

    2007-08-28

    An all-aqueous, stepwise deposition process with silk fibroin protein for the assembly of nanoscale layered controlled release coatings was exploited. Model compounds, Rhodamine B, Even Blue and Azoalbumin, representing small molecule drugs and therapeutically relevant proteins were incorporated in the nanocoating process and their loading and release behavior was quantified. In addition, the structure and morphology of the coatings were characterized. Release studies in vitro showed that control of beta-sheet crystal content and the multilayer structure of the silk coatings correlated with the release properties of the incorporated compounds. In particular, higher crystallinity and a thicker silk capping layer suppressed the initial burst of release and prolonged the duration of release. These novel coatings and deposition approach provide a unique option to regulate structure and morphology, and thus release kinetics. The results also suggest these systems as a promising framework for surface engineering of biomaterials and medical devices to regulate the release of drugs, when considered with the all-aqueous process involved, the conformal nature of the coatings, the robust material properties of silk fibroin, and the degradability and biocompatibility of this family of protein. PMID:17628161

  14. Nanolayer Biomaterial Coatings of Silk Fibroin for Controlled Release

    PubMed Central

    Wang, Xianyan; Hu, Xiao; Daley, Andrea; Rabotyagova, Olena; Cebe, Peggy; Kaplan, David L.

    2009-01-01

    An all-aqueous, stepwise deposition process with silk fibroin protein for the assembly of nanoscale layered controlled release coatings was exploited. Model compounds, Rhodamine B, Even Blue and Azoalbumin, representing small molecule drugs and therapeutically relevant proteins were incorporated in the nanocoating process and their loading and release behavior was quantified. In addition, the structure and morphology of the coatings were characterized. Release studies in vitro showed that control of ?-sheet crystal content and the multilayer structure of the silk coatings correlated with the release properties of the incorporated compounds. In particular, higher crystallinity and a thicker silk capping layer suppressed the initial burst of release and prolonged the duration of release. These novel coatings and deposition approach provide a unique option to regulate structure and morphology, and thus release kinetics. The results also suggest these systems as a promising framework for surface engineering of biomaterials and medical devices to regulate the release of drugs, when considered with the all-aqueous process involved, the conformal nature of the coatings, the robust material properties of silk fibroin, and the degradability and biocompatibility of this family of protein. PMID:17628161

  15. Artificial fibrous proteins: A review

    Microsoft Academic Search

    H. Heslot

    1998-01-01

    Several kinds of natural fibrous proteins have been chosen as models: silk fibroin from Bombyx mori, silks from various species of spiders and collagens. The dragline silk of the spider Nephila clavipes is able to stretch by 30% before breaking and has a high tensile strength. It is stronger per unit weight than high tensile steel. Although the partial sequence

  16. Design and Optimization of Resorbable Silk Internal Fixation Devices

    NASA Astrophysics Data System (ADS)

    Haas, Dylan S.

    Limitations of current material options for internal fracture fixation devices have resulted in a large gap between user needs and hardware function. Metal systems offer robust mechanical strength and ease of implantation but require secondary surgery for removal and/or result in long-term complications (infection, palpability, sensitivity, etc.). Current resorbable devices eliminate the need for second surgery and long-term complications but are still associated with negative host response as well as limited functionality and more difficult implantation. There is a definitive need for orthopedic hardware that is mechanically capable of immediate fracture stabilization and fracture fixation during healing, can safely biodegrade while allowing complete bone remodeling, can be resterilized for reuse, and is easily implantable (self-tapping). Previous work investigated the use of silk protein to produce resorbable orthopedic hardware for non- load bearing fracture fixation. In this study, silk orthopedic hardware was further investigated and optimized in order to better understand the ability of silk as a fracture fixation system and more closely meet the unfulfilled market needs. Solvent-based and aqueous-based silk processing formulations were cross-linked with methanol to induce beta sheet structure, dried, autoclaved and then machined to the desired device/geometry. Silk hardware was evaluated for dry, hydrated and fatigued (cyclic) mechanical properties, in vitro degradation, resterilization, functionalization with osteoinductive molecules and implantation technique for fracture fixation. Mechanical strength showed minor improvements from previous results, but remains comparable to current resorbable fixation systems with the advantages of self-tapping ability for ease of implantation, full degradation in 10 months, ability to be resterilized and reused, and ability to release molecules for osteoinudction. In vivo assessment confirmed biocompatibility, showed improved bone deposition and remodeling with functionalization and showed promising feasibility of fracture fixations with minor adjustments to geometry. The proposed silk orthopedic hardware exhibits high potential as a resorbable fixation system that can bridge the gap between the current materials for internal fixation devices.

  17. Structure and gelation mechanism of silk hydrogels.

    PubMed

    Nagarkar, Shailesh; Nicolai, Taco; Chassenieux, Christophe; Lele, Ashish

    2010-04-21

    Silk fibroin was regenerated from cocoons produced by the silkworm Bombyx Mori. Light scattering showed that an aqueous solution of the regenerated silk fibroin (RSF) was made of individual proteins with a weight average molar mass of about 4 x 10(5) g mol(-1) and a hydrodynamic radius of about 10 nm. Gel formation of RSF in acidic solutions was investigated as a function of the pH (2-4), concentration (0.5-10 g L(-1)) and temperature (5-70 degrees C). The structure of the gels was studied using light scattering and confocal laser scanning microscopy. The structure was found to be self-similar from length scales of less than 15 nm up to length scales of about 1 microm, and characterized by a correlation length of a few microns. Gel formation was tracked using turbidity, rheology, light scattering and circular dichroism. Gelation involves the formation of self-similar aggregates with a growth rate that increases exponentially. The protein aggregation is correlated to, and perhaps caused by, the formation of beta-sheets, the fraction of which also increases exponentially with time. PMID:20358077

  18. Enhanced Cellular Adhesion on Titanium by Silk Functionalized with titanium binding and RGD peptides

    PubMed Central

    Vidal, Guillaume; Blanchi, Thomas; Mieszawska, Aneta J.; Calabrese, Rossella; Rossi, Claire; Vigneron, Pascale; Duval, Jean-Luc; Kaplan, David L.; Egles, Christophe

    2012-01-01

    Soft tissue adhesion on titanium represents a challenge for implantable materials. In order to improve adhesion at the cell/material interface we used a new approach based on the molecular recognition of titanium by specific peptides. Silk fibroin protein was chemically grafted with titanium binding peptide (TiBP) to increase adsorption of these chimeric proteins to the metal surface. Quartz Crystal Microbalance was used to quantify the specific adsorption of TiBP-functionalized silk and an increase in protein deposition by more than 35% was demonstrated due to the presence of the binding peptide. A silk protein grafted with TiBP and fibronectin-derived RGD peptide was then prepared. The adherence of fibroblasts on the titanium surface modified with the multifunctional silk coating demonstrated an increase in the number of adhering cells by 60%. The improved adhesion was demonstrated by Scanning Electron Microscopy and immunocytochemical staining of focal contact points. Chick embryo organotypic culture also revealed strong adhesion of endothelial cells expanding on the multifunctional silk-peptide coating. These results demonstrated that silk functionalized with TiBP and RGD represents a promising approach to modify cell-biomaterial interfaces, opening new perspectives for implantable medical devices, especially when reendothelialization is required. PMID:22975628

  19. Release and cellular acceptance of multiple drugs loaded silk fibroin particles.

    PubMed

    Shi, Pujiang; Goh, James C H

    2011-11-28

    In this article, silk fibroin particles with average diameter of 980 nm were fabricated via self assembly. Exceptional loading efficiency and release patterns of hydrophobic and protein drugs were observed. Furthermore, smoother release patterns were observed with increase loading of the hydrophobic and protein model drugs, only about 23% FITC-BSA and 34% RhB were released from the silk particles at their highest corresponding loading in 50 days. Most importantly, osteoblasts' viability was augmented during co-culture with silk fibroin particles, as shown by Alamar Blue assay. Attachment of the particles and delivery of model drugs to cells were confirmed by fluorescence images and flow cytometry. Hence, the silk fibroin particles could be potential biomaterial for application in controlled release and pharmaceutics. PMID:21920418

  20. Will silk fibroin nanofiber scaffolds ever hold a useful place in Translational Regenerative Medicine?

    PubMed Central

    Ubaldo, Armato; Ilaria, Dal Prà; Anna, Chiarini; Giuliano, Freddi

    2011-01-01

    Presently, some view silk fibroin-based biomaterials as obsolete, being outperformed by a host of newly discovered biomaterials. But several lines of evidence support the notion that silk fibroin proteins, especially those from B. mori and spiders and their recombinant forms, particularly in the form of electrospun nanofiber scaffolds, still represent promising tools for human tissue engineering/regeneration. Inevitably, the allure of recently reported biomaterials turns away many scientists and resources from the aim of more deeply elucidating the biological interactions of the various kinds of silk fibroin nanofiber scaffolds in vivo. But, even the biological features of newly reported biomaterials are not investigated in adequate depth. Hence, collaborative efforts among biomaterialists, biomedical experts, and private firms must be undertaken on a much greater scale than hitherto done to assess the real usefulness of silk fibroin proteins, thereby allowing or denying their useful introduction into the fields of Translational Regenerative Medicine. PMID:22928155

  1. Biomaterials from ultrasonication-induced silk fibroin-hyaluronic acid hydrogels.

    PubMed

    Hu, Xiao; Lu, Qiang; Sun, Lin; Cebe, Peggy; Wang, Xiaoqin; Zhang, Xiaohui; Kaplan, David L

