These are representative sample records from Science.gov related to your search topic.
For comprehensive and current results, perform a real-time search at Science.gov.
1

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

PubMed

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

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

2013-04-01

2

Optimization of the silk scaffold sericin removal process for retention of silk fibroin protein structure and mechanical properties.  

PubMed

In the process of removing sericin (degumming) from a raw silk scaffold, the fibroin structural integrity is often challenged, leading to mechanical depreciation. This study aims to identify the factors and conditions contributing to fibroin degradation during alkaline degumming and to perform an optimization study of the parameters involved to achieve preservation of fibroin structure and properties. The methodology involves degumming knitted silk scaffolds for various durations (5-90 min) and temperatures (60-100 degrees C). Mechanical agitation and use of the refreshed solution during degumming are included to investigate how these factors contribute to degumming efficiency and fibroin preservation. Characterizations of silk fibroin morphology, mechanical properties and protein components are determined by scanning electron microscopy (SEM), single fiber tensile tests and gel electrophoresis (SDS-PAGE), respectively. Sericin removal is ascertained via SEM imaging and a protein fractionation method involving SDS-PAGE. The results show that fibroin fibrillation, leading to reduced mechanical integrity, is mainly caused by prolonged degumming duration. Through a series of optimization, knitted scaffolds are observed to be optimally degummed and experience negligible mechanical and structural degradation when subjected to alkaline degumming with mechanical agitation for 30 min at 100 degrees C. PMID:20460689

Teh, Thomas K H; Toh, Siew-Lok; Goh, James C H

2010-05-11

3

Silk sericin-insulin bioconjugates: synthesis, characterization and biological activity.  

PubMed

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 properties of the silk sericin-insulin (SS-Ins) conjugates were determined by Enzyme-Linked Immunosorbent Assay (ELISA). The biological activities of SS-Ins bioconjugates were investigated in vitro and in vivo. The results in human serum in vitro indicated that the half-life of the synthesized SS-Ins derivatives was 2.3 and 2.7 times more than that of bovine serum albumin-insulin (BSA-Ins) conjugates and intact insulin, respectively. The pharmacological activity of SS-Ins bioconjugates lengthened to 21 h in mice in vivo, which was over 4 times longer than that of the native insulin. The immunogenicity of silk sericin and the antigenicity of SS-Ins derivatives were not observed in both rabbits and mice. The bioconjugation of insulin with silk sericin protein evidently improved both physicochemical and biological stability of the polypeptide. PMID:17034892

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

2006-10-27

4

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

PubMed Central

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

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

2014-01-01

5

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

NASA Astrophysics Data System (ADS)

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.

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

2014-11-01

6

Molecular orientation behavior of silk sericin film as revealed by ATR infrared spectroscopy.  

PubMed

This paper reports the structure-dependent molecular orientation behavior of sericin, an adhesive silk protein secreted by silkworm, Bombyx mori. Although application of sericin as a biomaterial is anticipated because of its unique characteristics, sericin's physicochemical properties remain unclear, mainly because of its vulnerability to heat or alkaline treatment during separation from fibroin threads. This study employed intact sericin obtained from fibroin-deficient mutant silkworm to investigate the relationship between molecular orientation and the secondary structure of sericin. Sericin films were artificially stretched after moistening with aqueous ethanol of various concentrations. The resulting molecular orientation was analyzed using polarized infrared spectroscopy. These analyses indicated that formation of aggregated strands among extended sericin chains induced by ethanol treatment is the key to generating molecular orientation. Strong intermolecular hydrogen bonds are inferred to allow aggregated strands' stretching-force transmission, thereby causing molecular orientation. PMID:16004444

Teramoto, Hidetoshi; Miyazawa, Mitsuhiro

2005-01-01

7

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

PubMed

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

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

2013-09-01

8

Production of silk sericin/silk fibroin blend nanofibers  

PubMed Central

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

2011-01-01

9

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

PubMed

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

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

2014-08-27

10

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

PubMed

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

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

2014-04-14

11

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

Microsoft Academic Search

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)

Biman B. Mandal; S. C. Kundu

2009-01-01

12

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

PubMed

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

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

2014-01-01

13

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

PubMed

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

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

2014-05-01

14

Structure and expression of the silk adhesive protein Ser2 in Bombyx mori Barbara Kludkiewicz a,b  

E-print Network

Structure and expression of the silk adhesive protein Ser2 in Bombyx mori Barbara Kludkiewicz a 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

Â?urovec, Michal

15

The characteristics of bacterial nanocellulose gel releasing silk sericin for facial treatment.  

PubMed

BackgroundRecently, naturally derived facial masks with beneficial biological properties have received increasing interest. In this study, silk sericin-releasing bacterial nanocellulose gel was developed to be applied as a bioactive mask for facial treatment.ResultsThe silk sericin-releasing bacterial nanocellulose gel produced at a pH of 4.5 had an ultrafine and extremely pure fiber network structure. The mechanical properties and moisture absorption ability of the gel were improved, compared to those of the commercially available paper mask. Silk sericin could be control-released from the gel. A peel test with porcine skin showed that the gel was less adhesive than the commercially available paper mask, which would be removed from the face more easily without pain. The in vitro cytotoxicity test showed that the gel was not toxic to L929 mouse fibroblast and HaCaT human keratinocyte cells. Furthermore, when implanted subcutaneously and evaluated according to ISO10993-6 standard, the gel was not irritant to tissue.ConclusionThe silk sericin-releasing bacterial nanocellulose gel had appropriate physical and biological properties and safety for the facial treatment application. PMID:25487808

Aramwit, Pornanong; Bang, Nipaporn

2014-12-01

16

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

NASA Astrophysics Data System (ADS)

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.

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

2009-09-01

17

Effect of Silk Protein Processing on Drug Delivery from Silk Films  

PubMed Central

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 silk film by protease. Model compounds spanning a range of physical-chemical properties generally showed 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

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

2013-01-01

18

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

E-print Network

Identification of a Novel Type of Silk Protein and Regulation of Its Expression* (Received The silk of lepidopteran insects has been studied ex- tensively as proteins of two categories: the fibroins, which are produced in the posterior section of silk glands, and the sericins, which are secreted

Â?urovec, Michal

19

Isolation of the smallest component of silk protein.  

PubMed Central

Silk proteins were solubilized from cocoons with ethylenediamine/cupric hydroxide solution. A series of polymers of the smallest component, detected by polyacrylamide-gel electrophoresis, could be converted into the smallest component by reduction and aminoethylation. Fibroin and sericin fractions were separated by precipitation of sericin at pH 5.5. On gel electrophoresis, sericin showed distinct bands but fibroin did not. The components of fibroin and sericin were fractionated by gel filtration on Sepharose 6B. The smallest component in the sericin fraction was purified by rechromatography and showed a single band on gel electrophoresis. Its mol. wt. was 24 000, and its amino acid composition was determined. Images Fig. 1. Fig. 2. Fig. 4. PMID:7396855

Tokutake, S

1980-01-01

20

Biomedical Applications of Mulberry Silk and its Proteins: A Review  

NASA Astrophysics Data System (ADS)

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.

Nivedita, S.; Sivaprasad, V.

2014-04-01

21

Bioengineered silk proteins to control cell and tissue functions.  

PubMed

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

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

2013-01-01

22

www.sciencejournal.in COMPARATIVE SILK PROTEIN EXPRESSION OF DIFFERENT HYBRID VARIETIES OF  

E-print Network

The present study was undertaken to evaluate production of silk proteins in Kashmiri hybrid varieties of Bombyx mori L. SKUA-R-1×SK28 and SK28 × SK30 against commercially available NB4D2 × SH6 and Mysore evolved CSR2 × CSR4 hybrids. In all the four hybrids, it was observed that the total silk proteins, fibroin and sericin concentrations were more in middle silk gland than in posterior silk gland. This is due to the fact that silk proteins are secreted in the posterior silk gland but stored in the middle silk gland till spinning. The protein concentration in the middle silk gland was highest in Mysore evolved Bombyx mori hybrids CSR2 × CSR4 and commercially used NB4D2 × SH6 followed by Kashmir evolved hybrids SKUA-R-1 × SK28 and SK28 × SK30. The fibroin concentration was observed more in the cocoons of CSR2 × CSR4 and NB4D2 × SH6 followed by SKUA-R-1 × SK28 and SK28 × SK30. The percentage of fibroin was more in CSR2 × CSR4 followed by SK28 × SK30, SKUA-R-1 × SK28 and NB4D2 × SH6. The sericin concentration was more in NB4D2 × SH6 as compared to other three hybrids. The molecular weight of fibroin and sericin proteins from the silk glands and the cocoons of these four Bombyx mori hybrids were same. The heavy chain fibroin proteins were 350 kDa and the light chain were 25 kDa. The sericin proteins were 66 kDa. KEY WORDS: Bombyx mori, cocoons, sericin, fibrion, silk glands

unknown authors

23

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

PubMed

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

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

2013-11-01

24

Production and properties of electrosprayed sericin nanopowder  

NASA Astrophysics Data System (ADS)

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.

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

2012-06-01

25

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

PubMed Central

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

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

2013-01-01

26

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

NASA Astrophysics Data System (ADS)

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

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

2006-02-01

27

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

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

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

2014-12-01

28

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

PubMed Central

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

2014-01-01

29

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

PubMed

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

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

2014-01-01

30

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

PubMed

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

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

2012-03-01

31

Silk-based biomaterials  

Microsoft Academic Search

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

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

2003-01-01

32

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

PubMed

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

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

2014-05-01

33

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

PubMed Central

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

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

2014-01-01

34

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

PubMed

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

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

2015-03-01

35

Stem cell-based tissue engineering with silk biomaterials  

Microsoft Academic Search

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

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

2006-01-01

36

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

PubMed

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

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

37

Assembly mechanism of recombinant spider silk proteins  

E-print Network

Assembly mechanism of recombinant spider silk proteins S. Rammensee*, U. Slotta , T. Scheibel October 5, 2007) Spider silk threads are formed by the irreversible aggregation of silk proteins engineered and recombinantly produced spider dragline silk proteins eADF3 (engineered Araneus diadematus

Bausch, Andreas

38

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

E-print Network

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

Â?urovec, Michal

39

Recombinant DNA production of spider silk proteins  

PubMed Central

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

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

2013-01-01

40

Recombinant DNA production of spider silk proteins.  

PubMed

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

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

2013-11-01

41

Effect of the silk protein sericin on cryopreserved rat islets  

Microsoft Academic Search

Background\\/purpose  Cryopreservation is necessary for the long-term storage of islet cells and to increase the practicality of clinical islet\\u000a transplantation. Fetal bovine serum (FBS) supplemented with 10% dimethyl sulfoxide (DMSO) is generally used as a freezing\\u000a medium for islet cells. However, FBS should ideally be avoided in cell culture and transplantation because of recent animal\\u000a health problems, such as bovine spongiform

Kenji Ohnishi; Makoto Murakami; Mitsuhiro Morikawa; Akio Yamaguchi

42

Micro-scale Processing of Silk Protein  

E-print Network

fiber based applications for silk protein such as microspheres for drug delivery and the molding of nano- andnano- to micro-size orifices can now easily be purchased, and have been used to extrude silk fibers (

Breslauer, David Nate

2010-01-01

43

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

PubMed

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

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

2015-01-01

44

Thermal crystallization mechanism of silk fibroin protein  

NASA Astrophysics Data System (ADS)

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.

Hu, Xiao

45

Assembly mechanism of recombinant spider silk proteins  

PubMed Central

Spider silk threads are formed by the irreversible aggregation of silk proteins in a spinning duct with dimensions of only a few micrometers. Here, we present a microfluidic device in which engineered and recombinantly produced spider dragline silk proteins eADF3 (engineered Araneus diadematus fibroin) and eADF4 are assembled into fibers. Our approach allows the direct observation and identification of the essential parameters of dragline silk assembly. Changes in ionic conditions and pH result in aggregation of the two proteins. Assembly of eADF3 fibers was induced only in the presence of an elongational flow component. Strikingly, eADF4 formed fibers only in combination with eADF3. On the basis of these results, we propose a model for dragline silk aggregation and early steps of fiber assembly in the microscopic regime. PMID:18445655

Rammensee, S.; Slotta, U.; Scheibel, T.; Bausch, A. R.

2008-01-01

46

Designing silk-silk protein alloy materials for biomedical applications.  

PubMed

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

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

2014-01-01

47

Effect of Processing on Silk-Based Biomaterials: Reproducibility and Biocompatibility  

PubMed Central

Silk fibroin has been successfully used as a biomaterial for tissue regeneration. In order to prepare silk fibroin biomaterials for human implantation a series of processing steps are required to purify the protein. Degumming to remove inflammatory sericin is a crucial step related to biocompatibility and variability in the material. Detailed characterization of silk fibroin degumming is reported. The degumming conditions significantly affected cell viability on the silk fibroin material and the ability to form three-dimensional porous scaffolds from the silk fibroin, but did not affect macrophage activation or ?-sheet content in the materials formed. Methods are also provided to determine the content of residual sericin in silk fibroin solutions and to assess changes in silk fibroin molecular weight. Amino acid composition analysis was used to detect sericin residuals in silk solutions with a detection limit between 1.0% and 10% wt/wt, while fluorescence spectroscopy was used to reproducibly distinguish between silk samples with different molecular weights. Both methods are simple and require minimal sample volume, providing useful quality control tools for silk fibroin preparation processes. PMID:21695778

Wray, Lindsay S.; Hu, Xiao; Gallego, Jabier; Georgakoudi, Irene; Omenetto, Fiorenzo G.; Schmidt, Daniel; Kaplan, David L.

2012-01-01

48

Protein-protein nanoimprinting of silk fibroin films.  

PubMed

Protein-protein imprinting of silk fibroin is introduced as a rapid, high-throughput method for the fabrication of nanoscale structures in silk films, through the application of heat and pressure. Imprinting on conformal surfaces is demonstrated with minor adjustments to the system, at resolutions comparable to other currently available nonplanar nanoimprint lithography techniques. PMID:23483712

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

2013-05-01

49

Structure of a Protein Superfiber: Spider Dragline Silk  

Microsoft Academic Search

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

Ming Xu; Randolph V. Lewis

1990-01-01

50

A Materiomics Approach to Spider Silk: Protein Molecules to Webs  

E-print Network

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

Buehler, Markus J.

51

Silk protein aggregation kinetics revealed by Rheo-IR.  

PubMed

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

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

2014-02-01

52

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

PubMed Central

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

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

2014-01-01

53

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

PubMed Central

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

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

2014-01-01

54

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

PubMed

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

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

2014-01-01

55

Variation in Protein Intake Induces Variation in Spider Silk Expression  

PubMed Central

Background It is energetically expensive to synthesize certain amino acids. The proteins (spidroins) of spider major ampullate (MA) silk, MaSp1 and MaSp2, differ in amino acid composition. Glutamine and proline are prevalent in MaSp2 and are expensive to synthesize. Since most orb web spiders express high proline silk they might preferentially attain the amino acids needed for silk from food and shift toward expressing more MaSp1 in their MA silk when starved. Methodology/Principal Findings We fed three spiders; Argiope aetherea, Cyrtophora moluccensis and Leucauge blanda, high protein, low protein or no protein solutions. A. aetherea and L. blanda MA silks are high in proline, while C. moluccesnsis MA silks are low in proline. After 10 days of feeding we determined the amino acid compositions and mechanical properties of each species' MA silk and compared them between species and treatments with pre-treatment samples, accounting for ancestry. We found that the proline and glutamine of A. aetherea and L. blanda silks were affected by protein intake; significantly decreasing under the low and no protein intake treatments. Glutmaine composition in C. moluccensis silk was likewise affected by protein intake. However, the composition of proline in their MA silk was not significantly affected by protein intake. Conclusions Our results suggest that protein limitation induces a shift toward different silk proteins with lower glutamine and/or proline content. Contradictions to the MaSp model lie in the findings that C. moluccensis MA silks did not experience a significant reduction in proline and A. aetherea did not experience a significant reduction in serine on low/no protein. The mechanical properties of the silks could not be explained by a MaSp1 expressional shift. Factors other than MaSp expression, such as the expression of spidroin-like orthologues, may impact on silk amino acid composition and spinning and glandular processes may impact mechanics. PMID:22363691

Blamires, Sean J.; Wu, Chun-Lin; Tso, I-Min

2012-01-01

56

Silks produced by insect labial glands  

PubMed Central

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

Sutherland, Tara

2008-01-01

57

Recombinant production of spider silk proteins.  

PubMed

Natural spider silk fibers combine extraordinary properties such as stability and flexibility which results in a toughness superseding that of all other fiber materials. As the spider's aggressive territorial behavior renders their farming not feasible, the biotechnological production of spider silk proteins (spidroins) is essential in order to investigate and employ them for applications. In order to accomplish this task, two approaches have been tested: firstly, the expression of partial cDNAs, and secondly, the expression of synthetic genes in several host organisms, including bacteria, yeast, plants, insect cells, mammalian cells, and transgenic animals. The experienced problems include genetic instability, limitations of the translational and transcriptional machinery, and low solubility of the produced proteins. Here, an overview of attempts to recombinantly produce spidroins will be given, and advantages and disadvantages of the different approaches and host organisms will be discussed. PMID:23415154

Heidebrecht, Aniela; Scheibel, Thomas

2013-01-01

58

IDENTIFICATION, CHARACTERIZATION AND ANTI-FUNGAL ACTVITIES OF SILK PROTEINS IN ASPERGILLUS FLAVUS  

E-print Network

IDENTIFICATION, CHARACTERIZATION AND ANTI-FUNGAL ACTVITIES OF SILK PROTEINS IN ASPERGILLUS FLAVUS 2006 #12;IDENTIFICATION, CHARACTERIZATION AND ANTI-FUNGAL ACTVITIES OF SILK PROTEINS IN ASPERGILLUS: IDENTIFICATION, CHARACTERIZATION AND ANTIFUNGAL ACTIVITIES OF SILK PROTEINS IN ASPERGILLUS FLAVUS RESISTANT

Ray, David

59

Production And Characterization Of Synthetic Spider Silks Based On Nephila Clavipes Major Ampullate Silk Proteins  

NASA Astrophysics Data System (ADS)

The extraordinary mechanical properties of orb-weaving spider silks have served spiders for over 400 million years. However, only in the late 20th century did we start to understand the molecular nature of spider silk that contributes to its incredible properties as biomaterials. Among all seven types of spider silks, major ampullate silk from typical orb-weaving spiders is the toughest of all, it consists of primarily two proteins: MaSp1 and MaSp2. Variable ratios and conserved motifs of these two proteins in all the native spider silks demonstrate the significant role of MaSp1 and MaSp2 in controlling the mechanical properties of the fiber. The amino acid sequences of the orb weaving spider silk proteins have remained almost unchanged for more than 100 million years. Interestingly, MaSp1 and MaSp2 are the only two components in all studied dragline silk fibers from these spiders. The mechanical properties of native dragline silk vary slightly between species, which are believed to relate to the ratio of MaSp1 to MaSp2 in the silk. Both of these facts clearly indicate the importance of these two proteins to the mechanical properties of the fiber. Various types of synthetic spider silk fibers have been produced and studied in an effort to mass-produce man-made fibers with qualities comparable to native spider silk. To investigate the roles of MaSp1 and MaSp2 in silk fiber, synthetic MaSp1 (major abundant protein in Nephila clavipes major ampullate silks) only fibers, MaSp1/MaSp2 protein mixture fibers and chimeric protein fibers with both MaSp1 and MaSp2 sequence features have been produced and tested for mechanical properties. Solid-State Nuclear Magnetic Resonance was used to characterize the structure of silk fibers and reveal the relation between fiber spatial structure and mechanical properties.

An, Bo

60

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

PubMed Central

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

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

2013-01-01

61

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

PubMed Central

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

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

2009-01-01

62

Bioengineered chimeric spider silk-uranium binding proteins.  

PubMed

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

Krishnaji, Sreevidhya Tarakkad; Kaplan, David L

2013-02-01

63

Bioengineered Chimeric Spider Silk-Uranium Binding Proteins  

PubMed Central

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

Krishnaji, Sreevidhya Tarakkad; Kaplan, David L.

2014-01-01

64

Engineered spider silk protein-based composites for drug delivery.  

PubMed

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

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

2013-10-01

65

Coatings and films made of silk proteins.  

PubMed

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

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

2014-09-24

66

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

PubMed Central

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

Scheibel, Thomas

2004-01-01

67

Recent advances in production of recombinant spider silk proteins.  

PubMed

Spider silk has been drawing much attention as a great biomaterial having many applications in biotechnology and biomedicine owing to its several desired material characteristics such as outstanding strength, toughness, and elasticity as well as biodegradability and biocompatibility. With various applications foreseeable in industry, there has been much effort to produce recombinant spider silk protein in large amounts. However, owing to the difficulties in its production using spiders, alternative host systems and engineering methods have been investigated to develop suitable production systems that can efficiently produce spider silk protein. Here, we review recent advances in production of spider silk proteins in various heterologous host systems with focus given on the development of metabolic and cellular engineering strategies. PMID:22521455

Chung, Hannah; Kim, Tae Yong; Lee, Sang Yup

2012-12-01

68

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

NASA Astrophysics Data System (ADS)

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.

Tian, M.; Lewis, R. V.

2006-02-01

69

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

Microsoft Academic Search

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

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

2009-01-01

70

Solution behavior of synthetic silk peptides and modified recombinant silk proteins  

NASA Astrophysics Data System (ADS)

Spider dragline silk from Nephila clavipes possesses impressive mechanical properties derived in part from repetitive primary sequence containing polyalanine regions that self-assemble into crystalline ?-sheets. In the present study, we have sought to understand more details of redox responses related to conformational transitions of modified silk peptides and a recombinant protein containing encoded methionine triggers. Regardless of the position of the methionine trigger relative to the polyalanine domain, chemical oxidation was rapid and slight increases in the ?-helical structure and decreases in the ?-sheet and random coil content were observed by CD and FTIR in the assembled silk-like peptides and the recombinant protein. CD results indicated that the decrease in ?-sheet and random coil conformations, coupled with the increase in helical content during oxidation, occurred during the first 30 min of the reaction. No further conformational changes occurred after this time and the response was independent of methionine trigger location relative to the penta-alanine domain. These results were confirmed with fluorescence studies. The design, processing and utility of these modified redox triggered silk-like peptides and proteins suggest a range of potential utility, from biomaterials to engineered surface coatings with chemically alterable secondary structure and, thus, properties.

Foo, C. Wong Po; Bini, E.; Huang, J.; Lee, S. Y.; Kaplan, D. L.

2006-02-01

71

Novel Assembly Properties of Recombinant Spider Dragline Silk Proteins  

Microsoft Academic Search

Spider dragline silk, which exhibits extraordinary strength and toughness, is primarily composed of two related proteins that largely consist of repetitive sequences. In most spiders, the repetitive region of one of these proteins is rich in prolines, which are not present in the repetitive region of the other [1]. The absence of prolines in one component was previously speculated to

Daniel Huemmerich; Thomas Scheibel; Fritz Vollrath; Shulamit Cohen; Uri Gat; Shmulik Ittah

2004-01-01

72

Silk-tropoelastin protein films for nerve guidance.  

PubMed

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

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

2015-03-01

73

Interfacial rheological properties of recombinant spider-silk proteins Cyrille Vzya  

E-print Network

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

Bausch, Andreas

74

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

NASA Astrophysics Data System (ADS)

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

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

2006-02-01

75

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

PubMed

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

Xu, Hanfu

2014-10-01

76

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

E-print Network

Study of Protein Conformation and Orientation in Silkworm and Spider Silk Fibers Using Raman for the first time to determine quantitatively the orientation of the -sheets in silk monofilaments from Bombyx, for systems with uniaxial symmetry such as silk, it is possible to determine the order parameters P2 and P4

Pezolet, Michel

77

A Materiomics Approach to Spider Silk: Protein Molecules to Webs  

NASA Astrophysics Data System (ADS)

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.

Tarakanova, Anna; Buehler, Markus J.

2012-02-01

78

Biomaterials 24 (2003) 401416 Silk-based biomaterials  

E-print Network

Biomaterials 24 (2003) 401­416 Silk-based biomaterials Gregory H. Altman, Frank Diaz, Caroline, Medford, MA 02155, USA Received 17 April 2002; accepted 19 June 2002 Abstract Silk from the silkworm biocompatibility problems have been reported for silkworm silk; however, contamination from residual sericin (glue

Lu, Helen H.

79

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

PubMed

Spiders spin high performance fibers with diverse biological functions and mechanical properties. Molecular and biochemical studies of spider prey wrapping silks have revealed the presence of the aciniform silk fibroin AcSp1-like. In our studies we demonstrate the presence of a second distinct polypeptide present within prey wrapping silk. Combining matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry and reverse genetics, we have isolated a novel gene called MiSp1-like and demonstrate that its protein product is a constituent of prey wrap silks from the black widow spider, Latrodectus hesperus. BLAST searches of the NCBInr protein database using the amino acid sequence of MiSp1-like revealed similarity to the conserved C-terminal domain of silk family members. In particular, MiSp1-like showed the highest degree of sequence similarity to the nonrepetitive C-termini of published orb-weaver minor ampullate fibroin molecules. Analysis of the internal amino acid sequence of the black widow MiSp1-like revealed polyalanine stretches interrupted by glycine residues and glycine-alanine couplets within MiSp1-like as well as repeats of the heptameric sequence AGGYGQG. Real-time quantitative PCR analysis demonstrates that the MiSp1-like gene displays a minor ampullate gland-restricted pattern of expression. Furthermore, amino acid composition analysis, coupled with scanning electron microscopy of raw wrapping silk, supports the assertion that minor ampullate silks are important constituents of black widow spider prey wrap silk. Collectively, our findings provide direct molecular evidence for the involvement of minor ampullate fibroins in swathing silks and suggest composite materials play an important role in the wrap attack process for cob-weavers. PMID:18376847

La Mattina, Coby; Reza, Ryan; Hu, Xiaoyi; Falick, Arnold M; Vasanthavada, Keshav; McNary, Shannon; Yee, Russell; Vierra, Craig A

2008-04-22

80

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

NASA Astrophysics Data System (ADS)

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.

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

2012-01-01

81

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

NASA Astrophysics Data System (ADS)

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.

