Article

Control of silicification by genetically engineered fusion proteins: silk-silica binding peptides (SiBPs)

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Abstract

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 composite silk-silica in two different bioinspired silicification systems: solution-solution and solution-solid. Condensed silica nanoscale particles (600-800 nm) were formed in the presence of the recombinant silk and chimeras, which were smaller than those formed by 15mer-SiBP chimeras [1], revealing that 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 solution system is controlled by the size of protein assemblies in solution. In the solution-solid interface system, silicification reactions were performed on the surface of films fabricated from the recombinant silk proteins and chimeras and then treated to induce β-sheet formation. A higher density of condensed silica formed on the films containing the lowest β-sheet content while the films with highest β-sheet content precipitated the lowest density of silica, revealing an inverse correlation between β-sheet secondary structure and 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.

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... The construct was cloned into commercially available pET30a( + ) vector (Novagen, San Diego, CA, USA), as described previously [38] . The recombinant construct was expressed in Escherichia coli strain BL21 Star (DE3) (Invitrogen, Grand Island, NY, USA) and purified as described previously [39] . Briefly, the recombinant strain was grown overnight at 37 °C in Lysogeny Broth (LB) medium. ...
... The resulting films were air-dried overnight in a fume hood. Then, the films were subjected to water vapor annealing using an isotemp vacuum oven for 24 h at room temperature [22,39] to induce the formation of β-sheets. Finally, the films were air-dried overnight in a fume hood at room temperature. ...
... To induce silica precipitation, R5-15mer-ch protein films were treated with pre-hydrolyzed tetraethyl orthosilicate (TEOS) in 100 mM bis-tris propane/citric acid buffer at pHs ranging from 3 to 10, according to protocols previously described [22,25,39] . For the SEM material characterization, a total of 1 mL of 30 mM TEOS at different pHs was added into each well containing a cover glass of a 12-well plate to cover the film formed in the cover glass for 1, 2 or 5 h at room temperature. ...
Article
Understanding the properties and behavior of biomineralized protein-based materials at the organic-inorganic interface is critical to optimize the performance of such materials for biomedical applications. To that end, this work investigates biomineralized protein-based films with applications for bone regeneration. These films were generated using a chimeric protein fusing the consensus repeat derived from the spider Nephila clavipes major ampullate dragline silk with the silica-promoting peptide R5 derived from the Cylindrotheca fusiformis silaffin gene. The effect of pH on the size of silica nanoparticles during their biomineralization on silk films was investigated, as well as the potential impact of nanoparticle size on the differentiation of human mesenchymal stem cells (hMSCs) into osteoblasts. To that end, induction of the integrin αV subunit and the osteogenic markers Runx2 transcription factor and Bone Sialoprotein (BSP) was followed. The results indicated that pH values of 7-8 during biomineralization maximized the coverage of the film surface by silica nanoparticles yielding nanoparticles ranging 200-500 nm and showing enhanced osteoinduction in gene expression analysis. Lower (3-5) or high (10) pH values led to lower biomineralization and poor coverage of the protein surfaces, showing reduced osteoinduction. Molecular dynamics simulations confirmed the activation of the integrin αVβ3 in contact with silica nanoparticles, correlating with the experimental data on the induction of osteogenic markers. This work sheds light on the optimal conditions for the development of fit-for-purpose biomaterial designs for bone regeneration, while the agreement between experimental and computational results shows the potential of computational methods to predict the expression of osteogenic markers for biomaterials. Statement of Significance The ability of biomineralized materials to induce hMSCs differentiation for bone tissue regeneration applications was analyzed. Biomaterials were created using a recombinant protein formed by the consensus repeat derived from the spider Nephila clavipes major ampullate dragline silk and the silica-promoting peptide R5 derived from the Cylindrotheca fusiformis silaffin gene. A combination of computational and experimental techniques revealed the optimal conditions for the synthesis of biomineralized silk-silica films with enhanced expression of markers related to bone regeneration.
... The R5 peptide (SSKKSGSYSGSKGSKRRIL) derived from silaffin was able to bind and deposit silica in a controlled fashion [84]. To gain control over biosilica formation, the R5 peptide was fused to bioengineered silks [85][86][87]. The fusion of a 15-mer protein to the R5 peptide generated nanocomposite materials containing silica particles with a narrow size distribution of 0.5-2 µm in diameter. ...
... Moreover, this hybrid silk was able to form films and fibers [85]. Film based on a recombinant silk 6-mer protein functionalized with the R5 peptide was reported to not only deposit biosilica on its surface but also control the size and rate of silica mineralization through control of the β-sheet content of the silk protein [86]. The modification of silk with the R5 peptide was the most promising functionalization compared with other modifications such as fusion to peptides A1 (SGSKGSKRRIL) and A3 (MSPHPHPRHHHT) [86]. ...
... Film based on a recombinant silk 6-mer protein functionalized with the R5 peptide was reported to not only deposit biosilica on its surface but also control the size and rate of silica mineralization through control of the β-sheet content of the silk protein [86]. The modification of silk with the R5 peptide was the most promising functionalization compared with other modifications such as fusion to peptides A1 (SGSKGSKRRIL) and A3 (MSPHPHPRHHHT) [86]. Furthermore, the 15-mer silk protein functionalized with the R5 peptide formed a composite silk/silica film that promoted the proliferation of hMSCs and their differentiation into an osteogenic lineage, which was indicated by the presence of osteogenic gene markers [87]. ...
Article
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The great mechanical properties, biocompatibility and biodegradability of silk-based materials make them applicable to the biomedical field. Genetic engineering enables the construction of synthetic equivalents of natural silks. Knowledge about the relationship between the structure and function of silk proteins enables the design of bioengineered silks that can serve as the foundation of new biomaterials. Furthermore, in order to better address the needs of modern biomedicine, genetic engineering can be used to obtain silk-based materials with new functionalities. Sequences encoding new peptides or domains can be added to the sequences encoding the silk proteins. The expression of one cDNA fragment indicates that each silk molecule is related to a functional fragment. This review summarizes the proposed genetic functionalization of silk-based materials that can be potentially useful for biomedical applications.
... Plasmids obtained from the recombinant DNA library were transformed into competent E. coli BL21* (New England Biolabs, Ipswich, MA) for expression as described in [56]. Briefly, transformants were cultured in a 1 L hyper-broth media (Sigma-Aldrich, Natick, MA, USA) containing a yeast extract medium 25 g/L and a 15% glucose nutrient mix as well as ampicillin (100 µg/mL) at 37 • C under aeration conditions until cultures reached the mid logarithmic growth phase (OD600 = 0.5-0.8); ...
... Ten Gaussian peaks were selected across the amide I absorption band (1720-1580 cm −1 ). Four secondary structure motifs were analyzed corresponding to β-sheet (1618-1629 cm −1 and 1697-1703 cm −1 ), α-helix (1658-1667 cm −1 ), β-turns (1668-1696 cm −1 ) and random coil (1630-1657 cm −1 ) [56]. Using the second derivative method, the relative area of the curves corresponding to each of the secondary structural motifs was calculated; relative areas were divided by the summed area of all the deconvoluted curves and transformed into percentages. ...
... Deconvolution of the amide I spectra showed that the addition of methanol resulted in a decreased peak at 1650 and 1660 cm −1 with an increased peak at 1620 cm −1 suggesting a change in structure from random coils and α-helix to a more ordered β-sheet structure ( Figure 3). This remains consistent with expectations, as the addition of methanol has been shown to induce β-sheet formation in many silk-related polypeptides [30,56]. The constructions from the library showed similar secondary structure compositions when compared to native spidroin proteins, as reported in previous publications. ...