    2010-11-01

    We report formation of biocompatible hydrogels using physically cross-linked biopolymers. Gelation of silk fibroin (from B. mori silkworm) aqueous solution was effected by ultrasonication and used to entrap blended, un-cross-linked, hyaluronic acid (HA) without chemical cross-linking. HA was formed into silk/HA blended hydrogels with different mixing ratios, forming homogeneous materials with stable swelling behavior when the HA content was less than 40 wt %. This is a novel approach to HA hydrogel systems, which otherwise require chemical cross-linking. Further, these systems exploit the beneficial material and biological properties of both polymers. Differential scanning calorimetry (DSC), temperature modulated DSC, and thermal gravimetric analysis were used to show that well-blended silk/HA hydrogel systems formed without macrophase separation. Fourier transform infrared spectroscopy was used to determine secondary structures from the amide I region of silk protein by spectral subtraction and Fourier-self-deconvolution. The ?-sheet crystal fraction of the silk protein increased with increase of HA content (26-35 wt %), which resulted in stable, crystalline features in the blend hydrogel materials, favorable features to support human mesenchymal stem cell attachment and proliferation. Scanning electron microscopy was used to characterize morphology. ?-Sheet content controlled the stability of the silk/HA hydrogel systems, with a minimum crystalline content needed to maintain a stable hydrogel system of ?26 wt %. This value is close to the ?-sheet content in pure silk fibroin hydrogels. These novel nonchemically cross-linked blend hydrogels may be useful for biomedical applications due to biocompatibility and the widespread utility of hydrogel systems. The attributes of HA in combination with the features of silk, offer a useful suite of properties, combining the mechanical integrity and slow degradation of silk with the control of water interactions and biological signaling of HA. PMID:20942397

  2. Silk Optics Mark Cronin-Golomb

    E-print Network

    Barthelat, Francois

    Technologies · Acquired by Allergan 2010 ­ Seri #12;1/27/14 3 Silk Materials ­ A Road to Sustainable High http://onlinelibrary.wiley.com/doi/10.1002/adma.201104477/full#fig2 Silk Materials ­ A Road1/27/14 1 Silk Optics Mark Cronin-Golomb Department of Biomedical Engineering Tufts University

  3. Discrimination of cultivated silk and wild silk by conventional instrumental analyses.

    PubMed

    Matsuyama, Yuji; Nagatani, Yoshiaki; Goto, Toshiyuki; Suzuki, Shinichi

    2013-09-10

    In Japan, recent trends have seen wild silk preferred over cultivated silk because of its texture. Some cases of fraud have occurred where cultivated silk garments are sold as wild silk. Samples from these cases, morphological observation using light microscope and polarized microscope have been conducted in forensic science laboratories. Sometimes scanning electron microscopy was also carried out. However, the morphology of silk shows quite wide variation, which makes it difficult to discriminate wild and cultivated silks by this method. In this report, silk discrimination was investigated using conventional instrumental analyses commonly available in forensic laboratories, such as Fourier-transfer infrared spectrometry (FT-IR), pyrolysis-gas chromatography/mass spectrometry (pyr-GC/MS) and differential thermal analysis (DTA). By FT-IR, cultivated and wild silk gave similar infrared spectra, but wild silk had a characteristic peak at 965 cm(-1) from the deformation vibration of the carbon-carbon double bond of the indole ring. Comparison of the pyrograms of cultivated and wild silk showed that wild silk had large indole and skatole peaks that cultivated silk did not, and these peaks might arise from tryptophan. The results of thermogravimetry/DTA showed that the endothermic peak was about 40 °C higher for wild silk than for cultivated silk. Using a combination of these results, cultivated and wild silk could be discriminated by common forensic instrumental techniques. PMID:23742990

  4. A green salt-leaching technique to produce sericin/PVA/glycerin scaffolds with distinguished characteristics for wound-dressing applications.

    PubMed

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

    2015-05-01

    Sericin/PVA/glycerin scaffolds could be fabricated using the freeze-drying technique; they showed good physical and biological properties and can be applied as wound dressings. However, freeze-drying is an energy- and time-consuming process with a high associated cost. In this study, an alternative, solvent-free, energy- and time-saving, low-cost salt-leaching technique is introduced as a green technology to produce sericin/PVA/glycerin scaffolds. We found that sericin/PVA/glycerin scaffolds were successfully fabricated without any crosslinking using a salt-leaching technique. The salt-leached sericin/PVA/glycerin scaffolds had a porous structure with pore interconnectivity. The sericin in the salt-leached scaffolds had a crystallinity that was as high as that of the freeze-dried scaffolds. Compared to the freeze-dried scaffolds with the same composition, the salt-leached sericin/PVA/glycerin scaffolds has larger pores, a lower Young's modulus, and faster rates of biodegradation and sericin release. When cultured with L929 mouse fibroblast cells, a higher number of cells were found in the salt-leached scaffolds. Furthermore, the salt-leached scaffolds were less adhesive to the wound, which would reduce pain upon removal. Therefore, salt-leached sericin/PVA/glycerin scaffolds with distinguished characteristics were introduced as another choice of wound dressing, and their production process was simpler, more energy efficient, and saved time and money compared to the freeze-dried scaffolds. PMID:25175958

  5. Loss of Posterior Silk Gland Transcription Specificity of Fibroin Light Chain Promoter due to Absence of 41 bp Sequence Containing Possible Inhibitor Binding Sites

    Microsoft Academic Search

    Ting-Qing GUO; Jian-Yang WANG; Sheng-Peng WANG; Xiu-Yang GUO; Ke-Wei HUANG; Jun-Ting HUANG; Chang-De LU

    2005-01-01

    The gene encoding fibroin light chain protein (FibL) is specifically expressed in the posterior silk gland of silkworm and repressed in other tissues. The binding sites of several transcription factors involved in the silk gland transcription specificity of fibl promoter have been recognized, including SGFB, PSGF and BMFA. Here we report the leak expression of the enhanced green fluorescent protein

  6. Electrospinning of chitosan/sericin/PVA nanofibers incorporated with in situ synthesis of nano silver.

    PubMed

    Hadipour-Goudarzi, Elmira; Montazer, Majid; Latifi, Masoud; Aghaji, Ali Akbar Ghare

    2014-11-26

    Here, chitosan/sericin/poly(vinyl alcohol) as a biodegradable nanofibrous membrane was prepared through electrospinning with and without silver nitrate. The influences of spinning conditions including volume ratio of chitosan and sericin, voltage and spinning distance at constant feed rate on the fiber morphology and size distribution were examined by SEM and Image J software. The FT-IR spectrum and EDAX were used to indicate the chemical structure of nanofibrous membrane. In addition, the effect of AgNO3 on the nanofibers diameter and its antibacterial activity was investigated. The optimum conditions obtained with chitosan:sericin (50:50, v/v), 22 kV voltage, 10 cm spinning distance at 0.25 mL/h feed rate to prepare nanofibers with small diameter and narrow size distribution without beads. The mean diameter of nanofibers was about 180 nm while introducing AgNO3 led to smaller nanofibers diameter about 95 nm. Moreover, the presence of AgNO3 produced an excellent antibacterial activity against Escherchia coli. PMID:25256480

  7. Modulation and Stabilization of Silk Fibroin-Coated Oil-in-Water Emulsions

    Microsoft Academic Search

    Jia-Jia Rao; Zhong-Min Chen; Bing-Can Chen

    Summary The purpose of this study is to prepare and characterize stable oil-in-water emulsions containing droplets coated with silk fibroin. Silk fibroin, a native edible fibrous protein ori- ginating from silkworm cocoons, was used to prepare 10 % (by mass) corn oil-in-water emulsions at ambient temperature (pH=7.0, 10 mM phosphate buffer). Emulsions with relatively small mean particle diameter (d32=0.47 mm)

  8. Structure modifications induced in silk fibroin by enzymatic treatments. A Raman study

    NASA Astrophysics Data System (ADS)

    Monti, Patrizia; Freddi, Giuliano; Sampaio, Sandra; Tsukada, Masuhiro; Taddei, Paola

    2005-06-01

    Raman spectroscopy was used to investigate various enzyme-catalyzed reactions onto silk fibroin, i.e. the biodegradation of Tussah ( Antheraea pernyi) silk fibroin films by a proteolytic enzyme, the oxidation of domestic ( Bombyx mori) silk fibroin by mushroom tyrosinase and the subsequent grafting of chitosan onto oxidized silk. The spectra of Tussah silk fibroin films exposed to a bacterial protease for different times demonstrated that the cleavage of sensitive peptide bonds in the amorphous glycine-rich domains resulted in the loss of various amino acid residues (Tyr, Trp, Asp, etc.). The bands attributed to the crystalline alanine-rich sequences increased in intensity, and the ?-sheet molecular conformation was not affected by biodegradation. Following oxidation with mushroom tyrosinase, the tyrosine bands of Bombyx mori fibroin decreased in intensity but did not disappear. The increase of the I853/ I829 intensity ratio indicated that the Tyr residues not accessible to the enzyme were located in a strongly hydrophobic environment. Raman spectroscopy provided evidence that chitosan was effectively grafted onto oxidized silk, probably via the Schiff-base mechanism, as shown by the behavior of the imine band at about 1646 cm -1. Grafting chitosan onto silk fibroin resulted in a ?-sheet?random coil conformational transition of the protein component in the bioconjugated product.