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

2014-07-01

82

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

PubMed Central

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

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

2014-01-01

83

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

PubMed

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

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

2014-01-01

84

Engineering aqueous fiber assembly into silk-elastin-like protein polymers.  

PubMed

Self-assembled peptide/protein nanofibers are valuable 1D building blocks for creating complex structures with designed properties and functions. It is reported that the self-assembly of silk-elastin-like protein polymers into nanofibers or globular aggregates in aqueous solutions can be modulated by tuning the temperature of the protein solutions, the size of the silk blocks, and the charge of the elastin blocks. A core-sheath model is proposed for nanofiber formation, with the silk blocks in the cores and the hydrated elastin blocks in the sheaths. The folding of the silk blocks into stable cores--affected by the size of the silk blocks and the charge of the elastin blocks--plays a critical role in the assembly of silk-elastin nanofibers. Furthermore, enhanced hydrophobic interactions between the elastin blocks at elevated temperatures greatly influence the nanoscale features of silk-elastin nanofibers. PMID:24798978

Zeng, Like; Jiang, Linan; Teng, Weibing; Cappello, Joseph; Zohar, Yitshak; Wu, Xiaoyi

2014-07-01

85

Properties and antityrosinase activity of sericin from various extraction methods.  

PubMed

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

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

2010-02-01

86

Silk: A Potential Medium for Tissue Engineering  

PubMed Central

Objective: Human skin is a complex bilayered organ that serves as a protective barrier against the environment. The loss of integrity of skin by traumatic experiences such as burns and ulcers may result in considerable disability or ultimately death. Therefore, in skin injuries, adequate dermal substitutes are among primary care targets, aimed at replacing the structural and functional properties of native skin. To date, there are very few single application tissue-engineered dermal constructs fulfilling this criterion. Silk produced by the domestic silkworm, Bombyx mori, has a long history of use in medicine. It has recently been increasingly investigated as a promising biomaterial for dermal constructs. Silk contains 2 fibrous proteins, sericin and fibroin. Each one exhibits unique mechanical and biological properties. Methods: Comprehensive review of randomized-controlled trials investigating current dermal constructs and the structures and properties of silk-based constructs on wound healing. Results: This review revealed that silk-fibroin is regarded as the most promising biomaterial, providing options for the construction of tissue-engineered skin. Conclusion: The research available indicates that silk fibroin is a suitable biomaterial scaffold for the provision of adequate dermal constructs. PMID:18997857

Sobajo, Cassandra; Behzad, Farhad; Yuan, Xue-Feng; Bayat, Ardeshir

2008-01-01

87

Stability of Silk and Collagen Protein Materials in Space  

NASA Astrophysics Data System (ADS)

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.

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

2013-12-01

88

Stability of Silk and Collagen Protein Materials in Space  

PubMed Central

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

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

2013-01-01

89

Stability of silk and collagen protein materials in space.  

PubMed

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

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

2013-01-01

90

Glycopolymer functionalization of engineered spider silk protein-based materials for improved cell adhesion.  

PubMed

Silk protein-based materials are promising biomaterials for application as tissue scaffolds, due to their processability, biocompatibility, and biodegradability. The preparation of films composed of an engineered spider silk protein (eADF4(C16)) and their functionalization with glycopolymers are described. The glycopolymers bind proteins found in the extracellular matrix, providing a biomimetic coating on the films that improves cell adhesion to the surfaces of engineered spider silk films. Such silk-based materials have potential as coatings for degradable implantable devices. PMID:24700586

Hardy, John G; Pfaff, André; Leal-Egaña, Aldo; Müller, Axel H E; Scheibel, Thomas R

2014-07-01

91

High yield recombinant silk-like protein production in transgenic plants through protein targeting  

Microsoft Academic Search

DP1B is a synthetic analogue of spider dragline silk protein. It can be spun to form silk fiber. Previously, it had been expressed in transgenic plants, showing the general feasibility of the plant-based DP1B production. However, success of such a plant-based platform requires a great increase of DP1B productivity in plant cells to reduce production cost. This report describes a

Jianjun Yang; Leslie A. Barr; Stephen R. Fahnestock; Zhan-Bin Liu

2005-01-01

92

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

PubMed

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

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

2014-01-01

93

Molecular Architecture and Evolution of a Modular Spider Silk Protein Gene  

NASA Astrophysics Data System (ADS)

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

Hayashi, Cheryl Y.; Lewis, Randolph V.

2000-02-01

94

Composition of Silk  

Microsoft Academic Search

IT is well known that the sericin component of silk is soluble in water, and the fibroin is soluble in certain acids and a variety of salt solutions. The latter can be dialysed to leave a solution of fibroin in water1,2, from which water-soluble films can be cast. Ambrose et al.1 have shown by infra-red methods that these are in

F. Happey; A. J. Hyde

1952-01-01

95

Electroresponsive aqueous silk protein as "smart" mechanical damping fluid.  

PubMed

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

Jose, Rod R; Elia, Roberto; Tien, Lee W; Kaplan, David L

2014-05-14

96

Biological responses to spider silk-antibiotic fusion protein  

PubMed Central

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

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

2011-01-01

97

Nanoparticle self-assembly by a highly stable recombinant spider wrapping silk protein subunit.  

PubMed

Artificial spider silk proteins may form fibers with exceptional strength and elasticity. Wrapping silk, or aciniform silk, is the toughest of the spider silks, and has a very different protein composition than other spider silks. Here, we present the characterization of an aciniform protein (AcSp1) subunit named W1, consisting of one AcSp1 199 residue repeat unit from Argiope trifasciata. The structural integrity of recombinant W1 is demonstrated in a variety of buffer conditions and time points. Furthermore, we show that W1 has a high thermal stability with reversible denaturation at ?71°C and forms self-assembled nanoparticle in near-physiological conditions. W1 therefore represents a highly stable and structurally robust module for protein-based nanoparticle formation. PMID:23994530

Xu, Lingling; Tremblay, Marie-Laurence; Orrell, Kathleen E; Leclerc, Jérémie; Meng, Qing; Liu, Xiang-Qin; Rainey, Jan K

2013-10-01

98

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

PubMed

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

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

2013-02-01

99

Enhancing Analysis of Cells and Proteins by Fluorescence Imaging on Silk-Based Biomaterials: Modulating the Autofluorescence of Silk.  

PubMed

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

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

2014-10-17

100

Processing and modification of films made from recombinant spider silk proteins  

NASA Astrophysics Data System (ADS)

Protein films represent an interesting class of materials with various possibilities for applications. We investigated films made of two different synthetic spider silk proteins derived from the garden spider’s (Araneus diadematus) two dragline silk proteins ADF-3 and ADF-4. Protein films cast from hexafluoroisopropanol solutions displayed a predominantly ?-helical secondary structure. Processing such films with potassium phosphate or methanol resulted in a transition to a ?-sheet rich structure. While as-cast films could be dissolved in water, processed ?-sheet rich films were water insoluble. The chemical stability of processed films depended on the amino acid sequence of the respective protein employed. As a proof of principle, fluorescent probes or enzymes were covalently attached to the film surface. The presented approach provides a basis for designing tailor-made protein films using silk proteins as scaffold, in which the film properties can be controlled by genetic engineering of the underlying silks.

Huemmerich, D.; Slotta, U.; Scheibel, T.

2006-02-01

101

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

PubMed

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

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

2014-01-13

102

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

PubMed Central

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

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

2013-01-01

103

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

PubMed Central

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

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

2009-01-01

104

The critical role of water in spider silk and its consequence for protein Cameron P. Brown,*abcd  

E-print Network

The critical role of water in spider silk and its consequence for protein mechanics Cameron P, there is considerable interest in understanding, and replicating, spider silk's stress-processing mechanisms of the different regions in the spider silk fibre, and their relative contributions to stress processing. We

105

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

PubMed

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

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

2014-11-10

106

Sequence-structure correlations in the MaSp1 protein of N. clavipes dragline silk  

E-print Network

Silk is a hierarchically structured protein fiber with exceptional tensile strength and extensibility, making it one of the toughest and most versatile biocompatible materials. While experimental studies have shown that ...

Bratzel, Graham Hayden

2011-01-01

107

Utilizing conformational changes for patterning thin films of recombinant spider silk proteins.  

PubMed

Recombinant spider silk proteins mimicking the properties of dragline silk proteins represent a class of materials that hold great potential for future high-performance applications. Here we explore the self-assembly behavior of a recombinantly produced spider silk protein based on the dragline silk of the Araneus diadematus , eADF4 (C16), by selectively patterning its secondary structure using capillary transfer lithography and solvent-assisted microcontact molding. Two conformational transitions were observed, influenced by initial solvent composition: ?-helix/random coil conformation to a more densely packed ?-sheet conformation (by casting from 1,1,1,3,3,3-hexafluoro-propanol) and moderate initial ?-sheet content to higher ?-sheet content (casting from formic acid). Furthermore, by using the solvent-assisted microcontact molding technique, we were able to achieve a submicrometer spatial resolution and reveal fine details of morphological and mechanical changes in patterned regions and at interfaces. PMID:22947370

Young, Seth L; Gupta, Maneesh; Hanske, Christoph; Fery, Andreas; Scheibel, Thomas; Tsukruk, Vladimir V

2012-10-01

108

Processing of recombinant spider silk proteins into tailor-made materials for biomaterials applications.  

PubMed

Spider silk has extraordinary mechanical properties, is biocompatible and biodegradable, and therefore an ideal material for biomedical applications. However, a drawback for any application is the inhomogeneity of spider silk, as seen for other natural materials, as well as the low availability due to the cannibalism of most spiders. Recently, developed recombinant spider silk proteins ensure constant material properties, as well as scalable production, and further the processing into morphologies other than fibres. Biotechnology enables genetic modification, broadening the range of applications, such as implant coatings, scaffolds for tissue engineering, wound dressing devices as well as drug delivery systems. PMID:24657706

Schacht, Kristin; Scheibel, Thomas

2014-10-01

109

STUDIES ON THE POSTERIOR SILK GLAND OF THE SILKWORM BOMBIX MORI  

PubMed Central

Ultracentrifugal analyses of the native silk proteins extracted from the various parts of the middle silk gland of the mature silkworm have revealed that there exist four components with S°20,w values of 10S, 9–10S, 9S, and 4S in the extract. It is suggested that the fastest 10S component is the native fibroin synthesized in the posterior silk gland and transferred to the middle silk gland to be stored there, while the slower three components probably correspond to inner, middle, and outer sericins which were synthesized in the posterior, middle, and anterior portion of the middle silk gland, respectively. Native fibroin solution was prepared from the most posterior part of the middle silk gland. Ultracentrifugal analyses have shown that the solution contains considerable amounts of aggregates in addition to the main 10S component. Treatment with lithium bromide (LiBr), urea, or guanidine hydrochloride solution up to 6 M all have failed to dissociate the 10S component. From the sedimentation equilibrium analyses and partial specific volume of 0.716, the molecular weight of the 10S component of the native fibroin solution was found to be between 3.2 – 4.2 x 105, with a tendency to lie fairly close to 3.7 x 105. PMID:5459009

Tashiro, Yutaka; Otsuki, Eiichi

1970-01-01

110

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

PubMed

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

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

2013-03-01

111

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

PubMed Central

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

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

2012-01-01

112

Nanostructure and molecular mechanics of spider dragline silk protein assemblies  

E-print Network

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 ...

Keten, Sinan

113

Native-sized spider silk proteins synthesized in planta via intein-based multimerization.  

PubMed

The synthesis of native-sized proteins is a pre-requisite for exploiting the potential of spider silk as a bio-based material. The unique properties of spider silk, such as extraordinary tensile strength and elasticity, result from the highly repetitive nature of spider silk protein motifs. The present report describes the combination of spider silk flagelliform protein (FLAG) production in the endoplasmic reticulum of tobacco plant leaf cells with an intein-based posttranslational protein fusion technology. The repeated ligation of FLAG monomers resulted in the formation of large multimers. This method avoids the need for highly repetitive transgenes, which may result in a higher genetic and transcriptional stability. Here we show, for the first time, the production of synthetic, high molecular weight spider silk proteins larger than 250 kDa based on the assembly of protein monomers via intein-mediated trans-splicing in planta. The resulting multimeric structures form microfibers, thereby demonstrating their great potential as a biomaterial. PMID:23001519

Hauptmann, Valeska; Weichert, Nicola; Menzel, Matthias; Knoch, Dominic; Paege, Norman; Scheller, Jürgen; Spohn, Uwe; Conrad, Udo; Gils, Mario

2013-04-01

114

Transglutamination allows production and characterization of native-sized ELPylated spider silk proteins from transgenic plants.  

PubMed

In the last two decades it was shown that plants have a great potential for production of specific heterologous proteins. But high cost and inefficient downstream processing are a main technical bottleneck for the broader use of plant-based production technology especially for protein-based products, for technical use as fibres or biodegradable plastics and also for medical applications. High-performance fibres from recombinant spider silks are, therefore, a prominent example. Spiders developed rather different silk materials that are based on proteins. These spider silks show excellent properties in terms of elasticity and toughness. Natural spider silk proteins have a very high molecular weight, and it is precisely this property which is thought to give them their strength. Transgenic plants were generated to produce ELPylated recombinant spider silk derivatives. These fusion proteins were purified by Inverse Transition Cycling (ITC) and enzymatically multimerized with transglutaminase in vitro. Layers produced by casting monomers and multimers were characterized using atomic force microscopy (AFM) and AFM-based nanoindentation. The layered multimers formed by mixing lysine- and glutamine-tagged monomers were associated with the highest elastic penetration modulus. PMID:24237483

Weichert, Nicola; Hauptmann, Valeska; Menzel, Matthias; Schallau, Kai; Gunkel, Philip; Hertel, Thomas C; Pietzsch, Markus; Spohn, Uwe; Conrad, Udo

2014-02-01

115

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

PubMed

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

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

2015-02-01

116

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

PubMed

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 (600-800nm) were formed in the presence of the recombinant silk and chimeras, which were smaller than those formed by 15mer-SiBP chimeras [1], 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

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

2015-03-01

117

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

PubMed

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

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

2014-05-01

118

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

PubMed Central

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

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

2009-01-01

119

Silk and collagen scaffolds for tendon reconstruction.  

PubMed

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

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

2014-04-01

120

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

PubMed

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

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

2015-01-28

121

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

PubMed Central

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

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

2014-01-01

122

Microphase Separation Controlled Beta Sheet Crystallization Kinetics in Silk Fibroin Protein.  

NASA Astrophysics Data System (ADS)

We investigate the mechanism of isothermal crystallization kinetics of beta-sheet crystals in silk multiblock fibrous proteins. The Avrami analysis kinetic theory, for studies of synthetic polymer crystal growth, is for the first time extended to investigate protein self-assembly in beta-sheet rich Bombyx mori silk fibroin samples, using time-resolved Fourier transform infrared spectroscopy, differential scanning calorimetry and synchrotron real-time wide-angle X-ray scattering. Results indicate formation of beta sheet crystals in silk proteins is different from the 3-D spherulitic crystal growth found in synthetic homopolymers. Observations by scanning electron microscopy support the view that the protein structures vary during the different stages of crystal growth, and show a microphase separation pattern after chymotrypsin enzyme biodegradation. We present a model to explain the crystallization of the multiblock silk fibroin protein, by analogy to synthetic block copolymers. This model could be widely applicable in other proteins with multiblock (i.e., crystallizable and non-crystallizable) domains.

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

2009-03-01

123

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

PubMed Central

Silk production has independently evolved in numerous arthropod lineages, such as Lepidoptera, the moths and butterflies. Lepidopteran larvae (caterpillars) synthesize silk proteins in modified salivary glands and spin silk fibers into protective tunnels, escape lines, and pupation cocoons. Molecular sequence data for these proteins are necessary to determine critical features of their function and evolution. To this end, we constructed an expression library from the silk glands of the ghost moth, Hepialus californicus, and characterized light chainfibroin and heavy chain fibroin gene transcripts. The predicted H. californicus silk fibroins share many elements with other lepidopteran and trichopteran fibroins, such as conserved placements of cysteine, aromatic, and polar amino acid residues. Further comparative analyses were performed to determine site-specific signatures of selection and to assess whether fibroin genes are informative as phylogenetic markers. We found that purifying selection has constrained mutation within the fibroins and that light chain fibroin is a promising molecular marker. Thus, by characterizing the H. californicus fibroins, we identified key functional amino acids and gained insight into the evolutionary processes that have shaped these adaptive molecules. PMID:20458474

Mita, Kazuei; Sehnal, Frantisek; Hayashi, Cheryl Y.

2010-01-01

124

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

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

Vogt, Richard G.

125

Tunable Self-Assembly of Genetically Engineered Silk-Elastin-Like Protein Polymers  

PubMed Central

Silk-elastin-like protein polymers (SELPs), consisting of the repeating units of silk and elastin blocks, combine a set of outstanding physical and biological properties of silk and elastin. Due to the unique properties, SELPs have been widely fabricated into various materials for the applications in drug delivery and tissue engineering. However, little is known about the fundamental self-assembly characteristics of these remarkable polymers. Here we propose a two-step self-assembly process of SELPs in aqueous solution for the first time and report the importance of the ratio of silk to elastin blocks in a SELP’s repeating unit on the assembly of the SELP. Through precise tuning of the ratio of silk to elastin, various structures including nanoparticles, hydrogels and nanofibers could be generated either reversibly or irreversibly. This assembly process might provide opportunities to generate innovative smart materials for biosensors, tissue engineering and drug delivery. Furthermore, the newly developed SELPs in this study may be potentially useful as biomaterials for controlled drug delivery and biomedical engineering. PMID:21955178

Xia, Xiao-Xia; Xu, Qiaobing; Hu, Xiao; Qin, Guokui; Kaplan, David L.

2011-01-01

126

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

SciTech Connect

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.

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

127

Structural changes of thin films from recombinant spider silk proteins upon post-treatment  

NASA Astrophysics Data System (ADS)

Engineering of spider silk proteins offers the possibility to control their molecular sequence and thus their material properties. Spin coating was used to prepare films of engineered spider silk protein derived from the garden spider’s ( Araneus diadematus) dragline silk protein ADF-4. A conformational transition from ?-helix to ?-sheet-rich structures upon methanol treatment of the films was detected by external reflection IR spectroscopy. We present direct evidence for this structural transformation using grazing-incidence X-ray diffraction (GIXRD) and small-angle scattering (GISAXS). The protein film structure after the methanol treatment consists mainly of ?-sheet polyalanine crystals dispersed in an amorphous protein matrix. The GIXRD intensity profiles show Bragg peaks from ?-sheet polyalanine crystallites having an average size of 7.5 nm. The non-uniform and large crystal size distributions within the film were explained based on the protein composition. The effect of the chemical nature of the interface on the protein film structure was investigated as well.

Metwalli, E.; Slotta, U.; Darko, C.; Roth, S. V.; Scheibel, T.; Papadakis, C. M.

2007-11-01

128

Protein unfolding versus ?-sheet separation in spider silk nanocrystals  

NASA Astrophysics Data System (ADS)

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

Alam, Parvez

2014-03-01

129

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

NASA Astrophysics Data System (ADS)

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).

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

2013-10-01

130

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

PubMed

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

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

131

Air filter devices including nonwoven meshes of electrospun recombinant spider silk proteins.  

PubMed

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

Lang, Gregor; Jokisch, Stephan; Scheibel, Thomas

2013-01-01

132

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

PubMed Central

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

Lang, Gregor; Jokisch, Stephan; Scheibel, Thomas

2013-01-01

133

The N-terminal domains of spider silk proteins assemble ultrafast and protected from charge screening.  

PubMed

Web spiders assemble spidroin monomers into silk fibres of unrivalled tensile strength at remarkably high spinning speeds of up to 1 m s(-1). The spidroin N-terminal domain contains a charge-driven, pH-sensitive relay that controls self-association by an elusive mechanism. The underlying kinetics have not yet been reported. Here we engineer a fluorescence switch into the isolated N-terminal domain from spidroin 1 of the major ampullate gland of the nursery web spider E. australis that monitors dimerization. We observe ultrafast association that is surprisingly insensitive to salt, contrasting the classical screening effects in accelerated, charged protein interfaces. To gain deeper mechanistic insight, we mutate each of the protonatable residue side chains and probe their contributions. Two vicinal aspartic acids are critically involved in an unusual process of accelerated protein association that is protected from screening by electrolytes, potentially facilitating the rapid synthesis of silk fibres by web spiders. PMID:24240554

Schwarze, Simone; Zwettler, Fabian U; Johnson, Christopher M; Neuweiler, Hannes

2013-01-01

134

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

PubMed Central

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

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

2014-01-01

135

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

PubMed

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

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

2014-01-01

136

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

PubMed

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

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

2014-01-01

137

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

PubMed Central

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

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

138

Spider dragline silk.  

E-print Network

??Spider dragline silk possesses several desirable features of a biomaterial; it has extraordinary mechanical properties, is biocompatible and biodegradable. It consists of large proteins, major… (more)

Rising, Anna

2007-01-01

139

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

NASA Astrophysics Data System (ADS)

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

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

2005-10-01

140

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

PubMed

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

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

141

Protein Secondary Structure and Orientation in Silk as Revealed by Raman Spectromicroscopy  

PubMed Central

Taking advantage of recent advances in polarized Raman microspectroscopy, and based on a rational decomposition of the amide I band, the conformation and orientation of proteins have been determined for cocoon silks of the silkworms Bombyx mori and Samia cynthia ricini and dragline silks of the spiders Nephila clavipes and Nephila edulis. This study distinguished between band components due to ?-sheets, ?-turns, 31-helices, and unordered structure for the four fibers. For B. mori, the ?-sheet content is 50%, which matches the proportion of residues that form the GAGAGS fibroin motifs. For the Nephila dragline and S. c. ricini cocoon, the ?-sheet content (36–37% and 45%, respectively) is higher than the proportion of residues that belong to polyalanine blocks (18% and 42%, respectively), showing that adjacent GGA motifs are incorporated into the ?-sheets. Nephila spidroins contain fewer ?-sheets and more flexible secondary structures than silkworm fibroins. The amorphous polypeptide chains are preferentially aligned parallel to the fiber direction, although their level of orientation is much lower than that of ?-sheets. Overall, the results show that the four silks exhibit a common molecular organization, with mixtures of different amounts of ?-sheets and flexible structures, which are organized with specific orientation levels. PMID:17277183

Lefèvre, Thierry; Rousseau, Marie-Eve; Pézolet, Michel

2007-01-01

142

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

PubMed Central

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

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

143

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

PubMed

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

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

144

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

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

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

2015-01-01

145

Silk as a Biomaterial  

PubMed Central

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

Vepari, Charu

2009-01-01

146

Silk-functionalized titanium surfaces for enhancing osteoblast functions and reducing bacterial adhesion.  

PubMed

It would be ideal to have implants which can simultaneously inhibit bacterial adhesion and promote osteoblast functions. In this work, titanium surfaces were modified with poly(methacrylic acid) (P(MAA)) followed by immobilization of silk sericin. Firstly a trichlorosilane coupling agent, which is an atom transfer radical polymerization (ATRP) initiator, was immobilized on the oxidized titanium surface to facilitate the surface-initiated ATRP of methacrylic acid sodium salt (MAAS). The pendant carboxyl end groups of the grafted and partially protonated MAA chains were subsequently coupled with silk sericin via carbodiimide chemistry. The functionalized Ti surfaces were characterized by X-ray photoelectron spectroscopy, and assayed for osteoblast cell functions and bacterial adhesion. The covalently immobilized MAA brushes significantly reduce the adhesion of the two bacterial strains (Staphylococcus aureus and Staphylococcus epidermidis) tested. The silk sericin-immobilized surfaces, at the same time, promote osteoblast cells' adhesion, proliferation, and alkaline phosphatase activity. Thus, the P(MAA) and silk sericin functionalized Ti surfaces have potential applications combating biomaterial-centered infection and promoting osseointegration. PMID:18829101

Zhang, Fan; Zhang, Zhengbiao; Zhu, Xiulin; Kang, En-Tang; Neoh, Koon-Gee

2008-12-01

147

Degumming of silk fabric with several proteases.  

PubMed

A crêpe silk fabric was treated with different alkaline (3374-L, GC 897-H), neutral (3273-C), and acid (EC 3.4 23.18) proteases with the aim to study their effectiveness as degumming agents. Proteases were used under optimum conditions of pH and temperature, while enzyme dosage (0.05-2 U/g fabric) and treatment time (5-240 min) were changed in order to study the kinetics of sericin removal. Degumming loss with soap and alkali was 27 wt.%. The maximum amount of sericin removed in 1 h was 17.6, 24, and 19 wt.% for 3374-L (2 U/g fabric), GC 897-H (1U/g fabric), and 3273-C (0.1 U/g fabric), respectively. Under the experimental conditions adopted, EC 3.4 23.18 was almost ineffective as a degumming agent. Degumming loss increased as a function of the treatment time, reaching a value of 25 wt.% with 1 U/g fabric of 3374-L. The morphological analysis showed that sericin was completely removed from the warp yarns of the crêpe fabric, while the highly twisted weft yarns still exhibited the presence of sericin deposits within the most internal parts of the close fibre texture. The chromatographic pattern of soluble sericin peptides changed as a function of the kind of enzyme used, enzyme dosage, and treatment time. A mixture of peptides from 5 to 20 kDa in weight, with a weight-average molecular weight of about 12 kDa was obtained. PMID:14636714

Freddi, Giuliano; Mossotti, Raffaella; Innocenti, Riccardo

2003-12-01

148

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

PubMed Central

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

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

2013-01-01

149

Structure-property relationship of regenerated spider silk protein nano/microfibrous scaffold fabricated by electrospinning.  