Article
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The properties of native spider silk vary within and across species due to the presence of different genes containing conserved repetitive core domains encoding a variety of silk proteins. Previous studies seeking to understand the function and material properties of these domains focused primarily on the analysis of dragline silk proteins, MaSp1 and MaSp2. Our work seeks to broaden the mechanical properties of silk-based biomaterials by establishing two libraries containing genes from the repetitive core region of the native Latrodectus hesperus silk genome (Library A: genes masp1, masp2, tusp1, acsp1; Library B: genes acsp1, pysp1, misp1, flag). The expressed and purified proteins were analyzed through Fourier Transform Infrared Spectrometry (FTIR). Some of these new proteins revealed a higher portion of β-sheet content in recombinant proteins produced from gene constructs containing a combination of masp1/masp2 and acsp1/tusp1 genes than recombinant proteins which consisted solely of dragline silk genes (Library A). A higher portion of β-turn and random coil content was identified in recombinant proteins from pysp1 and flag genes (Library B). Mechanical characterization of selected proteins purified from Library A and Library B formed into films was assessed by Atomic Force Microscopy (AFM) and suggested Library A recombinant proteins had higher elastic moduli when compared to Library B recombinant proteins. Both libraries had higher elastic moduli when compared to native spider silk proteins. The preliminary approach demonstrated here suggests that repetitive core regions of the aforementioned genes can be used as building blocks for new silk-based biomaterials with varying mechanical properties.
... The enzymatic gelation system of SF hydrogels was used to crosslink tyrosine residues in silk protein through HRP and H2O2 [38]. Tyrosine residues of the SF aqueous solution formed tight bonds between each SF surface through the addition of HRP and H2O2, so these bonding reactions have a chance to occur between the SiNPs@NFs, SF aqueous solution and both of them in the composite hydrogels [38,42,43]. The transparency of the composite hydrogels was decreased with increasing concentrations of the homogenized SiNPs@NFs added (Figure 1C). ...
... Therefore, segmentally distribution of SiNPs within SF-NFs by electrospinning could boost up the compressive strength of the composite hydrogels since native bones structured with ordered mineral crystals in ECM fibrils was mimicked by the structure with the segmental distribution of SiNPs clusters into SF fibrils [8,24]. Moreover, SF was reported that could bind with SiNPs to generate silk-silica peptides through silicification, which would promote strength enhancement of the (SiNPs@NFs)5%-SF hydrogel [42,43,45]. ...
Article
Various materials are utilized as artificial substitutes for bone repair. In this study, a silk fibroin (SF) hydrogel reinforced by short silica nanoparticles (SiNPs)‐distributed‐silk fibroin nanofibers (SiNPs@NFs), which exhibits a superior osteoinductive property, is fabricated for treating bone defects. SF acts as the base part of the composite scaffold to mimic the extracellular matrix (ECM), which is the organic component of a native bone. The distribution of SiNPs clusters within the composite hydrogel partially mimics the distribution of mineral crystals within the ECM. Incorporation of SiNPs@NFs enhances the mechanical properties of the composite hydrogel. In addition, the composite hydrogel provides a biocompatible microenvironment for cell adhesion, proliferation, and osteogenic differentiation in vitro. In vivo studies confirm that the successful repair is achieved with the formation of a large amount of new bone in the large‐sized cranial defects that are treated with the composite hydrogel. In conclusion, the SiNPs@NFs‐reinforced‐hydrogel fabricated in this study has the potential for use in bone tissue engineering.
... After FSD, the deconvoluted spectra were fitted with 11 Gaussian peaks to determine the secondary structure components. The details of this method were described in our previous works [22][23][24]. ...
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Statement of significance: 3D bioprinting has emerged as a technology that can produce biologically relevant structures in defined geometries with microscale resolution. Techniques for fabrication of free-standing structures by printing into granular gel media has been demonstrated previously, however, these methods require crosslinking agents and post-processing steps on printed structures. Our method utilizes one-step gelation of silk fibroin within a suspension of synthetic nanoclay (Laponite), with no need for additional crosslinking compounds or post processing of the material. This new method allows for in situ physical crosslinking of pure aqueous silk fibroin into defined geometries produced through freeform 3D printing.
... 24 Silk−silica fusion proteins were designed as biomaterials for bone regeneration. 25 However, conventional recombinant protein methods are limited by generally tedious and lowyielding cloning and biosynthesis methods because of the repetitive nature and length of the silk sequences. In addition, the serial design process, where new genetic/protein constructs are prepared and characterized in sequential fashion, also leads to a slower, trial-and-error process to reach specific functional goals for the materials. ...
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Tailored biomaterials with tunable functional properties are crucial for a variety of task-specific applications ranging from healthcare to sustainable, novel bio–nanodevices. To generate polymeric materials with predictive functional outcomes, exploiting designs from nature while morphing them toward non-natural systems offers an important strategy. Silks are Nature’s building blocks and are produced by arthropods for a variety of uses that are essential for their survival. Due to the genetic control of encoded protein sequence, mechanical properties, biocompatibility, and biodegradability, silk proteins have been selected as prototype models to emulate for the tunable designs of biomaterial systems.
... For example, a pentatyrosine protag was shown to allow tyrosinase-mediated covalent coupling of an IgG-binding protein G or a human glycoprotein, ApoH, with both polysaccharides (Wu et al., 2009) and silk fibroin from Bombyx mori (Wu et al., 2020); a polyglutamine tag facilitated the assembly of proteins onto both gelatin (Liu et al., 2015) and spider silk (Wu et al., 2017); and a polylysine tag was added to enzymes for covalent tethering onto engineered tobacco mosaic virus-derived virus like particles (Bhokisham et al., 2020). Other peptide tags of varied amino acid composition enable binding onto solid materials such as gold (Tamerler et al., 2006;Adams et al., 2015;Terrell et al., 2021), silver (Sedlak et al., 2012), silicon (Zhou et al., 2015), as well as various hydrophobic surfaces (Tanaka et al., 2006) through non-covalent interactions. Methodologies that allow protein attachment to various substrates have created new possibilities to construct devices with diverse functions introduced by the assembled proteins. ...
Article
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Biofabrication utilizes biological materials and biological means, or mimics thereof, for assembly. When interfaced with microelectronics, electrobiofabricated assemblies enable exquisite sensing and reporting capabilities. We recently demonstrated that thiolated polyethylene glycol (PEG-SH) could be oxidatively assembled into a thin disulfide crosslinked hydrogel at an electrode surface; with sufficient oxidation, extra sulfenic acid groups are made available for covalent, disulfide coupling to sulfhydryl groups of proteins or peptides. We intentionally introduced a polycysteine tag (5xCys-tag) consisting of five consecutive cysteine residues at the C-terminus of a Streptococcal protein G to enable its covalent coupling to an electroassembled PEG-SH film. We found, however, that its expression and purification from E. coli was difficult, owing to the extra cysteine residues. We developed a redox-based autoinduction methodology that greatly enhanced the yield, especially in the soluble fraction of E. coli extracts. The redox component involved the deletion of oxyRS, a global regulator of the oxidative stress response and the autoinduction component integrated a quorum sensing (QS) switch that keys the secreted QS autoinducer-2 to induction. Interestingly, both methods helped when independently employed and further, when used in combination (i.e., autodinduced oxyRS mutant) the results were best—we found the highest total yield and highest yield in the soluble fraction. We hypothesize that the production host was less prone to severe metabolic perturbations that might reduce yield or drive sequestration of the -tagged protein into inclusion bodies. We expect this methodology will be useful for the expression of many such Cys-tagged proteins, ultimately enabling a diverse array of functionalized devices.
... The inspiration for such materials is often drawn from biology, including the concept of hierarchical self-assembly of biopolymers to generate the materials' structure. Furthermore, embedding active biomolecules-often proteins-that are capable of functioning with unparalleled affinity and specificity has the potential to create bioactive materials (Huang et al., 2011;Nileb€ ack et al., 2017;Tsai et al., 2015;Zhou et al., 2015). Proteins offer many advantages in forming the scaffold, or basic structure, in these materials. ...
Chapter
The development of functionalized materials is needed to enable diverse applications. Protein-based materials are typically biocompatible and biodegradable and can exhibit a wide variety of useful mechanical properties. Most importantly, gene fusion enables facile incorporation of active proteins into the materials. However, many protocols rely on denaturing conditions to stimulate materials formation. These conditions would be expected to inactivate any appended functional proteins. This chapter describes methods to create protein fibers and films in a mild aqueous buffer near neutral pH. This facile, inexpensive single-pot approach to materials assembly does not require any special equipment. Also included in this chapter are methods to fuse fibers to form fiber bundles, and to use fibers for cell culture. Although these methods were developed to generate materials from the Drosophila Hox transcription factor Ultrabithorax, they may also work for other self-assembling proteins, many of which have sequence features in common with Ubx.