  9. Rate-Dependent Behavior of the Amorphous Phase of Spider Dragline Silk

    PubMed Central

    Patil, Sandeep P.; Markert, Bernd; Gräter, Frauke

    2014-01-01

    The time-dependent stress-strain behavior of spider dragline silk was already observed decades ago, and has been attributed to the disordered sequences in silk proteins, which compose the soft amorphous matrix. However, the actual molecular origin and magnitude of internal friction within the amorphous matrix has remained inaccessible, because experimentally decomposing the mechanical response of the amorphous matrix from the embedded crystalline units is challenging. Here, we used atomistic molecular dynamics simulations to obtain friction forces for the relative sliding of peptide chains of Araneus diadematus spider silk within bundles of these chains as a representative unit of the amorphous matrix in silk fibers. We computed the friction coefficient and coefficient of viscosity of the amorphous phase to be in the order of 10?6 Ns/m and 104 Ns/m2, respectively, by extrapolating our simulation data to the viscous limit. Finally, we used a finite element method for the amorphous phase, solely based on parameters derived from molecular dynamics simulations including the newly determined coefficient of viscosity. With this model the time scales of stress relaxation, creep, and hysteresis were assessed, and found to be in line with the macroscopic time-dependent response of silk fibers. Our results suggest the amorphous phase to be the primary source of viscosity in silk and open up the avenue for finite element method studies of silk fiber mechanics including viscous effects. PMID:24896131

  10. Rate-dependent behavior of the amorphous phase of spider dragline silk.

    PubMed

    Patil, Sandeep P; Markert, Bernd; Gräter, Frauke

    2014-06-01

    The time-dependent stress-strain behavior of spider dragline silk was already observed decades ago, and has been attributed to the disordered sequences in silk proteins, which compose the soft amorphous matrix. However, the actual molecular origin and magnitude of internal friction within the amorphous matrix has remained inaccessible, because experimentally decomposing the mechanical response of the amorphous matrix from the embedded crystalline units is challenging. Here, we used atomistic molecular dynamics simulations to obtain friction forces for the relative sliding of peptide chains of Araneus diadematus spider silk within bundles of these chains as a representative unit of the amorphous matrix in silk fibers. We computed the friction coefficient and coefficient of viscosity of the amorphous phase to be in the order of 10(-6) Ns/m and 10(4) Ns/m(2), respectively, by extrapolating our simulation data to the viscous limit. Finally, we used a finite element method for the amorphous phase, solely based on parameters derived from molecular dynamics simulations including the newly determined coefficient of viscosity. With this model the time scales of stress relaxation, creep, and hysteresis were assessed, and found to be in line with the macroscopic time-dependent response of silk fibers. Our results suggest the amorphous phase to be the primary source of viscosity in silk and open up the avenue for finite element method studies of silk fiber mechanics including viscous effects. PMID:24896131

  11. Impact of processing parameters on the haemocompatibility of Bombyx mori silk films.

    PubMed

    Seib, F Philipp; Maitz, Manfred F; Hu, Xiao; Werner, Carsten; Kaplan, David L

    2012-02-01

    Silk has traditionally been used for surgical sutures due to its lasting strength and durability; however, the use of purified silk proteins as a scaffold material for vascular tissue engineering goes beyond traditional use and requires application-orientated biocompatibility testing. For this study, a library of Bombyx mori silk films was generated and exposed to various solvents and treatment conditions to reflect current silk processing techniques. The films, along with clinically relevant reference materials, were exposed to human whole blood to determine silk blood compatibility. All substrates showed an initial inflammatory response comparable to polylactide-co-glycolide (PLGA), and a low to moderate haemostasis response similar to polytetrafluoroethylene (PTFE) substrates. In particular, samples that were water annealed at 25 °C for 6 h demonstrated the best blood compatibility based on haemostasis parameters (e.g. platelet decay, thrombin-antithrombin complex, platelet factor 4, granulocytes-platelet conjugates) and inflammatory parameters (e.g. C3b, C5a, CD11b, surface-associated leukocytes). Multiple factors such as treatment temperature and solvent influenced the biological response, though no single physical parameter such as ?-sheet content, isoelectric point or contact angle accurately predicted blood compatibility. These findings, when combined with prior in vivo data on silk, support a viable future for silk-based vascular grafts. PMID:22079005

  12. Variation in the material properties of spider dragline silk across species

    NASA Astrophysics Data System (ADS)

    Swanson, B. O.; Blackledge, T. A.; Beltrán, J.; Hayashi, C. Y.

    2006-02-01

    Spiders produce high performance fibers that compare favorably with the best manmade fibers in strength and toughness. The amino acid sequences of silk proteins have been determined for a number of silk types and species, revealing extensive variation. This variation in sequence is hypothesized to confer different material properties. However, the material properties of silk have been characterized from only a few ecologically similar species, even though spiders are extremely diverse. Using a Nano Bionix® tensile tester, we measured mechanical properties of one type of silk, the dragline, from a broad sample of spider species. These taxa included orb-weavers and representatives of other lineages of true spiders that do not spin aerial capture webs. We found that all of the species sampled produce high-performance dragline fibers, suggesting that the remarkable properties of dragline silk predate the origin of the aerial orb-web. However, we report significant variation in all of the material properties measured. Furthermore, material properties tend not to be correlated, implying that different properties may have been selected upon in different spider lineages. We suggest that the spectrum of dragline silk sequences and material properties that have been produced over evolutionary time provides a rich resource for the design of biomimetic silk fibers.

  13. Macroporous silk fibroin cryogels.

    PubMed

    Ak, Fatih; Oztoprak, Zeynep; Karakutuk, Ilknur; Okay, Oguz

    2013-03-11

    Silk fibroin cryogels with remarkable properties were obtained from frozen fibroin solutions (4.2-12.6%) at subzero temperatures between -5 and -22 °C. This was achieved by the addition of ethylene glycol diglycidyl ether (EGDE) into the cryogelation system. EGDE triggers the conformational transition of fibroin from random coil to ?-sheet structure and hence fibroin gelation. One of the unique features of fibroin cryogels is their elasticity that allows them to resist complete compression without any crack development, during which water inside the cryogel is removed. The compressed cryogel immediately swells during unloading to recover its original shape. The scaffolds obtained by freeze-drying of the cryogels consist of regular, interconnected pores of diameters ranging from 50 to 10 ?m that could be regulated by the synthesis parameters. The mechanical compressive strength and the modulus of the scaffolds increase with decreasing pore diameter, that is, with decreasing gelation temperature or, with increasing fibroin or EGDE concentrations in the feed. The scaffolds produced at 12.6% fibroin exhibit a very high compressive modulus (50 MPa) making them good candidates as bone scaffold materials. PMID:23360211

  14. Analytical markers for silk degradation: comparing historic silk and silk artificially aged in different environments.

    PubMed

    Vilaplana, Francisco; Nilsson, Johanna; Sommer, Dorte V P; Karlsson, Sigbritt

    2015-02-01

    Suitable analytical markers to assess the degree of degradation of historic silk textiles at molecular and macroscopic levels have been identified and compared with silk textiles aged artificially in different environments, namely (i) ultraviolet (UV) exposure, (ii) thermo-oxidation, (iii) controlled humidity and (iv) pH. The changes at the molecular level in the amino acid composition, the formation of oxidative moieties, crystallinity and molecular weight correlate well with the changes in the macroscopic properties such as brightness, pH and mechanical properties. These analytical markers are useful to understand the degradation mechanisms that silk textiles undergo under different degradation environments, involving oxidation processes, hydrolysis, chain scission and physical arrangements. Thermo-oxidation at high temperatures proves to be the accelerated ageing procedure producing silk samples that most resembled the degree of degradation of early seventeenth-century silk. These analytical markers will be valuable to support the textile conservation tasks currently being performed in museums to preserve our heritage. PMID:25492090

  15. Identification of Highly Active Genes from the Posterior Silk Gland of the Oak Silkworm, Antheraea yamamai, Through Expressed Sequence Tag (EST) Strategy

    Microsoft Academic Search

    Jae-Pil Jeon; Jae-Sam Hwang; Jeong-Hae Lee; Sang-Mong Lee; Hyun-Ah Kang; Uik Sohn

    2000-01-01

    Gene discovery from highly specialized tissues is important for understanding of development and tissue-dependent gene regulation in organism. To identify highly active genes from the posterior silk gland of the oak silkworm, Antheraea yamamai, which is specialized for the production of silk proteins, we constructed an oligo (dT) primed directional cDNA library from the posterior silk gland of the final

  16. Folate conjugated silk fibroin nanocarriers for targeted drug delivery.

    PubMed

    Subia, Bano; Chandra, Sourov; Talukdar, Sarmistha; Kundu, Subhas C

    2014-02-01

    Disease treatment processes mainly focus on the development of nontoxic, biodegradable, non-immunogenic, biocompatible materials capable of controlled and long-term release of biomolecules. In this work silk protein fibroin from non-mulberry tropical tasar silkworm, Antheraea mylitta, is used to prepare nanoparticles as a drug delivery system. Folate is a vitamin, which is brought into healthy and cancerous cells by folate receptors. The efficiency of silk fibroin-folate nanoparticles loaded with anticancer drug doxorubicin was evaluated by analysing the cell viability, proliferation and endocytosis. Consequently the effects of pro-inflammatory responses by cytokines such as TNF-?, IL-1? and nitric oxide were checked by stimulating the macrophages with folate conjugated silk fibroin nanoparticles. The fibroin-folate nanocarriers are nontoxic, easily taken up by cells and capable of sustained drug release. Nanoparticles loaded with anticancer drug doxorubicin target cancer cells. The results show that silk fibroin-folate nanoparticles may serve as promising nanocarriers for different biomedical and nanotechnology applications in cancer research. PMID:24345855

  17. Response of Human Corneal Fibroblasts on Silk Film Surface Patterns

    PubMed Central

    Gil, Eun Seok; Park, Sang Huyg; Marchant, Jeff; Omenetto, Fiorenzo; Kaplan, David L.

    2011-01-01

    Transparent, biodegradable, mechanically robust, and surface-patterned silk films were evaluated for the effect of surface morphology on human corneal fibroblast (hCF) cell proliferation, orientation, and ECM deposition and alignment. A series of dimensionally different surface groove patterns were prepared from optically graded glass substrates followed by casting poly(dimethylsiloxane) (PDMS) replica molds. The features on the patterned silk films showed an array of asymmetric triangles and displayed 37–342 nm depths and 445–3 582 nm widths. hCF DNA content on all patterned films were not significantly different from that on flat silk films after 4 d in culture. However, the depth and width of the grooves influenced cell alignment, while the depth differences affected cell orientation; overall, deeper and narrower grooves induced more hCF orientation. Over 14 d in culture, cell layers and actin filament organization demonstrated that confluent hCFs and their cytoskeletal filaments were oriented along the direction of the silk film patterned groove axis. Collagen type V and proteoglycans (decorin and biglycan), important markers of corneal stromal tissue, were highly expressed with alignment. Understanding corneal stromal fibroblast responses to surface features on a protein-based biomaterial applicable in vivo for corneal repair potential suggests options to improve corneal tissue mimics. Further, the approaches provide fundamental biomaterial designs useful for bioengineering oriented tissue layers, an endemic feature in most biological tissue structures that lead to critical tissue functions. PMID:20301120

  18. A quicker degradation rate is yielded by a novel kind of transgenic silk fibroin consisting of shortened silk fibroin heavy chains fused with matrix metalloproteinase cleavage sites.