PubMed

The regenerated Araneus ventricosus spider dragline silk protein fibrous scaffold with moderate strength and flexibility was fabricated by electrospinning and post treatment with 90 vol % acetone. The effect of collection method on the morphology of regenerated spider silk protein (RSSP) fibrous scaffold, the effects of the post treatment solvents and their concentrations on the molecular conformation, crystallinity and mechanical properties were studied. The results show that the morphology was affected by the solvent used in the coagulation bath. The molecular conformation, crystallinity and mechanical property of this scaffold were strongly affected by the kind of post treatment solvent and slightly influenced by its concentration when it was higher than 50 vol %. The degradation rate of this scaffold was very slow and resulting in little pH change of the degradation medium within 5 months. PC 12 cells were cultured on the electrospun RSSP fibrous scaffold and in its extraction fluid to examine the changes of PC 12 cells after different times of culture. The results show that the electrospun RSSP fibrous scaffold had good biocompatibility with PC 12 cells. PMID:24288266

Yu, Qiaozhen; Xu, Shuiling; Zhang, Hong; Gu, Li; Xu, Yepei; Ko, Frank

2014-11-01

150

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

PubMed

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

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

2014-12-01

151

A novel marine silk  

NASA Astrophysics Data System (ADS)

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

Kronenberger, Katrin; Dicko, Cedric; Vollrath, Fritz

2012-01-01

152

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

Microsoft Academic Search

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

Thomas Scheibel

2004-01-01

153

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

PubMed

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

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

2015-01-12

154

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

NASA Astrophysics Data System (ADS)

Production of brand new protein-based materials with precise control over the amino acid sequences at single residue level has been made possible by genetic engineering, through which artificial genes can be developed that encode protein-based materials with desired features. As an example, silk-elastinlike protein polymers (SELPs), composed of tandem repeats of amino acid sequence motifs from Bombyx mori (silkworm) silk and mammalian elastin, have been produced in this approach. SELPs have been studied extensively in the past two decades, however, the fundamental mechanism governing the self-assembly process to date still remains largely unresolved. Further, regardless of the unprecedented success when exploited in areas including drug delivery, gene therapy, and tissue augmentation, SELPs scaffolds as a three-dimensional cell culture model system are complicated by the inability of SELPs to provide the embedded tissue cells with appropriate biochemical stimuli essential for cell survival and function. In this dissertation, it is reported that the self-assembly of silk-elastinlike protein polymers (SELPs) into nanofibers in aqueous solutions can be modulated by tuning the curing temperature, the size of the silk blocks, and the charge of the elastin blocks. A core-sheath model was proposed for nanofiber formation, with the silk blocks in the cores and the hydrated elastin blocks in the sheaths. The folding of the silk blocks into stable cores -- affected by the size of the silk blocks and the charge of the elastin blocks -- plays a critical role in the assembly of silk-elastin nanofibers. The assembled nanofibers further form nanofiber clusters on the microscale, and the nanofiber clusters then coalesce into nanofiber micro-assemblies, interconnection of which eventually leads to the formation of three-dimensional scaffolds with distinct nanoscale and microscale features. SELP-Collagen hybrid scaffolds were also fabricated to enable independent control over the scaffolds' biochemical input and matrix stiffness. It is reported herein that in the hybrid scaffolds, collagen provides essential biochemical cues needed to promote cell attachment and function while SELP imparts matrix stiffness tunability. To obtain tissue-specificity in matrix stiffness that spans over several orders of magnitude covering from soft brain to stiff cartilage, the hybrid SELP-Collagen scaffolds were crosslinked by transglutaminase at physiological conditions compatible for simultaneous cell encapsulation. The effect of the increase in matrix stiffness induced by such enzymatic crosslinking on cellular viability and proliferation was also evaluated using in vitro cell assays.

Zeng, Like

155

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

NASA Astrophysics Data System (ADS)

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

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

2008-03-01

156

Quantification of the physiochemical constraints on the export of spider silk proteins by Salmonella type III secretion  

Microsoft Academic Search

BACKGROUND: The type III secretion system (T3SS) is a molecular machine in gram negative bacteria that exports proteins through both membranes to the extracellular environment. It has been previously demonstrated that the T3SS encoded in Salmonella Pathogenicity Island 1 (SPI-1) can be harnessed to export recombinant proteins. Here, we demonstrate the secretion of a variety of unfolded spider silk proteins

Daniel M Widmaier; Christopher A Voigt

2010-01-01

157

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

PubMed

Using atomistic simulations, we demonstrate that ?-sheet, an essential component of spider silk protein, has a thermal conductivity 1-2 orders of magnitude higher than that of some other protein structures reported in the literature. In contrast to several other nanostructured materials of similar bundled/layered structures (e.g. few-layer graphene and bundled carbon nanotubes), the ?-sheet is found to uniquely feature enhanced thermal conductivity with an increased number of constituting units, i.e. ?-strands. Phonon analysis identifies inter-?-strand hydrogen bonding as the main contributor to the intriguing phenomenon, which prominently influences the state of phonons in both low- and high-frequency regimes. A thermal resistance model further verifies the critical role of hydrogen bonding in thermal conduction through ?-sheet structures. PMID:24811747

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

2014-07-21

158

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

PubMed

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

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

2014-09-01

159

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

PubMed

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

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

2014-12-10

160

The critical role of water in spider silk and its consequence for protein mechanics  

NASA Astrophysics Data System (ADS)

Due to its remarkable mechanical and biological properties, there is considerable interest in understanding, and replicating, spider silk's stress-processing mechanisms and structure-function relationships. Here, we investigate the role of water in the nanoscale mechanics of the different regions in the spider silk fibre, and their relative contributions to stress processing. We propose that the inner core region, rich in spidroin II, retains water due to its inherent disorder, thereby providing a mechanism to dissipate energy as it breaks a sacrificial amide-water bond and gains order under strain, forming a stronger amide-amide bond. The spidroin I-rich outer core is more ordered under ambient conditions and is inherently stiffer and stronger, yet does not on its own provide high toughness. The markedly different interactions of the two proteins with water, and their distribution across the fibre, produce a stiffness differential and provide a balance between stiffness, strength and toughness under ambient conditions. Under wet conditions, this balance is destroyed as the stiff outer core material reverts to the behaviour of the inner core.

Brown, Cameron P.; MacLeod, Jennifer; Amenitsch, Heinz; Cacho-Nerin, Fernando; Gill, Harinderjit S.; Price, Andrew J.; Traversa, Enrico; Licoccia, Silvia; Rosei, Federico

2011-09-01

161

Evolution of repetitive proteins: spider silks from Nephila clavipes (Tetragnathidae) and Araneus bicentenarius (Araneidae).  

PubMed

Spider silks are highly repetitive proteins, characterized by regions of polyalanine and glycine-rich repeating units. We have obtained two variants of the Spidroin 1 (NCF-1) silk gene sequence from Nephila clavipes. One sequence (1726 bp) was from a cloned cDNA, and the other (1951 bp) was from PCR of genomic DNA. When these sequences are compared with each other and the previously published Spidroin 1 sequence, there are differences due to sequence rearrangements, as well as single base substitutions. These variations are similar to those that have been reported from other highly repetitive genes, and probably represent the results of unequal cross-overs. We have also obtained 708 bp of sequence from pCR of genomic DNA from Araneus biocentenarius. This sequence shows considerable similarity to a dragline sequence (ADF-3) from A. diadematus, as well as Spidroin 2 (NCF-2) from N. clavipes. Minor but consistent differences in the repeating unit sequence between A. bicentenarius and A. diadematus suggest that concerted evolution or gene conversion processes are acting to maintain similarity among repeat units within a single gene. PMID:9654736

Beckwitt, R; Arcidiacono, S; Stote, R

1998-03-01

162

Hydrogen bonding-assisted thermal conduction in ?-sheet crystals of spider silk protein  

NASA Astrophysics Data System (ADS)

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

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

2014-06-01

163

Histochemical and ultrastructural evidence of lipid secretion by the silk gland of the sugarcane borer Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae).  

PubMed

The silk gland in Lepidoptera larvae is responsible for the silk production used for shelter or cocoon construction. The secretion of fibroin and sericin by the different silk gland regions are well established. There are few attempts to detect lipid components in the insect silk secretion, although the presence of such element may contribute to the resistance of the shelter to wet environment. This study characterizes the glandular region and detects the presence of lipid components in the secretion of the silk gland of Diatraea saccharalis(Fabricius). The silk gland was submitted to histochemical procedure for lipid detection or conventionally prepared for ultrastructural analyses. Lipid droplets were histochemically detected in both the apical cytoplasm of cell of the anterior region and in the lumen among the microvilli. Ultrastructural analyses of the anterior region showed lipid material, visualized as myelin-like structures within the vesicular Golgi complex and in the apical secretory globules, mixed up with the sericin; similar material was observed into the lumen, adjacent to the microvilli. Lipids were not detected in the cells neither in the lumen of the posterior region. Our results suggest that the silk produced by D. saccharalis has a minor lipid content that is secreted by the anterior region together with the sericin. PMID:18060296

Victoriano, Eliane; Pinheiro, Daniela O; Gregório, Elisa A

2007-01-01

164

Wet-spinning of recombinant silk-elastin-like protein polymer fibers with high tensile strength and high deformability.  

PubMed

A recombinant silk-elastin-like protein copolymer SELP-47K containing tandemly repeated amino acid sequence blocks from silk, GAGAGS, and elastin, GVGVP, was fabricated into microdiameter fibers using a wet-spinning technique. Raman spectral analysis revealed the formation of antiparallel beta-sheet crystals of the silk-like blocks. Dry SELP-47K fibers display the dependence of mechanical properties such as Young's modulus on fiber diameter, suggesting more oriented and crystallized molecular chains in small-diameter fibers. Additionally, a brittle fracture mode was identified for dry fibers by SEM analysis of fracture surfaces. Hydration dramatically influenced the mechanical behavior of SELP-47K fibers. In contrast to the high tensile strength and limited strains to failure of dry fibers, fully hydrated SELP-47K fibers possessed strains to failure as high as 700%. Furthermore, upon chemical cross-linking, a tensile mechanical strength up to 20 MPa was achieved in hydrated fibers without compromising their high deformability. By combing the silk- and elastin-derived sequences into a single SELP-47K protein polymer, we demonstrated that protein fibers with high tensile strength and high deformability can be fabricated. PMID:19186950

Qiu, Weiguo; Teng, Weibing; Cappello, Joseph; Wu, Xiaoyi

2009-03-01

165

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

PubMed

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

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

2013-03-01

166

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

PubMed Central

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

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

2013-01-01

167

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

NASA Astrophysics Data System (ADS)

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.

Liivak, Oskar

2000-09-01

168

Spider Silk Fibers Spun from Soluble Recombinant Silk Produced in Mammalian Cells  

Microsoft Academic Search

Spider silks are protein-based ``biopolymer'' filaments or threads secreted by specialized epithelial cells as concentrated soluble precursors of highly repetitive primary sequences. Spider dragline silk is a flexible, lightweight fiber of extraordinary strength and toughness comparable to that of synthetic high-performance fibers. We sought to ``biomimic'' the process of spider silk production by expressing in mammalian cells the dragline silk

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

2002-01-01

169

Analysis of aqueous glue coating proteins on the silk fibers of the cob weaver, Latrodectus hesperus.  

PubMed

Elucidation of the molecular composition and physical properties of spider glue is necessary to understand its function in the mechanics of the web and prey capture. Previous reports have indicated that components of the adhesive coating contain inorganic molecules, phosphorylated glycoproteins, lipids, and organic low-molecular mass (LMM) compounds. Using a proteomic strategy, we have investigated the viscid, aqueous components that coat different silk fiber types from the black widow spider, Latrodectus hesperus. After in-solution tryptic digestion of the aqueous protein material extracted from egg case sacs, gumfooted lines, and the web scaffolding connection joints, followed by peptide analysis using MALDI tandem TOF mass spectrometry, we demonstrate that these fibers are coated with common peptides. Utilizing a reverse genetics approach, we have isolated the cDNAs encoding two distinct fiber coating products, which we have named spider coating peptide 1 and 2 (SCP-1 and SCP-2). Secreted forms of SCP-1 and SCP-2 contain 36 and 19 amino acids, respectively, and their primary sequences display no significant similarities to ensemble repeat units from traditional fibroins. Quantitative real-time reverse transcription PCR analyses show that these mRNAs are chiefly produced by the aggregate gland. Biochemical studies also demonstrate that the SCP-1 peptide has intrinsic metal binding properties, suggesting a role of peptide-metal ion interactions with the fiber constituents to enhance thread performance. Collectively, these investigations are the first to reveal a novel role for the aggregate gland in the production of peptides that coat spider silk threads. PMID:17311422

Hu, Xiaoyi; Yuan, Jing; Wang, Xiaodong; Vasanthavada, Keshav; Falick, Arnold M; Jones, Patrick R; La Mattina, Coby; Vierra, Craig A

2007-03-20

170

Quantification of the physiochemical constraints on the export of spider silk proteins by Salmonella type III secretion  

PubMed Central

Background The type III secretion system (T3SS) is a molecular machine in gram negative bacteria that exports proteins through both membranes to the extracellular environment. It has been previously demonstrated that the T3SS encoded in Salmonella Pathogenicity Island 1 (SPI-1) can be harnessed to export recombinant proteins. Here, we demonstrate the secretion of a variety of unfolded spider silk proteins and use these data to quantify the constraints of this system with respect to the export of recombinant protein. Results To test how the timing and level of protein expression affects secretion, we designed a hybrid promoter that combines an IPTG-inducible system with a natural genetic circuit that controls effector expression in Salmonella (psicA). LacO operators are placed in various locations in the psicA promoter and the optimal induction occurs when a single operator is placed at the +5nt (234-fold) and a lower basal level of expression is achieved when a second operator is placed at -63nt to take advantage of DNA looping. Using this tool, we find that the secretion efficiency (protein secreted divided by total expressed) is constant as a function of total expressed. We also demonstrate that the secretion flux peaks at 8 hours. We then use whole gene DNA synthesis to construct codon optimized spider silk genes for full-length (3129 amino acids) Latrodectus hesperus dragline silk, Bombyx mori cocoon silk, and Nephila clavipes flagelliform silk and PCR is used to create eight truncations of these genes. These proteins are all unfolded polypeptides and they encompass a variety of length, charge, and amino acid compositions. We find those proteins fewer than 550 amino acids reliably secrete and the probability declines significantly after ~700 amino acids. There also is a charge optimum at -2.4, and secretion efficiency declines for very positively or negatively charged proteins. There is no significant correlation with hydrophobicity. Conclusions We show that the natural system encoded in SPI-1 only produces high titers of secreted protein for 4-8 hours when the natural psicA promoter is used to drive expression. Secretion efficiency can be high, but declines for charged or large sequences. A quantitative characterization of these constraints will facilitate the effective use and engineering of this system. PMID:20973967

2010-01-01

171

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

PubMed

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

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

172

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

PubMed Central

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

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

2011-01-01

173

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

NASA Astrophysics Data System (ADS)

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.

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

2012-02-01

174

A Juvenile Hormone Transcription Factor Bmdimm-Fibroin H Chain Pathway Is Involved in the Synthesis of Silk Protein in Silkworm, Bombyx mori.  

PubMed

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

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

175

Silk inverse opals  

NASA Astrophysics Data System (ADS)

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

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

2012-12-01

176

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.

177

Nanostructure and molecular mechanics of spider dragline silk  

E-print Network

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

Buehler, Markus J.

178

Non-mulberry silk gland fibroin protein 3-D scaffold for enhanced differentiation of human mesenchymal stem cells into osteocytes.  

PubMed

This study investigates the potential of three-dimensional (3-D) scaffolds of wild non-mulberry tropical tasar silk gland fibroin protein as substratum for osteogenic differentiation of human mesenchymal stem cells (hMSCs). The novelty of the study lies in the fabrication of scaffolds from non-bioengineered silk fibroin directly extracted from the glands of non-mulberry tropical tasar silkworms using sodium dodecyl sulfate dissolution protocol and its osteogenic application using single- and double-seeding methods. The scaffolds were mechanically robust and showed homogenous pore distribution within the scaffold. hMSCs were seeded on the scaffolds and were cultured for up to 28days under static conditions in osteogenic media. Osteogenic differentiation of hMSCs seeded on fibroin scaffolds resulted in extensive mineralization with the formation of large calcium nodules, higher alkaline phosphatase activity and intense von Kossa staining. Real-time studies revealed higher transcript levels for osteopontin (OS) and bone sialoprotein (IBSP) under double-seeded conditions as compared to single-seeded scaffolds. Histological analysis showed the development of osteoblastic cells and large calcified nodules. The development and spreading of nuclei and actin filaments on fibroin matrices were revealed through confocal studies. The results suggest the suitability of non-mulberry silk-fibroin protein 3-D scaffolds as natural biomaterial for potential in vitro bone-tissue engineering applications. PMID:19345621

Mandal, Biman B; Kundu, S C

2009-09-01

179

Hydrophobic Drug-Triggered Self-Assembly of Nanoparticles from Silk-Elastin-Like Protein Polymers for Drug Delivery  

PubMed Central

Silk-elastin-like protein polymers (SELPs) combine the mechanical and biological properties of silk and elastin. These properties have led to the development of various SELP-based materials for drug delivery. However, SELPs have rarely been developed into nanoparticles, partially due to the complicated fabrication procedures, nor assessed for potential as an anticancer drug delivery system. We have recently constructed a series of SELPs (SE8Y, S2E8Y, and S4E8Y) with various ratios of silk to elastin blocks and described their capacity to form micellar-like nanoparticles upon thermal triggering. In this study, we demonstrate that doxorubicin, a hydrophobic antitumor drug, can efficiently trigger the self-assembly of SE8Y (SELPs with silk to elastin ratio of 1:8) into uniform micellar-like nanoparticles. The drug can be loaded in the SE8Y nanoparticles with an efficiency around 6.5% (65 ng doxorubicin/?g SE8Y), S2E8Y with 6%, and S4E8Y with 4%, respectively. In vitro studies with HeLa cell lines demonstrate that the protein polymers are not cytotoxic (IC50 > 200 ?g/mL), while the doxorubicin-loaded SE8Y nanoparticles showed a 1.8-fold higher cytotoxicity than the free drug. Confocal laser scanning microscopy (CLSM) and flow cytometry indicate significant uptake of the SE8Y nanoparticles by the cells and suggest internalization of the nanoparticles through endocytosis. This study provides an all-aqueous, facile method to prepare nanoscale, drug-loaded SELPs packages with potential for tumor cell treatments. PMID:24527851

2015-01-01

180

Hydrophobic drug-triggered self-assembly of nanoparticles from silk-elastin-like protein polymers for drug delivery.  

PubMed

Silk-elastin-like protein polymers (SELPs) combine the mechanical and biological properties of silk and elastin. These properties have led to the development of various SELP-based materials for drug delivery. However, SELPs have rarely been developed into nanoparticles, partially due to the complicated fabrication procedures, nor assessed for potential as an anticancer drug delivery system. We have recently constructed a series of SELPs (SE8Y, S2E8Y, and S4E8Y) with various ratios of silk to elastin blocks and described their capacity to form micellar-like nanoparticles upon thermal triggering. In this study, we demonstrate that doxorubicin, a hydrophobic antitumor drug, can efficiently trigger the self-assembly of SE8Y (SELPs with silk to elastin ratio of 1:8) into uniform micellar-like nanoparticles. The drug can be loaded in the SE8Y nanoparticles with an efficiency around 6.5% (65 ng doxorubicin/?g SE8Y), S2E8Y with 6%, and S4E8Y with 4%, respectively. In vitro studies with HeLa cell lines demonstrate that the protein polymers are not cytotoxic (IC50 > 200 ?g/mL), while the doxorubicin-loaded SE8Y nanoparticles showed a 1.8-fold higher cytotoxicity than the free drug. Confocal laser scanning microscopy (CLSM) and flow cytometry indicate significant uptake of the SE8Y nanoparticles by the cells and suggest internalization of the nanoparticles through endocytosis. This study provides an all-aqueous, facile method to prepare nanoscale, drug-loaded SELPs packages with potential for tumor cell treatments. PMID:24527851

Xia, Xiao-Xia; Wang, Ming; Lin, Yinan; Xu, Qiaobing; Kaplan, David L

2014-03-10

181

Tip-induced micropatterning of silk fibroin protein using in situ solution atomic force microscopy.  

PubMed

Silk fibroin (SF) is a promising candidate for a variety of application in the fields of tissue engineering, drug delivery, and biomedical optics. Recent research has already begun to explore the construction of nano- and micropatterned SF films under ambient environment. The structure and biocompatibility of SF are dependent on SF state (solution or solid) and the method of drying the SF solution to prepare various biomaterials such as films, sponges, and fibers. Therefore, it is important to explore the construction of SF nano- and micropatterns under aqueous solution. This paper reports a novel application of atomic force microscopy (AFM) under liquid for direct deposition of the relatively hydrophobic protein SF onto hydrophilic mica. We demonstrate that the AFM tip, in either the contact or the tapping mode, can fabricate SF micropatterns on mica with controlled surface topography. We show that the deposition process requires a mechanical force-induced SF sol-gel transition followed by a transfer to the mica surface at the tip-surface contact, and the efficiency of this process depends on not only AFM operation mode but also the SF bulk concentration, the SF amount on mica, and the AFM tip spring constant. PMID:23276203

Zhong, Jian; Ma, Mengjia; Zhou, Juan; Wei, Daixu; Yan, Zhiqiang; He, Dannong

2013-02-01

182

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

PubMed

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. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:988-994, 2015. PMID:25546241

Doblhofer, Elena; Scheibel, Thomas

2015-03-01

183

High level biosynthesis of a silk-elastin-like protein in E. coli.  

PubMed

Silk-elastin-like proteins (SELPs) have enormous potential for use as customizable biomaterials in numerous biomedical and materials applications, yet success in harnessing this potential has been limited by the lack of a commercially viable industrially relevant production process. We have developed a scalable fed-batch production approach which enables a SELP volumetric productivity of 4.3 g L(-1) with E. coli BL21(DE3). This is the highest SELP productivity reported to date and is 50-fold higher than that reported by other groups. As compared to typical fed-batch processes, high preinduction growth rates and low inducer and oxygen concentrations are allowed whereas reduced postinduction feeding rates are preferred. Limiting factors were identified and productivity was found to be strongly influenced by a trade-off between the rate of production and plasmid stability. The process developed is robust, reproducible, and applicable to scale up to the industrial level and moves these biopolymers a step closer to the marketplace. PMID:24884240

Collins, Tony; Barroca, Mário; Branca, Fernando; Padrão, Jorge; Machado, Raul; Casal, Margarida

2014-07-14

184

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

PubMed Central

The development of a spider silk-manufacturing process is of great interest. However, there are serious problems with natural manufacturing through spider farming, and standard recombinant protein production platforms have provided limited progress due to their inability to assemble spider silk proteins into fibers. Thus, we used piggyBac vectors 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 proteins integrated in an extremely stable manner. Furthermore, these composite fibers were, on average, tougher than the parental silkworm silk fibers and as tough as native dragline spider silk fibers. These results demonstrate that silkworms can be engineered to manufacture composite silk fibers containing stably integrated spider silk protein sequences, which significantly improve the overall mechanical properties of the parental silkworm silk fibers. PMID:22215590

Teulé, Florence; Miao, Yun-Gen; Sohn, Bong-Hee; Kim, Young-Soo; Hull, J. Joe; Fraser, Malcolm J.; Lewis, Randolph V.; Jarvis, Donald L.

2012-01-01

185

Spider silk as rubber  

NASA Astrophysics Data System (ADS)

The silks produced by spiders are exceptional structural materials. Although their tensile strengths are similar to those of cellulose, collagen and chitin, their extensibilities are considerably greater1,2. Thus, the energy required to break spider silk (that is, its toughness) can be 10 times greater than for these other biological materials. The silks are crystalline proteins, with substantial portions of their polypeptide chains forming crystallites of stacked ? -pleated sheets3-6. However, the extensibility of silk is attributed to noncrystalline regions, which appear amorphous on X-ray diffraction7,8. The mechanical properties and macromolecular conformation of these amorphous regions have been the subject of much speculation1,2,7-10, but little direct experimentation. Here we show that these regions can behave as rubber networks and exhibit rubber-like or entropy elasticity.

Gosline, John M.; Denny, Mark W.; Demont, M. Edwin

1984-06-01

186

Characterizing the Secondary Protein Structure of Black Widow Dragline Silk Using Solid-State NMR & X-ray Diffraction  

PubMed Central

This study provides a detailed secondary structural characterization of major ampullate dragline silk from Latrodectus hesperus (black widow) spiders. X-ray diffraction results show that the structure of black widow major ampullate silk fibers is comprised of stacked ?-sheet nanocrystallites oriented parallel to the fiber axis and an amorphous region with oriented (anisotropic) and isotropic components. The combination of two-dimensional (2D) 13C-13C through-space and through-bond solid-state NMR experiments provide chemical shifts that are used to determine detailed information about amino acid motif secondary structure in black widow spider dragline silk. Individual amino acids are incorporated into different repetitive motifs that make up the majority of this protein-based biopolymer. From the solid-state NMR measurements, we assign distinct secondary conformations to each repetitive amino acid motif and hence to the amino acids that make up the motifs. Specifically, alanine is incorporated in ?-sheet (poly(Alan) and poly(Gly-Ala)), 31-helix (poly(Gly-Gly-Xaa), and ?-helix (poly(Gln-Gln-Ala-Tyr)) components. Glycine is determined to be in ?-sheet (poly(Gly-Ala)) and 31-helical (poly(Gly-Gly-Xaa)) regions, while serine is present in ?-sheet (poly(Gly-Ala-Ser)), 31-helix (poly(Gly-Gly-Ser)), and ?-turn (poly(Gly-Pro-Ser)) structures. These various motif-specific secondary structural elements are quantitatively correlated to the primary amino acid sequence of major ampullate spidroin 1 and 2 (MaSp1 and MaSp2) and are shown to form a self-consistent model for black widow dragline silk. PMID:24024617

Jenkins, Janelle E.; Sampath, Sujatha; Butler, Emily; Kim, Jihyun; Henning, Robert W.; Holland, Gregory P.; Yarger, Jeffery L.

2013-01-01

187

Characterizing the secondary protein structure of black widow dragline silk using solid-state NMR and X-ray diffraction.  

