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

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.     

Expression and characterization of chimeric spidroins from flagelliform-aciniform repetitive modules

Spiders can produce up to seven different types of silks or glues with different mechanical properties. Of these, flagelliform (Flag) silk is the most elastic, and aciniform (AcSp1) silk is the toughest. To produce a chimeric spider silk (spidroin) Flag_R-AcSp1_R, we fused one repetitive module of flagelliform silk from Araneus ventricosus and one repetitive module of aciniform silk from Argiope trifasciata. The recombinant protein expressed in E. coli formed silk-like fibers by manual-drawing. CD analysis showed that the secondary structure of Flag_R-AcSp1_R spidroin remained stable during the gradual reduction of pH from 7.0 to 5.5. The spectrum of FTIR indicated that the secondary structure of Flag_R-AcSp1_R changed from α-helix to β-sheet. The conformation change of Flag_R-AcSp1_R was similar to other spidroins in the fiber formation process. SEM analysis revealed that the mean diameter of the fibers was around 1 ~ 2 μm, and the surface was smooth and uniform. The chimeric fibers exhibited superior toughness (~33.1 MJ/m³) and tensile strength (~261.4 MPa). This study provides new insight into design of chimeric spider silks with high mechanical properties.     

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

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

Conserved C-terminal domain of spider tubuliform spidroin 1 contributes to extensibility in synthetic fibers

Spider silk is renowned for its extraordinary mechanical properties, having a balance of high tensile strength and extensibility. To date, the majority of studies have focused on the production of dragline silks from synthetic spider silk gene products. Here we report the first mechanical analysis of synthetic egg case silk fibers spun from the Latrodectus hesperus tubuliform silk proteins, TuSp1 and ECP-2. We provide evidence that recombinant ECP-2 proteins can be spun into fibers that display mechanical properties similar to other synthetic spider silks. We also demonstrate that silks spun from recombinant thioredoxin-TuSp1 fusion proteins that contain the conserved C-terminal domain exhibit increased extensibility and toughness when compared to the identical fibers spun from fusion proteins lacking the C-terminus. Mechanical analyses reveal that the properties of synthetic tubuliform silks can be modulated by altering the postspin draw ratios of the fibers. Fibers subject to increased draw ratios showed elevated tensile strength and decreased extensibility but maintained constant toughness. Wide-angle X-ray diffraction studies indicate that postdrawn fibers containing the C-terminal domain of TuSp1 have more amorphous content when compared to fibers lacking the C-terminus. Taken together, these studies demonstrate that recombinant tubuliform spidroins that contain the conserved C-terminal domain with embedded protein tags can be effectively spun into fibers, resulting in similar tensile strength but increased extensibility relative to nontagged recombinant dragline silk proteins spun from equivalently sized proteins.     

The elaborate structure of spider silk

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.     

Effect of loading rate on mechanical properties and fracture morphology of spider silk

Spider silks have been shown to have impressive mechanical properties. In order to assess the effect of extension rate, both quasi-static and high-rate tensile properties were determined for single fibers of major (MA) and minor (MI) ampullate single silk from the orb weaving spider Nephila clavipes . Low rate tests have been performed using a DMA Q800 at 10(-3) s(-1), while high rate analysis was done at 1700 s(-1) utilizing a miniature Kolsky bar apparatus. Rate effects exhibited by both respective silk types are addressed, and direct comparison of the tensile response between the two fibers is made. The fibers showed major increases in toughness at the high extension rate. Mechanical properties of these organic silks are contrasted to currently employed ballistic fibers and examination of fiber fracture mechanisms are probed via scanning electron microscope, revealing a globular rupture surface topography for both rate extremums.     

Recent advances in production of recombinant spider silk proteins

1. Download : Download high-res image (94KB)
2. Download : Download full-size image

Highlights? Spider silk a biopolymer of great strength, toughness, and elasticity as well as biodegradability and biocompatibility. ? Various host systems ranging from bacteria to animal systems have been employed for the production of recombinant spider silk proteins. ? Ultra-high molecular weight spider silk protein showing Kevlar strength could be produced in E. coli by systems metabolic engineering. ? Transgenic silkworms producing recombinant or chimera spider silk have great potential for actual production in large scale.     