... One interesting class of biomaterials is bio-inspired silica, where the polycondensation of silicic acid is directed by the presence of a polycationic templating molecule or by enzymes [1][2][3][4][5][6] . In vitro, large amounts of proteins can be properly encapsulated in silica by simply mixing cationic catalyst with the silicic acid and the protein itself 7 or, to increase the immobilization yield, the protein can be fused to a biosilica-promoting peptide [8][9][10][11][12][13][14] . The materials obtained by such natural processes can be easily tuned into different morphologies by the use of slight perturbations of the experimental conditions 10,15 , and can be easily endowed with peculiar chemical properties, such as fluorescence or catalytic activity by the incorporation of proteins 9 . ...
Article
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Proton-detection in solid-state NMR, enabled by high magnetic fields (> 18 T) and fast magic angle spinning (> 50â kHz), allows for the acquisition of traditional (1) H-(15) N experiments on systems that are too big to be observed in solution. Among those, proteins entrapped in a bioinspired silica matrix are an attractive target that is receiving a large share of attention. We demonstrate that (1) H-detected SSNMR provides a novel approach to the rapid assessment of structural integrity in proteins entrapped in bioinspired silica.
... The features of the designed scaffolds were tuned based on the variables of silk constructs (silk source or number of consensus repeats), peptide identity, and the synthesis approach (chemical or genetic) [137] . In general, this strategy could be useful for controlling the fabrication of biomaterials for medical applications [138] . Gueo et al. developed a new approach for the design of biomaterial grafts using the continuous gradients of silica precipitation on the engineered silk-based composites. ...
Article
The rational design and controllable synthesis of functional silica-based materials have gained increased interest in a variety of biomedical and biotechnological applications due to their unique properties. The current review shows that marine organisms, such as siliceous sponges and diatoms, could be the inspiration for the fabrication of advanced biohybrid materials. Several biomolecules were involved in the molecular mechanism of biosilicification in vivo. Mimicking their behavior, functional silica-based biomaterials have been generated via biomimetic and bioinspired silicification in vitro. Additionally, several advanced technologies were developed for in vitro and in vivo immobilization of biomolecules with potential applications in biocatalysis, biosensors, bioimaging, and immunoassays. A thin silica layer could coat a single living cell or virus as a protective shell offering new opportunities in biotechnology and nanomedicine fields. Promising nanotechnologies have been developed for drug encapsulation and delivery in a targeted and controlled manner, in particular for poorly soluble hydrophobic drugs. Moreover, biomimetic silica, as a morphogenetically active biocompatible material, has been utilized in the field of bone regeneration and in the development of biomedical implantable devices. Statement of significance In nature, silica-based biomaterials, such as diatom frustules and sponge spicules, with high mechanical and physical properties were created under biocompatible conditions. The fundamental knowledge underlying the molecular mechanisms of biosilica formation could inspire engineers and chemists to design novel hybrid biomaterials using molecular biomimetic strategies. The production of such biohybrid materials brings the biosilicification field closer to practical applications. This review starts with the biosilicification process of sponges and diatoms with recently updated researches. Then, this article covers recent advances in the design of silica-based biomaterials and their potential applications in the fields of biotechnology and nanomedicine, highlighting several promising technologies for encapsulation of functional proteins and living cells, drug delivery and the preparation of scaffolds for bone regeneration.
... 11−13 Those enabled probing the two-way communications between the environmental stimuli and biological signals in the devices. Another intriguing example is the bioinspired silicification 14,15 by harnessing the biomimetic silica polycondensation process derived from diatoms. The silica binding peptides were engineered with biomolecules forming an interface to guide the nanosilica deposition on the designated localities. ...
Article
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Silk fibroin produced from silkworms has been intensively utilized as a scaffold material for a variety of biotechnological applications owing to its remarkable mechanical strength, extensibility, biocompatibility, and ease of biofunctionalization. In this research, we engineered silk as a novel trap platform capable of capturing microorganisms. Specifically, we first fabricated the silk material into a silk sponge by lyophilization, yielding a 3D scaffold with porous microstructures. The sponge stability in water was significantly improved by ethanol treatment with elevated β-sheet content and crystallinity of silk. Next, we biofunctionalized the silk sponge with a poly-specific microbial targeting molecule, ApoH (apolipoprotein H), to enable a novel silk-based microbial trap. The recombinant ApoH engineered with an additional penta-tyrosine was assembled onto the silk sponge through the horseradish peroxidase (HRP) mediated dityrosine cross-linking. Last, the ApoH-decorated silk sponge was demonstrated to be functional in capturing our model microorganism targets, E. coli and norovirus-like particles. We envision that this biofabricated silk platform, capable of trapping a variety of microbial entities, could serve as a versatile scaffold for rapid isolation and enrichment of microbial samples toward future diagnostics and therapeutics. This strategy, in turn, can expedite advancing future biodevices with functionality and sustainability.
... Recently, Perry and coworkers investigated the influence of connection point of the silica-binding peptide, located at either the N-or C-terminus of the silk protein. 423 Although both chimeric constructs supported cell viability and differentiation of hMSCs in vitro, the N-terminal location of the silica-binding sequence supported a higher potential to induce silica growth, which translated into a greater propensity to promote hMSC differentiation. Recently, Kaplan and co-workers 424 extended this approach to create fusions of the spider-silk inspired domain with a HAP-binding sequence, and again, these authors explored the impact of fusing the peptide at either the C-or Nterminus, or both, on the formation of crystalline HAP. ...
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In vertebrates and invertebrates biomineralization is controlled by the cell and the proteins they produce. A large number of these proteins are intrinsically disordered, gaining some secondary structure when they interact with their binding partners. These partners include the component ions of the mineral being deposited, the crystals themselves, the template on which the initial crystals form, and other intrinsically disordered proteins and peptides. This review speculates why intrinsically disordered proteins are so important for biomineralization, providing illustrations from the SIBLING (small integrin binding N-glycosylated) proteins and their peptides. It is concluded that the flexible structure, and the ability of the intrinsically disordered proteins to bind to a multitude of surfaces is crucial, but details on the precise-interactions, energetics and kinetics of binding remain to be determined.
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Biomineralization at the organic–inorganic interface is critical to many biology material functions in vitro and in vivo. Recombinant silk–silica fusion peptides are organic–inorganic hybrid material systems that can be effectively used to study and control biologically mediated mineralization due to the genetic basis of sequence control. However, to date, the mechanisms by which these functionalized silk–silica proteins trigger the differentiation of human mesenchymal stem cells (hMSCs) to osteoblasts remain unknown. To address this challenge, silk–silica surfaces are analyzed for silica–hMSC receptor binding and activation, and the intracellular pathways involved in the induction of osteogenesis on these bioengineered biomaterials. The induction of gene expression of αVβ3 integrin, all three mitogen-activated protein kinsases, as well as c-Jun, runt-related transcription factor 2, and osteoblast marker genes is demonstrated upon growth of the hMSCs on the silk–silica materials. This induction of key markers of osteogenesis correlates with the content of silica on the materials. Moreover, computational simulations are performed for silk/silica-integrin binding which show activation of αVβ3 integrin in contact with silica. This integrated computational and experimental approach provides insight into interactions that regulate osteogenesis toward more efficient biomaterial designs.