PubMed

This study provides a detailed secondary structural characterization of major ampullate dragline silk from Latrodectus hesperus (black widow) spiders. X-ray diffraction results show that the structure of black widow major ampullate silk fibers is comprised of stacked ?-sheet nanocrystallites oriented parallel to the fiber axis and an amorphous region with oriented (anisotropic) and isotropic components. The combination of two-dimensional (2D) (13)C-(13)C through-space and through-bond solid-state NMR experiments provide chemical shifts that are used to determine detailed information about the amino acid motif secondary structure in black widow spider dragline silk. Individual amino acids are incorporated into different repetitive motifs that make up the majority of this protein-based biopolymer. From the solid-state NMR measurements, we assign distinct secondary conformations to each repetitive amino acid motif and, hence, to the amino acids that make up the motifs. Specifically, alanine is incorporated in ?-sheet (poly(Alan) and poly(Gly-Ala)), 3(1)-helix (poly(Gly-Gly-Xaa), and ?-helix (poly(Gln-Gln-Ala-Tyr)) components. Glycine is determined to be in ?-sheet (poly(Gly-Ala)) and 3(1)-helical (poly(Gly-Gly-X(aa))) regions, while serine is present in ?-sheet (poly(Gly-Ala-Ser)), 3(1)-helix (poly(Gly-Gly-Ser)), and ?-turn (poly(Gly-Pro-Ser)) structures. These various motif-specific secondary structural elements are quantitatively correlated to the primary amino acid sequence of major ampullate spidroin 1 and 2 (MaSp1 and MaSp2) and are shown to form a self-consistent model for black widow dragline silk. PMID:24024617

Jenkins, Janelle E; Sampath, Sujatha; Butler, Emily; Kim, Jihyun; Henning, Robert W; Holland, Gregory P; Yarger, Jeffery L

2013-10-14

188

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

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

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

2012-01-01

189

De novo engineering of reticular connective tissue in vivo by silk fibroin nonwoven materials  

Microsoft Academic Search

Biologically tolerated biomaterials are the focus of intense research. In this work, we examined the biocompatibility of three-dimensional (3D) nonwovens of sericin-deprived, Bombyx mori silk fibroin (SF) in ?-sheet form implanted into the subcutaneous tissue of C57BL6 mice, using sham-operated mice as controls. Both groups of mice similarly healed with no residual problem. Macroarray analysis showed that an early (day

Ilaria Dal Pra; Giuliano Freddi; Jasminka Minic; Anna Chiarini; Ubaldo Armato

2005-01-01

190

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...

191

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

PubMed

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

Pahlevan, Mahdi; Toivakka, Martti; Alam, Parvez

2014-08-01

192

Effect of sodium chloride on the structure and stability of spider silk's N-terminal protein domain.  

PubMed

A spider's ability to store silk protein solutions at high concentration is believed to be related to the protein's terminal domains. It has been suggested that a shift in salt concentration and pH can have a significant influence on the assembly process. Based on experimental data, a model has been proposed in which the N-terminal domain exists as a monomer during storage and assembles into a homodimer upon spinning. Here we perform a systematic computational study using atomistic, coarse-grained and well-tempered metadynamics simulation to understand how the NaCl concentration in the solution affects the N-terminal domain of the silk protein. Our results show that a high salt concentration, as found during storage, weakens key salt bridges between the monomers, inducing a loss in bond energy by 28.6% in a single salt bridge. As a result dimer formation is less likely as 35.5% less energy is required to unfold the dimer by mechanical force. Conversely, homodimer formation appears to be more likely at low salt concentrations as the salt bridge stays at the lower energy state. The link between salt concentration, structure and stability of the N-terminal domain provides a possible mechanism that prevents premature fiber formation during storage. PMID:23833703

Gronau, Greta; Qin, Zhao; Buehler, Markus J

2013-03-01

193

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

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

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

1997-06-01

194

Characterization and real-time imaging of gene expression of adenovirus embedded silk-elastinlike protein polymer hydrogels.  

PubMed

Transient expression levels, vector dissemination and toxicities associated with adenoviral vectors have prompted the usage of matrices for localized and controlled gene delivery. Two recombinant silk-elastinlike protein polymer analogues, SELP-47K and SELP-415K, consisting of different lengths and ratios of silk and elastin units, were previously shown to be injectable hydrogels capable of matrix-mediated controlled adenoviral gene delivery. Reported here is a study of spatiotemporal control over adenoviral gene expression with these SELP analogues in a human tumor xenograft model of head and neck cancer using whole animal imaging. Real-time images of viral expression levels indicate that polymer concentration and polymer structure are predominant factors that affect viral release and, thus, viral transfection. Decrease in polymer concentration and increase in polymer elastin content results in greater release, probably due to changes in the network structure of the hydrogel. To better understand this relationship, macro- and microstructural properties of the hydrogels were analyzed using dynamic mechanical analysis (DMA) and transmission electron microscopy (TEM). The results confirm that the concentration and the elastin content of the protein polymer affect the pore size of the hydrogel by changing the physical constraints of the SELP fibril network and the degree of hydration of the SELP fibrils. The potential to modulate viral release using SELP hydrogel delivery vehicles that can be injected intratumorally by minimally invasive techniques holds significant promise for the delivery of therapeutic viruses. PMID:18763804

Cresce, Arthur von Wald; Dandu, Ramesh; Burger, Angelika; Cappello, Joseph; Ghandehari, Hamidreza

2008-01-01

195

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

E-print Network

: spider silks from Nephila clavipes (Tetragnathidae) and Araneus bicentenarius (Araneidae)1 Richard Center, Natick, MA 01760, USA Received 21 May 1997; accepted 29 September 1997 Abstract Spider silks have obtained two variants of the Spidroin 1 (NCF-1) silk gene sequence from Nephila clavipes. One

Beckwitt, Richard

196

Silk-I is a Hydrate  

NASA Astrophysics Data System (ADS)

In order to study the thermodynamics of supramolecular self-assembly phenomena in crystallizable segments of Bombyx mori silkworm silk fibroin, the osmotic stress method was applied. Precise control of the protein and LiBr concentration enables us to manipulate the phases of the silk fibroin, thus the method provided a means for the direct investigation of microscopic and thermodynamic details of these intermolecular interactions in aqueous media. Under the experimental osmotic pressure range of 0.2-7.6MPa applied by poly(ethylene glycol), it is apparent that as osmotic pressure increases, silk fibroin molecules are crowded together to form a water-soluble crystalline mesophase (silk-I), and then with further increase in osmotic pressure become anti-parallel beta-sheet structure (silk-II). A partial ternary phase diagram of water-silk fibroin-LiBr was constructed based on X-ray results. The microscopic data clearly indicate that silk-I is a hydrated structure since water of hydration is found to be lost in the conversion to silk-II. A rough estimate of the number of water molecules lost by the structure upon converting from silk-I to silk-II has been made, and found to be about 2.2 per [GAGAGS] hexapeptide. The phase diagram suggests that processes, such as natural, regenerated or biomimetic silk fibroin spinning, that convert random coil fibroin to silk-II are likely to require a silk-I intermediate structure. This intermediate is necessary for the pre-alignment of the fibroin molecules and to shed water. We tried to utilize hydrated silk-I mesophase to pre-structure the silk fibroin by applying osmotic pressure, for the wet-spinning of silk fibroin.

Sohn, Sungkyun; Strey, Helmut H.; Gido, Samuel P.

2004-03-01

197

Functional design of spider's silk  

NASA Astrophysics Data System (ADS)

The orb-web weaving spiders produce a broad range of high performance structural fibers (i.e. silks) with mechanical properties that are superbly matched to their function. Our interest in these materials stems both from an interest in the biology of the spiders and the design of their webs and also from a desire to discover principles of mechanical design of protein-based structural materials that can guide the development of novel bio-engineered materials. All spiders produce silks, but the orb-web weaving spiders are unique in their ability to produce seven different silks, each from distinct gland/spinneret complexes. Considering the wide diversity of spider species, there is likely to be an enormous range of material properties available in spider silk. However, at present, we only have information on two species of spiders, and only two of their seven silks have been studied in any detail. These are: (1) the silk produced by the major ampullate gland, which forms the safety-line or dragline of the spider and also is used to form the frame of its orb-web, and (2) the viscid silk produced by the flagelliform gland, which forms the glue-covered catching spiral of the web. In this paper we describe several aspects of the mechanical design of the dragline and viscid silks produced by the spider Araneus diadematus.

Gosline, John; Guerette, Paul; Ortlepp, Christine

1996-02-01

198

Conformational transitions in model silk peptides.  

PubMed Central

Protein structural transitions and beta-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 beta-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 beta-sheet form, were characterized using Fourier transform infrared spectroscopy, circular dichroism, and electron diffraction. Polymorphs resembling the silk I, silk II (beta-sheet), and silk III (threefold polyglycine II-like helix) crystal structures were identified for the peptide fibroin C (GAGAGS repetitive sequence). Two peptides based on silk amorphous sequences, fibroin A (GAGAGY) and fibroin V (GDVGGAGATGGS), crystallized as silk I under most conditions. Methanol treatment of fibroin A resulted in a gradual transition from silk I to silk II, with an intermediate state involving a high proportion of beta-turns. Attenuated total reflectance Fourier transform infrared spectroscopy has been used to observe conformational changes as the peptides adsorb from solution onto a hydrophobic surface. Fibroin C has a beta-strand structure in solution but adopts a silk I-like structure upon adsorption, which when dried on the ZnSe crystal contains silk III crystallites. PMID:10777765

Wilson, D; Valluzzi, R; Kaplan, D

2000-01-01

199

The Effect of Sericin from Various Extraction Methods on Cell Viability and Collagen Production  

PubMed Central

Silk sericin (SS) can accelerate cell proliferation and attachment; however, SS can be extracted by various methods, which result in SS exhibiting different physical and biological properties. We found that SS produced from various extraction methods has different molecular weights, zeta potential, particle size and amino acid content. The MTT assay indicated that SS from all extraction methods had no toxicity to mouse fibroblast cells at concentrations up to 40 ?g/mL after 24 h incubation, but SS obtained from some extraction methods can be toxic at higher concentrations. Heat-degraded SS was the least toxic to cells and activated the highest collagen production, while urea-extracted SS showed the lowest cell viability and collagen production. SS from urea extraction was severely harmful to cells at concentrations higher than 100 ?g/mL. SS from all extraction methods could still promote collagen production in a concentration-dependent manner, even at high concentrations that are toxic to cells. PMID:20559510

Aramwit, Pornanong; Kanokpanont, Sorada; Nakpheng, Titpawan; Srichana, Teerapol

2010-01-01

200

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

PubMed

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

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

201

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

PubMed

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

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

2012-12-10

202

Reproducing Natural Spider Silks’ Copolymer Behavior in Synthetic Silk Mimics  

PubMed Central

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

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

2012-01-01

203

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

PubMed

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

Humenik, Martin; Magdeburg, Michael; Scheibel, Thomas

2014-06-01

204

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

PubMed

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

Correa-Garhwal, Sandra M; Garb, Jessica E

2014-12-01

205

An innovative bi-layered wound dressing made of silk and gelatin for accelerated wound healing.  

PubMed

In this study, the novel silk fibroin-based bi-layered wound dressing was developed. Wax-coated silk fibroin woven fabric was introduced as a non-adhesive layer while the sponge made of sericin and glutaraldehyde-crosslinked silk fibroin/gelatin was fabricated as a bioactive layer. Wax-coated silk fibroin fabrics showed improved mechanical properties compared with the non-coated fabrics, but less adhesive than the commercial wound dressing mesh. This confirmed by results of peel test on both the partial- and full-thickness wounds. The sericin-silk fibroin/gelatin spongy bioactive layers showed homogeneous porous structure and controllable biodegradation depending on the degree of crosslinking. The bi-layered wound dressings supported the attachment and proliferation of L929 mouse fibroblasts, particularly for the silk fibroin/gelatin ratio of 20/80 and 0.02% GA crosslinked. Furthermore, we proved that the bi-layered wound dressings promoted wound healing in full-thickness wounds, comparing with the clinically used wound dressing. The wounds treated with the bi-layered wound dressings showed the greater extent of wound size reduction, epithelialization, and collagen formation. The superior properties of the silk fibroin-based bi-layered wound dressings compared with those of the clinically used wound dressings were less adhesive and had improved biological functions to promote cell activities and wound healing. This novel bi-layered wound dressing should be a good candidate for the healing of full-thickness wounds. PMID:22771972

Kanokpanont, Sorada; Damrongsakkul, Siriporn; Ratanavaraporn, Juthamas; Aramwit, Pornanong

2012-10-15

206

Silk: Caterpillars  

NSDL National Science Digital Library

If you listen closely, you can hear the sounds of a critical element in the production of silk--silkworm caterpillars dining on mulberry leaves. This episode of the two-minute radio show Pulse of the Planet focuses on how silkworms are raised. In the episode, available here in MP3 audio and text formats, a guest biochemist describes the silk farming process, noting the silkworms' dependence on large quantities of human-supplied mulberry leaves. The host points out how much silkworms grow in length and mass as evidence for why so many leaves are needed. Copyright 2005 Eisenhower National Clearinghouse

Planet, Pulse O.

2005-11-23

207

Composition and Hierarchical Organisation of a Spider Silk  

PubMed Central

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

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

2007-01-01

208

The elaborate structure of spider silk  

PubMed Central

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

Römer, Lin

2008-01-01

209

Microdissection of black widow spider silk-producing glands.  

PubMed

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of developing offspring. Spiders accomplish these tasks by spinning several distinct fiber types that have diverse mechanical properties. Such specialization of fiber types has occurred through the evolution of different silk-producing glands, which function as small biofactories. These biofactories manufacture and store large quantities of silk proteins for fiber production. Through a complex series of biochemical events, these silk proteins are converted from a liquid into a solid material upon extrusion. Mechanical studies have demonstrated that spider silks are stronger than high-tensile steel. Analyses to understand the relationship between the structure and function of spider silk threads have revealed that spider silk consists largely of proteins, or fibroins, that have block repeats within their protein sequences. Common molecular signatures that contribute to the incredible tensile strength and extensibility of spider silks are being unraveled through the analyses of translated silk cDNAs. Given the extraordinary material properties of spider silks, research labs across the globe are racing to understand and mimic the spinning process to produce synthetic silk fibers for commercial, military and industrial applications. One of the main challenges to spinning artificial spider silk in the research lab involves a complete understanding of the biochemical processes that occur during extrusion of the fibers from the silk-producing glands. Here we present a method for the isolation of the seven different silk-producing glands from the cobweaving black widow spider, which includes the major and minor ampullate glands [manufactures dragline and scaffolding silk], tubuliform [synthesizes egg case silk], flagelliform [unknown function in cob-weavers], aggregate [makes glue silk], aciniform [synthesizes prey wrapping and egg case threads] and pyriform [produces attachment disc silk]. This approach is based upon anesthetizing the spider with carbon dioxide gas, subsequent separation of the cephalothorax from the abdomen, and microdissection of the abdomen to obtain the silk-producing glands. Following the separation of the different silk-producing glands, these tissues can be used to retrieve different macromolecules for distinct biochemical analyses, including quantitative real-time PCR, northern- and western blotting, mass spectrometry (MS or MS/MS) analyses to identify new silk protein sequences, search for proteins that participate in the silk assembly pathway, or use the intact tissue for cell culture or histological experiments. PMID:21248709

Jeffery, Felicia; La Mattina, Coby; Tuton-Blasingame, Tiffany; Hsia, Yang; Gnesa, Eric; Zhao, Liang; Franz, Andreas; Vierra, Craig

2011-01-01

210

Microdissection of Black Widow Spider Silk-producing Glands  

PubMed Central

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of developing offspring 1,2. Spiders accomplish these tasks by spinning several distinct fiber types that have diverse mechanical properties. Such specialization of fiber types has occurred through the evolution of different silk-producing glands, which function as small biofactories. These biofactories manufacture and store large quantities of silk proteins for fiber production. Through a complex series of biochemical events, these silk proteins are converted from a liquid into a solid material upon extrusion. Mechanical studies have demonstrated that spider silks are stronger than high-tensile steel 3. Analyses to understand the relationship between the structure and function of spider silk threads have revealed that spider silk consists largely of proteins, or fibroins, that have block repeats within their protein sequences 4. Common molecular signatures that contribute to the incredible tensile strength and extensibility of spider silks are being unraveled through the analyses of translated silk cDNAs. Given the extraordinary material properties of spider silks, research labs across the globe are racing to understand and mimic the spinning process to produce synthetic silk fibers for commercial, military and industrial applications. One of the main challenges to spinning artificial spider silk in the research lab involves a complete understanding of the biochemical processes that occur during extrusion of the fibers from the silk-producing glands. Here we present a method for the isolation of the seven different silk-producing glands from the cobweaving black widow spider, which includes the major and minor ampullate glands [manufactures dragline and scaffolding silk] 5,6, tubuliform [synthesizes egg case silk] 7,8, flagelliform [unknown function in cob-weavers], aggregate [makes glue silk], aciniform [synthesizes prey wrapping and egg case threads] 9 and pyriform [produces attachment disc silk] 10. This approach is based upon anesthetizing the spider with carbon dioxide gas, subsequent separation of the cephalothorax from the abdomen, and microdissection of the abdomen to obtain the silk-producing glands. Following the separation of the different silk-producing glands, these tissues can be used to retrieve different macromolecules for distinct biochemical analyses, including quantitative real-time PCR, northern- and western blotting, mass spectrometry (MS or MS/MS) analyses to identify new silk protein sequences, search for proteins that participate in the silk assembly pathway, or use the intact tissue for cell culture or histological experiments. PMID:21248709

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

2011-01-01

211

Research Article Antheraea pernyi Silk Fiber: A Potential Resource for Artificially Biospinning Spider Dragline Silk  

E-print Network

License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The outstanding properties of spider dragline silk are likely to be determined by a combination of the primary sequences and the secondary structure of the silk proteins. Antheraea pernyi silk has more similar sequences to spider dragline silk than the silk from its domestic counterpart, Bombyx mori. This makes it much potential as a resource for biospinning spider dragline silk. This paper further verified its possibility as the resource from the mechanical properties and the structures of the A. pernyi silks prepared by forcible reeling. It is surprising that the stress-strain curves of the A. pernyi fibers show similar sigmoidal shape to those of spider dragline silk. Under a controlled reeling speed of 95 mm/s, the breaking energy was 1.04 × 10 5 J/kg, the tensile strength was 639 MPa and the initial modulus was 9.9 GPa. It should be noted that this breaking energy of the A. pernyi silk approaches that of spider dragline silk. The tensile properties, the optical orientation and the ?-sheet structure contents of the silk fibers are remarkably increased by raising the spinning speeds up to 95 mm/s. 1.

Yaopeng Zhang; Hongxia Yang; Huili Shao; Xuechao Hu

2010-01-01

212

Mechanism of silk processing in insects and spiders  

Microsoft Academic Search

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

Hyoung-Joon Jin; David L. Kaplan

2003-01-01

213

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

SciTech Connect

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.

Tsuboi, Yasuyuki; Goto, Masaharu; Itaya, Akira

2001-06-15

214

Molecular nanosprings in spider capture-silk threads  

NASA Astrophysics Data System (ADS)

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.

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

2003-04-01

215

Modeling the Strength of ?-sheet Structures in Silk Crystals and Protein Molecules  

NASA Astrophysics Data System (ADS)

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

Grubb, David

2012-02-01

216

Post-secretion processing influences spider silk performance.  

PubMed

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

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

2012-10-01

217

Post-secretion processing influences spider silk performance  

PubMed Central

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

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

2012-01-01

218

Spider Silk Fibers Spun from Soluble Recombinant Silk Produced in Mammalian Cells  

NASA Astrophysics Data System (ADS)

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

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

2002-01-01

219

Functionalized silk biomaterials for wound healing.  

PubMed

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

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

2013-01-01

220

Hydrodynamical properties of recombinant spider silk proteins: Effects of pH, salts and shear, and implications for the spinning process.  

PubMed

We have investigated the effect of pH, salts and shear on the hydrodynamical diameter of recombinant major ampullate (MA) rMaSpI silk proteins in solution as a function of time using (1) H solution NMR spectroscopy. The results indicate that the silk proteins in solution are composed of two diffusing populations, a high proportion of "native" solubilized proteins and a small amount of high molecular weight oligomers. Similar results are observed with the MA gland content. Salts help maintaining the proteins in a compact form in solution over time and inhibit aggregation, the absence of salts triggering protein assembly leading to a gel state. Moreover, the aggregation kinetics of rMaSpI at low salt concentration accelerates as the pH is close to the isoelectric point of the proteins, suggesting that the pH decrease tends to slow down aggregation. The data also support the strong impact of shear on the spinning process and suggest that the assembly is driven by a nucleation conformational conversion mechanism. Thus, the adjustment of the physicochemical conditions in the ampulla seems to promote a stable, long term storage. In addition, the optimization of protein conformation as well as their unfolding and aggregation propensity in the duct leads to a specifically organized structure. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 582-593, 2013. PMID:23794371

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

2013-09-01

221

Hypoxia and Amino Acid Supplementation Synergistically Promote the Osteogenesis of Human Mesenchymal Stem Cells on Silk Protein Scaffolds  

PubMed Central

Tailoring tissue engineering strategies to match patient- and tissue-specific bone regeneration needs offers to improve clinical outcomes. As a step toward this goal, osteogenic outcomes and metabolic parameters were assessed when varying inputs into the bone formation process. Silk protein scaffolds seeded with human mesenchymal stem cells in osteogenic differentiation media were used to study in vitro osteogenesis under varied conditions of amino acid (lysine and proline) concentration and oxygen level. The cells were assessed to probe how the microenvironment impacted metabolic pathways and thus osteogenesis. The most favorable osteogenesis outcomes were found in the presence of low (5%) oxygen combined with high lysine and proline concentrations during in vitro cultivation. This same set of culture conditions also showed the highest glucose consumption, lactate synthesis, and certain amino acid consumption rates. On the basis of these results and known pathways, a holistic metabolic model was derived which shows that lysine and proline supplements as well as low (5%) oxygen levels regulate collagen matrix synthesis and thereby rates of osteogenesis. This study establishes early steps toward a foundation for patient- and tissue-specific matches between metabolism, repair site, and tissue engineering approaches toward optimized bone regeneration. PMID:20673134

Sengupta, Sejuti; Park, Sang-Hyug; Patel, Atur; Carn, Julia; Lee, Kyongbum

2010-01-01

222

Silk fibroin microparticles as carriers for delivery of human recombinant bone morphogenetic protein-2: in vitro and in vivo bioactivity.  

PubMed

The in vitro and in vivo efficiency of fibroin microparticles as a delivery carrier for bone morphogenetic protein-2 (BMP-2) was evaluated. BMP-2 was encapsulated in silk fibroin particles that were produced by a simple and very mild processing method. The dose-response of BMP-2-loaded fibroin particles was examined in C2C12 cells, after 5 days of culture. The BMP-2 retained most of its activity as observed by the increase in alkaline phosphatase activity, which was much higher when BMP-2 was encapsulated into the particles rather than just surface-adsorbed. After 2 weeks of culture, increased mineralization was observed with BMP-2-loaded particles in comparison to soluble added growth factor. No significant cytotoxicity was detected. When implanted in a rat ectopic model, bone formation was observed by in vivo micro-computed tomography after 2 and 4 weeks postimplantation, with particles loaded with 5 or 12.5?microg BMP-2. An increase in bone density was observed over time. Histology revealed further evidence of ectopic bone formation, observed by strong alizarin red staining and osteocalcin immunostaining. Our findings show that fibroin microparticles may present an interesting option for future clinical applications in the bone tissue engineering field, and therefore, further studies have been planned. PMID:19958078

Bessa, Paulo César; Balmayor, Elizabeth R; Hartinger, Joachim; Zanoni, Gerald; Dopler, Daniela; Meinl, Alexandra; Banerjee, Asmita; Casal, Margarida; Redl, Heinz; Reis, Rui L; van Griensven, Martijn

2010-10-01

223

Spider Webs and Silks.  

ERIC Educational Resources Information Center

Compares the attributes of the silk from spiders with those of the commercially harvested silk from silkworms. Discusses the evolution, design, and effectiveness of spider webs; the functional mechanics of the varieties of silk that can be produced by the same spider; and the composite, as well as molecular, structure of spider silk thread. (JJK)

Vollrath, Fritz

1992-01-01

224

The promotion of osteochondral repair by combined intra-articular injection of parathyroid hormone-related protein and implantation of a bi-layer collagen-silk scaffold.  

PubMed

The repair of osteochondral defects can be enhanced with scaffolds but is often accompanied with undesirable terminal differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Parathyroid hormone-related protein (PTHrP) has been shown to inhibit aberrant differentiation, but administration at inappropriate time points would have adverse effects on chondrogenesis. This study aims to develop an effective tissue engineering strategy by combining PTHrP and collagen-silk scaffold for osteochondral defect repair. The underlying mechanisms of the synergistic effect of combining PTHrP administration with collagen-silk scaffold implantation for rabbit knee joint osteochondral defect repair were investigated. In vitro studies showed that PTHrP treatment significantly reduced Alizarin Red staining and expression of terminal differentiation-related markers. This is achieved in part through blocking activation of the canonical Wnt/?-catenin signaling pathway. For the in vivo repair study, intra-articular injection of PTHrP was carried out at three different time windows (4-6, 7-9 and 10-12 weeks) together with implantation of a bi-layer collagen-silk scaffold. Defects treated with PTHrP at the 4-6 weeks time window exhibited better regeneration (reconstitution of cartilage and subchondral bone) with minimal terminal differentiation (hypertrophy, ossification and matrix degradation), as well as enhanced chondrogenesis (cell shape, Col2 and GAG accumulation) compared with treatment at other time windows. Furthermore, the timing of PTHrP administration also influenced PTHrP receptor expression, thus affecting the treatment outcome. Our results demonstrated that intra-articular injection of PTHrP at 4-6 weeks post-injury together with collagen-silk scaffold implantation is an effective strategy for inhibiting terminal differentiation and enhancing chondrogenesis, thus improving cartilage repair and regeneration in a rabbit model. PMID:23702148

Zhang, Wei; Chen, Jialin; Tao, Jiadong; Hu, Changchang; Chen, Longkun; Zhao, Hongshi; Xu, Guowei; Heng, Boon C; Ouyang, Hong Wei

2013-08-01

225

Biodegradation of Silk Biomaterials  

PubMed Central

Silk fibroin from the silkworm, Bombyx mori, has excellent properties such as biocompatibility, biodegradation, non-toxicity, adsorption properties, etc. As a kind of ideal biomaterial, silk fibroin has been widely used since it was first utilized for sutures a long time ago. The degradation behavior of silk biomaterials is obviously important for medical applications. This article will focus on silk-based biomaterials and review the degradation behaviors of silk materials. PMID:19468322

Cao, Yang; Wang, Bochu

2009-01-01

226

Silk film biomaterials for ocular surface repair  

NASA Astrophysics Data System (ADS)

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.