    PubMed

    Huang, Guoping; Yang, Danfeng; Sun, Chunfeng; Huang, Jianping; Chen, Keping; Zhang, Chunxia; Chen, Huiqing; Yao, Qin

    2014-08-01

    Degradation performance of silk fibroin is an important property for its medical applications. Herein we constructed a shortened silk fibroin heavy chain protein fused with a matrix metalloproteinase cleavage site (SSFH-MMP) along with a glutathione S-transferase tag ahead. The digestion assay shows it can be cut by matrix metalloproteinase-2 (MMP-2) at its MMP cleavage site. Furthermore, we introduced the SSFH-MMP into silk fibroin by genetic modification of silkworms in order to increase the degradation rate of the silk fibroin. After acquisition of a race of transgenic silkworms with the coding sequence of the MMP cleavage site in their genomic DNA, we tested some properties of their silk fibroin designated TSF-MMP. The results show that the TSF-MMP has MMP cleavage sites and yields a quicker degradation rate during dilution in MMP-2 enzyme buffer or implantation into tumor tissues compared with that of normal silk fibroin. Moreover, the TSF-MMP is in vitro non-toxic to human bone marrow mesenchymal stem cells (hBM-MSCs) indicating that the TSF-MMP may become a biomaterial with a quicker degradation rate for its medical applications. PMID:24801061

  19. A lepidopterous cocoon evidence for silk in the

    E-print Network

    Panagiotakopulu, Eva

    A lepidopterous cocoon evidence for silk in the Age from Thera and Aegean Bronze E. PANAGIOTAKOPULU of silk?And if silk, where did the stuff, or knowledge of cultivating the silk-worms, come from):420-29 #12;A LEPIDOPTEROUS COCOON FROM THERA & EVIDENCE FOR SILK IN THE AEGEAN BRONZE AGE 421

  20. Size-dependent mechanical properties of beta-structures in protein materials

    E-print Network

    Keten, Sinan

    2010-01-01

    Protein materials such as spider silk can be exceptionally strong, and they can stretch tremendously before failure. Notably, silks are made entirely of proteins, which owe their structure and stability to weak molecular ...

  1. Ca2+ and endoplasmic reticulum Ca2+-ATPase regulate the formation of silk fibers with favorable mechanical properties.

    PubMed

    Wang, Xin; Li, Yi; Xie, Kang; Yi, Qiying; Chen, Quanmei; Wang, Xiaohuan; Shen, Hong; Xia, Qingyou; Zhao, Ping

    2015-02-01

    Calcium ions (Ca(2+)) are crucial for the conformational transition of silk fibroin in vitro, and silk fibroin conformations correlate with the mechanical properties of silk fibers. To investigate the relationship between Ca(2+) and mechanical properties of silk fibers, CaCl2 was injected into silkworms (Bombyx mori). Fourier-transform infrared spectroscopy (FTIR) analysis and mechanical testing revealed that injection of CaCl2 solution (7.5mg/g body weight) significantly increased the levels of ?-helix and random coil structures of silk proteins. In addition, extension of silk fibers increased after CaCl2 injection. In mammals, sarcoplasmic reticulum Ca(2+)-ATPase in muscle and endoplasmic reticulum Ca(2+)-ATPase in other tissues (together denoted by SERCA) are responsible for calcium balance. Therefore, we analyzed the expression pattern of silkworm SERCA (BmSERCA) in silk glands and found that BmSERCA was abundant in the anterior silk gland (ASG). After injection of thapsigargin (TG) to block SERCA activity, silkworms showed a silk-spinning deficiency and their cocoons had higher calcium content compared to that of controls. Moreover, FTIR analysis revealed that the levels of ?-helix and ?-sheet structures increased in silk fibers from TG-injected silkworms compared to controls. The results provide evidence that BmSERCA has a key function in calcium transportation in ASG that is related to maintaining a suitable ionic environment. This ionic environment with a proper Ca(2+) concentration is crucial for the formation of silk fibers with favorable mechanical performances. PMID:25602367

  2. Crystal growth of calcium carbonate in silk fibroin/sodium alginate hydrogel

    NASA Astrophysics Data System (ADS)

    Ming, Jinfa; Zuo, Baoqi

    2014-01-01

    As known, silk fibroin-like protein plays a pivotal role during the formation of calcium carbonate (CaCO3) crystals in the nacre sheets. Here, we have prepared silk fibroin/sodium alginate nanofiber hydrogels to serve as templates for calcium carbonate mineralization. In this experiment, we report an interesting finding of calcium carbonate crystal growth in the silk fibroin/sodium alginate nanofiber hydrogels by the vapor diffusion method. The experimental results indicate calcium carbonate crystals obtained from nanofiber hydrogels with different proportions of silk fibroin/sodium alginate are mixture of calcite and vaterite with unusual morphologies. Time-dependent growth study was carried out to investigate the crystallization process. It is believed that nanofiber hydrogels play an important role in the process of crystallization. This study would help in understanding the function of organic polymers in natural mineralization, and provide a novel pathway in the design and synthesis of new materials related unique morphology and structure.

  3. Silk-based nanocomplexes with tumor-homing peptides for tumor-specific gene delivery.

    PubMed

    Numata, Keiji; Mieszawska-Czajkowska, Aneta J; Kvenvold, Laura A; Kaplan, David L

    2012-01-01

    Nanoscale complexes of recombinant silk molecules containing THPs with DNA are designed as less cytotoxic and highly target-specific gene carriers. Genetically engineered silk proteins containing poly(L-lysine) domains to interact with pDNA and the THP to bind to specific tumorigenic cells for target-specific pDNA delivery are prepared, followed by in vitro transfection into MDA-MB-435 melanoma cells, highly metastatic human breast tumor MDA-MB-231?cells, and non-tumorigenic MCF-10A breast epithelial cells. The silk/poly(L-lysine) block copolymer containing Lyp1 (ML-Lyp1) shows significant differences from silk/poly(L-lysine) block copolymer containing F3 (ML-F3) in cytotoxicity to MCF10A cells. ML-F3 is the most promising candidate for target delivery into tumorigenic cells. PMID:22052706

  4. Pancreatic Islet Survival and Engraftment Is Promoted by Culture on Functionalized Spider Silk Matrices

    PubMed Central

    Johansson, Ulrika; Dekki Shalaly, Nancy; Zaitsev, Sergei V.; Berggren, Per-Olof; Hedhammar, My

    2015-01-01

    Transplantation of pancreatic islets is one approach for treatment of diabetes, however, hampered by the low availability of viable islets. Islet isolation leads to disruption of the environment surrounding the endocrine cells, which contributes to eventual cell death. The reestablishment of this environment is vital, why we herein investigated the possibility of using recombinant spider silk to support islets in vitro after isolation. The spider silk protein 4RepCT was formulated into three different formats; 2D-film, fiber mesh and 3D-foam, in order to provide a matrix that can give the islets physical support in vitro. Moreover, cell-binding motifs from laminin were incorporated into the silk protein in order to create matrices that mimic the natural cell environment. Pancreatic mouse islets were thoroughly analyzed for adherence, necrosis and function after in vitro maintenance on the silk matrices. To investigate their suitability for transplantation, we utilized an eye model which allows in vivo imaging of engraftment. Interestingly, islets that had been maintained on silk foam during in vitro culture showed improved revascularization. This coincided with the observation of preserved islet architecture with endothelial cells present after in vitro culture on silk foam. Selected matrices were further evaluated for long-term preservation of human islets. Matrices with the cell-binding motif RGD improved human islet maintenance (from 36% to 79%) with preserved islets architecture and function for over 3 months in vitro. The islets established cell-matrix contacts and formed vessel-like structures along the silk. Moreover, RGD matrices promoted formation of new, insulin-positive islet-like clusters that were connected to the original islets via endothelial cells. On silk matrices with islets from younger donors (<35 year), the amount of newly formed islet-like clusters found after 1 month in culture were almost double compared to the initial number of islets added. PMID:26090859

  5. The other prey-capture silk: Fibres made by glow-worms (Diptera: Keroplatidae) comprise cross-?-sheet crystallites in an abundant amorphous fraction.

    PubMed

    Walker, Andrew A; Weisman, Sarah; Trueman, Holly E; Merritt, David J; Sutherland, Tara D

    2015-09-01

    Glow-worms (larvae of dipteran genus Arachnocampa) are restricted to moist habitats where they capture flying prey using snares composed of highly extensible silk fibres and sticky mucus droplets. Little is known about the composition or structure of glow-worm snares, or the extent of possible convergence between glow-worm and arachnid capture silks. We characterised Arachnocampa richardsae silk and mucus using X-ray scattering, Fourier transform infrared spectroscopy and amino acid analysis. Silk but not mucus contained crystallites of the cross-?-sheet type, which occur in unrelated insect silks but have not been reported previously in fibres used for prey capture. Mucus proteins were rich in Gly (28.5%) and existed in predominantly a random coil structure, typical of many adhesive proteins. In contrast, the silk fibres were unusually rich in charged and polar residues, particularly Lys (18.1%), which we propose is related to their use in a highly hydrated state. Comparison of X-ray scattering, infrared spectroscopy and amino acid analysis data suggests that silk fibres contain a high fraction of disordered protein. We suggest that in the native hydrated state, silk fibres are capable of extension via deformation of both disordered regions and cross-?-sheet crystallites, and that high extensibility is an adaptation promoting successful prey capture. This study illustrates the rich variety of protein motifs that are available for recruitment into biopolymers, and how convergently evolved materials can nevertheless be based on fundamentally different protein structures. PMID:26006749

  6. Silk fibroin\\/chitosan–hyaluronic acid versus silk fibroin scaffolds for tissue engineering: promoting cell proliferations in vitro

    Microsoft Academic Search

    Tze-Wen Chung; Yu-Lin Chang

    2010-01-01

    The feasibility of silk fibroin protein (SF) scaffolds for tissue engineering applications to promote cell proliferation has\\u000a been demonstrated, as well as the ability to mimic natural extra-cellular matrix (ECM), SF\\/chitosan (CS), a polysaccharide,\\u000a scaffolds for tissue engineering. However, the response of cells to SF\\/CS–hyaluronic acid (SF\\/CS–HA) scaffolds has not been\\u000a examined, which this study attempts to do and then

  7. Silk fibroin membranes from solvent-crystallized silk fibroin\\/gelatin blends: Effects of blend and solvent composition

    Microsoft Academic Search

    Eun S. Gil; David J. Frankowski; Samuel M. Hudson; Richard J. Spontak

    2007-01-01

    Protein membranes have been prepared by mixing gelatin (G) with Bombyx mori silk fibroin (SF) and using aqueous methanol (MeOH) to induce SF crystallization. Amorphous blends of these polymers appear quasi-homogeneous, as discerned from visual observation, electron microscopy and Fourier-transform infrared (FTIR) spectroscopy. Upon subsequent exposure to aqueous MeOH, SF undergoes a conformational change from random-coil to ?-sheet. This transformation

  8. Translational enhancement of recombinant protein synthesis in transgenic silkworms by a 5'-untranslated region of polyhedrin gene of Bombyx mori Nucleopolyhedrovirus.