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Vaccine design ushered in the era of nanotechnology, as the vaccine is being developed toward particulate formulation. We have previously shown that the attenuated pneumolysin mutant (ΔA146PLY) was a safe and effective pneumococcal vaccine candidate. Here, to further optimize the formulation, we fused calcium phosphate (CaP) binding domains with ΔA146PLY so that the biocompatible CaP can mineralize with the protein automatically, allowing simple production of nanoparticle antigen during preparation. We fabricated four different nanoparticles, and then we compared the characteristics of different CaP-ΔA146PLY nanoparticles and demonstrated the influence of CaP binding domains on the size, shape and surface calcium content of the nanoparticles. It was found that these self-biomineralized CaP-ΔA146PLY nanoparticles varied in their capacity to induce BMDCs and splenocytes production of cytokines. We further demonstrated that, compared to free proteins, nanoparticle antigens induced more efficient humoral and cellular immune responses which was strong enough to protect mice from both pneumonia and sepsis infection. Also, the integration of CaP to protein has no significant impairment on body weight of animals, and subcutaneous injection of ΔA146PLY-peptides@CaP nanoparticles did not lead to permanent formation of nodules in the skin relative to Alum adjuvant formulated antigens. Together, our data sufficiently suggest that soluble ΔA146PLY vaccine candidate could be processed into nanoparticles by self-biomineralization of CaP, the immunogenicity of which could be efficiently improved by the CaP binding domains and biomineralization.
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Metallo-oxide (MO)-based bioinorganic nanocomposites promise unique structures, physicochemical properties, and novel biochemical functionalities, and within the past decade, investment in research on materials such as ZnO, TiO2, SiO2, and GeO2 has significantly increased. Besides traditional approaches, the synthesis, shaping, structural patterning, and postprocessing chemical functionalization of the materials surface is inspired by strategies which mimic processes in nature. Would such materials deliver new technologies? Answering this question requires the merging of historical knowledge and current research from different fields of science. Practically, we need an effective defragmentation of the research area. From our perspective, the superficial accounting of material properties, chemistry of the surfaces, and the behavior of biomolecules next to such surfaces is a problem. This is particularly of concern when we wish to bridge between technologies in vitro and biotechnologies in vivo. Further, besides the potential practical technological efficiency and advantages such materials might exhibit, we have to consider the wider long-term implications of material stability and toxicity. In this contribution, we present a critical review of recent advances in the chemistry and engineering of MO-based biocomposites, highlighting the role of interactions at the interface and the techniques by which these can be studied. At the end of the article, we outline the challenges which hamper progress in research and extrapolate to developing and promising directions including additive manufacturing and synthetic biology that could benefit from molecular level understanding of interactions occurring between inanimate (abiotic) and living (biotic) materials.
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Surgical site infections (SSI) represent a serious health problem that occur after invasive surgery, thus new antimicrobial biomaterials able to prevent SSI are needed. Silks are natural biopolymers with excellent biocompatibility, low immunogenicity and controllable biodegradability. Spider silk‐based materials can be bioengineered and functionalized with specific peptides, such as antimicrobial peptides, creating innovative polymers. Herein, we explored new drug‐free multifunctional silk films with antimicrobial properties, specifically tailored to hamper microbial infections. Different spider silk domains derived from the dragline sequence of the spider Nephila clavipes (6mer and 15mer, 27 and 41 kDa proteins, respectively) were fused with the two antimicrobial peptides, Hepcidin (Hep) and Human Neutrophil peptide 1 (HNP1). The self‐assembly features of the spider silk domains (β‐sheets) were maintained after functionalization. The bioengineered 6mer‐HNP1 protein demonstrated inhibitory effects against microbial pathogens. Silk‐based films with 6mer‐HNP1 and different contents of silk fibroin (SF) significantly reduced bacterial adhesion and biofilm formation, whereas higher bacterial counts were found on the films prepared with 6mer or SF alone. The silk‐based films showed no cytotoxic effects on human foreskin fibroblasts. The positive cellular response, together with structural and antimicrobial properties, highlight the potential of these multifunctional silk‐based films as new materials for preventing SSI.
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Animal silks, consisting of pure protein components, offer an extraordinary combination of strength, elongation, and toughness, exceeding most engineered materials. The secret to this success is their unique nanoarchitectures formed through the hierarchical self‐assembly of silk proteins. This natural process contrasts the production of artificial silk materials, which usually are directly constructed as bulk structures from silk fibroin (SF) molecules. A variety of fabrication strategies to control nanostructures of silks or to create functional materials from silk nanoscale building blocks have been developed in the recent years. These emerging fabrication strategies offer an opportunity to tailor the structure of SF at the nanoscale and provide a promising route to produce structurally and functionally optimized silk nanomaterials. Herein, the critical roles of silk nanoarchitectures in property and function of natural silk fibers are reviewed and the strategies of utilization of these silk nanobuilding blocks are outlined. Further, the state‐of‐the‐art techniques to create silk nanoarchitectures and to generate silk‐based nanocomponents are summarized. An effective approach to constructing sophisticated silk functional nanocomposites with promising applications in regenerative medicine, drug delivery, and optical and electronic device designs is provided. Further, such insights suggest templates to consider for other material systems. A critical summary of the state of the art in the field of design, fabrication, and function of silk‐based nanomaterials is provided in this review. The structure–property–function relationships of nanobuilding blocks in silk fibers and the emerging strategies for fabrication and use of natural and regenerated silk fibroin–based nanomaterials are highlighted.
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Silk combines biological properties, such as non-toxicity and biodegradability, with physico-chemical ones, for example, mechanical strength. Base on molecular engineering, nowadays also new non-silk functions can be implemented in silk materials. Driven by rational design and ingenuity, innovative recombinant silk proteins can be designed with a plethora of functions to address biomedical and technological challenges. Herein, we review advances in engineering silk proteins for tailored functions at the molecular level. Insights are provided in genetically engineered silk fusions with functional or other structural proteins and in hybrids with DNA. In such novel materials, self-assembly features of silk are combined and utilized with expedient properties of the additional components. The availability of functionalized silk materials is opening routes toward a whole set of novel applications not achievable with natural silk or other polymers.
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Diatoms produce intricately patterned silica structures under ambient conditions, a process initiated by post‐translationally modified silaffin peptides that nucleate silicic acid. Designing these peptides would enable the production of silica nanostructures with desired properties; however, the functional effects of modifications are poorly understood. Here, Escherichia coli is used to express and modify recombinant silaffin R5 peptide from the diatom Cylindrotheca fusiformis . A library of 38 enzymes is tested for R5 modifications in vitro, from which active methyltransferases, kinases, acetyltransferases, oxidases, and myristoyltransferases are identified from diatoms, humans, yeast, and bacteria. Modified R5 peptides are used for silica precipitation and the impacts on particle size, shape, porosity, and surface area are quantified. In vivo pathways are designed to coexpress R5 and a modifying enzyme and the resulting peptide is used to nucleate silica nanostructures with controlled size (100–3500 nm), porosity (20–635 m² g⁻¹), or embedded with melanin. It is found that phosphorylation reduces the need for inorganic phosphate during silica synthesis. The simultaneous methylation and phosphorylation of R5 leads to smaller particles requiring less inorganic phosphate. Inspired by diatoms, the use of post‐translationally modified peptides will enable the control of silica morphology under ambient conditions, with potential applications in electronics and photonics.
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Among protein immobilization strategies, encapsulation in bioinspired silica is increasingly popular. Encapsulation offers high yields and the solid support is created through a protein-catalyzed polycondensation reaction that occurs under mild conditions. An integrated strategy is reported for the characterization of both the protein and bioinspired silica scaffold generated by the encapsulation of enzymes with an external silica-forming promoter or with the promoter expressed as a fusion to the enzyme. This strategy is applied to the catalytic domain of matrix metalloproteinase 12. Analysis reveals that the structure of the protein encapsulated by either method is not significantly altered with respect to the native form. The structural features of silica obtained by either strategy are also similar, but differ from those obtained by other approaches. In case of the covalently linked R5-enzyme construct, immobilization yields are higher. Encapsulation through a fusion protein, therefore, appears to be the method of choice.
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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/µg 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>200µg/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.
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The remarkable mechanical characteristics of the spider silk protein major ampullate spidroin protein suggest this polymer as a promising biomaterial to consider for the fabrication of scaffolds for bone regeneration. Herein, a new functionalized spider silk-bone sialoprotein fusion protein was designed, cloned, expressed, purified and the osteogenic activity studied. Bone sialoprotein (BSP) is a multi-domain protein with the ability to induce cell attachment and differentiation and the deposition of calcium phosphates (CaP). Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) was used to assess the secondary structure of the fusion protein. In vitro mineralization studies demonstrated that this new fusion protein with BSP retained the ability to induce the deposition of CaP. Studies in vitro indicated that human mesenchymal stem cells had significant improvement towards osteogenic outcomes when cultivated in the presence of the new fusion protein vs. silk alone. The present work demonstrates the potential of this new fusion protein for future applications in bone regeneration.