Lawrence, Brian David

227

Silk layering as studied with neutron reflectivity.  

PubMed

Neutron reflectivity (NR) measurements of ultrathin surface films (below 30 nm) composed of Bombyx mori silk fibroin protein in combination with atomic force microscopy and ellipsometry were used to reveal the internal structural organization in both dry and swollen states. Reconstituted aqueous silk solution deposited on a silicon substrate using the spin-assisted layer-by-layer (SA-LbL) technique resulted in a monolayer silk film composed of random nanofibrils with constant scattering length density (SLD). However, a vertically segregated ordering with two different regions has been observed in dry, thicker, seven-layer SA-LbL silk films. The vertical segregation of silk multilayer films indicates the presence of a different secondary structure of silk in direct contact with the silicon oxide surface (first 6 nm). The layered structure can be attributed to interfacial ?-sheet crystallization and the formation of well-developed nanofibrillar nanoporous morphology for the initially deposited silk surface layers with the preservation of less dense, random coil secondary structure for the layers that follow. This segregated structure of solid silk films defines their complex nonuniform behavior in the D(2)O environment with thicker silk films undergoing delamination during swelling. For a silk monolayer with an initial thickness of 6 nm, we observed the increase in the effective thickness by 60% combined with surprising decrease in density. Considering the nanoporous morphology of the hydrophobic silk layer, we suggested that the apparent increase in its thickness in liquid environment is caused by the air nanobubble trapping phenomenon at the liquid-solid interface. PMID:22697306

Wallet, Brett; Kharlampieva, Eugenia; Campbell-Proszowska, Katie; Kozlovskaya, Veronika; Malak, Sidney; Ankner, John F; Kaplan, David L; Tsukruk, Vladimir V

2012-08-01

228

Statistical approaches for investigating silk properties  

NASA Astrophysics Data System (ADS)

Amino acid repeats or motifs have engendered interest because of their significance for protein physical characteristics as well as folding properties. Spider dragline silk proteins are unique because they are composed of long repetitive sections and relatively short non-repetitive sections that are known to interact to generate the very peculiar mechanical and solubility properties of silk. Computational analysis compared with in vitro measurements suggest that the silks achieve their unique pattern of extreme solubility inside the spider glands/complete insolubility outside by correlating their repetitive hydrophobic regions through a type of stochastic resonance, generated by the addition of the non-repetitive sequences to a basically periodic hydrophobicity pattern.

Zbilut, J. P.; Scheibel, T.; Huemmerich, D.; Webber, C. L., Jr.; Colafranceschi, M.; Giuliani, A.

2006-02-01

229

Phosphorylated silk fibroin matrix for methotrexate release.  

PubMed

Silk-based matrix was produced for delivery of a model anticancer drug, methotrexate (MTX). The calculation of net charge of silk fibroin and MTX was performed to better understand the electrostatic interactions during matrix formation upon casting. Silk fibroin films were cast at pH 7.2 and pH 3.5. Protein kinase A was used to prepare phosphorylated silk fibroin. The phosphorylation content of matrix was controlled by mixing at specific ratios the phosphorylated and unphosphorylated solutions. In vitro release profiling data suggest that the observed interactions are mainly structural and not electrostatical. The release of MTX is facilitated by use of proteolytic enzymes and higher pHs. The elevated ?-sheet content and crystallinity of the acidified-cast fibroin solution seem not to favor drug retention. All the acquired data underline the prevalence of structural interactions over electrostatical interactions between methotrexate and silk fibroin. PMID:25435334

Volkov, Vadim; Sárria, Marisa P; Gomes, Andreia C; Cavaco-Paulo, Artur

2015-01-01

230

Continuous production of flexible fibers from transgenically produced honeybee silk proteins.  

PubMed

Flexible and solvent stable fibers are produced after concentrated recombinant honeybee protein solutions are extruded into a methanol bath, dried, drawn in aqueous methanol, then covalently cross-linked using dry heat. Proteins in solution are predominantly coiled coil. Significant levels of non-orientated ß-sheets form during drying or after coagulation in aqueous methanol. Drawing generally aligns the coiled coil component parallel with the fibre axis and ß-sheet component perpendicular to the fiber axis. The fibres are readily handled, stable in the strong protein denaturants, urea and guanidinium, and suitable for a range of applications such as weaving and knitting. PMID:23881528

Poole, Jacinta; Church, Jeffrey S; Woodhead, Andrea L; Huson, Mickey G; Sriskantha, Alagacone; Kyratzis, Ilias L; Sutherland, Tara D

2013-10-01

231

Silkmapin of Hyriopsis cumingii, a novel silk-like shell matrix protein involved in nacre formation.  

PubMed

Understanding the role of matrix proteins in nacre formation and biomineralization in mollusks is important for the pearl industry. In this study, the gene encoding the novel Hyriopsis cumingii shell matrix protein silkmapin was characterized. The gene encodes a protein of 30.89kDa in which Gly accounts for 34.41% of the amino acid content, and the C-terminal region binds Ca(2+). Secondary structure prediction indicated a predominantly ?-fold and a structure typical of filamentous proteins. Real-time quantitative PCR and in situ hybridization showed that silkmapin was expressed in epithelial cells at the edge and pallial of mantle tissue, indicated that silkmapin play roles in the shell nacreous and prismatic layer formation. Further real-time PCR results indicated an involvement in pearl formation via nucleation of calcium carbonate prior to formation of the nacre. PMID:25447895

Liu, Xiaojun; Dong, Shaojian; Jin, Can; Bai, Zhiyi; Wang, Guiling; Li, Jiale

2015-01-25

232

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

PubMed

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, (13)C 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

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

2014-01-01

233

Brown recluse spider's nanometer scale ribbons of stiff extensible silk.  

PubMed

The silk of the recluse spider features a ribbon-like morphology unlike any other spider silk or synthetically spun polymer fiber. These protein ribbons represent free-standing polymer films with a thickness of about 50 nm. Stress-strain characterization of individual fibers via atomic force microscopy reveals that these ribbons, only a few molecular layers of protein thin, rival the mechanical performance of the best silks. PMID:24352987

Schniepp, Hannes C; Koebley, Sean R; Vollrath, Fritz

2013-12-23

234

Silk fibroin rods for sustained delivery of breast cancer therapeutics.  

PubMed

A silk-protein based reservoir rod was developed for zero-order and long-term sustained drug delivery applications. Silk reservoir rod formulations were processed in three steps. First, a regenerated silk fibroin solution, rich in random-coil content was transformed into a tubular silk film with controllable dimensions, uniform film morphology and a structure rich in silk II, ?-sheet content via "film-spinning." Second, the drug powder was loaded into swollen silk tubes followed by tube end clamping. Last, clamped silk tube ends were sealed completely via dip coating. Anastrozole, an FDA approved active ingredient for the treatment of breast cancer, was used as a model drug to investigate viability of the silk reservoir rod technology for sustained drug delivery. The in vitro and in vivo pharmacokinetic data (in a female Sprague-Dawley rat model) analyzed via liquid chromatography-tandem mass spectroscopy indicated zero-order release for 91 days. Both in vitro and in vivo anastrozole release rates could be controlled simply by varying silk rod dimensions. The swelling behavior of silk films and zero-order anastrozole release kinetics indicated practically immediate film hydration and formation of a linear anastrozole concentration gradient along the silk film thickness. The dependence of anastrozole release rate on the overall silk rod dimensions was in good agreement with an essentially diffusion-controlled sustained release from a reservoir cylindrical geometry. In vivo results highlighted a strong in vitro-in vivo pharmacokinetic correlation and a desirable biocompatibility profile of silk reservoir rods. During a 6-month implantation in rats, the apparent silk molecular weight values decreased gradually, while rod dry mass and ?-sheet crystal content values remained essentially constant, providing a suitable timeframe for controlled, long-term sustained delivery applications. Overall, the silk reservoir rod may be a viable candidate for sustained delivery of breast cancer therapeutics. PMID:25009069

Yucel, Tuna; Lovett, Michael L; Giangregorio, Raffaella; Coonahan, Erin; Kaplan, David L

2014-10-01

235

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

PubMed

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

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

2014-07-01

236

History of Silk  

NSDL National Science Digital Library

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.

0000-00-00

237

Artificial spider silk.  

E-print Network

??Spider dragline silk is Nature’s high-performance fiber that outperforms the best man-made materials by displaying extraordinary mechanical properties. In addition, spider silk is biocompatible and… (more)

Grip, Stefan

2008-01-01

238

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

PubMed

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

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

2014-12-01

239

Permeability of silk microcapsules made by the interfacial adsorption of Kevin D. Hermanson,a  

E-print Network

Permeability of silk microcapsules made by the interfacial adsorption of proteinw Kevin D interfaces to assemble recombinant silk proteins into thin-shelled microcapsules. These microcapsules and functionalizability. Recently, the assembly of recombinant spider-silk proteins at an emulsion interface has been used

Bausch, Andreas

240

Super Spider Silk  

E-print Network

superstrong silk thread by injecting spider thread genes into silkworm eggs for a stronger and more elastic pair of socks. The hybrid thread is called spider silk and is expected to be twice as strong and stretchy as conventional silk thread. In addition...

Hacker, Randi; Tsutsui, William

2008-02-06

241

Stylized Silk Painting.  

ERIC Educational Resources Information Center

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)

Skophammer, Karen

1998-01-01

242

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

PubMed

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

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

2013-01-01

243

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

PubMed Central

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

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

2013-01-01

244

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

NASA Astrophysics Data System (ADS)

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.

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

2001-03-01

245

Amino Acid Composition-Dependent Elasticity of SPIDER Silk  

NASA Astrophysics Data System (ADS)

Spider silk exhibits excellent mechanical features in both toughness and extensibility. In recent years considerable investigations have focused on it. The understanding of spider silk protein is important for the development bionic silk. In this paper, we study by Monte Carlo simulation the force-of-extension property of spider silk proteins as a function of the residue composition for major and minor ampullate glands of typical Araneid orb weaver as well as that for one artificial spider silk. The results are also compared with those from a designed protein whose amino acid composition is uniform. The results clearly show that the major and minor ampullate gland proteins are much tougher than the designed protein, whereas the artificial protein, as a model of a nature spider silk, does have good mechanical properties. Our simulation reveals that mechanical property of a spider silk protein is dependent on its amino acid composition and that an excellent result of natural evolution is manifest in the composition of the spider silk protein.

Zhang, Yong; Dai, Luru; Ou-Yang, Zhong-Can

246

Silk fibroin biomaterials for tissue regenerations.  

PubMed

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

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

2013-04-01

247

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

PubMed Central

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

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

2012-01-01

248

Mechanism of silk processing in insects and spiders  

NASA Astrophysics Data System (ADS)

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.

Jin, Hyoung-Joon; Kaplan, David L.

2003-08-01

249

Silk-based delivery systems of bioactive molecules  

PubMed Central

Silks are biodegradable, biocompatible, self-assemblying proteins that can also be tailored via genetic engineering to contain specific chemical features, offering utility for drug and gene delivery. Silkworm silk has been used in biomedical sutures for decades and has recently achieved Food and Drug Administration approval for expanded biomaterials device utility. With the diversity and control of size, structure and chemistry, modified or recombinant silk proteins can be designed and utilized in various biomedical application, such as for the delivery of bioactive molecules. This review focuses on the biosynthesis and applications of silk-based multi-block copolymer systems and related silk protein drug delivery systems. The utility of these systems for the delivery of small molecule drugs, proteins and genes are reviewed. PMID:20298729

Numata, Keiji; Kaplan, David L

2010-01-01

250

Structure-function-property-design interplay in biopolymers: spider silk.  

PubMed

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

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

2014-04-01

251

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

E-print Network

Construction of Silk Fiber Core in Lepidoptera Frantisek Sehnal* and Michal Zurovec Entomological silk fibers depend on amino acid repeats in the principal protein, heavy chain fibroin (H. Introduction Silk fibers are produced from various types of ectodermal glands in the mites, spiders

Â?urovec, Michal

252

9422 Stratospheric ice catalyzes chlorine reactions 9428 Fusing silk and silica  

E-print Network

9422 Stratospheric ice catalyzes chlorine reactions 9428 Fusing silk and silica 9482 Identifying, and Mario J. Molina (see pages 9422­9427) CHEMISTRY Fusing silk and silica An engineered protein combining the flexibility and tensile strength of spider silk with the resilience of silica has been developed. The result

McFadden, Geoff

253

Unique Molecular Architecture of Silk Fibroin in the Waxmoth, Galleria mellonella*  

E-print Network

Unique Molecular Architecture of Silk Fibroin in the Waxmoth, Galleria mellonella* Received, 370 05 C eske´ Budejovice, Czech Republic Proteins of silk fibers are characterized by reitera- tions in higher order assemblies vary in all spider and lepidopteran silks analyzed so far. This paper describes

Â?urovec, Michal

254

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

255

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

E-print Network

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

Rogers, John A.

256

Sequence conservation in the C-terminal region of spider silk proteins (Spidroin) from Nephila clavipes (Tetragnathidae) and Araneus bicentenarius (Araneidae).  

PubMed

The polymerase chain reaction (PCR) has been used to amplify the portion of the Spidroin 1 gene that codes for the C-terminal part of the silk protein of the spider Nephila clavipes. Along with some substitution mutations of minor consequence, the PCR-derived sequence reveals an additional base missing from the previously published Nephila Spidroin 1 sequence. Comparison of the PCR-derived sequence with the equivalent region of Spidroin 2 indicates that the insertion of this single base results in greatly increased similarity in the resulting amino acid sequences of Spidroin 1 and Spidroin 2 (75% over 97 amino acids). The same PCR primers also amplified a fragment of the same length from Araneus bicentenarius. This sequence is also very similar to Spidroin 1 of Nephila (71% over 238 bases excluding the PCR primers, which translates into 76% over 79 amino acids). PMID:8120021

Beckwitt, R; Arcidiacono, S

1994-03-01

257

Biomedical applications of chemically-modified silk fibroin  

PubMed Central

Silk proteins belong to a class of unique, high molecular weight, block copolymer-like proteins that have found widespread use in biomaterials and regenerative medicine. The useful features of these proteins, including self-assembly, robust mechanical properties, biocompatibility and biodegradability can be enhanced through a variety of chemical modifications. These modifications provide chemical handles for the attachment of growth factors, cell binding domains and other polymers to silk, expanding the range of cell and tissue engineering applications attainable. This review focuses on the chemical reactions that have been used to modify the amino acids in silk proteins, and describes their utility in biomedical applications. PMID:20161439

Murphy, Amanda R.

2009-01-01

258

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

PubMed Central

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

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

2012-01-01

259

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

NASA Astrophysics Data System (ADS)

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.

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

2012-10-01

260

Antimicrobial functionalized genetically engineered spider silk  

PubMed Central

Genetically engineered fusion proteins offer potential as multifunctional biomaterials for medical use. Fusion or chimeric proteins can be formed using recombinant DNA technology by combining nucleotide sequences encoding different peptides or proteins that are otherwise not found together in nature. In the present study, three new fusion proteins were designed, cloned and expressed and assessed for function, by combining the consensus sequence of dragline spider silk with three different antimicrobial peptides. The human antimicrobial peptides human neutrophil defensin 2 (HNP-2), human neutrophil defensins 4 (HNP-4) and hepcidin were fused to spider silk through bioengineering. The spider silk domain maintained its self-assembly features, a key aspect of these new polymeric protein biomaterials, allowing the formation of ?-sheets to lock in structures via physical interactions without the need for chemical cross-linking. These new functional silk proteins were assessed for antimicrobial activity against Gram - Escherichia coli and Gram + Staphylococcus aureus and microbicidal activity was demonstrated. Dynamic light scattering was used to assess protein aggregation to clarify the antimicrobial patterns observed. Attenuated-total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and circular dichroism (CD) were used to assess the secondary structure of the new recombinant proteins. In vitro cell studies with a human osteosarcoma cell line (SaOs-2) demonstrated the compatibility of these new proteins with mammalian cells. PMID:21458065

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

2011-01-01

261

Early events in the evolution of spider silk genes.  

PubMed

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

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

2012-01-01

262

Early Events in the Evolution of Spider Silk Genes  

PubMed Central

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

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

2012-01-01

263

Water-insoluble Silk Films with Silk I Structure  

SciTech Connect

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.

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

2010-01-01

264

Bone Tissue Engineering with Premineralized Silk Scaffolds  

PubMed Central

Silks fibroin biomaterials are being explored as novel protein-based systems for cell and tissue culture. In the present study, biomimetic growth of calcium phosphate on porous silk fibroin polymeric scaffolds was explored to generate organic/inorganic composites as scaffolds for bone tissue engineering. Aqueous-derived silk fibroin scaffolds were prepared with the addition of polyaspartic acid during processing, followed by the controlled deposition of calcium phosphate by exposure to CaCl2 and Na2HPO4. These mineralized protein-composite scaffolds were subsequently seeded with human bone marrow stem cells (hMSC) and cultured in vitro for 6 weeks under osteogenic conditions with or without BMP-2. The extent of osteoconductivity was assessed by cell numbers, alkaline phosphatase and calcium deposition, along with immunohistochemistry for bone related outcomes. The results suggest increased osteoconductive outcomes with an increase in initial content of apatite and BMP-2 in the silk fibroin porous scaffolds. The premineralization of these highly porous silk fibroin protein scaffolds provided enhanced outcomes for the bone tissue engineering. PMID:18387349

Kim, Hyeon Joo; Kim, Ung-Jin; Kim, Hyun Suk; Li, Chunmei; Wada, Masahisa; Leisk, Gary G.; Kaplan, David L.

2009-01-01

265

Vortex-Induced Injectable Silk Fibroin Hydrogels  

PubMed Central

Abstract 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 oscillatory rheology experiments collected under the same assembly conditions as the circular dichroism experiments indicated that the increase in ?-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? ? 70 kPa for 5.2 wt % protein concentration). Vortex-induced, ?-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

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

2009-01-01

266

Biofabrication of Cell-Loaded 3D Spider Silk Constructs.  

PubMed

Biofabrication is an emerging and rapidly expanding field of research in which additive manufacturing techniques in combination with cell printing are exploited to generate hierarchical tissue-like structures. Materials that combine printability with cytocompatibility, so called bioinks, are currently the biggest bottleneck. Since recombinant spider silk proteins are non-immunogenic, cytocompatible, and exhibit physical crosslinking, their potential as a new bioink system was evaluated. Cell-loaded spider silk constructs can be printed by robotic dispensing without the need for crosslinking additives or thickeners for mechanical stabilization. Cells are able to adhere and proliferate with good viability over at least one week in such spider silk scaffolds. Introduction of a cell-binding motif to the spider silk protein further enables fine-tuned control over cell-material interactions. Spider silk hydrogels are thus a highly attractive novel bioink for biofabrication. PMID:25640578

Schacht, Kristin; Jüngst, Tomasz; Schweinlin, Matthias; Ewald, Andrea; Groll, Jürgen; Scheibel, Thomas

2015-02-23

267

Art on Silk Hoops  

ERIC Educational Resources Information Center

Painting on silk has a magic all its own. Versions of painting on silk can be found throughout the world from Japan and Europe to the United States. Themes for the paintings can be most any type of design or imagery. Applying the liquid dyes is exciting, as the vivid liquid colors flow and blend into the fabric. The process captures students'…

Padrick, Deborah

2012-01-01

268

An experimental confirmation of thermal transitions in native and regenerated spider silks.  

PubMed

Biological structures such as spider silks are formed by proteins. The physical properties of such proteins are determined by environmental conditions such as temperature and humidity. In this paper, we confirm the thermal transitions that take place in spider silks using differential scanning calorimetry and study how the interaction of spider silk proteins with water affects the onset temperatures for these thermal processes. Native fibres and regenerated films of dragline silk and egg sac silk from Argiope argentata spiders were used to study thermal transitions of protein based structures. For the first time, differential scanning calorimetry (DSC) tests were carried out with spider silk samples of relatively large mass (10mg). Previous attempts of DSC tests applied to spider silk samples failed to detect thermal transitions in a conclusive way. The tests reported here, however, show thermal transitions on both natural and regenerated samples that are in agreement with results from dynamic mechanical analysis (DMA) tests reported in the literature. The water content on spider silks seems to lower the temperatures at which such thermal transitions take place. The results also confirm that the amorphous regions of native and regenerated spider silk and silk worm silk give rise to similar thermal transitions. PMID:23827592

Torres, Fernando G; Troncoso, Omar P; Torres, Carlos; Cabrejos, Wilson

2013-04-01

269

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,...

270

Liquid crystalline spinning of spider silk  

NASA Astrophysics Data System (ADS)

Spider silk has outstanding mechanical properties despite being spun at close to ambient temperatures and pressures using water as the solvent. The spider achieves this feat of benign fibre processing by judiciously controlling the folding and crystallization of the main protein constituents, and by adding auxiliary compounds, to create a composite material of defined hierarchical structure. Because the `spinning dope' (the material from which silk is spun) is liquid crystalline, spiders can draw it during extrusion into a hardened fibre using minimal forces. This process involves an unusual internal drawdown within the spider's spinneret that is not seen in industrial fibre processing, followed by a conventional external drawdown after the dope has left the spinneret. Successful copying of the spider's internal processing and precise control over protein folding, combined with knowledge of the gene sequences of its spinning dopes, could permit industrial production of silk-based fibres with unique properties under benign conditions.

Vollrath, Fritz; Knight, David P.

2001-03-01

271

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

PubMed

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

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

2015-02-01

272

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

PubMed

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

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

2013-10-01

273

An Unlikely Silk: The Composite Material of Green Lacewing Cocoons  

SciTech Connect

Spiders routinely produce multiple types of silk; however, common wisdom has held that insect species produce one type of silk each. This work reports that the green lacewing (Mallada signata, Neuroptera) produces two distinct classes of silk. We identified and sequenced the gene that encodes the major protein component of the larval lacewing cocoon silk and demonstrated that it is unrelated to the adult lacewing egg-stalk silk. The cocoon silk protein is 49 kDa in size and is alanine rich (>40%), and it contains an {alpha}-helical secondary structure. The final instar lacewing larvae spin protein fibers of {approx}2 {mu}m diameter to construct a loosely woven cocoon. In a second stage of cocoon construction, the insects lay down an inner wall of lipids that uses the fibers as a scaffold. We propose that the silk protein fibers provide the mechanical strength of the composite lacewing cocoon whereas the lipid layer provides a barrier to water loss during pupation.

Weisman, Sarah; Trueman, Holly E.; Mudie, Stephen T.; Church, Jeffrey S.; Sutherland, Tara D.; Haritos, Victoria S. (CSIRO/MSE); (CSIRO)

2009-01-15

274

Molecular dynamics analysis of supercontraction in spider dragline silk  

E-print Network

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 ...

Batty, Laura

2013-01-01

275

Unraveled mechanism in silk engineering: Fast reeling induced silk toughening  

NASA Astrophysics Data System (ADS)

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.

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

2009-08-01

276

Quantifying Osteogenic Cell Degradation of Silk Biomaterials  

PubMed Central

The degradation of silk protein films by human mesenchymal stem cells (hMSCs), osteoblasts and osteoclasts, cells involved in osteogenic functions in normal and diseased bone, was assessed in vitro. The involvement of specific matrix metalloproteinases (MMPs) and integrin signaling in the degradation process was determined. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to quantitatively compare degradation by the different cell types using surface patterned silk films. Osteoblasts and osteoclasts demonstrated significant degradation of the silk films in vitro in comparison to the hMSCs and the film controls without cells. The osteoclasts degraded the silk films the most and also generated the highest level of MMPs 1 and 2. The osteoblasts upregulated integrins ?5 and ?1 while the osteoclasts upregulated integrins ?2 and ?1. There was significant contrast in responses on the silk matrices between osteogenic cells vs undifferentiated hMSCs to illustrate in vitro the role of cell type on matrix remodeling. These are important issues in matching biomaterial matrix features and studies in vitro to remodeling in vivo, in both normal and disease tissue systems. Cell populations and niche factors impact tissue regeneration, wound healing and physiological state and the ability to better understand the role of different cell types is critical to overall regenerative outcomes. PMID:21105641

Sengupta, Sejuti; Park, Sang-Hyug; Seok, Gil Eun; Patel, Atur; Numata, Keiji; Lu, Chia-Li; Kaplan, David L.

2010-01-01

277

Clay-Enriched Silk Biomaterials for Bone Formation  

PubMed Central

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

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

2011-01-01

278

Greatly Increased Toughness of Infiltrated Spider Silk  

NASA Astrophysics Data System (ADS)

In nature, tiny amounts of inorganic impurities, such as metals, are incorporated in the protein structures of some biomaterials and lead to unusual mechanical properties of those materials. A desire to produce these biomimicking new materials has stimulated materials scientists, and diverse approaches have been attempted. In contrast, research to improve the mechanical properties of biomaterials themselves by direct metal incorporation into inner protein structures has rarely been tried because of the difficulty of developing a method that can infiltrate metals into biomaterials, resulting in a metal-incorporated protein matrix. We demonstrated that metals can be intentionally infiltrated into inner protein structures of biomaterials through multiple pulsed vapor-phase infiltration performed with equipment conventionally used for atomic layer deposition (ALD). We infiltrated zinc (Zn), titanium (Ti), or aluminum (Al), combined with water from corresponding ALD precursors, into spider dragline silks and observed greatly improved toughness of the resulting silks. The presence of the infiltrated metals such as Al or Ti was verified by energy-dispersive x-ray (EDX) and nuclear magnetic resonance spectra measured inside the treated silks. This result of enhanced toughness of spider silk could potentially serve as a model for a more general approach to enhance the strength and toughness of other biomaterials.