    PubMed

    Iizuka, Masashi; Tomita, Masahiro; Shimizu, Katsuhiko; Kikuchi, Yutaka; Yoshizato, Katsutoshi

    2008-06-01

    Previously, we established a method to produce recombinant proteins (r-proteins) in cocoons of germline transgenic silkworms, and showed that a step(s) in post-transcription processes was rate-limiting in obtaining a high yield of r-proteins. In this study, we examined whether the 5'-untranslated region (5'-UTR) of the polyhedrin gene (pol) of nucleopolyhedrovirus (NPV) has a translational enhancer activity in the r-protein expression by middle silk gland (MSG) cells of silkworm Bombyx mori (Bm). Sericin 1 gene (ser1) promoter-driven transformation vectors were constructed in which pol5'-UTRs of NPVs isolated from four different species, Bm, Spodoptera frugiperda, Ectropis oblique, and Malacosoma neustria, were each placed upstream of a reporter gene. Transient expression assays in MSGs showed that these pol5'-UTRs all enhanced the protein expression of reporter genes, and the pol5'-UTR of Bm NPV (pol5'-UTR/Bm) was the most effective among them. Thus, transgenic silkworms were generated, which bore the ser1 promoter-driven His-tagged secretory EGFP (sEGFP-His) gene under the control of pol5'-UTR/Bm. The synthesis of sEGFP-His proteins in MSGs of the transgenic worms was approximately 1.5-fold higher than that in those bearing null vectors. However, its mRNA expression levels were 67% of the control worms, indicating that the pol5'-UTR/Bm specifically enhanced the translational level. In conclusion, pol5'-UTR/Bm increased the yield of r-protein production in transgenic silkworms by enhancing the translational activity and this 5'-UTR could be useful for the mass production of r-proteins in germline transgenic silkworms. PMID:18640598

  9. Thromboelastometric and platelet responses to silk biomaterials

    PubMed Central

    Kundu, Banani; Schlimp, Christoph J.; Nürnberger, Sylvia; Redl, Heinz; Kundu, S. C.

    2014-01-01

    Silkworm's silk is natural biopolymer with unique properties including mechanical robustness, all aqueous base processing and ease in fabrication into different multifunctional templates. Additionally, the nonmulberry silks have cell adhesion promoting tri-peptide (RGD) sequences, which make it an immensely potential platform for regenerative medicine. The compatibility of nonmulberry silk with human blood is still elusive; thereby, restricts its further application as implants. The present study, therefore, evaluate the haematocompatibility of silk biomaterials in terms of platelet interaction after exposure to nonmulberry silk of Antheraea mylitta using thromboelastometry (ROTEM). The mulberry silk of Bombyx mori and clinically used Uni-Graft W biomaterial serve as references. Shortened clotting time, clot formation times as well as enhanced clot strength indicate the platelet mediated activation of blood coagulation cascade by tested biomaterials; which is comparable to controls. PMID:24824624

  10. Non-equilibrium silk fibroin adhesives

    Microsoft Academic Search

    Tuna Yucel; Nikola Kojic; Gary G. Leisk; Tim J. Lo; David L. Kaplan

    2010-01-01

    Regenerated silkworm silk solutions formed metastable, soft-solid-like materials (e-gels) under weak electric fields, displaying interesting mechanical characteristics such as dynamic adhesion and strain stiffening. Raman spectroscopy, in situ electric field dynamic oscillatory rheology and polarized optical microscopy indicated that silk fibroin electrogelation involved intermolecular self-assembly of silk molecules into amorphous, micron-scale, micellar structures and the formation of relatively long lifetime,

  11. Variation of mechanical properties with amino acid content in the silk of Nephila clavipes.

    PubMed

    Zax, David B; Armanios, Daniel E; Horak, Sally; Brodowski, Chris; Malowniak, Chris; Yang, Zhitong

    2004-01-01

    In this paper, we explore the impact of dietary deprivation, where spiders are provided diets missing one or more of the amino acids, on the properties of the spider dragline silk spun after one month on the diet. Cohorts of female N. clavipes spiders were selected for diets deprived of alanine (Ala) and glycine (Gly), arginine (Arg), leucine (Leu), or tyrosine (Tyr), and their silk was harvested twice weekly during the one-month course of the diet. Significant mechanical differences are observed after as little as 6 days on the diet. Utilizing conventional tensile testing methods, single fibers were strained to break so as to study the influence of diet on the stress/strain properties. Diets deprived of Ala and Gly appear to most directly impact the load-bearing foundation of dragline silk. Diets deprived of Arg, Tyr, and possibly Leu reduce the strength of the silk, and diets missing Tyr and Leu reduce the strain-to-failure. Observations obtained from ESEM photos of the fracture interfaces after tensile testing illustrate the fracture mechanics of spider silk. Both solid-state NMR and amino acid analysis of the digested protein suggest, however, that the relationship between diet and amino acid incorporation into the silk fiber is not straightforward. PMID:15132654

  12. Effect of ?-sheet crystalline content on mass transfer in silk films.

    PubMed

    Karve, Kiran A; Gil, Eun Seok; McCarthy, Stephen P; Kaplan, David L

    2011-11-01

    The material properties of silk are favorable for drug delivery due to the ability to control material structure and morphology under ambient, aqueous processing conditions. Mass transport of compounds with varying physical-chemical characteristics was studied in silk fibroin films with control of ?-sheet crystalline content. Two compounds, vitamin B12 and fluorescein isothiocynate (FITC) labeled lysozyme were studied in a diffusion apparatus to determine transport through silk films. The films exhibited size exclusion phenomenon with permeability coefficients with contrasting trends with increases in ?-sheet crystallinity. The size exclusion phenomenon observed with the two model compounds was characterized by contrasting trends in permeability coefficients of the films as a function of ?-sheet crystallinity. The diffusivity of the compounds was examined in the context of free volume theory. Apart from the ?-sheet crystallinity, size of the compound and its interactions with silk influenced mass transfer. Diffusivity of vitamin B12 was modeled to define a power law relationship with ?-sheet crystallinity. The results of the study demonstrate that diffusion of therapeutic agents though silk fibroin films can be directed to match a desired rate by modulating secondary structure of the silk proteins. PMID:22135474

  13. Unexpected behavior of irradiated spider silk links conformational freedom to mechanical performance.

    PubMed

    Perea, G Belén; Solanas, Concepción; Plaza, Gustavo R; Guinea, Gustavo V; Jorge, Inmaculada; Vázquez, Jesús; Pérez Mateos, Jorge M; Marí-Buyé, Núria; Elices, Manuel; Pérez-Rigueiro, José

    2015-06-28

    Silk fibers from Argiope trifasciata and Nephila inaurata orb-web weaving spiders were UV irradiated to modify the molecular weight of the constituent proteins. Fibers were characterized either as forcibly silked or after being subjected to maximum supercontraction. The effect of irradiation on supercontraction was also studied, both in terms of the percentage of supercontraction and the tensile properties exhibited by irradiated and subsequently supercontracted fibers. The effects of UV exposure at the molecular level were assessed by polyacrylamide gel electrophoresis and mass spectrometry. It is shown that UV-irradiated fibers show a steady decrease in their main tensile parameters, most notably, tensile strength and strain. The combination of the mechanical and biochemical data suggests that the restricted conformational freedom of the proteins after UV irradiation is critical in the reduction of these properties. Consequently, an adequate topological organization of the protein chains emerges as a critical design principle in the performance of spider silk. PMID:25994594

  14. A comparison of convergently evolved insect silks that share ?-sheet molecular structure.

    PubMed

    Church, Jeffrey S; Woodhead, Andrea L; Walker, Andrew A; Sutherland, Tara D

    2014-06-01

    Raspy crickets produce silk webs that are used to build shelters. These webs have been found to consist of both fiber and film components. Raman spectra obtained from both components were found to be very similar for a given species. The protein structure of the fibers and films produced by both species was predominately ?-sheet with lesser amounts of ?-turns, unordered and ?-helical protein also detected. The orientation of the ?-sheet backbone in the fiber was determined to be parallel to the fiber axis. Compared to cocoon and dragline silk the orientation distribution exhibits a significant randomly orientated protein component. Amino acid analysis confirmed the presence of glycine, serine, and alanine in both species, which are known to form antiparallel ?-sheet structures. Both species, although at significantly different concentrations, where found to contain proline. This amino acid is uncommon in insect silks, and likely involved in increasing fiber elasticity. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 630-639, 2014. PMID:24170682