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

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.     

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

Hybrid silks hold a great potential as specific biomaterials due to its controlled mechanical properties. To produce fibers with tunable properties, here we firstly made chimeric proteins in vitro, called W2C4CT and W2C8CT, with ligation of MaSp repetitive modules (C) with AcSp modules (W) by intein trans splicing technology from smaller precursors without final yield reduction. Intein mediated chimeric proteins form fibers at a low concentration of 0.4 mg/mL in 50 mM K₃PO₄ pH 7.5 just drawn by hand. Hybrid fibers show smoother surface, and also have stronger chemical resistance as compared with fibers from W2CT (W fibers) and mixture of W2CT/C8CT (MHF8 fibers). Fibers from chimeric protein W2C4CT (HFH4) have improved mechanical properties than W fibers; however, with more C modules W2C8CT fibers (HFH8) properties decreased, indicates the length proportion of various modules is very important and should be optimized for fibers with specific properties. Generally, hybrid silks generated via chimeric proteins, which can be simplified by intein trans splicing, has greater potential to produce fibers with tunable properties. Our research shows that intein mediated directional protein ligation is a novel way to make large chimeric spider silk proteins and hybrid silks. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 385–392, 2016.     

Efflux and Influx of Erythrocyte Water

Rabbit erythrocytes were washed in buffered NaCl solutions isotonic with rabbit serum (Δ^t -0.558°C.) and suspended in buffered NaCl solutions of tonicity equidistant from intracellular tonicity (Δ^t = -0.558°C. ± 0.112°C.) of varying pH and incubated at varying temperatures. After incubation, the freezing point depression (Δ^t) was measured on the supernatant. Change in the Δ^t measured change in the water content of the extracellular solutions—water being withdrawn by erythrocytes (W_I) from the hypotonic solutions and added (W_E) to the hypertonic solutions. W_E was always less than W_I and was inversely proportional to the pH in the range 6.5–8.0. W_E was significantly increased by lowering the temperature of the cell suspension to 4°C. W_I was increased by raising or lowering the pH or raising the temperature of the cell suspension. W_E x W_I ≠ k. W_E and W_I were affected differently by changes in pH and temperature. It was concluded that W_E and W_E were probably under different physicochemical control.     

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

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.     

Protein Fiber Linear Dichroism for Structure Determination and Kinetics in a Low-Volume, Low-Wavelength Couette Flow Cell

High-resolution structure determination of soluble globular proteins relies heavily on x-ray crystallography techniques. Such an approach is often ineffective for investigations into the structure of fibrous proteins as these proteins generally do not crystallize. Thus investigations into fibrous protein structure have relied on less direct methods such as x-ray fiber diffraction and circular dichroism. Ultraviolet linear dichroism has the potential to provide additional information on the structure of such biomolecular systems. However, existing systems are not optimized for the requirements of fibrous proteins. We have designed and built a low-volume (200 μL), low-wavelength (down to 180 nm), low-pathlength (100 μm), high-alignment flow-alignment system (couette) to perform ultraviolet linear dichroism studies on the fibers formed by a range of biomolecules. The apparatus has been tested using a number of proteins for which longer wavelength linear dichroism spectra had already been measured. The new couette cell has also been used to obtain data on two medically important protein fibers, the all-β-sheet amyloid fibers of the Alzheimer's derived protein Aβ and the long-chain assemblies of α₁-antitrypsin polymers.     