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Diatom cell walls are regarded as a paradigm for controlled production of nanostructured silica, but the mechanisms allowing biosilicification to proceed at ambient temperature at high rates have remained enigmatic. A set of polycationic peptides (called silaffins) isolated from diatom cell walls were shown to generate networks of silica nanospheres within seconds when added to a solution of silicic acid. Silaffins contain covalently modified lysine-lysine elements. The first lysine bears a polyamine consisting of 6 to 11 repeats of the N-methyl-propylamine unit. The second lysine was identified as ɛ-N,N-dimethyl- lysine. These modifications drastically influence the silica-precipitating activity of silaffins.
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Two silica-precipitating peptides, silaffin-1A1 and-1A2, both encoded by thesil1 gene from the diatom Cylindrotheca fusiformis, were extracted from cell walls and purified to homogeneity. The chemical structures were determined by protein chemical methods combined with mass spectrometry. Silaffin-1A1 and -1A2 consist of 15 and 18 amino acid residues, respectively. Each peptide contains a total of four lysine residues, which are all found to be post-translationally modified. In silaffin-1A2 the lysine residues are clustered in two pairs in which the ε-amino group of the first residue is linked to a linear polyamine consisting of 5 to 11N-methylated propylamine units, whereas the second lysine is converted to ε-N,N-dimethyllysine. Silaffin-1A1 contains only a single lysine pair exhibiting the same structural features. One of the two remaining lysine residues was identified as ε-N,N,N-trimethyl-δ-hydroxylysine, a lysine derivative containing a quaternary ammonium group. The fourth lysine residue again is linked to a long-chain polyamine. Silaffin-1A1 is the first peptide shown to contain ε-N,N,N-trimethyl-δ-hydroxylysine. In vitro, both peptides precipitate silica nanospheres within seconds when added to a monosilicic acid solution.
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Silica skeletal architectures in diatoms are characterized by remarkable morphological and nanostructural details. Silk proteins from spiders and silkworms form strong and intricate self-assembling fibrous biomaterials in nature. We combined the features of silk with biosilica through the design, synthesis, and characterization of a novel family of chimeric proteins for subsequent use in model materials forming reactions. The domains from the major ampullate spidroin 1 (MaSp1) protein of Nephila clavipes spider dragline silk provide control over structural and morphological details because it can be self-assembled through diverse processing methods including film casting and fiber electrospinning. Biosilica nanostructures in diatoms are formed in aqueous ambient conditions at neutral pH and low temperatures. The R5 peptide derived from the silaffin protein of Cylindrotheca fusiformis induces and regulates silica precipitation in the chimeric protein designs under similar ambient conditions. Whereas mineralization reactions performed in the presence of R5 peptide alone form silica particles with a size distribution of 0.5-10 microm in diameter, reactions performed in the presence of the new fusion proteins generate nanocomposite materials containing silica particles with a narrower size distribution of 0.5-2 microm in diameter. Furthermore, we demonstrate that composite morphology and structure could be regulated by controlling processing conditions to produce films and fibers. These results suggest that the chimeric protein provides new options for processing and control over silica particle sizes, important benefits for biomedical and specialty materials, particularly in light of the all aqueous processing and the nanocomposite features of these new materials.
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OPTIMIZER is an on-line application that optimizes the codon usage of a gene to increase its expression level. Three methods of optimization are available: the ‘one amino acid–one codon’ method, a guided random method based on a Monte Carlo algorithm, and a new method designed to maximize the optimization with the fewest changes in the query sequence. One of the main features of OPTIMIZER is that it makes it possible to optimize a DNA sequence using pre-computed codon usage tables from a predicted group of highly expressed genes from more than 150 prokaryotic species under strong translational selection. These groups of highly expressed genes have been predicted using a new iterative algorithm. In addition, users can use, as a reference set, a pre-computed table containing the mean codon usage of ribosomal protein genes and, as a novelty, the tRNA gene-copy numbers. OPTIMIZER is accessible free of charge at http://genomes.urv.es/OPTIMIZER.
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Considerable research has been directed toward identifying the mechanisms involved in biosilicification to understand and possibly mimic the process for the production of superior silica-based materials while simultaneously minimizing pollution and energy costs. Molecules isolated from diatoms and, most recently sponges, thought to be key to this process contain polyamines with a propylamine backbone and variable levels of methylation. In a chemical approach to understanding the role of amine (especially propylamine) structures in silicification we have explored three key structural features: (i) the degree of polymerization, (ii) the level of amine methylation, and (iii) the size of the amine chain spacers. In this article, we show that there are two factors critical to their function: the ability of the amines to produce microemulsions and the presence of charged and uncharged amine groups within a molecule, with the latter feature helping to catalyze silicic acid condensation by a proton donor/acceptor mechanism. The understanding of amine–silicate interactions obtained from this study has enabled the controlled preparation of hollow and nonporous siliceous materials under mild conditions (circumneutral pH, room temperature, and in all aqueous systems) possibly compatible with the conditions used by biosystems. The “rules” identified from our study were further used predictively to modulate the activity of a given amine. We believe that the outcomes of the present contribution will form the basis for an approach to controlling the growth of inorganic materials by using tailor-made organic molecules. • bioinspired materials • biosilica • hollow particles
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Biosilicification is an evolutionarily old and widespread type of biomineralization both in unicellular and multicellular organisms, including sponges, diatoms, radiolarians, choanoflagellates, and higher plants. In the last few years combined efforts in molecular biology, cell biology, and inorganic and analytical chemistry have allowed the first insight into the molecular mechanisms by which these organisms form an astonishing variety of siliceous structures that cannot be achieved by chemical methods. Here we report about the present stage of knowledge on structure, biochemical composition, and mechanisms of biosilica formation, focusing our attention particularly on sponges because of the enormous (nano)biotechnological potential of the enzymes involved in this process.
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The brain remains one of the most important but least understood tissues in our body, in part because of its complexity as well as the limitations associated with in vivo studies. Although simpler tissues have yielded to the emerging tools for in vitro 3D tissue cultures, functional brain-like tissues have not. We report the construction of complex functional 3D brain-like cortical tissue, maintained for months in vitro, formed from primary cortical neurons in modular 3D compartmentalized architectures with electrophysiological function. We show that, on injury, this brain-like tissue responds in vitro with biochemical and electrophysiological outcomes that mimic observations in vivo. This modular 3D brain-like tissue is capable of real-time nondestructive assessments, offering previously unidentified directions for studies of brain homeostasis and injury.
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Emulating corneal stromal tissue is believed to be the most challenging step in bioengineering an artificial human cornea because of the difficulty in reproducing its highly ordered microstructure, the key to the robust biomechanical properties and optical transparency of this tissue. We conducted a comparative study to assess the feasibility of human corneal stromal stem cells (hCSSCs) and human corneal fibroblasts (hCFs) in the generation of human corneal stromal tissue on groove-patterned silk substrates. In serum-free keratocyte differentiation medium, hCSSCs successfully differentiated into keratocytes secreting multilayered lamellae with orthogonally-oriented collagen fibrils, in a pattern mimicking human corneal stromal tissue. The constructs were 90-100 μm thick, containing abundant cornea-specific extracellular matrix (ECM) components, including keratan sulfate, lumican, and keratocan. In contrast, hCFs tended to differentiate into myofibroblasts that deposited less organized collagen in a pattern resembling that of corneal scar tissue. RGD surface coupling coupling was an essential factor in enhancing cell attachment, orientation, proliferation, differentiation and ECM deposition on the silk substratum. These results demonstrated that an approach of combining hCSSCs with an RGD surface-coupled patterned silk film offers a powerful tool to develop highly ordered collagen fibril-based constructs for corneal regeneration and corneal stromal tissue repair.