  15. Complex gene expression in the dragline silk producing glands of the Western black widow (Latrodectus hesperus)

    PubMed Central

    2013-01-01

    Background Orb-web and cob-web weaving spiders spin dragline silk fibers that are among the strongest materials known. Draglines are primarily composed of MaSp1 and MaSp2, two spidroins (spider fibrous proteins) expressed in the major ampullate (MA) silk glands. Prior genetic studies of dragline silk have focused mostly on determining the sequence of these spidroins, leaving other genetic aspects of silk synthesis largely uncharacterized. Results Here, we used deep sequencing to profile gene expression patterns in the Western black widow, Latrodectus hesperus. We sequenced millions of 3?-anchored “tags” of cDNAs derived either from MA glands or control tissue (cephalothorax) mRNAs, then associated the tags with genes by compiling a reference database from our newly constructed normalized L. hesperus cDNA library and published L. hesperus sequences. We were able to determine transcript abundance and alternative polyadenylation of each of three loci encoding MaSp1. The ratio of MaSp1:MaSp2 transcripts varied between individuals, but on average was similar to the estimated ratio of MaSp1:MaSp2 in dragline fibers. We also identified transcription of TuSp1 in MA glands, another spidroin family member that encodes the primary component of egg-sac silk, synthesized in tubuliform glands. In addition to the spidroin paralogs, we identified 30 genes that are more abundantly represented in MA glands than cephalothoraxes and represent new candidates for involvement in spider silk synthesis. Conclusions Modulating expression rates of MaSp1 variants as well as MaSp2 and TuSp1 could lead to differences in mechanical properties of dragline fibers. Many of the newly identified candidate genes likely encode secreted proteins, suggesting they could be incorporated into dragline fibers or assist in protein processing and fiber assembly. Our results demonstrate previously unrecognized transcript complexity in spider silk glands. PMID:24295234

  16. NMR study of the structures of repeated sequences, GAGXGA (X = S, Y, V), in Bombyx mori liquid silk.

    PubMed

    Suzuki, Yu; Yamazaki, Toshimasa; Aoki, Akihiro; Shindo, Heisaburo; Asakura, Tetsuo

    2014-01-13

    The silk fibroin stored in the silk gland of the Bombyx mori silkworm, called "liquid silk", is spun out and converted into the silk fiber with extremely high strength and high toughness. Therefore it is important to determine the silk structure before spinning called Silk I at an atomic level to clarify the fiber formation mechanism. We proposed the repeated type II ?-turn structure as Silk I in the solid state with the model peptide (AG)15 and several solid state NMR techniques previously. In this paper, the solution structure of native "liquid silk" was determined with solution NMR, especially for tandem repeated sequences with (GAGXGA)n (X = S, Y, V) and GAASGA motifs in the B. mori silk fibroin. The assignment of the (13)C, (15)N, and (1)H solution NMR spectra for the repetitive sequence motifs was achieved, and the chemical shifts were obtained. The program, TALOS-N, to predict the backbone torsion angles from the chemical shifts of proteins was applied to these motifs with (13)C?, (13)C?, (13)CO, (1)H?, (1)HN, and (15)N chemical shifts. The twenty-five best matches of torsion angles (?, ?) were well populated and mainly fell into the regions for typical type II ?-turn structures in the (?, ?) map for the GAGXGA (X = S, Y, V) motifs. In contrast, (?, ?) plots for motif GAASGA were scattered, indicating that the motif is in a disordered structure. Furthermore, inter-residue HN-H? NOE cross peaks between i-th and (i+2)th residues in GAGXGA (X = S, Y, V) motifs were observed, supporting the repeated type II ?-turn structure. Thus, we could show the presence of the repeated type II ?-turn structure in "liquid silk". PMID:24266784

  17. Development and characterization of silk fibroin coated quantum dots

    NASA Astrophysics Data System (ADS)

    Nathwani, B. B.; Needham, C.; Mathur, A. B.; Meissner, K. E.

    2008-02-01

    Recent progress in the field of semiconductor nanocrystals or Quantum Dots (QDs) has seen them find wider acceptance as a tool in biomedical research labs. As produced, high quality QDs, synthesized by high temperature organometallic synthesis, are coated with a hydrophobic ligand. Therefore, they must be further processed to be soluble in water and to be made biocompatible. To accomplish this, the QDs are generally coated with a synthetic polymer (eg. block copolymers) or the hydrophobic surface ligands exchanged with hydrophilic material (eg. thiols). Advances in this area have enabled the QDs to experience a smooth transition from being simple inorganic fluorophores to being smart sensors, which can identify specific cell marker proteins and help in diagnosis of diseases such as cancer. In order to improve the biocompatibility and utility of the QDs, we report the development of a procedure to coat QDs with silk fibroin, a fibrous crystalline protein extracted from Bombyx Mori silkworm. Following the coating process, we characterize the size, quantum yield and two-photon absorption cross section of the silk coated QDs. Additionally, the results of biocompatibility studies carried out to compare the properties of these QD-silks with conventional QDs are presented. These natural polymer coatings on QDs could enhance the intracellular delivery and enable the use of these nanocrystals as an imaging tool for studying subcellular machinery at the molecular level.

  18. Construction of transgenic silkworm spinning antibacterial silk with fluorescence.

    PubMed

    Li, Zhen; Jiang, Yue; Cao, Guangli; Li, Jingzhi; Xue, Renyu; Gong, Chengliang

    2015-01-01

    A targeting vector consisting of a fusion gene of the green fluorescent protein (GFP) gene gfp and the antimicrobial peptide cecropin gene cec flanked by pieces of the 5' and 3' sequences of the fibroin L chain gene fib-L of the silkworm (Bombyx mori) and a negative selection DsRed marker gene driven by the baculovirus immediate early gene 1 (i.e.-1) promoter, was used to target the silkworm genome in order to explore the possibility of improving the performance of silk. A transgenic silkworm with a green fluorescent cocoon was obtained and PCR analysis of its genome confirmed that the target genes had been integrated into the silkworm genome correctly. Furthermore, in the posterior silk glands of the G6 generation transformation silkworm, a band representing the fusion protein Fib-L-GFP-Cec with a molecular mass of 68.7 kDa was detected by western blotting with an antibody against GFP. An investigation of the number of bacteria attached to a cocoon showed the transgenic silkworm cocoon possessed antibacterial properties. These results suggested the performance of silk can be improved by modifying the fibroin gene. PMID:25223857

  19. Isolation and characterisation of sericin antifreeze peptides and molecular dynamics modelling of their ice-binding interaction.

    PubMed

    Wu, Jinhong; Rong, Yuzhi; Wang, Zhengwu; Zhou, Yanfu; Wang, Shaoyun; Zhao, Bo

    2015-05-01

    This study aimed to isolate and characterise a novel sericin antifreeze peptide and investigate its ice-binding molecular mechanism. The thermal hysteresis activity of ice-binding sericin peptides (I-SP) was measured and their activity reached as high as 0.94 °C. A P4 fraction, with high hypothermia protective activity and inhibition activity of ice recrystallisation, was obtained from I-SP, and a purified sericin peptide, named SM-AFP, with the sequence of TTSPTNVSTT and a molecular weight of 1009.50 Da was then isolated from the P4 fraction. Treatment of Lactobacillus delbrueckii Subsp. bulgaricus LB340 LYO with 100 ?g/ml synthetic SM-AFP led to 1.4-fold increased survival (p < 0.05). Finally, an SM-AFP/ice binding model was constructed and results of molecular dynamics simulation suggested that the binding of SM-AFP with ice and prevention of ice crystal growth could be attributed to hydrogen bond formation, hydrophobic interaction and non-bond interactions. Sericin peptides could be developed into beneficial cryoprotectants and used in frozen food processing. PMID:25529728

  20. Silk Film Topography Directs Collective Epithelial Cell Migration

    PubMed Central

    Rosenblatt, Mark I.

    2012-01-01

    The following study provides new insight into how surface topography dictates directed collective epithelial cell sheet growth through the guidance of individual cell movement. Collective cell behavior of migrating human corneal limbal-epithelial cell sheets were studied on highly biocompatible flat and micro-patterned silk film surfaces. The silk film edge topography guided the migratory direction of individual cells making up the collective epithelial sheet, which resulted in a 75% increase in total culture elongation. This was due to a 3-fold decrease in cell sheet migration rate efficiency for movement perpendicular to the topography edge. Individual cell migration direction is preferred in the parallel approach to the edge topography where localization of cytoskeletal proteins to the topography’s edge region is reduced, which results in the directed growth of the collective epithelial sheet. Findings indicate customized biomaterial surfaces may be created to direct both the migration rate and direction of tissue epithelialization. PMID:23185573

  1. Secrets of the Silk Road

    NSDL National Science Digital Library

    Secrets of the Silk Road is another worthy addition to the growing body of Web-based information about the Silk Road (see, for example, The International Dunhuang Project, founded in 1998 and mentioned in several Scout Reports), launched by the Bowers Museum of Santa Ana, California and presented by University of Pennsylvania Museum of Archaeology and Anthropology. This site includes artifacts documenting the history of the Silk Road in the vast Tarim Basin in the Far Western Xinjiang Uyghur Autonomous Region of China. Three well-preserved mummies from the Tarim Basin have been brought to the United States for the exhibition, along with lavish grave goods. Visitors to the website can view images of "The Beauty of Xiaohe," a female mummy from 1800-1500 BCE and other treasures ranging from a Chrysanthemum Shaped Dessert from the 5th-3rd century BCE, to gold objects such as mask from 5th-6th century CE or the Gold Plaque with Lion Design, 5th-3rd Century BCE.