Lee, Seung-Mo; Pippel, Eckhard; Gösele, Ulrich; Dresbach, Christian; Qin, Yong; Chandran, C. Vinod; Bräuniger, Thomas; Hause, Gerd; Knez, Mato

2009-04-01

279

Liquid crystalline spinning of spider silk  

Microsoft Academic Search

Spider silk has outstanding mechanical properties despite being spun at close to ambient temperatures and pressures using water as the solvent. The spider achieves this feat of benign fibre processing by judiciously controlling the folding and crystallization of the main protein constituents, and by adding auxiliary compounds, to create a composite material of defined hierarchical structure. Because the 'spinning dope'

Fritz Vollrath; David P. Knight

2001-01-01

280

Lyophilized Silk Fibroin Hydrogels for the Sustained Local Delivery of Therapeutic Monoclonal Antibodies  

PubMed Central

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

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

2011-01-01

281

Atomistic model of the spider silk nanostructure  

NASA Astrophysics Data System (ADS)

Spider silk is an ultrastrong and extensible self-assembling biopolymer that outperforms the mechanical characteristics of many synthetic materials including steel. Here we report atomic-level structures that represent aggregates of MaSp1 proteins from the N. Clavipes silk sequence based on a bottom-up computational approach using replica exchange molecular dynamics. We discover that poly-alanine regions predominantly form distinct and orderly beta-sheet crystal domains while disorderly structures are formed by poly-glycine repeats, resembling 31-helices. These could be the molecular source of the large semicrystalline fraction observed in silks, and also form the basis of the so-called "prestretched" molecular configuration. Our structures 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.

Keten, Sinan; Buehler, Markus J.

2010-04-01

282

Peptide backbone orientation and dynamics in spider dragline silk and two-photon excitation in nuclear magnetic and quadrupole resonance  

Microsoft Academic Search

In the first part of the dissertation, spider dragline silk is studied by solid state NMR techniques. The dependence of NMR frequency on molecular orientation is exploited using the DECODER experiment to determine the orientation of the protein backbone within the silk fibre. Practical experimental considerations require that the silk fibres be wound about a cylindrical axis perpendicular to the

Philip Thomas Eles

2005-01-01

283

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

E-print Network

Molecular and Mechanical Characterization of Aciniform Silk: Uniformity of Iterated Sequence Modules in a Novel Member of the Spider Silk Fibroin Gene Family Cheryl Y. Hayashi,* Todd A. Blackledge to seven different protein-based silks/glues that have diverse physical properties. The fibroin sequences

Blackledge, Todd

284

Blueprint for a high-performance biomaterial: full-length spider dragline silk genes.  

PubMed

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

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

2007-01-01

285

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

PubMed Central

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

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

2007-01-01

286

A three-dimensional multiporous fibrous scaffold fabricated with regenerated spider silk protein/poly(l-lactic acid) for tissue engineering.  

PubMed

An axially aligned three-dimensional (3-D) fibrous scaffold was fabricated with regenerated spider silk protein (RSSP)/poly (l-lactic acid) (PLLA) through electrospinning and post treatment. The morphology, mechanical and degradation properties of the scaffold were controlled through the weight ratio of RSSP to PLLA, the thickness of the scaffold and the treatment time. The scaffold with a weight ratio of 2:3 (RSSP:PLLA) had a nanoleaves-on-nanofibers hierarchical nanostructure; the length and thickness of the nanoleaves were about 400 and 30 nm, respectively. The holes of the scaffolds ranged from hundreds of nanometers to several microns. The scaffold showed an ideal mechanical property that it was stiff when dry, but became soft once hydrated in the culture medium. Its degradation rate was very slow in the first 2 months, and then accelerated in the following 2 months. The pH values of the degradation mediums of all the samples remained in the range of 7.40-7.12 during degradation for 6 months. It had good biocompatibility with PC 12 cells. The aligned hierarchical nanostructure could guide the directions of the axon extension. This scaffold has a potential application in Tissue Engineering and controlled release. This study provides a method to produce synthetic or natural biodegradable polymer scaffold with tailored morphology, mechanical, and degradation properties. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2014. PMID:24825592

Yu, Qiaozhen; Sun, Chengjun

2014-05-14

287

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

PubMed

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

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

288

Highly tunable elastomeric silk biomaterials.  

PubMed

Elastomeric, fully degradable and biocompatible biomaterials are rare, with current options presenting significant limitations in terms of ease of functionalization and tunable mechanical and degradation properties. We report a new method for covalently crosslinking tyrosine residues in silk proteins, via horseradish peroxidase and hydrogen peroxide, to generate highly elastic hydrogels with tunable properties. The tunable mechanical properties, gelation kinetics and swelling properties of these new protein polymers, in addition to their ability to withstand shear strains on the order of 100%, compressive strains greater than 70% and display stiffness between 200 - 10,000 Pa, covering a significant portion of the properties of native soft tissues. Molecular weight and solvent composition allowed control of material mechanical properties over several orders of magnitude while maintaining high resilience and resistance to fatigue. Encapsulation of human bone marrow derived mesenchymal stem cells (hMSC) showed long term survival and exhibited cell-matrix interactions reflective of both silk concentration and gelation conditions. Further biocompatibility of these materials were demonstrated with in vivo evaluation. These new protein-based elastomeric and degradable hydrogels represent an exciting new biomaterials option, with a unique combination of properties, for tissue engineering and regenerative medicine. PMID:25395921

Partlow, Benjamin P; Hanna, Craig W; Rnjak-Kovacina, Jelena; Moreau, Jodie E; Applegate, Matthew B; Burke, Kelly A; Marelli, Benedetto; Mitropoulos, Alexander N; Omenetto, Fiorenzo G; Kaplan, David L

2014-08-01

289

Unravelling the biodiversity of nanoscale signatures of spider silk fibres.  

PubMed

Living organisms are masters at designing outstanding self-assembled nanostructures through a hierarchical organization of modular proteins. Protein-based biopolymers improved and selected by the driving forces of molecular evolution are among the most impressive archetypes of nanomaterials. One of these biomacromolecules is the myriad of compound fibroins of spider silks, which combine surprisingly high tensile strength with great elasticity. However, no consensus on the nano-organization of spider silk fibres has been reached. Here we explore the biodiversity of spider silk fibres, focusing on nanoscale characterization with high-resolution atomic force microscopy. Our results reveal an evolution of the nanoroughness, nanostiffness, nanoviscoelastic, nanotribological and nanoelectric organization of microfibres, even when they share similar sizes and shapes. These features are related to unique aspects of their molecular structures. The results show that combined nanoscale analyses of spider silks may enable the screening of appropriate motifs for bioengineering synthetic fibres from recombinant proteins. PMID:24345771

Silva, Luciano P; Rech, Elibio L

2013-01-01

290

Unravelling the biodiversity of nanoscale signatures of spider silk fibres  

NASA Astrophysics Data System (ADS)

Living organisms are masters at designing outstanding self-assembled nanostructures through a hierarchical organization of modular proteins. Protein-based biopolymers improved and selected by the driving forces of molecular evolution are among the most impressive archetypes of nanomaterials. One of these biomacromolecules is the myriad of compound fibroins of spider silks, which combine surprisingly high tensile strength with great elasticity. However, no consensus on the nano-organization of spider silk fibres has been reached. Here we explore the biodiversity of spider silk fibres, focusing on nanoscale characterization with high-resolution atomic force microscopy. Our results reveal an evolution of the nanoroughness, nanostiffness, nanoviscoelastic, nanotribological and nanoelectric organization of microfibres, even when they share similar sizes and shapes. These features are related to unique aspects of their molecular structures. The results show that combined nanoscale analyses of spider silks may enable the screening of appropriate motifs for bioengineering synthetic fibres from recombinant proteins.

Silva, Luciano P.; Rech, Elibio L.

2013-12-01

291

An Australian webspinner species makes the finest known insect silk fibers  

SciTech Connect

Aposthonia gurneyi, an Australian webspinner species, is a primitive insect that constructs and lives in a silken tunnel which screens it from the attentions of predators. The insect spins silk threads from many tiny spines on its forelegs to weave a filmy sheet. We found that the webspinner silk fibers have a mean diameter of only 65 nm, an order of magnitude smaller than any previously reported insect silk. The purpose of such fine silk may be to reduce the metabolic cost of building the extensive tunnels. At the molecular level, the A. gurneyi silk has a predominantly beta-sheet protein structure. The most abundant clone in a cDNA library produced from the webspinner silk glands encoded a protein with extensive glycine-serine repeat regions. The GSGSGS repeat motif of the A. gurneyi silk protein is similar to the well-known GAGAGS repeat motif found in the heavy fibroin of silkworm silk, which also has beta-sheet structure. As the webspinner silk gene is unrelated to the silk gene of the phylogenetically distant silkworm, this is a striking example of convergent evolution.

Okada, Shoko; Weisman, Sarah; Trueman, Holly E.; Mudie, Stephen T.; Haritos, Victoria S.; Sutherland, Tara D. (CSIRO/MSE); (CSIRO)

2009-01-15

292

Recombinant spider silk genetically functionalized with affinity domains.  

PubMed

Functionalization of biocompatible materials for presentation of active protein domains is an area of growing interest. Herein, we describe a strategy for functionalization of recombinant spider silk via gene fusion to affinity domains of broad biotechnological use. Four affinity domains of different origin and structure; the IgG-binding domains Z and C2, the albumin-binding domain ABD, and the biotin-binding domain M4, were all successfully produced as soluble silk fusion proteins under nondenaturing purification conditions. Silk films and fibers produced from the fusion proteins were demonstrated to be chemically and thermally stable. Still, the bioactive domains are concluded to be folded and accessible, since their respective targets could be selectively captured from complex samples, including rabbit serum and human plasma. Interestingly, materials produced from mixtures of two different silk fusion proteins displayed combined binding properties, suggesting that tailor-made materials with desired stoichiometry and surface distributions of several binding domains can be produced. Further, use of the IgG binding ability as a general mean for presentation of desired biomolecules could be demonstrated for a human vascular endothelial growth factor (hVEGF) model system, via a first capture of anti-VEGF IgG to silk containing the Z-domain, followed by incubation with hVEGF. Taken together, this study demonstrates the potential of recombinant silk, genetically functionalized with affinity domains, for construction of biomaterials capable of presentation of almost any desired biomolecule. PMID:24678858

Jansson, Ronnie; Thatikonda, Naresh; Lindberg, Diana; Rising, Anna; Johansson, Jan; Nygren, Per-Åke; Hedhammar, My

2014-05-12

293

Piriform Spider Silk Sequences Reveal Unique Repetitive Elements  

PubMed Central

Orb-weaving spider silk fibers are assembled from very large, highly repetitive proteins. The repeated segments contain, in turn, short, simple repetitive amino acid motifs that account for the physical and mechanical properties of the assembled fiber. Of the six orb-weaver silk fibroins, the piriform silk that makes the attachment discs, which lashes the joints of the web and attaches dragline silk to surfaces has not been previously characterized. Piriform silk protein cDNAs were isolated from phage libraries of three species, A. trifasciata, N. clavipes, and N. cruentata. The deduced amino acid sequences from these genes revealed two new repetitive motifs: an alternating proline motif where every other amino acid is proline, and a glutamine-rich motif of 6 to 8 amino acids. Similar to other spider silk proteins, the repeated segments are large (>200 amino acids) and highly homogenized within a species. There is also substantial sequence similarity across the genes from the three species with particular conservation of the repetitive motifs. Northern blot analysis revealed that the messenger RNA is larger than 11kb and is expressed exclusively in the piriform glands of the spider. Phylogenetic analysis of the C-terminal regions of the new proteins with published spidroins robustly shows that the pirifom sequences form an ortholog group. PMID:20954740

Perry, David J.; Bittencourt, Daniela; Siltberg-Liberles, Jessica; Rech, Elibio L.; Lewis, Randolph V.

2010-01-01

294

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

PubMed

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

Wang, Jia; Chen, Gefei; Meng, Qing

2014-08-01

295

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

PubMed

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:25507484

Wang, Jia; Chen, Gefei; Meng, Qing

2014-08-01

296

Rheological characterization of hydrogels formed by recombinantly produced spider silk  

NASA Astrophysics Data System (ADS)

Many fibrous proteins such as spider silks exhibit impressive mechanical properties and are highly biocompatible leading to many potential biomaterial applications. For applications such as tissue engineering, polymer hydrogels have been proposed as an effective means of producing porous but stable scaffolds. Here, nanofiber-based hydrogels were produced from engineered and recombinantly produced spider silk proteins. The silk nanofibers are stable semi-flexible polymers which assemble into hydrogel networks. We studied the hydrogel rheology and determined the concentration dependence of the elastic modulus. AFM images indicate that the nanofibers might assemble into branch-like structures, which would also be consistent with the measured rheological behavior. Since the developed spider silk hydrogels are stable over weeks and show a high elastic modulus at low volume fractions, they are well suited for a broad variety of applications.

Rammensee, S.; Huemmerich, D.; Hermanson, K. D.; Scheibel, T.; Bausch, A. R.

2006-02-01

297

Spider genomes provide insight into composition and evolution of venom and silk.  

PubMed

Spiders are ecologically important predators with complex venom and extraordinarily tough silk that enables capture of large prey. Here we present the assembled genome of the social velvet spider and a draft assembly of the tarantula genome that represent two major taxonomic groups of spiders. The spider genomes are large with short exons and long introns, reminiscent of mammalian genomes. Phylogenetic analyses place spiders and ticks as sister groups supporting polyphyly of the Acari. Complex sets of venom and silk genes/proteins are identified. We find that venom genes evolved by sequential duplication, and that the toxic effect of venom is most likely activated by proteases present in the venom. The set of silk genes reveals a highly dynamic gene evolution, new types of silk genes and proteins, and a novel use of aciniform silk. These insights create new opportunities for pharmacological applications of venom and biomaterial applications of silk. PMID:24801114

Sanggaard, Kristian W; Bechsgaard, Jesper S; Fang, Xiaodong; Duan, Jinjie; Dyrlund, Thomas F; Gupta, Vikas; Jiang, Xuanting; Cheng, Ling; Fan, Dingding; Feng, Yue; Han, Lijuan; Huang, Zhiyong; Wu, Zongze; Liao, Li; Settepani, Virginia; Thøgersen, Ida B; Vanthournout, Bram; Wang, Tobias; Zhu, Yabing; Funch, Peter; Enghild, Jan J; Schauser, Leif; Andersen, Stig U; Villesen, Palle; Schierup, Mikkel H; Bilde, Trine; Wang, Jun

2014-01-01

298

Spider genomes provide insight into composition and evolution of venom and silk  

PubMed Central

Spiders are ecologically important predators with complex venom and extraordinarily tough silk that enables capture of large prey. Here we present the assembled genome of the social velvet spider and a draft assembly of the tarantula genome that represent two major taxonomic groups of spiders. The spider genomes are large with short exons and long introns, reminiscent of mammalian genomes. Phylogenetic analyses place spiders and ticks as sister groups supporting polyphyly of the Acari. Complex sets of venom and silk genes/proteins are identified. We find that venom genes evolved by sequential duplication, and that the toxic effect of venom is most likely activated by proteases present in the venom. The set of silk genes reveals a highly dynamic gene evolution, new types of silk genes and proteins, and a novel use of aciniform silk. These insights create new opportunities for pharmacological applications of venom and biomaterial applications of silk. PMID:24801114

Sanggaard, Kristian W.; Bechsgaard, Jesper S.; Fang, Xiaodong; Duan, Jinjie; Dyrlund, Thomas F.; Gupta, Vikas; Jiang, Xuanting; Cheng, Ling; Fan, Dingding; Feng, Yue; Han, Lijuan; Huang, Zhiyong; Wu, Zongze; Liao, Li; Settepani, Virginia; Thøgersen, Ida B.; Vanthournout, Bram; Wang, Tobias; Zhu, Yabing; Funch, Peter; Enghild, Jan J.; Schauser, Leif; Andersen, Stig U.; Villesen, Palle; Schierup, Mikkel H; Bilde, Trine; Wang, Jun

2014-01-01

299

Characterization and assembly of a GFP-tagged cylindriform silk into hexameric complexes.  

PubMed

Spider silk has been studied extensively for its attractive mechanical properties and potential applications in medicine and industry. The production of spider silk, however, has been lagging behind for lack of suitable systems. Our approach focuses on solving the production of spider silk by designing, expressing, purifying and characterizing the silk from cylindriform glands. We show that the cylindriform silk protein, in contrast to the commonly used dragline silk protein, is fully folded and stable in solution. With the help of GFP as a fusion tag we enhanced the expression of the silk protein in Escherichia coli and could optimize the downstream processing. Secondary structures analysis by circular dichroism and FTIR shows that the GFP-silk fusion protein is predominantly ?-helical, and that pH can trigger a ?- to ?-transition resulting in aggregation. Structural analysis by small angle X-ray scattering suggests that the GFP-Silk exists in the form of a hexamer in solution. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 378-390, 2014. PMID:23955662

Oster, Carl; Bonde, Johan Svensson; Bülow, Leif; Dicko, Cedric

2014-04-01

300

Electrospinning of chitosan/sericin/PVA nanofibers incorporated with in situ synthesis of nano silver.  

PubMed

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

Hadipour-Goudarzi, Elmira; Montazer, Majid; Latifi, Masoud; Aghaji, Ali Akbar Ghare

2014-11-26

301

Molecular and Mechanical Properties of Major Ampullate Silk of the Black Widow Spider, Latrodectus h esperus  

Microsoft Academic Search

Molecular and material properties of major ampullate silk were studied for the cobweb-building black widow spider Latrodectus hesperus. Material properties were measured by stretching the silk to breaking. The strength was 1.0 ( 0.2 GPa, and the extensibility was 34 ( 8%. The secondary structure of the major ampullate silk protein was studied using carbon-13 NMR spectroscopy. Alanine undergoes a

Barbara A. Lawrence; Craig A. Vierra; Anne M. F. Moore

2004-01-01

302

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

PubMed

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

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

2005-03-01

303

Functionalized spider silk spheres as drug carriers for targeted cancer therapy.  

PubMed

Bioengineered spider silk is a biomaterial that combines the properties of self-assembly, biocompatibility and biodegradability with reasonable accessibility and a simple purification procedure. Moreover, genetic engineering enables the functionalization of silk by adding the peptide coding sequences of the desired attribute. Hybrids composed of Her2 binding peptides (H2.1 or H2.2) and bioengineered silk MS1 (based on the MaSp1 sequence from N. clavipes) were designed. Bioengineered silks were expressed in a bacterial system and purified using a tag-free thermal method. The hybrid silks with N-terminal functionalization were bound more efficiently to cells that were overexpressing Her2 than those with the C-terminal fusion. Moreover, the functionalization did not impede the self-assembly property of bioengineered silk, enabling the processing of silk proteins into spheres. The binding domains were exposed on the surface of the spheres, because the functionalized particles specifically bound and internalized into Her2-overexpressing cells. The binding of the functionalized spheres to Her2-positive cells was significantly higher compared with the control sphere and Her2-negative cell binding. Silk spheres were loaded with doxorubicin and showed pH-dependent drug release. The silk spheres were not cytotoxic, unless they were loaded with the drug doxorubicin. This study indicates the ability of drug-loaded functionalized spider silk spheres to serve as a carrier for targeted cancer therapy. PMID:24963985

Florczak, Anna; Mackiewicz, Andrzej; Dams-Kozlowska, Hanna

2014-08-11

304

Hierarchical Chain Model of Spider Capture Silk Elasticity  

NASA Astrophysics Data System (ADS)

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.,

Nat. Mater. 2, 278 (2003)1476-1122
]. 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 supramodules 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.

Zhou, Haijun; Zhang, Yang

2005-01-01

305

Hierarchical Chain Model of Spider Capture Silk Elasticity  

E-print Network

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.

Haijun Zhou; Yang Zhang

2004-12-20

306

Stabilization of Enzymes in Silk Films  

PubMed Central

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

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

2009-01-01

307

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

PubMed

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

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

2014-05-01

308

Electrospun Silk Biomaterial Scaffolds for Regenerative Medicine  

PubMed Central

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

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

2009-01-01

309

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

E-print Network

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

Rogers, John A.

310

Materials: Surprising strength of silkworm silk  

NASA Astrophysics Data System (ADS)

Commercial silkworm silk is presumed to be much weaker and less extensible than spider dragline silk, which has been hailed as a 'super-fibre'. But we show here that the mechanical properties of silkworm silks can approach those of spider dragline silk when reeled under controlled conditions. We suggest that silkworms might be able to produce threads that compare well with spider silk by changing their spinning habits, rather than by having their silk genes altered.

Shao, Zhengzhong; Vollrath, Fritz

2002-08-01

311

Sericin cream reduces pruritus in hemodialysis patients: a randomized, double-blind, placebo-controlled experimental study  

PubMed Central

Background Uremic pruritus (UP) is a significant complication in ESRD patients and substantially impairs their quality of life. UP is considered to be a skin manifestation of chronic inflammation. Because sericin can suppress the release of pro-inflammatory cytokines, the purpose of this study was to investigate the short-term safety and efficacy of sericin cream for treating UP in hemodialysis patients. Methods This study used a double-blind design to investigate the effects of random topical administration of sericin cream and cream base (placebo) on either the right or left extremities of hemodialysis patients for 6?weeks. Skin hydration, irritation and pigmentation were evaluated every 2?weeks using Skin Diagnostic SD27. The visual analog scale for itching was also evaluated every 2?weeks, and the Kidney Disease Quality of Life Short Form was performed on the day of each patient’s enrollment and after 6?weeks of treatment. Results Fifty dialysis patients were enrolled, 47 of which completed the study. The hydration of the skin of the patients’ extremities increased significantly after administration of sericin cream; significant differences were found between sericin treatment and control after 6?weeks of treatment (p?=?0.041 for arms and p?=?0.022 for legs, respectively). Moreover, a significant difference was also found in skin irritation between the two treatments (p?=?0.013 for arms and p?=?0.027 for legs, respectively). At the end of the study, the skin pigmentation level was significantly reduced on both the arms (p?=?0.032) and legs (p?=?0.021) of the sericin-treated side compared with the side treated with cream base. The mean itching score decreased significantly from moderate to severe at the time of enrollment to mild pruritus after 6?weeks of treatment (p?=?0.002). A better quality of life was found in all domains tested although statistically significant differences before and after treatment was found only in the patients’ pain scores, the effect of kidney disease on daily life, sleep quality and symptoms or problems related to kidney disease. Conclusions We conclude that sericin cream has a high potential for reducing UP in hemodialysis patients. The trial registration number of this study is ISRCTN16019033; its public title is “sericin cream reduces pruritus in hemodialysis patients”. PMID:23006933

2012-01-01

312

Ingrowth of human mesenchymal stem cells into porous silk particle reinforced silk composite scaffolds: An in vitro study.  

PubMed

Silk fibroin protein is biodegradable and biocompatible, exhibiting excellent mechanical properties for various biomedical applications. However, porous three-dimensional (3-D) silk fibroin scaffolds, or silk sponges, usually fall short in matching the initial mechanical requirements for bone tissue engineering. In the present study, silk sponge matrices were reinforced with silk microparticles to generate protein-protein composite scaffolds with desirable mechanical properties for in vitro osteogenic tissue formation. It was found that increasing the silk microparticle loading led to a substantial increase in the scaffold compressive modulus from 0.3 MPa (non-reinforced) to 1.9 MPa for 1:2 (matrix:particle) reinforcement loading by dry mass. Biochemical, gene expression, and histological assays were employed to study the possible effects of increasing composite scaffold stiffness, due to microparticle reinforcement, on in vitro osteogenic differentiation of human mesenchymal stem cells (hMSCs). Increasing silk microparticle loading increased the osteogenic capability of hMSCs in the presence of bone morphogenic protein-2 (BMP-2) and other osteogenic factors in static culture for up to 6 weeks. The calcium adsorption increased dramatically with increasing loading, as observed from biochemical assays, histological staining, and microcomputer tomography (?CT) analysis. Specifically, calcium content in the scaffolds increased by 0.57, 0.71, and 1.27 mg (per ?g of DNA) from 3 to 6 weeks for matrix to particle dry mass loading ratios of 1:0, 1:1, and 1:2, respectively. In addition, ?CT imaging revealed that at 6 weeks, bone volume fraction increased from 0.78% for non-reinforced to 7.1% and 6.7% for 1:1 and 1:2 loading, respectively. Our results support the hypothesis that scaffold stiffness may strongly influence the 3-D in vitro differentiation capabilities of hMSCs, providing a means to improve osteogenic outcomes. PMID:20656075

Rockwood, Danielle N; Gil, Eun Seok; Park, Sang-Hyug; Kluge, Jonathan A; Grayson, Warren; Bhumiratana, Sarindr; Rajkhowa, Rangam; Wang, Xungai; Kim, Sung Jun; Vunjak-Novakovic, Gordana; Kaplan, David L

2011-01-01

313

Ingrowth of Human Mesenchymal Stem Cells into Porous Silk Particle Reinforced Silk Composite Scaffolds: An In Vitro Study  

PubMed Central

Silk fibroin protein is biodegradable and biocompatible, exhibiting excellent mechanical properties for various biomedical applications. However, porous 3D silk fibroin scaffolds, or silk sponges, usually fall short in matching the initial mechanical requirements for bone tissue engineering. In the present study, silk sponge matrices were reinforced with silk microparticles to generate protein-protein composite scaffolds with desirable mechanical properties for in vitro osteogenic tissue formation. It was found that increasing the silk microparticle loading led to a substantial increase in the scaffold compressive modulus from 0.3 MPa (nonreinforced) to 1.9 MPa for 1:2 (matrix:particle) reinforcement loading by dry mass. Biochemical, gene expression, and histological assays were employed to study the possible effects of increasing composite scaffold stiffness, due to microparticle reinforcement, on in vitro osteogenic differentiation of human mesenchymal stem cells (hMSCs). Increasing silk microparticle loading increased the osteogenic capability of hMSCs in the presence of bone morphogenic protein-2 (BMP-2) and other osteogenic factors in static culture for up to six weeks. The calcium adsorption increased dramatically with increasing loading, as observed from biochemical assays, histological staining, and microCT (?CT) analysis. Specifically, calcium content in the scaffolds increased by 0.57, 0.71, and 1.27 mg (per ?g of DNA) from 3 to 6 weeks for matrix to particle dry mass loading ratios of 1:0, 1:1 and 1:2, respectively. In addition, ?CT imaging revealed that at 6 weeks, bone volume fraction increased from 0.78% for nonreinforced to 7.1% and 6.7% for 1:1 and 1:2 loading, respectively. Our results support the hypothesis that scaffold stiffness may strongly influence the 3D in vitro differentiation capabilities of hMSCs, providing a means to improve osteogenic outcomes. PMID:20656075

Rockwood, Danielle N.; Gil, Eun Seok; Park, Sang-Hyug; Kluge, Jonathan A.; Grayson, Warren; Bhumiratana, Sarindr; Rajkhowa, Rangam; Wang, Xungai; Kim, Sung Jun; Vunjak-Novakovic, Gordana; Kaplan, David L

2010-01-01

314

Spider silk reduces insect herbivory.  