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There is critical clinical demand for tissue-engineered (TE), three-dimensional (3D) constructs for tissue repair and organ replacements. Current efforts toward this goal are prone to necrosis at the core of larger constructs because of limited oxygen and nutrient diffusion. Therefore, critically sized 3D TE constructs demand an immediate vascular system for sustained tissue function upon implantation. To address this challenge the goal of this project was to develop a strategy to incorporate microchannels into a porous silk TE scaffold that could be fabricated reproducibly using microfabrication and soft lithography. Silk is a suitable biopolymer material for this application because it is mechanically robust, biocompatible, slowly degrades in vivo, and has been used in a variety of TE constructs. We report the fabrication of a silk-based TE scaffold that contains an embedded network of porous microchannels. Enclosed porous microchannels support endothelial lumen formation, a critical step toward development of the vascular niche, while the porous scaffold surrounding the microchannels supports tissue formation, demonstrated using human mesenchymal stem cells. This approach for fabricating vascularized TE constructs is advantageous compared to previous systems, which lack porosity and biodegradability or degrade too rapidly to sustain tissue structure and function. The broader impact of this research will enable the systemic study and development of complex, critically-sized engineered tissues, from regenerative medicine to in vitro tissue models of disease states.
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We synthesized and characterized a new family of di-block copolymers based on the amino acid sequences of Nephila clavipes major ampulate dragline spider silk, having the form HABn and HBAn (n=1-3), comprising an alanine-rich hydrophobic block, A, a glycine-rich hydrophilic block, B, and a histidine tag, H. The reversing heat capacities, Cp(T), for temperatures below and above the glass transition, Tg, were measured by temperature modulated differential scanning calorimetry. For the solid state, we then calculated the heat capacities of our novel block copolymers based on the vibrational motions of the constituent poly(amino acid)s, whose heat capacities are known or can be estimated from the ATHAS Data Bank. For the liquid state, the heat capacity was estimated by using the rotational and translational motions in the polymer chain. Excellent agreement was found between the measured and calculated values of the heat capacity, showing that this method can serve as a standard by which to assess the Cp for other biologically inspired block copolymers. The fraction of beta sheet crystallinity of spider silk block copolymers was also determined by using the predicted Cp, and was verified by wide angle X-ray diffraction and Fourier transform infrared spectroscopy. The glass transition temperatures of spider silk block copolymer were fitted by Kwei's equation and the results indicate that attractive interaction exists between the A-block and B-block.
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Genetically engineered spider silk-like block copolymers were studied to determine the influence of polyalanine domain size on secondary structure. The role of polyalanine block distribution on -sheet formation was explored using FT-IR and WAXS. The number of polyalanine blocks had a direct effect on the formation of crystalline -sheets, reflected in the change in crystallinity index as the blocks of polyalanines increased. WAXS analysis confirmed the crystalline nature of the sample with the largest number of polyalanine blocks. This approach provides a platform for further exploration of the role of specific amino acid chemistries in regulating the assembly of -sheet secondary structures, leading to options to regulate material properties through manipulation of this key component in spider silks.
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Composite or hybrid materials are commonly found in Nature, formed through the concentration and subsequent nucleation of ions upon organic templates that are most often protein based. Examples include the deposition of calcium containing salts in bone, teeth and the inner ear and iron oxide structures in magnetotactic bacteria. Biological organisms use a limited number of metal ions, the principal ones being calcium and iron, with lesser amounts of strontium, and barium. The ability to utilize other ions to generate composites offers the possibility of new material properties. New materials incorporating silver would be useful in the context of antimicrobial functions. Therefore, in the present study, a new route to such functionalized biomaterials is reported. Genetically engineered fusion proteins are created by the incorporation of nucleotides corresponding to short silver binding peptides identified by a combinatorial biopanning process into the consensus sequence of silk from the spider, Nephila clavipes. The resulting chimeric silk–silver binding proteins nucleated Ag ions from a solution of silver nitrate while the silk protein provided a stable template material which could be processed into films, fibers, and three-dimensional scaffolds. The silk films inhibited microbial growth of both Gram-positive and Gram-negative microrganisms on agar plates and in liquid culture, thus highlighting the potential of these chimeric material systems as antimicrobial biomedical coatings.
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The aim of the study was to determine the extent and mechanism of influence on silica condensation that is presented by a range of known silicifying recombinant chimeras (R5: SSKKSGSYSGSKGSKRRIL; A1: SGSKGSKRRIL; and Si4-1: MSPHPHPRHHHT and repeats thereof) attached at the N-terminus end of a 15-mer repeat of the 32 amino acid consensus sequence of the major ampullate dragline Spindroin 1 (Masp1) Nephila clavipes spider silk sequence ([SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQG](15)X). The influence of the silk/chimera ratio was explored through the adjustment of the type and number of silicifying domains (denoted X above), and the results were compared with their non-chimeric counterparts and the silk from Bombyx mori. The effect of pH (3-9) on reactivity was also explored. Optimum conditions for rate and control of silica deposition were determined, and the solution properties of the silks were explored to determine their mode(s) of action. For the silica-silk-chimera materials formed there is a relationship between the solution properties of the chimeric proteins (ability to carry charge), the pH of reaction, and the solid state materials that are generated. The region of colloidal instability correlates with the pH range observed for morphological control and coincides with the pH range for the highest silica condensation rates. With this information it should be possible to predict how chimeric or chemically modified proteins will affect structure and morphology of materials produced under controlled conditions and extend the range of composite materials for a wide spectrum of uses in the biomedical and technology fields.
Article
Novel protein chimeras constituted of "silk" and a silica-binding peptide (KSLSRHDHIHHH) were synthesized by genetic or chemical approaches and their influence on silica-silk based chimera composite formation evaluated. Genetic chimeras were constructed from 6 or 15 repeats of the 32 amino acid consensus sequence of Nephila clavipes spider silk ([SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQG](n)) to which one silica binding peptide was fused at the N terminus. For the chemical chimera, 28 equiv of the silica binding peptide were chemically coupled to natural Bombyx mori silk after modification of tyrosine groups by diazonium coupling and EDC/NHS activation of all acid groups. After silica formation under mild, biomaterial-compatible conditions, the effect of peptide addition on the properties of the silk and chimeric silk-silica composite materials was explored. The composite biomaterial properties could be related to the extent of silica condensation and to the higher number of silica binding sites in the chemical chimera as compared with the genetically derived variants. In all cases, the structure of the protein/chimera in solution dictated the type of composite structure that formed with the silica deposition process having little effect on the secondary structural composition of the silk-based materials. Similarly to our study of genetic silk based chimeras containing the R5 peptide (SSKKSGSYSGSKGSKRRIL), the role of the chimeras (genetic and chemical) used in the present study resided more in aggregation and scaffolding than in the catalysis of condensation. The variables of peptide identity, silk construct (number of consensus repeats or silk source), and approach to synthesis (genetic or chemical) can be used to "tune" the properties of the composite materials formed and is a general approach that can be used to prepare a range of materials for biomedical and sensor-based applications.
Article
We report a study of self-assembled beta-pleated sheets in B. mori silk fibroin films using thermal analysis and infrared spectroscopy. B. mori silk fibroin may stand as an exemplar of fibrous proteins containing crystalline beta-sheets. Materials were prepared from concentrated solutions (2-5 wt% fibroin in water) and then dried to achieve a less ordered state without beta-sheets. Crystallization of beta-pleated sheets was effected either by heating the films above the glass transition temperature (T-g) and holding isothermally or by exposure to methanol. The fractions of secondary structural components including random coils, alpha-helices, beta-pleated sheets, turns, and side chains were evaluated using Fourier self-deconvolution (FSD) of the infrared absorbance spectra. The silk fibroin films were studied thermally using temperature-modulated differential scanning calorimetry (TMDSC) to obtain the reversing heat capacity. The increment of the reversing heat capacity Delta C-p0(T-g) at the glass transition for the less ordered, noncrystalline, silk fibroin is found to be 0.478 +/- 0.005 J/(g K). As crystalline beta-sheets form, the heat capacity increment at Tg is systematically decreased. We find that the heat capacity increment from the TMDSC trace is linearly well correlated ( negatively) with beta-sheet content phi(C) determined from FSD, yielding Delta C-p = 0.475-0.494 phi(C). The correlation allows the beta-sheet content to be determined from a direct measurement of the heat capacity increment at T-g. This type of analysis can serve as an alternative to X-ray methods and may have wide applicability to other crystalline beta-sheet forming proteins.