  2. Characterization of silk spun by the embiopteran, Antipaluria urichi

    Microsoft Academic Search

    Matthew A. Collin; Jessica E. Garb; Janice S. Edgerly; Cheryl Y. Hayashi

    2009-01-01

    Silks are renowned for being lightweight materials with impressive mechanical properties. Though moth and spider silks have received the most study, silk production has evolved in many other arthropods. One insect group that has been little investigated is Embioptera (webspinners). Embiopterans produce silk from unique tarsal spinning structures during all life stages. We characterize the molecular and mechanical properties of

  3. Silk fibroin aerogels: potential scaffolds for tissue engineering applications.

    PubMed

    Mallepally, Rajendar R; Marin, Michael A; Surampudi, Vasudha; Subia, Bano; Rao, Raj R; Kundu, Subhas C; McHugh, Mark A

    2015-01-01

    Silk fibroin (SF) is a natural protein, which is derived from the Bombyx mori silkworm. SF based porous materials are extensively investigated for biomedical applications, due to their biocompatibility and biodegradability. In this work, CO2 assisted acidification is used to synthesize SF hydrogels that are subsequently converted to SF aerogels. The aqueous silk fibroin concentration is used to tune the morphology and textural properties of the SF aerogels. As the aqueous fibroin concentration increases from 2 to 6?wt%, the surface area of the resultant SF aerogels increases from 260 to 308?m(2)?g(-1) and the compressive modulus of the SF aerogels increases from 19.5 to 174?kPa. To elucidate the effect of the freezing rate on the morphological and textural properties, SF cryogels are synthesized in this study. The surface area of the SF aerogels obtained from supercritical CO2 drying is approximately five times larger than the surface area of SF cryogels. SF aerogels exhibit distinct pore morphology compared to the SF cryogels. In vitro cell culture studies with human foreskin fibroblast cells demonstrate the cytocompatibility of the silk fibroin aerogel scaffolds and presence of cells within the aerogel scaffolds. The SF aerogels scaffolds created in this study with tailorable properties have potential for applications in tissue engineering. PMID:25953953

  4. Silk-Road Scholarship Program Background The Silk-Road Scholarship Program is jointly funded by the SBS Cultural Foundation

    E-print Network

    Choi, Sunghyun

    Silk-Road Scholarship Program Background The Silk-Road Scholarship Program is jointly funded fee and living expenses. Students from countries located in the area of the Silk-Road may apply. We are currently enrolling or who graduated from the eligible universities of the Silk-Road Scholarship Program

  5. Structural study of Bombyx mori silk fibroin during processing for regeneration

    NASA Astrophysics Data System (ADS)

    Ha, Sung-Won

    Bombyx mori silk fibroin has excellent mechanical properties combined with flexibility, tissue compatibility, and high oxygen permeability in the wet condition. This important material should be dissolved and regenerated to be utilized as useful forms such as gel, film, fiber, powder, or non-woven. However, it has long been a problem that the regenerated fibroin materials show poor mechanical properties and brittleness. These problems were technically solved by improving a fiber processing method reported here. The regenerated fibroin fibers showed much better mechanical properties compared to the original silk fibers. This improved technique for the fiber processing of Bombyx mori silk fibroin may be used as a model system for other semi-crystalline fiber forming proteins, becoming available through biotechnology. The physical and chemical properties of the regenerated fibers were characterized by SinTechRTM tensile testing, X-ray diffraction, solid state 13C NMR spectroscopy, and SEM. Unlike synthetic polymers, the molecular weight distribution of Bombyx mori silk fibroin is mono-disperse because silk fibroin is synthesized from DNA template. Genetic studies have revealed the entire amino acid sequence of Bombyx mori silk fibroin. It is known that the crystalline silk II structure is composed of hexa-amino acid sequences, GAGAGS. However, in the amino acid sequence of Bombyx mori silk fibroin heavy chain, there are present 11 chemically irregular but evolutionarily conserved sequences with about 31 amino acid residues (irregular GT˜GT sequences). The structure and role of these irregular sequences have remained unknown. One of the most frequently appearing irregular sequences was synthesized by a peptide synthesizer. The three-dimensional structure of this irregular silk peptide was studied by the high resolution two-dimensional NMR technique. The three-dimensional structure of this peptide shows that it makes a turn or loop structure (distorted O shape), which means the proceeding backbone direction is changed 180° by this sequence. This may facilitate the beta-sheet formation of the crystal forming building blocks, GAGAGS/GY˜GY sequences, in fibroin heavy chain. It may also facilitate the solubilization of the fibroin heavy chain within the silk gland.

  6. Selection of silk-binding peptides by phage display

    Microsoft Academic Search

    Yoko Nomura; Vandana Sharma; Asami Yamamura; Yohei Yokobayashi

    2011-01-01

    Peptides that bind to silkworm-derived silk fibroin fiber were selected from a phage-displayed random peptide library. The\\u000a selected silk-binding peptides contained a consensus sequence QSWS which is important for silk-binding as confirmed by binding\\u000a assays using phage and synthetic peptides. With further optimization, we anticipate that the silk-binding peptides will be\\u000a useful for functionalization of silk for biomaterial applications.

  7. Selection of silk-binding peptides by phage display.

    PubMed

    Nomura, Yoko; Sharma, Vandana; Yamamura, Asami; Yokobayashi, Yohei

    2011-05-01

    Peptides that bind to silkworm-derived silk fibroin fiber were selected from a phage-displayed random peptide library. The selected silk-binding peptides contained a consensus sequence QSWS which is important for silk-binding as confirmed by binding assays using phage and synthetic peptides. With further optimization, we anticipate that the silk-binding peptides will be useful for functionalization of silk for biomaterial applications. PMID:21259034

  8. Spider Silks-Biomimetics Beyond Silk Fibers: Hydrogels, films & Adhesives from Aqueous Recombinant Spider Silk dopes: A Synchrotron X-Ray Nano-Structural Study

    NASA Astrophysics Data System (ADS)

    Sampath, Sujatha; Jones, Justin; Harris, Thomas; Lewis, Randolph

    2015-03-01

    With a combination of high strength and extensibility, spider silk's (SS) mechanical properties surpass those of any man made fiber. The superior properties are due to the primary protein composition and the complex hierarchical structural organization from nanoscale to macroscopic length scales. Considerable progress has been made to synthetically mimic the production of fibers based on SS proteins. We present synchrotron x-ray micro diffraction (SyXRD) results on new fibers and gels (hydrogels, lyogels) from recombinant SS protein water-soluble dopes. Novelty in these materials is two-fold: water based rather than widely used HFIP acid synthesis, makes them safe in medical applications (replacement for tendons & ligaments). Secondly, hydrogels morphology render them as excellent carriers for targeted drug delivery biomedical applications. SyXRD results reveal semi-crystalline structure with ordered beta-sheets and relatively high degree of axial orientation in the fibers, making them the closest yet to natural spider silks. SyXRD on the gels elucidate the structural transformations during the self-recovery process through mechanical removal and addition of water. Studies correlating the observed structural changes to mechanical properties are underway.

  9. Production, structure and in vitro degradation of electrospun honeybee silk nanofibers

    PubMed Central

    Wittmer, Corinne R.; Hu, Xiao; Gauthier, Pierre-Chanel; Weisman, Sarah; Kaplan, David L.; Sutherland, Tara D.

    2012-01-01

    Honeybees produce silken cocoons containing four related fibrous proteins. High levels of each of the honeybee silk proteins can be produced recombinantly by fermentation in E. coli. In this study we have used electrospinning to fabricate a single recombinant honeybee silk protein, AmelF3, into nanofibres of around 200 nm diameter. Infrared spectroscopy found that the molecular structure of the nanofibres was predominantly coiled coil, essentially the same as native honeybee silk. Mats of the honeybee nanofibres were treated with methanol or by water annealing, which increased their ß-sheet content and rendered them water-insensitive. The insoluble mats were degraded by protease on a time scale of hours to days. The protease gradually released proteins from the solid state and these were subsequently rapidly degraded into small peptides without the accumulation of partial degradation products. Cell culture assays demonstrated that the mats allowed survival, attachment and proliferation of fibroblasts. These results indicate that honeybee silk proteins meet many prerequisites for use as a biomaterial. PMID:21689795

  10. Spider silk gut: development and characterization of a novel strong spider silk fiber.

    PubMed

    Jiang, Ping; Marí-Buyé, Núria; Madurga, Rodrigo; Arroyo-Hernández, María; Solanas, Concepción; Gañán, Alfonso; Daza, Rafael; Plaza, Gustavo R; Guinea, Gustavo V; Elices, Manuel; Cenis, José Luis; Pérez-Rigueiro, José

    2014-01-01

    Spider silk fibers were produced through an alternative processing route that differs widely from natural spinning. The process follows a procedure traditionally used to obtain fibers directly from the glands of silkworms and requires exposure to an acid environment and subsequent stretching. The microstructure and mechanical behavior of the so-called spider silk gut fibers can be tailored to concur with those observed in naturally spun spider silk, except for effects related with the much larger cross-sectional area of the former. In particular spider silk gut has a proper ground state to which the material can revert independently from its previous loading history by supercontraction. A larger cross-sectional area implies that spider silk gut outperforms the natural material in terms of the loads that the fiber can sustain. This property suggests that it could substitute conventional spider silk fibers in some intended uses, such as sutures and scaffolds in tissue engineering. PMID:25475975

  11. Surface modification of silk fibroin fabric using layer-by-layer polyelectrolyte deposition and heparin immobilization for small-diameter vascular prostheses.