PubMed

The role of predators in food webs extends beyond their ability to kill and consume prey. Such trait-mediated effects occur when signals of the predator influence the behaviour of other animals. Because all spiders are silk-producing carnivores, we hypothesized that silk alone would signal other arthropods and enhance non-lethal effects of spiders. We quantified the herbivory inflicted by two beetle species on green bean plants (Phaseolus vulgaris) in the presence of silkworm silk and spider silk along with no silk controls. Single leaflets were treated and enclosed with herbivores in the laboratory and field. Another set of leaflets were treated and left to experience natural herbivory in the field. Entire plants in the field were treated with silk and enclosed with herbivores or left exposed to herbivory. In all cases, the lowest levels of herbivory occurred with spider silk treatments and, in general, silkworm silk produced intermediate levels of leaf damage. These results suggest that silk may be a mechanism for the trait-mediated impacts of spiders and that it might contribute to integrated pest management programmes. PMID:23193048

Rypstra, Ann L; Buddle, Christopher M

2013-02-23

315

Relationships between supercontraction and mechanical properties of spider silk  

NASA Astrophysics Data System (ADS)

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.

Liu, Yi; Shao, Zhengzhong; Vollrath, Fritz

2005-12-01

316

Transdermal Delivery Devices: Fabrication, Mechanics and Drug Release from Silk**  

PubMed Central

Microneedles are a relatively simple, minimally invasive and painless approach to deliver drugs across the skin. However, there remain limitations with this approach because of the materials most commonly utilized for such systems. Silk protein, with tunable and biocompatibility properties, is a useful biomaterial to overcome the current limitations with microneedles. Silk devices preserve drug activity, offer superior mechanical properties and biocompatibility, can be tuned for biodegradability, and can be processed under aqueous, benign conditions. In the present work, we report the fabrication of dense microneedle arrays from silk with different drug release kinetics. The mechanical properties of the microneedle patches are tuned by post-fabrication treatments or by loading the needles with silk microparticles to increase capacity and mechanical strength. Drug release is further enhanced by the encapsulation of the drugs in the silk matrix and coating with a thin dissolvable drug layer. The microneedles are used on human cadaver skin and drugs were delivered successfully. The various attributes demonstrated suggest that silk-based microneedle devices can provide significant benefit as a platform material for transdermal drug delivery. PMID:23653252

Raja, Waseem K.; MacCorkle, Scott; Diwan, Izzuddin M.; Abdurrob, Abdurrahman; Lu, Jessica; Omenetto, Fiorenzo G.; Kaplan, David L.

2013-01-01

317

Molecular mechanics of silk nanostructures under varied mechanical loading.  

PubMed

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

Bratzel, Graham; Buehler, Markus J

2012-06-01

318

Silk constructs for delivery of muskuloskeletal therapeutics  

PubMed Central

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 render SF so exciting for biomedical applications. his pattern along with the versatility of this biopolymer have 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

Meinel, Lorenz; Kaplan, David L.

2012-01-01

319

Preparation of hexagonal GeO? particles with particle size and crystallinity controlled by peptides, silk and silk-peptide chimeras.  

PubMed

We demonstrate the use of silk based proteins to control the particle/crystallite size during GeO2 formation, using a bio-mimetic approach at circumneutral pH and ambient temperature. Multicrystalline GeO2 was prepared from germanium tetraethoxide (TEOG) in the presence of different silk-based proteins: Bombyx mori silk (native silk) and two chimeric proteins prepared by linking a germania binding peptide (Ge28: HATGTHGLSLSH) with Bombyx mori silk via chemical coupling at different peptide loadings (silk-Ge28 10% and silk-Ge28 50%). The mineralisation activity of the silk-based proteins was compared with that of peptide Ge28 as a control system. GeO2 mineralisation was investigated in water and in citric acid/bis-tris propane buffer at pH 6. Morphology, particle size, crystallinity, water and organic content of the materials obtained were analysed to study the effect of added biomolecules and mineralisation environment on material properties. In the presence of silk additives well-defined cube-shape hybrid materials composed of hexagonal germania and up to ca. 5 wt% organic content were obtained. The cubic particles ranged from 0.4 to 1.4 ?m in size and were composed of crystalline domains in the range 35-106 nm depending on the additive used and synthesis conditions. The organic material incorporated in the mineral did not appear to affect the unit cell dimensions. The silk and chimeric proteins in water promote material formation and crystal growth, possibly via an effective ion-channelling mechanism, however further studies are needed to assert to what extent the presence of the silk impacts on nucleation and growth stages. The germania binding peptide alone did not have any significant effect on reaction rate, yield or the material's properties compared to the blank. Interestingly, the peptide content in the silk chimeras tested did not affect mineralisation. The presence of buffer inhibited mineral condensation rate and yield. The use of silk-based biomolecules allows control of crystallite/particle size of hybrid materials opening up opportunities for bio-inspired approaches to be applied for the synthesis of functional germania based devices and materials. PMID:25300352

Boix, Estefania; Puddu, Valeria; Perry, Carole C

2014-11-28

320

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

PubMed Central

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

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

2013-01-01

321

Evidence of ?-helical coiled coils and ?-sheets in hornet silk.  

PubMed

?-Helical coiled coil and ?-sheet complexes are essential structural building elements of silk proteins produced by different species of the Hymenoptera. Beside X-ray scattering at wide and small angles we applied cryo-electron diffraction and microscopy to demonstrate the presence and the details of such structures in silk of the giant hornet Vespa mandarinia japonica. Our studies on the assembly of the fibrous silk proteins and their internal organization in relation to the primary chain structure suggest a 172 Å pitch supercoil consisting of four intertwined alanine-rich ?-helical strands. The axial periodicity may adopt even multiples of the pitch value. Coiled coil motifs form the largest portion of the hornet silk structure and are aligned nearly parallel to the cocoon fiber axis in the same way as the membrane-like parts of the cocoon are molecularly orientated in the spinning direction. Supercoils were found to be associated with ?-crystals, predominantly localized in the l-serine-rich chain sequences terminating each of the four predominant silk proteins. Such ?-sheet blocks are considered resulting from transformation of random coil molecular sequences due to the action of elongational forces during the spinning process. PMID:24345346

Kameda, Tsunenori; Nemoto, Takashi; Ogawa, Tetsuya; Tosaka, Masatoshi; Kurata, Hiroki; Schaper, Andreas K

2014-03-01

322

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

PubMed

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

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

2011-11-01

323

Shear-induced rigidity in spider silk glands  

NASA Astrophysics Data System (ADS)

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.

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

2012-09-01

324

Synthetic Spider Silk Production on a Laboratory Scale  

PubMed Central

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation biomaterials with high performance properties. The development of these new structural materials must be rapid, cost-efficient and involve processing methodologies and products that are environmentally friendly and sustainable. Spiders spin a multitude of different fiber types with diverse mechanical properties, offering a rich source of next generation engineering materials for biomimicry that rival the best manmade and natural materials. Since the collection of large quantities of natural spider silk is impractical, synthetic silk production has the ability to provide scientists with access to an unlimited supply of threads. Therefore, if the spinning process can be streamlined and perfected, artificial spider fibers have the potential use for a broad range of applications ranging from body armor, surgical sutures, ropes and cables, tires, strings for musical instruments, and composites for aviation and aerospace technology. In order to advance the synthetic silk production process and to yield fibers that display low variance in their material properties from spin to spin, we developed a wet-spinning protocol that integrates expression of recombinant spider silk proteins in bacteria, purification and concentration of the proteins, followed by fiber extrusion and a mechanical post-spin treatment. This is the first visual representation that reveals a step-by-step process to spin and analyze artificial silk fibers on a laboratory scale. It also provides details to minimize the introduction of variability among fibers spun from the same spinning dope. Collectively, these methods will propel the process of artificial silk production, leading to higher quality fibers that surpass natural spider silks. PMID:22847722

Hsia, Yang; Gnesa, Eric; Pacheco, Ryan; Kohler, Kristin; Jeffery, Felicia; Vierra, Craig

2012-01-01

325

Synthetic spider silk production on a laboratory scale.  

PubMed

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation biomaterials with high performance properties. The development of these new structural materials must be rapid, cost-efficient and involve processing methodologies and products that are environmentally friendly and sustainable. Spiders spin a multitude of different fiber types with diverse mechanical properties, offering a rich source of next generation engineering materials for biomimicry that rival the best manmade and natural materials. Since the collection of large quantities of natural spider silk is impractical, synthetic silk production has the ability to provide scientists with access to an unlimited supply of threads. Therefore, if the spinning process can be streamlined and perfected, artificial spider fibers have the potential use for a broad range of applications ranging from body armor, surgical sutures, ropes and cables, tires, strings for musical instruments, and composites for aviation and aerospace technology. In order to advance the synthetic silk production process and to yield fibers that display low variance in their material properties from spin to spin, we developed a wet-spinning protocol that integrates expression of recombinant spider silk proteins in bacteria, purification and concentration of the proteins, followed by fiber extrusion and a mechanical post-spin treatment. This is the first visual representation that reveals a step-by-step process to spin and analyze artificial silk fibers on a laboratory scale. It also provides details to minimize the introduction of variability among fibers spun from the same spinning dope. Collectively, these methods will propel the process of artificial silk production, leading to higher quality fibers that surpass natural spider silks. PMID:22847722

Hsia, Yang; Gnesa, Eric; Pacheco, Ryan; Kohler, Kristin; Jeffery, Felicia; Vierra, Craig

2012-01-01

326

Isolation and characterisation of sericin antifreeze peptides and molecular dynamics modelling of their ice-binding interaction.  

PubMed

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 1009.50Da 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

Wu, Jinhong; Rong, Yuzhi; Wang, Zhengwu; Zhou, Yanfu; Wang, Shaoyun; Zhao, Bo

2015-05-01

327

Nanomaterial building blocks based on spider silk-oligonucleotide conjugates.  

PubMed

Self-assembling protein nanofibrils are promising structures for the "bottom-up" fabrication of bionanomaterials. Here, the recombinant protein eADF4(C16), a variant of Araneus diadematus dragline silk ADF4, which self-assembles into nanofibrils, and short oligonucleotides were modified for site-specific azide-alkyne coupling. Corresponding oligonuleotide-eADF4(C16) "click" conjugates were hybridized in linear or branched fashion according to the designed complementarities of the DNA moieties. Self-assembly properties of higher ordered structures of the spider silk-DNA conjugates were dominated by the silk component. Assembled ?-sheet rich conjugate fibrils were similar in appearance to fibrils of unmodified eADF4(C16) but enabled the specific attachment of neutravidin-modified gold nanoparticles on their surface directed by complementary biotin-oligonucleotides, providing the basis for functionalization of such conjugates. PMID:24405063

Humenik, Martin; Scheibel, Thomas

2014-02-25

328

Characterization of recombinantly produced spider flagelliform silk domains.  

PubMed

The capture spiral of a spider's orb web is made of flagelliform silk, providing high elasticity and an outstanding toughness, perfectly suited for trapping prey. Flagelliform silk comprises mainly one single protein (FLAG) with an estimated molecular weight of 360kDa. We engineered constructs mimicking distinct domains of FLAG (eFLAG) and produced them recombinantly to analyze the structure-function relationship of FLAG domains and assembly properties of FLAG. While in solution the small carboxy-terminal domain is structured, domains from the repetitive core region adopt a conformation typical for intrinsically unstructured proteins. To investigate the influence of the respective domains on solubility and assembly, we tested the aggregation behaviour of individual domains and domain ensembles in presence of conditions known to trigger silk assembly. Both, the length of the repetitive core domain as well as the presence of the carboxy-terminal non-repetitive domain showed impact on eFLAG aggregation. PMID:20045468

Heim, Markus; Ackerschott, Christian B; Scheibel, Thomas

2010-05-01

329

Nutrient Deprivation Induces Property Variations in Spider Gluey Silk  

PubMed Central

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

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

2014-01-01

330

Nutrient deprivation induces property variations in spider gluey silk.  

PubMed

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

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

2014-01-01

331

NMR Studies of Molecular Orientation and Dynamics in Spider silk  

NASA Astrophysics Data System (ADS)

Spider dragline silk has a unique combination of strength and extensibility that has been difficult to achieve in synthetic polymer fibres and has inspired industrial efforts to produce genetically engineered analogues. In light of these efforts elsewhere, we describe solid-state NMR experiments that elucidate the molecular structure and dynamics of this remarkable material. These experiments include the use of a 2-D exchange NMR experiment known as DECODER in which the sample is reoriented through a discrete angle during the mixing time. This experiment allows a reconstruction of the orientation distribution of the protein backbone. Our data is well described by a two-component distribution where the protein backbones of both components are preferentially aligned along the silk fibre. This experiment is also sensitive to molecular motion on a wide range of time-scales, and is employed to study changes in the silk as a function of fibre extension and hydration. Hydrated silk undergoes a remarkable phenomena known as supercontraction where fibres shrink by up to 50% in length while swelling in diameter. DECODER NMR of fully and partially supercontracted silk reveals that supercontraction occurs through a process of local phase transitions where water disrupts inter- and intra-chain hydrogen bonds.

Michal, Carl; Eles, Philip

2004-05-01

332

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

PubMed Central

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

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

2010-01-01

333

Accelerated In Vitro Degradation of Optically Clear Low ?-Sheet Silk Films by Enzyme-Mediated Pretreatment  

PubMed Central

Purpose: To design patterned, transparent silk films with fast degradation rates for the purpose of tissue engineering corneal stroma. Methods: ?-sheet (crystalline) content of silk films was decreased significantly by using a short water annealing time. Additionally, a protocol combining short water annealing time with enzymatic pretreatment of silk films with protease XIV was developed. Results: Low ?-sheet content (17%–18%) and enzymatic pretreatment provided film stability in aqueous environments and accelerated degradation of the silk films in the presence of human corneal fibroblasts in vitro. The results demonstrate a direct relationship between reduced ?-sheet content and enzymatic pretreatment, and overall degradation rate of the protein films. Conclusions: The novel protocol developed here provides new approaches to modulate the regeneration rate of silk biomaterials for corneal tissue regeneration needs. Translational Relevance: Patterned silk protein films possess desirable characteristics for corneal tissue engineering, including optical transparency, biocompatibility, cell alignment, and tunable mechanical properties, but current fabrication protocols do not provide adequate degradation rates to match the regeneration properties of the human cornea. This novel processing protocol makes silk films more suitable for the construction of human corneal stroma tissue and a promising way to tune silk film degradation properties to match corneal tissue regeneration. PMID:24049717

Shang, Ke; Rnjak-Kovacina, Jelena; Lin, Yinan; Hayden, Rebecca S.; Tao, Hu; Kaplan, David L.

2013-01-01

334

Silk electrogel coatings for titanium dental implants.  

PubMed

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

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

2014-11-25

335

Micromolar biosensing of nitric oxide using myoglobin immobilized in a synthetic silk film.  

PubMed

In this work we investigate the use of coiled-coil silk proteins, produced in recombinant Escherichia coli, as a new material for immobilizing biosensors. Myoglobin was embedded in transparent honeybee silk protein films. Immobilized myoglobin maintained a high affinity for nitric oxide (KD NO=52 µM) and good sensitivity with a limit of detection of 5 µM. The immobilized myoglobin-silk protein film was stable and could be stored as a dry film at room temperature for at least 60 days. The effect of immobilization on the structure of myoglobin was fully investigated using UV/visible, Fourier Transform Infrared and Raman spectroscopy, which indicated a weakening in the strength of the iron-histidine bond. This study demonstrates that recombinant coiled-coil silk proteins provide a safe and environmentally friendly alternative to sol-gels for stabilizing heme proteins for use as optical biosensors. PMID:25014754

Rapson, Trevor D; Church, Jeffrey S; Trueman, Holly E; Dacres, Helen; Sutherland, Tara D; Trowell, Stephen C

2014-12-15

336

Increasing silk fibre strength through heterogeneity of bundled fibrils.  

PubMed

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

Cranford, Steven W

2013-05-01

337

Increasing silk fibre strength through heterogeneity of bundled fibrils  

PubMed Central

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

Cranford, Steven W.

2013-01-01

338

Uncovering spider silk nanocrystalline variations that facilitate wind-induced mechanical property changes.  

PubMed

Spider major ampullate (MA) silk varies in mechanical properties when spun in different environments. Amino acid compositional changes induced by variations in MaSp1 and MaSp2 expression, and various biochemical and physiological glandular processes induce silk property variability. Quantifying the contributions of these mechanisms on silk variability may facilitate the development of silk biomimetics. Wind is a medium that induces variations in MA silk mechanics. We exposed the spider Cyclosa mulmeinensis to wind and measured the amino acid composition, tensile mechanics, and crystalline structure of its MA silk using HPLC, tensile tests, and X-ray diffraction. We found the mechanical properties of MA silks from spiders exposed to wind to differ from unexposed spiders. The amino acid compositions did not differ, but X-ray diffraction found a lower crystal density and greater ?-sheet alignment relative to the fiber axis in the silks of spiders exposed to wind. We found no evidence that the mechanical property variations were a product of profound changes to the alignment of the protein within the amorphous region. We conclude that variations in the density and alignment of the crystalline ?-sheets, probably accompanied by some alignment change in the amorphous region as a result of "stretching" during spinning of the silk, probably explains the mechanical property variations that we found across treatment subgroups. As C. mulmeinensis MA silk increases both in strength and elasticity when the spiders are exposed to wind, bioengineers may consider it as a model for the development of high-performance silk biomimetics. PMID:23947397

Blamires, Sean J; Wu, Chao-Chia; Wu, Chung-Lin; Sheu, Hwo-Shuenn; Tso, I-Min

2013-10-14

339

A green salt-leaching technique to produce sericin/PVA/glycerin scaffolds with distinguished characteristics for wound-dressing applications.  

PubMed

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. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2014. PMID:25175958

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

2014-08-30

340

Silk Properties Determined by Gland-Specific Expression of a Spider Fibroin Gene Family  

NASA Astrophysics Data System (ADS)

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.

Guerette, Paul A.; Ginzinger, David G.; Weber, Bernhard H. F.; Gosline, John M.

1996-04-01

341

Smart assembly of polymer fibers: lessons from major ampullate spider silk  

NASA Astrophysics Data System (ADS)

Studies of major ampullate silk (MAS), especially the secretions and fibers produced by the spider Nephila clavipes (golden orb weaver), have yielded several results of potential value to the materials scientist/engineer. There are lessons to be learned about synthesis, processing and microstructural design of high-tensile polymer fibers. The 'smart' aspect of silk production in nature concerns the ability of the spider to rapidly process a concentrated, viscous aqueous solution of silk protein (stored in the gland) into water-insoluble fiber on demand. This process centers on the assembly of a shear-sensitive supramolecular liquid crystalline phase by aggregation of the solubilized globular protein molecules.

Viney, Christopher

1996-02-01

342

Physical characterization of functionalized spider silk: electronic and sensing properties  

NASA Astrophysics Data System (ADS)

This work explores functional, fundamental and applied aspects of naturally harvested spider silk fibers. Natural silk is a protein polymer where different amino acids control the physical properties of fibroin bundles, producing, for example, combinations of ?-sheet (crystalline) and amorphous (helical) structural regions. This complexity presents opportunities for functional modification to obtain new types of material properties. Electrical conductivity is the starting point of this investigation, where the insulating nature of neat silk under ambient conditions is described first. Modification of the conductivity by humidity, exposure to polar solvents, iodine doping, pyrolization and deposition of a thin metallic film are explored next. The conductivity increases exponentially with relative humidity and/or solvent, whereas only an incremental increase occurs after iodine doping. In contrast, iodine doping, optimal at 70?°C, has a strong effect on the morphology of silk bundles (increasing their size), on the process of pyrolization (suppressing mass loss rates) and on the resulting carbonized fiber structure (that becomes more robust against bending and strain). The effects of iodine doping and other functional parameters (vacuum and thin film coating) motivated an investigation with magic angle spinning nuclear magnetic resonance (MAS-NMR) to monitor doping-induced changes in the amino acid-protein backbone signature. MAS-NMR revealed a moderate effect of iodine on the helical and ?-sheet structures, and a lesser effect of gold sputtering. The effects of iodine doping were further probed by Fourier transform infrared (FTIR) spectroscopy, revealing a partial transformation of ?-sheet-to-amorphous constituency. A model is proposed, based on the findings from the MAS-NMR and FTIR, which involves iodine-induced changes in the silk fibroin bundle environment that can account for the altered physical properties. Finally, proof-of-concept applications of functionalized spider silk are presented for thermoelectric (Seebeck) effects and incandescence in iodine-doped pyrolized silk fibers, and metallic conductivity and flexibility of micron-sized gold-sputtered silk fibers. In the latter case, we demonstrate the application of gold-sputtered neat spider silk to make four-terminal, flexible, ohmic contacts to organic superconductor samples.

Steven, Eden; Park, Jin Gyu; Paravastu, Anant; Branco Lopes, Elsa; Brooks, James S.; Englander, Ongi; Siegrist, Theo; Kaner, Papatya; Alamo, Rufina G.

2011-10-01

343

Analysis of a new type of major ampullate spider silk gene, MaSp1s.  

PubMed

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

Han, Leng; Zhang, Lei; Zhao, Tianfu; Wang, Yujun; Nakagaki, Masao

2013-05-01

344

In situ Raman spectroscopic study of Al-infiltrated spider dragline silk under tensile deformation.  

PubMed

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

Lee, Seung-Mo; Pippel, Eckhard; Moutanabbir, Oussama; Kim, Jae-Hyun; Lee, Hak-Joo; Knez, Mato

2014-10-01

345

Controlled hierarchical assembly of spider silk-DNA chimeras into ribbons and raft-like morphologies.  

PubMed

Spider silk-DNA conjugates comprising the recombinant spider silk protein eADF4(C16) and short oligonucleotides were arranged in a linear antiparallel and parallel as well as in a branched manner via designed complementarity of the DNA moieties. After cross-? fibril self-assembly, temperature-induced annealing of the DNA moieties triggered fibril association into ribbons, composed of aligned nanofibrils, and rafts composed of ribbons ordered into sharply bordered, squared fibrous microstructures. The formation of the superstructures was clearly dependent on the individual silk-DNA conjugate. A combination of 5'-conjugated silk moieties via complementary nucleic acids enhanced fibril association, whereas mixing complementary 5'- and 3'-silk conjugates inhibited the formation of higher-order structures. PMID:24924514

Humenik, Martin; Drechsler, Markus; Scheibel, Thomas

2014-07-01

346

Identification and dynamics of polyglycine II nanocrystals in Argiope trifasciata flagelliform silk  

NASA Astrophysics Data System (ADS)

Spider silks combine a significant number of desirable characteristics in one material, including large tensile strength and strain at breaking, biocompatibility, and the possibility of tailoring their properties. Major ampullate gland silk (MAS) is the most studied silk and their properties are explained by a double lattice of hydrogen bonds and elastomeric protein chains linked to polyalanine ?-nanocrystals. However, many basic details regarding the relationship between composition, microstructure and properties in silks are still lacking. Here we show that this relationship can be traced in flagelliform silk (Flag) spun by Argiope trifasciata spiders after identifying a phase consisting of polyglycine II nanocrystals. The presence of this phase is consistent with the dominant presence of the -GGX- and -GPG- motifs in its sequence. In contrast to the passive role assigned to polyalanine nanocrystals in MAS, polyglycine II nanocrystals can undergo growing/collapse processes that contribute to increase toughness and justify the ability of Flag to supercontract.

Perea, G. B.; Riekel, C.; Guinea, G. V.; Madurga, R.; Daza, R.; Burghammer, M.; Hayashi, C.; Elices, M.; Plaza, G. R.; Pérez-Rigueiro, J.

2013-10-01

347

Identification and dynamics of polyglycine II nanocrystals in Argiope trifasciata flagelliform silk.  

PubMed

Spider silks combine a significant number of desirable characteristics in one material, including large tensile strength and strain at breaking, biocompatibility, and the possibility of tailoring their properties. Major ampullate gland silk (MAS) is the most studied silk and their properties are explained by a double lattice of hydrogen bonds and elastomeric protein chains linked to polyalanine ?-nanocrystals. However, many basic details regarding the relationship between composition, microstructure and properties in silks are still lacking. Here we show that this relationship can be traced in flagelliform silk (Flag) spun by Argiope trifasciata spiders after identifying a phase consisting of polyglycine II nanocrystals. The presence of this phase is consistent with the dominant presence of the -GGX- and -GPG- motifs in its sequence. In contrast to the passive role assigned to polyalanine nanocrystals in MAS, polyglycine II nanocrystals can undergo growing/collapse processes that contribute to increase toughness and justify the ability of Flag to supercontract. PMID:24162473

Perea, G B; Riekel, C; Guinea, G V; Madurga, R; Daza, R; Burghammer, M; Hayashi, C; Elices, M; Plaza, G R; Pérez-Rigueiro, J

2013-01-01

348

Identification and dynamics of polyglycine II nanocrystals in Argiope trifasciata flagelliform silk  

PubMed Central

Spider silks combine a significant number of desirable characteristics in one material, including large tensile strength and strain at breaking, biocompatibility, and the possibility of tailoring their properties. Major ampullate gland silk (MAS) is the most studied silk and their properties are explained by a double lattice of hydrogen bonds and elastomeric protein chains linked to polyalanine ?-nanocrystals. However, many basic details regarding the relationship between composition, microstructure and properties in silks are still lacking. Here we show that this relationship can be traced in flagelliform silk (Flag) spun by Argiope trifasciata spiders after identifying a phase consisting of polyglycine II nanocrystals. The presence of this phase is consistent with the dominant presence of the –GGX– and –GPG– motifs in its sequence. In contrast to the passive role assigned to polyalanine nanocrystals in MAS, polyglycine II nanocrystals can undergo growing/collapse processes that contribute to increase toughness and justify the ability of Flag to supercontract. PMID:24162473

Perea, G. B.; Riekel, C.; Guinea, G. V.; Madurga, R.; Daza, R.; Burghammer, M.; Hayashi, C.; Elices, M.; Plaza, G. R.; Pérez-Rigueiro, J.

2013-01-01

349

Silk materials--a road to sustainable high technology.  