Article
We constructed a fusion protein (GOx-R5) consisting of R5 (a polypeptide component of silaffin) and glucose oxidase (GOx) that was expressed in Pichia pastoris. Silaffin proteins are responsible for the formation of a silica-based cell matrix of diatoms, and synthetic variants of the R5 protein can perform silicification in vitro[1]. GOx secreted by P. pastoris was self-immobilized (biosilicification) in a pH 5 citric buffer using 0.1M tetramethoxysilane as a silica source. This self-entrapment property of GOx-R5 was used to immobilize GOx on a graphite rod electrode. An electric cell designed as a biosensor was prepared to monitor the glucose concentrations. The electric cell consisted of an Ag/AgCl reference electrode, a platinum counter electrode, and a working electrode modified with poly(neutral red) (PNR)/GOx/Nafion. Glucose oxidase was immobilized by fused protein on poly(neutral red) and covered by Nafion to protect diffusion to the solution. The morphology of the resulting composite PNR/GOx/Nafion material was analyzed by scanning electron microscopy (SEM). This amperometric transducer was characterized electrochemically using cyclic voltammetry and amperometry in the presence of glucose. An image produced by scanning electron microscopy supported the formation of a PNR/GOx complex and the current was increased to 1.58 μA cm(-1) by adding 1mM glucose at an applied potential of -0.5 V. The current was detected by way of PNR-reduced hydrogen peroxide, a product of the glucose oxidation by GOx. The detection limit was 0.67mM (S/N=3). The biosensor containing the graphite rod/PNR/GOx/Nafion detected glucose at various concentrations in mixed samples, which contained interfering molecules. In this study, we report the first expression of R5 fused to glucose oxidase in eukaryotic cells and demonstrate an application of self-entrapped GOx to a glucose biosensor.
Article
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. Because of 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.
Article
The catalytic effect of various sequential peptides for silica biomineralization has been studied. In peptide sequence design, lysine (K) and histidine (H) were selected as the standard amino acids and aspartic acid (D) was selected to promote the charge relay effects, such as in the enzyme active site. Therefore, homopolypeptides (K(10) and H(10)), block polypeptides (K(5)D(5) and H(5)D(5)), and alternate polypeptides [(KD)(5) and (HD)(5)] were designed, and the dehydration reaction ability of trimethylethoxysilane was investigated as a quantitative model of silica mineralization. The catalytic activity per basic residue of alternate polypeptide was the highest because of the charge relay effects at the surface of the peptide. In silica mineralization using tetraethoxysilane, spherical silica particles were obtained, and their size is related to the catalytic activities of the peptides in the model systems. From these results, the effect of the functional group combination by the peptide sequence design enables the control of the efficiency of mineralization and preparation of specific inorganic materials.
Article
We present a simple and effective method to obtain refined control of the molecular structure of silk biomaterials through physical temperature-controlled water vapor annealing (TCWVA). The silk materials can be prepared with control of crystallinity, from a low content using conditions at 4 °C (α helix dominated silk I structure), to highest content of ∼60% crystallinity at 100 °C (β-sheet dominated silk II structure). This new physical approach covers the range of structures previously reported to govern crystallization during the fabrication of silk materials, yet offers a simpler, green chemistry, approach with tight control of reproducibility. The transition kinetics, thermal, mechanical, and biodegradation properties of the silk films prepared at different temperatures were investigated and compared by Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), uniaxial tensile studies, and enzymatic degradation studies. The results revealed that this new physical processing method accurately controls structure, in turn providing control of mechanical properties, thermal stability, enzyme degradation rate, and human mesenchymal stem cell interactions. The mechanistic basis for the control is through the temperature-controlled regulation of water vapor to control crystallization. Control of silk structure via TCWVA represents a significant improvement in the fabrication of silk-based biomaterials, where control of structure-property relationships is key to regulating material properties. This new approach to control crystallization also provides an entirely new green approach, avoiding common methods that use organic solvents (methanol, ethanol) or organic acids. The method described here for silk proteins would also be universal for many other structural proteins (and likely other biopolymers), where water controls chain interactions related to material properties.
Article
The biomimetic design of silk/silica fusion proteins was carried out, combining the self assembling domains of spider dragline silk (Nephila clavipes) and silaffin derived R5 peptide of Cylindrotheca fusiformis that is responsible for silica mineralization. Genetic engineering was used to generate the protein-based biomaterials incorporating the physical properties of both components. With genetic control over the nanodomain sizes and chemistry, as well as modification of synthetic conditions for silica formation, controlled mineralized silk films with different silica morphologies and distributions were successfully generated; generating 3D porous networks, clustered silica nanoparticles (SNPs), or single SNPs. Silk serves as the organic scaffolding to control the material stability and multiprocessing makes silk/silica biomaterials suitable for different tissue regenerative applications. The influence of these new silk-silica composite systems on osteogenesis was evaluated with human mesenchymal stem cells (hMSCs) subjected to osteogenic differentiation. hMSCs adhered, proliferated, and differentiated towards osteogenic lineages on the silk/silica films. The presence of the silica in the silk films influenced osteogenic gene expression, with the upregulation of alkaline phosphatase (ALP), bone sialoprotein (BSP), and collagen type 1 (Col 1) markers. Evidence for early bone formation as calcium deposits was observed on silk films with silica. These results indicate the potential utility of these new silk/silica systems towards bone regeneration.
Article
Osteoinductive and biodegradable composite biomaterials for bone regeneration were prepared by combining silk fibroin with silica particles. The influence of these composite systems on osteogenesis was evaluated with human mesenchymal stem cells (hMSCs) subjected to osteogenic differentiation. hMSCs adhered, proliferated, and differentiated towards osteogenic lineages on silk/silica films. The addition of the silica to the silk films influenced gene expression leading to upregulation of bone sialoprotein (BSP) and collagen type 1 (Col 1) osteogenic markers. Evidence for early bone formation in the form of collagen fibers and apatite nodules was obtained on the silk/silica films. Collagen fibers were closely associated with apatite deposits and overall collagen content was higher for the silica containing samples. Also, smaller sized silica particles (24 nm-2 μm) with large surface area facilitated silica biodegradation in vitro through particle dissolution, leading to ∼5-fold decrease in silica content over 10 weeks. These results indicate the suitability of silk/silica composite system towards bone regeneration, where degradation/remodeling rates of the organic and inorganic components can be controlled.
Article
Silk proteins self-assemble into mechanically robust material structures that are also biodegradable and non-cytotoxic, suggesting utility for gene delivery. Since silk proteins can also be tailored in terms of chemistry, molecular weight and other design features via genetic engineering, further control of this system for gene delivery can be considered. In the present study, silk-based block copolymers were bioengineered with poly(L-lysine) domains for gene delivery. Ionic complexes of these silk-polylysine based block copolymers with plasmid DNA (pDNA) were prepared for gene delivery to human embryonic kidney (HEK) cells. The material systems were characterized by agarose gel electrophoresis, atomic force microscopy, and dynamic light scattering. The polymers self-assembled in solution and complexed plasmid DNA through ionic interactions. The pDNA complexes with 30 lysine residues prepared at a polymer/nucleotide ratio of 10 and with a solution diameter of 380 nm showed the highest efficiency for transfection. The pDNA complexes were also immobilized on silk films and demonstrated direct cell transfection from these surfaces. The results demonstrate the potential of bioengineered silk proteins as a new family of highly tailored gene delivery systems.