    PubMed

    Elahi, M Fazley; Guan, Guoping; Wang, Lu; Zhao, Xinzhe; Wang, Fujun; King, Martin W

    2015-03-01

    There is an urgent need to develop a biologically active implantable small-diameter vascular prosthesis with long-term patency. Silk-fibroin-based small-diameter vascular prosthesis is a promising candidate having higher patency rate; however, the surface modification is indeed required to improve its further hemocompatibility. In this study, silk fibroin fabric was modified by a two-stage process. First, the surface of silk fibroin fabric was coated using a layer-by-layer polyelectrolyte deposition technique by stepwise dipping the silk fibroin fabric into a solution of cationic poly(allylamine hydrochloride) (PAH) and anionic poly(acrylic acid) (PAA) solution. The dipping procedure was repeated to obtain the PAH/PAA multilayers deposited on the silk fibroin fabrics. Second, the polyelectrolyte-deposited silk fibroin fabrics were treated in EDC/NHS-activated low-molecular-weight heparin (LMWH) solution at 4 °C for 24 h, resulting in immobilization of LMWH on the silk fibroin fabrics surface. Scanning electron microscopy, atomic force microscopy, and energy-dispersive X-ray data revealed the accomplishment of LMWH immobilization on the polyelectrolyte-deposited silk fibroin fabric surface. The higher the number of PAH/PAA coating layers on the silk fibroin fabric, the more surface hydrophilicity could be obtained, resulting in a higher fetal bovine serum protein and platelets adhesion resistance properties when tested in vitro. In addition, compared with untreated sample, the surface-modified silk fibroin fabrics showed negligible loss of bursting strength and thus reveal the acceptability of polyelectrolytes deposition and heparin immobilization approach for silk-fibroin-based small-diameter vascular prostheses modification. PMID:25671295

  12. Preparation of Conducting Fibres from Cellulose and Silk by Polypyrrole Coating

    Microsoft Academic Search

    S. Hossein Hosseini; Ali Pairovi

    cellulose fibre (e.g., cotton) and a protein fibre (e.g., silk), after under going pre- treatment and activating stages, were coated by conductive polymers such as pyr- role by two methods: Vapour and liquid phases. The value of penetration of con- ductive polymer into fibres by scanning electron microscope (SEM) was determined. Then assessments were made on physical properties and optimization

  13. Inducing ?-sheets formation in synthetic spider silk fibers by aqueous post-spin stretching.

    PubMed

    An, Bo; Hinman, Michael B; Holland, Gregory P; Yarger, Jeffery L; Lewis, Randolph V

    2011-06-13

    As a promising biomaterial with numerous potential applications, various types of synthetic spider silk fibers have been produced and studied in an effort to produce man-made fibers with mechanical and physical properties comparable to those of native spider silk. In this study, two recombinant proteins based on Nephila clavipes Major ampullate Spidroin 1 (MaSp1) consensus repeat sequence were expressed and spun into fibers. Mechanical test results showed that fiber spun from the higher molecular weight protein had better overall mechanical properties (70 KD versus 46 KD), whereas postspin stretch treatment in water helped increase fiber tensile strength significantly. Carbon-13 solid-state NMR studies of those fibers further revealed that the postspin stretch in water promoted protein molecule rearrangement and the formation of ?-sheets in the polyalanine region of the silk. The rearrangement correlated with improved fiber mechanical properties and indicated that postspin stretch is key to helping the spider silk proteins in the fiber form correct secondary structures, leading to better quality fibers. PMID:21574576

  14. The conserved C-termini contribute to the properties of spider silk fibroins

    Microsoft Academic Search

    Alexander Sponner; Wolfram Vater; Winfried Rommerskirch; Fritz Vollrath; Eberhard Unger; Frank Grosse; Klaus Weisshart

    2005-01-01

    Spider silk fibroins can adopt different structural states at high protein concentrations. They are soluble within the spinning dope of the glands, but readily converted into insoluble polymers upon extrusion. A contribution of the C-termini to the maintenance and conversion of these states is suggested by their predicted secondary structures and biochemical behavior in vitro. Special sequence parts endow the

  15. Inducing ?-Sheets Formation in Synthetic Spider Silk Fibers by Aqueous Post-Spin Stretching

    PubMed Central

    Hinman, Michael B.; Holland, Gregory P.; Yarger, Jeffery L.; Lewis, Randolph V.

    2012-01-01

    As a promising biomaterial with numerous potential applications, various types of synthetic spider silk fibers have been produced and studied in an effort to produce manmade fibers with mechanical and physical properties comparable to those of native spider silk. In this study, two recombinant proteins based on Nephila clavipes Major ampullate Spidroin 1 (MaSp1) consensus repeat sequence were expressed and spun into fibers. Mechanical test results showed that fiber spun from the higher molecular weight protein had better overall mechanical properties (70 KD versus 46 KD), whereas postspin stretch treatment in water helped increase fiber tensile strength significantly. Carbon-13 solid-state NMR studies of those fibers further revealed that the postspin stretch in water promoted protein molecule rearrangement and the formation of ?-sheets in the polyalanine region of the silk. The rearrangement correlated with improved fiber mechanical properties and indicated that postspin stretch is key to helping the spider silk proteins in the fiber form correct secondary structures, leading to better quality fibers. PMID:21574576

  16. In vitro chondrogenic differentiation of human adipose-derived stem cells with silk scaffolds

    PubMed Central

    Kim, Hyeon Joo; Park, Sang-Hyug; Durham, Jennah; Gimble, Jeffrey M; Kaplan, David L

    2012-01-01

    Human adipose-derived stem cells have shown chondrogenic differentiation potential in cartilage tissue engineering in combination with natural and synthetic biomaterials. In the present study, we hypothesized that porous aqueous-derived silk protein scaffolds would be suitable for chondrogenic differentiation of human adipose-derived stem cells. Human adipose-derived stem cells were cultured up to 6 weeks, and cell proliferation and chondrogenic differentiation were investigated and compared with those in conventional micromass culture. Cell proliferation, glycosaminoglycan, and collagen levels in aqueous-derived silk scaffolds were significantly higher than in micromass culture. Transcript levels of SOX9 and type II collagen were also upregulated in the cell–silk constructs at 6 weeks. Histological examination revealed that the pores of the silk scaffolds were filled with cells uniformly distributed. In addition, chondrocyte-specific lacunae formation was evident and distributed in the both groups. The results suggest the biodegradable and biocompatible three-dimensional aqueous-derived silk scaffolds provided an improved environment for chondrogenic differentiation compared to micromass culture. PMID:23316274

  17. Self-assembly of nucleic acids, silk and hybrid materials thereof

    NASA Astrophysics Data System (ADS)

    Humenik, Martin; Scheibel, Thomas

    2014-12-01

    Top-down approaches based on etching techniques have almost reached their limits in terms of dimension. Therefore, novel assembly strategies and types of nanomaterials are required to allow technological advances. Self-assembly processes independent of external energy sources and unlimited in dimensional scaling have become a very promising approach. Here, we highlight recent developments in self-assembled DNA-polymer, silk-polymer and silk-DNA hybrids as promising materials with biotic and abiotic moieties for constructing complex hierarchical materials in ‘bottom-up’ approaches. DNA block copolymers assemble into nanostructures typically exposing a DNA corona which allows functionalization, labeling and higher levels of organization due to its specific addressable recognition properties. In contrast, self-assembly of natural silk proteins as well as their recombinant variants yields mechanically stable ?-sheet rich nanostructures. The combination of silk with abiotic polymers gains hybrid materials with new functionalities. Together, the precision of DNA hybridization and robustness of silk fibrillar structures combine in novel conjugates enable processing of higher-order structures with nanoscale architecture and programmable functions.

  18. Processing and characterisation of a novel electropolymerized silk fibroin hydrogel membrane

    PubMed Central

    Wang, Hai-Yan; Zhang, Yu-Qing

    2014-01-01

    Silk fibroin can be made into various forms of biocompatible medical materials, including hydrogel due to its excellent properties. Here, we report a novel method for the preparation of electropolymerized silk fibroin hydrogel membrane (ESFHM), which is formed on a nanoporous film as a barrier using a homemade device at a higher DC voltage. Regenerated silk fibroin solution in Tris buffer (pH 6.55–7.55) was added into a reservoir with a negative charge, and the silk molecules migrated toward the positive charge at 80VDC, resulting in the formation of the ESFHM on the barrier film. Barrier film with a MWCO of 10?kDa is favourable to the formation of the ESFHM. Semi-transparent ESFHM with a swelling ratio of 1056.4% predominantly consisted of a mixture of ?-sheets and ?-helix crystalline structures. SEM studies revealed that the ESFHM consisted of a 3D mesh structure woven by a chain of silk fibroin nanoparticles with a size of approximately 30 nanometres, similar to a pearl necklace. In vitro studies indicated that the ESFHM was degradable and was sufficient for cell adhesion and growth. Thus, ESFHM is a promising candidate for loading bioactive protein and appropriate cells, as artificial skin or for use in transplantation. PMID:25154713

  19. Processing and characterisation of a novel electropolymerized silk fibroin hydrogel membrane.

    PubMed

    Wang, Hai-Yan; Zhang, Yu-Qing

    2014-01-01

    Silk fibroin can be made into various forms of biocompatible medical materials, including hydrogel due to its excellent properties. Here, we report a novel method for the preparation of electropolymerized silk fibroin hydrogel membrane (ESFHM), which is formed on a nanoporous film as a barrier using a homemade device at a higher DC voltage. Regenerated silk fibroin solution in Tris buffer (pH 6.55-7.55) was added into a reservoir with a negative charge, and the silk molecules migrated toward the positive charge at 80VDC, resulting in the formation of the ESFHM on the barrier film. Barrier film with a MWCO of 10?kDa is favourable to the formation of the ESFHM. Semi-transparent ESFHM with a swelling ratio of 1056.4% predominantly consisted of a mixture of ?-sheets and ?-helix crystalline structures. SEM studies revealed that the ESFHM consisted of a 3D mesh structure woven by a chain of silk fibroin nanoparticles with a size of approximately 30 nanometres, similar to a pearl necklace. In vitro studies indicated that the ESFHM was degradable and was sufficient for cell adhesion and growth. Thus, ESFHM is a promising candidate for loading bioactive protein and appropriate cells, as artificial skin or for use in transplantation. PMID:25154713

  20. Preliminary results of the application of a silk fibroin scaffold to otology.

    PubMed

    Levin, Brett; Redmond, Sharon Leanne; Rajkhowa, Rangam; Eikelboom, Robert Henry; Marano, Robert Jeffery; Atlas, Marcus David

    2010-03-01

    The surgical treatment to repair chronic tympanic membrane perforations is myringoplasty. Although multiple autologous grafts, allografts, and synthetic graft materials have been used over the years, no single graft material is superior for repairing all perforation types. Recently, the remarkable properties of silk fibroin protein have been studied, with biomedical and tissue engineering applications in mind, across a number of medical and surgical disciplines. The present study examines the use of silk fibroin for its potential suitability as an alternative graft in myringoplasty surgery by investigating the growth and proliferation of human tympanic membrane keratinocytes on a silk fibroin scaffold in vitro. Light microscopy, immunofluorescent staining, and confocal imaging all reveal promising preliminary results. The biocompatibility, transparency, stability, high tensile strength, and biodegradability of fibroin make this biomaterial an attractive option to study for this utility. PMID:20176279