PubMed

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

Tao, Hu; Kaplan, David L; Omenetto, Fiorenzo G

2012-06-01

350

Neural Responses to Electrical Stimulation on Patterned Silk Films  

PubMed Central

Peripheral nerve injury is a critical issue for trauma patients. Following injury, incomplete axon regeneration or misguided axon innervation into tissue will result in loss of sensory and motor functions. The objective of this study was to examine axon outgrowth and axon alignment in response to surface patterning and electrical stimulation. To accomplish our objective, metal electrodes with dimensions of 1.5 mm × 4 cm, were sputter coated onto micropatterned silk protein films, with surface grooves 3.5 ?m wide × 500 nm deep. P19 neurons were seeded on the patterned electronic silk films and stimulated at 120 mV, 1 kHz, for 45 minutes each day for 7 days. Responses were compared to neurons on flat electronic silk films, patterned silk films without stimulation, and flat silk films without stimulation. Significant alignment was found on the patterned film groups compared to the flat film groups. Axon outgrowth was greater (p < 0.05) on electronic films on day 5 and day 7 compared to the unstimulated groups. In conclusion, electrical stimulation, at 120 mV, 1 kHz, for 45 minutes daily, in addition to surface patterning, of 3.5 ?m wide × 500 nm deep grooves, offered control of nerve axon outgrowth and alignment. PMID:23401351

Hronik-Tupaj, Marie; Raja, Waseem Khan; Tang-Schomer, Min; Omenetto, Fiorenzo G.; Kaplan, David L.

2013-01-01

351

Linking naturally and unnaturally spun silks through the forced reeling of Bombyx mori.  

PubMed

The forced reeling of silkworms offers the potential to produce a spectrum of silk filaments, spun from natural silk dope and subjected to carefully controlled applied processing conditions. Here we demonstrate that the envelope of stress-strain properties for forced reeled silks can encompass both naturally spun cocoon silk and unnaturally processed artificial silk filaments. We use dynamic mechanical thermal analysis (DMTA) to quantify the structural properties of these silks. Using this well-established mechanical spectroscopic technique, we show high variation in the mechanical properties and the associated degree of disordered hydrogen-bonded structures in forced reeled silks. Furthermore, we show that this disorder can be manipulated by a range of processing conditions and even ameliorated under certain parameters, such as annealing under heat and mechanical load. We conclude that the powerful combination of forced reeling silk and DMTA has tied together native/natural and synthetic/unnatural extrusion spinning. The presented techniques therefore have the ability to define the potential of Bombyx-derived proteins for use in fibre-based applications and serve as a roadmap to improve fibre quality via post-processing. PMID:25242653

Mortimer, Beth; Guan, Juan; Holland, Chris; Porter, David; Vollrath, Fritz

2015-01-01

352

All-water-based electron-beam lithography using silk as a resist.  

PubMed

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). PMID:24658173

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

353

Physical Characterization of Functionalized Silk Material for Electronic Application and Devices  

NASA Astrophysics Data System (ADS)

Naturally harvested spider silk fibers are investigated for their physical properties under ambient, humidified, iodine-doped, pyrolized, sputtered gold and carbon nanotube coated conditions. The functional properties include: humidity activated conductivity; enhanced flexibility and carbon yield of pyrolized iodized silk fibers; full metallic conductivity and flexibility of micron-sized gold-sputtered silk fibers; and high strain sensitivity of carbon nanotube coated silk fibers. Magic angle spinning nuclear magnetic resonance (MAS-NMR) and Fourier transform infrared spectroscopy (FTIR) are used to explore the nature of ambient and functionalized spider silk fiber, and significant changes in amino acid-protein backbone signature are correlated with gold sputtering, and iodine-doped conditions. The application of gold-sputtered neat spider silk fibers for making four terminal flexible, clean, ohmic contacts to organic superconductor samples and carbon nanotube coated silk fibers for heart pulse monitoring sensor are demonstrated. The role of silk thin film in organic thin film transistor will be briefly discussed.

Steven, Eden; Jobiliong, Eric; Park, Jin Gyu; Paravastu, Anant; Davidson, Michael; Baird, Michelle; Alamo, Rufina; Kaner, Papatya; Brooks, James; Siegrist, Theo

2012-02-01

354

All-water-based electron-beam lithography using silk as a resist  

NASA Astrophysics Data System (ADS)

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).

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

355

Silk matrix for tissue engineered anterior cruciate ligaments  

Microsoft Academic Search

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,

Gregory H Altman; Rebecca L Horan; Helen H Lu; Jodie Moreau; Ivan Martin; John C Richmond; David L Kaplan

2002-01-01

356

Total X-Ray Scattering of Spider Dragline Silk  

NASA Astrophysics Data System (ADS)

Total x-ray scattering measurements of spider dragline silk fibers from Nephila clavipes, Argiope aurantia, and Latrodectus hesperus all yield similar structure factors, with only small variations between the different species. Wide-angle x-ray scattering from fibers orientated perpendicular to the beam shows a high degree of anisotropy, and differential pair distribution functions obtained by integrating over wedges of the equatorial and meridian planes indicate that, on average, the majority (95%) of the atom-atom correlations do not extend beyond 1 nm. Futhermore, the atom-atom correlations between 1 and 3 nm are not associated with the most intense diffraction peaks at Q=1-2Å-1. Disordered molecular orientations along the fiber axis are consistent with proteins in similar structural arrangements to those in the equatorial plane, which may be associated with the silk’s greater flexibility in this direction.

Benmore, C. J.; Izdebski, T.; Yarger, J. L.

2012-04-01

357

Carbon nanotubes on a spider silk scaffold.  

PubMed

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

Steven, Eden; Saleh, Wasan R; Lebedev, Victor; Acquah, Steve F A; Laukhin, Vladimir; Alamo, Rufina G; Brooks, James S

2013-01-01

358

Carbon nanotubes on a spider silk scaffold  

NASA Astrophysics Data System (ADS)

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.

Steven, Eden; Saleh, Wasan R.; Lebedev, Victor; Acquah, Steve F. A.; Laukhin, Vladimir; Alamo, Rufina G.; Brooks, James S.

2013-09-01

359

Carbon nanotubes on a spider silk scaffold  

PubMed Central

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

Steven, Eden; Saleh, Wasan R.; Lebedev, Victor; Acquah, Steve F. A.; Laukhin, Vladimir; Alamo, Rufina G.; Brooks, James S.

2013-01-01

360

Structure to function: Spider silk and human collagen  

NASA Astrophysics Data System (ADS)

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.

Rabotyagova, Olena S.

361

Nephila clavipes Flagelliform silk-like GGX motifs contribute to extensibility and spacer motifs contribute to strength in synthetic spider silk fibers.  

PubMed

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

Adrianos, Sherry L; Teulé, Florence; Hinman, Michael B; Jones, Justin A; Weber, Warner S; Yarger, Jeffery L; Lewis, Randolph V

2013-06-10

362

Nephila clavipes Flagelliform Silk-like GGX Motifs Contribute to Extensibility and Spacer Motifs Contribute to Strength in Synthetic Spider Silk Fibers  

PubMed Central

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

Adrianos, Sherry L.; Teulé, Florence; Hinman, Michael B.; Jones, Justin A.; Weber, Warner S.; Yarger, Jeffery L.; Lewis, Randolph V.

2013-01-01

363

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

PubMed Central

Background Degummed silk fibroin from Bombyx mori (silkworm) has potential carrier capabilities for drug delivery in humans; however, the processing methods have yet to be comparatively analyzed to determine the differential effects on the silk protein properties, including crystalline structure and activity. Methods In this study, we treated degummed silk with four kinds of calcium-alcohol solutions, and performed secondary structure measurements and enzyme activity test to distinguish the differences between the regenerated fibroins and degummed silk fibroin. Results Gel electrophoresis analysis revealed that Ca(NO3)2-methanol, Ca(NO3)2-ethanol, or CaCl2-methanol treatments produced more lower molecular weights of silk fibroin than CaCl2-ethanol. X-ray diffraction and Fourier-transform infrared spectroscopy showed that CaCl2-ethanol produced a crystalline structure with more silk I (?-form, type II ?-turn), while the other treatments produced more silk II (?-form, anti-parallel ?-pleated sheet). Solid-State 13C cross polarization and magic angle spinning-nuclear magnetic resonance measurements suggested that regenerated fibroins from CaCl2-ethanol were nearly identical to degummed silk fibroin, while the other treatments produced fibroins with significantly different chemical shifts. Finally, enzyme activity test indicated that silk fibroins from CaCl2-ethanol had higher activity when linked to a known chemotherapeutic drug, L-asparaginase, than the fibroins from other treatments. Conclusions Collectively, these results suggest that the CaCl2-ethanol processing method produces silk fibroin with biomaterial properties that are appropriate for drug delivery. PMID:22676291

2012-01-01

364

A silk platform that enables electrophysiology and targeted drug delivery in brain astroglial cells  

PubMed Central

Astroglial cell survival and ion channel activity are relevant molecular targets for the mechanistic study of neural cell interactions with biomaterials and/or electronic interfaces. Astrogliosis is the most typical reaction to in-vivo brain implants and needs to be avoided by developing biomaterials that preserve astroglial cell physiological function. This cellular phenomenon is characterized by a proliferative state and altered expression of astroglial potassium (K+) channels. Silk is a natural polymer with potential for new biomedical applications due to its ability to support in vitro growth and differentiation of many cell types. We report on silk interactions with cultured neocortical astroglial cells. Astrocytes survival is similar when plated on silk-coated glass and on poly-D-lysine (PDL), a well-known polyionic substrate used to promote astroglial cell adhesion to glass surfaces. Comparative analyses of whole-cell and single-cell patch-clamp experiments reveal that silk- and PDL-coated cells display depolarized resting membrane potentials (? -40 mV), very high input resistance, and low specific conductance, with values similar to those of undifferentiated glial cells. Analysis of K+ channel conductance reveals that silk-astrocytes express large outwardly delayed rectifying K+ current (KDR). The magnitude of KDR in PDL- and silk-coated astrocytes is similar, indicating that silk does not alter the resting K+ current. We also demonstrate that guanosine-(GUO) embedded silk enables the direct modulation of astroglial K+ conductance in vitro. Astrocytes plated on GUO-embedded silk are more hyperpolarized and express inward rectifying K+ conductance (Kir). The K+ inward current increase and this is paralleled by upregulation and membrane-polarization of Kir4.1 protein signal. Collectively these results indicate that silk is a suitable biomaterial platform for the in vitro studies of astroglial ion channel responses and related physiology. PMID:20688390

Benfenati, Valentina; Toffanin, Stefano; Capelli, Raffaella; Camassa, Laura M. A.; Ferroni, Stefano; Kaplan, David L.; Omenetto, Fiorenzo G.; Muccini, Michele; Zamboni, Roberto

2010-01-01

365

Fifty Years Later: The Sequence, Structure and Function of Lacewing Cross-beta Silk  

SciTech Connect

Classic studies of protein structure in the 1950s and 1960s demonstrated that green lacewing egg stalk silk possesses a rare native cross-beta sheet conformation. We have identified and sequenced the silk genes expressed by adult females of a green lacewing species. The two encoded silk proteins are 109 and 67 kDa in size and rich in serine, glycine and alanine. Over 70% of each protein sequence consists of highly repetitive regions with 16-residue periodicity. The repetitive sequences can be fitted to an elegant cross-beta sheet structural model with protein chains folded into regular 8-residue long beta strands. This model is supported by wide-angle X-ray scattering data and tensile testing from both our work and the original papers. We suggest that the silk proteins assemble into stacked beta sheet crystallites bound together by a network of cystine cross-links. This hierarchical structure gives the lacewing silk high lateral stiffness nearly threefold that of silkworm silk, enabling the egg stalks to effectively suspend eggs and protect them from predators.

Weisman, Sarah; Okada, Shoko; Mudie, Stephen T.; Huson, Mickey G.; Trueman, Holly E.; Sriskantha, Alagacone; Haritos, Victoria S.; Sutherland, Tara D.; (CSIRO/MSE); (CSIRO)

2009-12-01

366

Untangling spider silk evolution with spidroin terminal domains  

PubMed Central

Background Spidroins are a unique family of large, structural proteins that make up the bulk of spider silk fibers. Due to the highly variable nature of their repetitive sequences, spidroin evolutionary relationships have principally been determined from their non-repetitive carboxy (C)-terminal domains, though they offer limited character data. The few known spidroin amino (N)-terminal domains have been difficult to obtain, but potentially contain critical phylogenetic information for reconstructing the diversification of spider silks. Here we used silk gland expression data (ESTs) from highly divergent species to evaluate the functional significance and phylogenetic utility of spidroin N-terminal domains. Results We report 11 additional spidroin N-termini found by sequencing ~1,900 silk gland cDNAs from nine spider species that shared a common ancestor > 240 million years ago. In contrast to their hyper-variable repetitive regions, spidroin N-terminal domains have retained striking similarities in sequence identity, predicted secondary structure, and hydrophobicity. Through separate and combined phylogenetic analyses of N-terminal domains and their corresponding C-termini, we find that combined analysis produces the most resolved trees and that N-termini contribute more support and less conflict than the C-termini. These analyses show that paralogs largely group by silk gland type, except for the major ampullate spidroins. Moreover, spidroin structural motifs associated with superior tensile strength arose early in the history of this gene family, whereas a motif conferring greater extensibility convergently evolved in two distantly related paralogs. Conclusions A non-repetitive N-terminal domain appears to be a universal attribute of spidroin proteins, likely retained from the origin of spider silk production. Since this time, spidroin N-termini have maintained several features, consistent with this domain playing a key role in silk assembly. Phylogenetic analyses of the conserved N- and C-terminal domains illustrate dramatic radiation of the spidroin gene family, involving extensive duplications, shifts in expression patterns and extreme diversification of repetitive structural sequences that endow spider silks with an unparalleled range of mechanical properties. PMID:20696068

2010-01-01

367

Optically probing torsional superelasticity in spider silks  

NASA Astrophysics Data System (ADS)

We investigate torsion mechanics of various spider silks using a sensitive optical technique. We find that spider silks are torsionally superelastic in that they can reversibly withstand great torsion strains of over 102-3 rotations per cm before failure. Among various silks from a spider, we find the failure twist-strain is greatest in the sticky capture silk followed by dragline and egg-case silk. Our in situ laser-diffraction measurements reveal that torsional strains on the silks induce a nano-scale transverse compression in its diameter that is linear and reversible. These unique torsional properties of the silks could find applications in silk-based materials and devices.

Kumar, Bhupesh; Thakur, Ashish; Panda, Biswajit; Singh, Kamal P.

2013-11-01

368

Optically probing torsional superelasticity in spider silks  

SciTech Connect

We investigate torsion mechanics of various spider silks using a sensitive optical technique. We find that spider silks are torsionally superelastic in that they can reversibly withstand great torsion strains of over 10{sup 2?3} rotations per cm before failure. Among various silks from a spider, we find the failure twist-strain is greatest in the sticky capture silk followed by dragline and egg-case silk. Our in situ laser-diffraction measurements reveal that torsional strains on the silks induce a nano-scale transverse compression in its diameter that is linear and reversible. These unique torsional properties of the silks could find applications in silk-based materials and devices.

Kumar, Bhupesh; Thakur, Ashish; Panda, Biswajit; Singh, Kamal P. [Department of Physical Sciences, IISER Mohali, Sector 81, Manauli, Mohali 140306 (India)] [Department of Physical Sciences, IISER Mohali, Sector 81, Manauli, Mohali 140306 (India)

2013-11-11

369

Discrimination of cultivated silk and wild silk by conventional instrumental analyses.  

PubMed

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

Matsuyama, Yuji; Nagatani, Yoshiaki; Goto, Toshiyuki; Suzuki, Shinichi

2013-09-10

370

Silk Optics Mark Cronin-Golomb  

E-print Network

1/27/14 1 Silk Optics Mark Cronin-Golomb Department of Biomedical Engineering Tufts University Bombyx mori Silk · Abundant · Easily processed · Biocompatible · Biodegradable · Non heavy chain ­GAGA(S,Y,A) repetitive beta sheet forming sequence. Self-assembly of silk Self assembly #12

Barthelat, Francois

371

Enhanced Cellular Adhesion on Titanium by Silk Functionalized with titanium binding and RGD peptides  

PubMed Central

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

Vidal, Guillaume; Blanchi, Thomas; Mieszawska, Aneta J.; Calabrese, Rossella; Rossi, Claire; Vigneron, Pascale; Duval, Jean-Luc; Kaplan, David L.; Egles, Christophe

2012-01-01

372

Controlled assembly: a prerequisite for the use of recombinant spider silk in regenerative medicine?  

PubMed

Recent biotechnological progress has enabled the production of spider silk proteins, spidroins, in heterologous hosts. Matrices based on recombinant spidroins support stem cell growth and are well tolerated when implanted in living tissue, thus the material is highly attractive for use in regenerative medicine. However, the matrices made are far from natural silk in terms of mechanical properties and are either spontaneously assembled, which results in heterogeneous products, or spun from harsh solvents with the concomitant risk of harmful remnants in the final products. If we could mimic the spider's aqueous silk spinning process we would likely obtain a material that had reproducible and better characteristics and that more easily could be transferred to clinical practice. Herein, the knowledge of the spiders' silk production system and the prerequisites for artificial spinning and assembly of recombinant proteins are reviewed and discussed in a biomedical context. PMID:24090990

Rising, Anna

2014-04-01

373

Structure and Properties of Nephila Clavipes Dragline Silk Polymer  

NASA Astrophysics Data System (ADS)

Silk, spun from an aqueous state at room temperature by a variety of organisms, is the most commonly spun extracellular fibrous protein. It comprises polypeptide chains with regions which can crystallize and regions which are predominantly amorphous. The polymer chains in the crystalline regions form anti-parallel pleated sheet structures with an orthorhombic unit cell. Dragline silk is a structural material produced by a variety of spiders. It has been genetically tailored to meet a specific purpose. Dragline silk exhibits high extensibility and tensile strength approaching that of high-strength synthetic fibers. The specific energy to break it can exceed some steels and synthetic fibers. Samples of Nephila clavipes (golden orb-weaver) dragline silk were extracted from live specimens and examined with a series of experimental techniques including optical, scanning electron, and atomic force microscopy, wide and small angle X-ray diffraction and birefringence compensation. Computer modeling of the mechanical properties of the crystallite was also performed. An assortment of features at a variety of length scales was observed by microscopy. These occur on both the as-spun and abraded silk surfaces. The silk was observed to undergo large deformations without evidence of failure, suggesting the absence of a microfibrillar structure. There was no conclusive evidence for either a microfibrillar or a skin core structure. Meridional and equatorial SAXD peaks were observed at Bragg spacings of 79 AA and 250 AA, respectively. Analysis of the WAXD patterns indicated that the silk belongs in Warwicker's category 3b and that the minimum dimensions of the crystals are approximately 38 AA in the molecular direction and 16 x 23 AA in the transverse directions. The crystal modulus was determined with WAXD to be 16.7 GPa, applying the assumption of uniform stress. This is lower than the 200 GPa modulus calculated with molecular modeling. These results and other factors indicate the inapplicability of the widely used assumption of uniform stress. The X-ray data show increased alignment of the crystals along the fiber axis with stress. The birefringence of the silk fibers increases monotonically to failure which indicates increasing molecular alignment. The change in birefringence appears to be governed by the amorphous contribution.

Mahoney, David Vincent

374

Identification and synthesis of novel biomaterials based on spider structural silk fibers  

NASA Astrophysics Data System (ADS)

The diversity in function and mechanical behavior of spider silks, and the ability to produce these silks recombinantly, have tremendous potential in creating a new class of biomimetic materials. Here we investigate the structural and mechanical properties of pyriform silks from the golden orb-weaver, Nephila clavipes. Nanoscale indentation measurements using atomic force microscopy on natural pyriform silk suggests that this biomaterial has high toughness that may be suitable for dissipating high amounts of mechanical energy. We also observed the occurrence of highly organized nanocrystals within the pyriform silk fibers that may contribute to the remarkable energy dissipation capability of these silks. It has been demonstrated that poly-(Gly-Ala) and poly-Ala stretches within the internal block repeat modules of dragline silk fibroins form nanocrystals, and these nanocrystalline structures may be responsible for the high extensibility of the dragline silks. In contrast, amino acid sequence analysis shows that PySp2 does not contain the same motifs. In the absence of poly-(Gly-Ala) and poly-Ala repeats, we hypothesized that PySp2 contains new protein motifs sufficient to polymerize into functional structures. To investigate the functional contributions of these novel motifs during pyriform fiber formation, we expressed different recombinant PySp2 fibroins with various segments spanning its block repeat units. We demonstrate that PySp2 recombinant proteins with the Pro-rich sub-block domain (PXP motifs, where X= sub-set of the amino acids A, L, or R) and/or the Ser + Gln + Ala-rich sub-block domain (QQSSVAQS motifs) are sufficient for artificial fiber formation. Moreover, we show that recombinant PySp2 proteins that contain a single block repeat unit can self-assemble into foam-like nanostructures. Collectively, our findings support the use of PySp2 recombinant proteins for a wide range of biomimetic materials with morphologies ranging from fibers to porous structures.

Hsia, Yang; Gnesa, Eric; Tang, Simon; Jeffery, Felicia; Geurts, Paul; Zhao, Liang; Franz, Andreas; Vierra, Craig

2011-11-01

375

Structure modifications induced in silk fibroin by enzymatic treatments. A Raman study  

NASA Astrophysics Data System (ADS)

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.

Monti, Patrizia; Freddi, Giuliano; Sampaio, Sandra; Tsukada, Masuhiro; Taddei, Paola

2005-06-01

376

Impact of processing parameters on the haemocompatibility of Bombyx mori silk films  

PubMed Central

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

Seib, F. Philipp; Maitz, Manfred F.; Hu, Xiao; Werner, Carsten; Kaplan, David L.

2013-01-01

377

Rate-dependent behavior of the amorphous phase of spider dragline silk.  

PubMed

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

Patil, Sandeep P; Markert, Bernd; Gräter, Frauke

2014-06-01

378

Variation in the material properties of spider dragline silk across species  

NASA Astrophysics Data System (ADS)

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.

Swanson, B. O.; Blackledge, T. A.; Beltrán, J.; Hayashi, C. Y.

2006-02-01

379

Analytical markers for silk degradation: comparing historic silk and silk artificially aged in different environments.  

PubMed

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

Vilaplana, Francisco; Nilsson, Johanna; Sommer, Dorte V P; Karlsson, Sigbritt

2014-12-10

380

A lepidopterous cocoon evidence for silk in the  

E-print Network

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

Panagiotakopulu, Eva

381

Silk-based nanocomplexes with tumor-homing peptides for tumor-specific gene delivery.  

PubMed

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

Numata, Keiji; Mieszawska-Czajkowska, Aneta J; Kvenvold, Laura A; Kaplan, David L

2012-01-01

382

Crystal growth of calcium carbonate in silk fibroin/sodium alginate hydrogel  

NASA Astrophysics Data System (ADS)

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.

Ming, Jinfa; Zuo, Baoqi

2014-01-01

383

Size-dependent mechanical properties of beta-structures in protein materials  

E-print Network

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 ...

Keten, Sinan

2010-01-01

384

Judaism and the Silk Route.  

ERIC Educational Resources Information Center

Demonstrates that the Judeans traveled along the Ancient Silk Route. Discusses the Iranian influence on the formation of Jewish religious ideas. Considers the development of Jewish trade networks, focusing on the Radanites (Jewish traders), the Jewish presence in the Far East, and the survival of Judaism in central Asia. (CMK)

Foltz, Richard

1998-01-01

385

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

E-print Network

Polarized light microscopy, variability in spider silk diameters, and the mechanical characterization of spider silk Todd A. Blackledge,a Richard A. Cardullo, and Cheryl Y. Hayashi Department of Biology, University of California, Riverside, California 92521, USA Abstract. Spider silks possess

Blackledge, Todd

386

Modification of Silk Fibroin Using Diazonium Coupling Chemistry and the Effects on hMSC Proliferation and Differentiation  

PubMed Central

A simple chemical modification method using diazonium coupling chemistry was developed to tailor the structure and hydrophilicity of silk fibroin protein. The extent of modification using several aniline derivatives was characterized using UV/vis and 1H NMR spectroscopy, and the resulting protein structure was analyzed with ATR-FTIR spectroscopy. Introduction of hydrophobic functional groups facilitated rapid conversion of the protein from a random coil to a ?-sheet structure, while addition of hydrophilic groups inhibited this process. hMSCs were grown on these modified silks to assess the biocompatibility of these materials. The hydrophilicity of the silk derivatives was found to affect the growth rate and morphology, but hMSCs were able to attach, proliferate and differentiate into an osteogenic lineage on all of the silk derivatives. PMID:18417206

Murphy, Amanda R.; John, Peter St.; Kaplan, David L.

2009-01-01

387

Fabrication and Biocompatibility of Electrospun Silk Biocomposites  

PubMed Central

Silk fibroin has attracted great interest in tissue engineering because of its outstanding biocompatibility, biodegradability and minimal inflammatory reaction. In this study, two kinds of biocomposites based on regenerated silk fibroin are fabricated by electrospinning and post-treatment processes, respectively. Firstly, regenerated silk fibroin/tetramethoxysilane (TMOS) hybrid nanofibers with high hydrophilicity are prepared, which is superior for fibroblast attachment. The electrospinning process causes adjacent fibers to ‘weld’ at contact points, which can be proved by scanning electron microscope (SEM). The water contact angle of silk/tetramethoxysilane (TMOS) composites shows a sharper decrease than pure regenerated silk fibroin nanofiber, which has a great effect on the early stage of cell attachment behavior. Secondly, a novel tissue engineering scaffold material based on electrospun silk fibroin/nano-hydroxyapatite (nHA) biocomposites is prepared by means of an effective calcium and phosphate (Ca–P) alternate soaking method. nHA is successfully produced on regenerated silk fibroin nanofiber within several min without any pre-treatments. The osteoblastic activities of this novel nanofibrous biocomposites are also investigated by employing osteoblastic-like MC3T3-E1 cell line. The cell functionality such as alkaline phosphatase (ALP) activity is ameliorated on mineralized silk nanofibers. All these results indicate that this silk/nHA biocomposite scaffold material may be a promising biomaterial for bone tissue engineering. PMID:24957869

Wei, Kai; Kim, Byoung-Suhk; Kim, Ick-Soo

2011-01-01

388