Article
The design, construction, and preliminary characterization of a novel family of spider silk-like block copolymers are described. The design was based on the assembly of individual spider silk modules, in particular, polyalanine (A) and glycine-rich (B) blocks, that display different phase behavior in aqueous solution. Spider silk was chosen as a model for these block copolymer studies based on its extraordinary material properties, such as toughness, biocompatibility, and biodegradability. Trends in spider silk-like block copolymer secondary structure and assembly behavior into specific material morphologies were determined as a function of the number of hydrophobic blocks, the presence of a hydrophilic purification tag and solvent effects. Structures and morphologies were assessed by Fourier transform infrared spectroscopy and scanning electron microscopy. In terms of structure, beta-sheet content increased with an increase in the number of polyalanine blocks, and the purification tag had significant impact on the secondary structure. In terms of morphology, spheres, rod-like structures, bowl-shaped micelles, and giant compound micelles were observed and the morphologies were linked with the size of the hydrophobic block, the presence of the purification tag, and the solvent environment. This study provides a basis for future designs of smart biomaterials based on spider silk chemistries, with controlled structure-architecture-function relationships.
Article
Synthetic genes encoding recombinant spider silk proteins have been constructed, cloned, and expressed. Protein sequences were derived from Nephila clavipes dragline silk proteins and reverse-translated to the corresponding DNA sequences. Codon selection was chosen to maximize expression levels in Escherichia coli. DNA "monomer" sequences were multimerized to encode high molecular weight synthetic spider silks using a "head-to-tail" construction strategy. Multimers were cloned into a prokaryotic expression vector and the encoded silk proteins were expressed in E. coli upon induction with IPTG. Four multimer, ranging in size from 14.7 to 41.3 kDa, were chosen for detailed analysis. These proteins were isolated by immobilized metal affinity chromatography and purified using reverse-phase HPLC. The composition and identity of the purified proteins were confirmed by amino acid composition analysis, N-terminal sequencing, laser desorption mass spectroscopy, and Western analysis using antibodies reactive to native spider dragline silk. Circular dichroism measurements indicate that the synthetic spider silks have substantial beta-sheet structure.
Article
A synthetic site-directed mutagenesis study of the non post-translationally modified silica precipitating R5 peptide reveals that the RRIL motif is critical in the formation of active silica precipitating assemblies.
Article
Spider silk fibers have remarkable mechanical properties that suggest the component proteins could be useful biopolymers for fabricating biomaterial scaffolds for tissue formation. Two bioengineered protein variants from the consensus sequence of the major component of dragline silk from Nephila clavipes were cloned and expressed to include RGD cell-binding domains. The engineered silks were characterized by CD and FTIR and showed structural transitions from random coil to insoluble beta-sheet upon treatment with methanol. The recombinant proteins were processed into films and fibers and successfully used as biomaterial matrixes to culture human bone marrow stromal cells induced to differentiate into bone-like tissue upon addition of osteogenic stimulants. The recombinant spider silk and the recombinant spider silk with RGD encoded into the protein both supported enhanced the differentiation of human bone marrow derived mesenchymal stem cells (hMSCs) to osteogenic outcomes when compared to tissue culture plastic. The recombinant spider silk protein without the RGD displayed enhanced bone related outcomes, measured by calcium deposition, when compared to the same protein with RGD. Based on comparisons to our prior studies with silkworm silks and RGD modifications, the current results illustrate the potential to bioengineer spider silk proteins into new biomaterial matrixes, while also highlighting the importance of subtle differences in silk sources and modes of presentation of RGD to cells in terms of tissue-specific outcomes.
Article
The cytotoxicity of 15-nm and 46-nm silica nanoparticles was investigated by using crystalline silica (Min-U-Sil 5) as a positive control in cultured human bronchoalveolar carcinoma-derived cells. Exposure to 15-nm or 46-nm SiO(2) nanoparticles for 48 h at dosage levels between 10 and 100 microg/ml decreased cell viability in a dose-dependent manner. Both SiO(2) nanoparticles were more cytotoxic than Min-U-Sil 5; however, the cytotoxicities of 15-nm and 46-nm silica nanoparticles were not significantly different. The 15-nm SiO(2) nanoparticles were used to determine time-dependent cytotoxicity and oxidative stress responses. Cell viability decreased significantly as a function of both nanoparticle dosage (10-100 microg/ml) and exposure time (24 h, 48 h, and 72 h). Indicators of oxidative stress and cytotoxicity, including total reactive oxygen species (ROS), glutathione, malondialdehyde, and lactate dehydrogenase, were quantitatively assessed. Exposure to SiO(2) nanoparticles increased ROS levels and reduced glutathione levels. The increased production of malondialdehyde and lactate dehydrogenase release from the cells indicated lipid peroxidation and membrane damage. In summary, exposure to SiO(2) nanoparticles results in a dose-dependent cytotoxicity in cultural human bronchoalveolar carcinoma-derived cells that is closely correlated to increased oxidative stress.
Article
Spider silks exhibit remarkable mechanical properties while dentin matrix protein 1 provides controlled nucleation and hydroxyapatite growth. In the present work, these two attributes were combined via genetic engineering to form a chimera, a clone encoding consensus repeats from the major protein in the spider dragline silk of Nephila clavipes fused to the carboxyl terminal domain of dentin matrix protein 1 (CDMP1). The objective was to exploit the self-assembly and material properties of silk proteins with controlled hydroxyapatite (HA) formation from CDMP1, for novel biomaterial composites. The purified recombinant protein retained native-silk like self-assembly properties and beta-sheet structure when formed into films and treated with methanol. When the chimeric protein in solution was incubated with CaCl(2,) the secondary structure shifted from random coil to alpha-helix and beta-sheet, due to the interactions between the CDMP1 domain and Ca(2+). The control protein without the CDMP1 domain did not undergo a similar transition. Films formed from the recombinant protein were mineralized using simulated body fluids and induced the formation of calcium-deficient carbonated HA, Ca(10)(PO(4))(6)(OH)(2) based on SEM, EDS, FTIR and TEM analysis. This mineral phase was not formed on the films formed from the control spider silk protein without the CDMP1 domain. Considering the osteoconductivity of HA and the novel material features of spider silks, these new hybrid systems offer potential as biomaterials for a number of potential applications.
Article
Many biological organisms contain specialized structures composed of inorganic materials. Cellular processes in vivo facilitate the organized assembly of mineral building blocks into complex structures. The structural hierarchy and complexity across a range of length scales are providing new ideas and concepts for materials chemistry. Proteins that direct biomineralization can be used to control the production of nanostructured materials and facilitate the fabrication of new structures. Here, we demonstrate that some of the silica-binding peptides isolated from a combinatorial phage peptide display library can be used in precipitating silica from a solution of silicic acid. The results described in this report demonstrate that peptides displayed by phages act as templates in inorganic material synthesis and provide a means of understanding how some of the biological systems may be carrying out materials chemistry in vivo.
Article
Biodegradable nanofibers have tremendous potential for tissue repair. However, the combined effects of nanofiber organization and immobilized bioactive factors on cell guidance are not well understood. In this study, we developed aligned and bioactive nanofibrous scaffolds by immobilizing extracellular matrix protein and growth factor onto nanofibers, which simulated the physical and biochemical properties of native matrix fibrils. The aligned nanofibers significantly induced neurite outgrowth and enhanced skin cell migration during wound healing compared to randomly oriented nanofibers. Furthermore, the immobilized biochemical factors (as efficient as soluble factors) synergized with aligned nanofibers to promote highly efficient neurite outgrowth but had less effect on skin cell migration. This study shed light on the relative importance of nanotopography and chemical signaling in the guidance of different cell behavior.
Article
Protein polymers (long-chain proteins in which a specific amino acid sequence "monomer" is repeated through the molecule) are found widely in nature, and these materials exhibit a diverse array of physical properties. One class of self-assembling proteins is hydrophobic-polar (HP) protein polymers capable of self-assembly under the appropriate solution conditions. We generated a chimeric protein consisting of an HP protein polymer monomer unit, EAK 1 (sequence n-AEAEAKAKAEAEAKAK-c), and a silaffin peptide, R5 (sequence: n-SSKKSGSYSGSKGSKRRIL-c). First identified in diatoms, silaffins represent a class of proteins and peptides capable of directing silica precipitation in vitro at neutral pH and ambient temperatures. The EAK 1-R5 chimera demonstrated self-assembly into hydrogels and the ability to direct silica precipitation in vitro. This chimera is capable of generating silica morphologies and feature sizes significantly different from those achievable with the R5 peptide alone, indicating that fusions of silaffins with self-assembling proteins may be a route to controlling the morphology of artificially produced silica matrices.