蜘蛛丝蛋白的模块结构性能和重组表达

蜘蛛丝是一类天然蛋白质纤维,具有独特的机械性能（高强度、高弹性和高断裂功等）和卓著的生物学特性（生物可降解性和与生物组织的相容性等）,在生物医学、材料、纺织和军事等领域都有着很高的潜在应用价值。综述了不同蜘蛛丝蛋白的模块结构特征及与其功能的关系,扼要介绍了目前利用各种基因工程方法表达重组蜘蛛丝蛋白的研究进展。     

Aciniform spidroin, a constituent of egg case sacs and wrapping silk fibers from the black widow spider Latrodectus hesperus

Spiders produce high performance fibers with diverse mechanical properties and biological functions. Molecular and biochemical studies of spider egg case silk have revealed that the main constituent of the large diameter fiber contains the fibroin TuSp1. Here we demonstrate by SDS-PAGE and protein silver staining the presence of a distinct approximately 300-kDa polypeptide that is found in solubilized egg case sacs. Combining matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry and reverse genetics, we have isolated a novel gene called AcSp1-like and demonstrate that its protein product is assembled into the small diameter fibers of egg case sacs and wrapping silks from the black widow spider, Latrodectus hesperus. BLAST searches of the NCBInr protein data base using the amino acid sequence of AcSp1-like revealed similarity to AcSp1, an inferred protein proposed to be a component of wrapping silk. However, the AcSp1-like protein was found to display more nonuniformity in its internal iterated repeat modules than the putative AcSp1 fibroin. Real time quantitative PCR analysis demonstrates that the AcSp1-like gene displays an aciniform gland-restricted pattern of expression. The amino acid composition of the fibroins extracted from the luminal contents of the aciniform glands was remarkably similar to the predicted amino acid composition of the AcSp1-like protein, which supports the assertion that AcSp1-like protein represents the major constituent stored within the aciniform gland. Collectively, our findings provide the first direct molecular evidence for the involvement of the aciniform gland in the production of a common fibroin that is assembled into the small diameter threads of egg case and wrapping silk of cob weavers.     

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

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.     

Effects of Cysteine Proteases on the Structural and Mechanical Properties of Collagen Fibers

Excessive cathepsin K (catK)-mediated turnover of fibrillar type I and II collagens in bone and cartilage leads to osteoporosis and osteoarthritis. However, little is known about how catK degrades compact collagen macromolecules. The present study is aimed to explore the structural and mechanical consequences of collagen fiber degradation by catK. Mouse tail type I collagen fibers were incubated with either catK or non-collagenase cathepsins. Methods used include scanning electron microscopy, protein electrophoresis, atomic force microscopy, and tensile strength testing. Our study revealed evidence of proteoglycan network degradation, followed by the progressive disassembly of macroscopic collagen fibers into primary structural elements by catK. Proteolytically released GAGs are involved in the generation of collagenolytically active catK-GAG complexes as shown by AFM. In addition to their structural disintegration, a decrease in the tensile properties of fibers was observed due to the action of catK. The Young''s moduli of untreated collagen fibers versus catK-treated fibers in dehydrated conditions were 3.2 ± 0.68 GPa and 1.9 ± 0.65 GPa, respectively. In contrast, cathepsin L, V, B, and S revealed no collagenase activity, except the disruption of proteoglycan-GAG interfibrillar bridges, which slightly decreased the tensile strength of fibers.     

悦目金蛛拖牵丝力学性能的变异

蜘蛛丝作为一种具有优良机械性能的天然动物蛋白纤维,其特有的结构和机械性能与其生物学功能密切相关。由大壶状腺纺出的拖牵丝在蜘蛛的行走、建网、捕食、逃生、繁殖等多种生命活动中均发挥了重要的功能,其机械性能会受到多种内外因素相互作用的影响。本文对在不同体重、不同猎物饲养和不同营养状态3种条件下人工抽出的悦目金蛛(Argiope amoena)拖牵丝与其不同单丝间的力学性能进行了比较研究。结果表明,悦目金蛛拖牵丝的力学性能在组间、组内不同个体,以及同一个体不同丝纤维间变异都较大。随着蜘蛛个体的增大,蛛丝横截面直径逐渐增大,这会使得蛛丝的力学性能更好,便于作为救命索的拖牵丝在遇到危险时承受蜘蛛体重;蜘蛛在经过1个月的饥饿后,蛛丝在屈服点附近的力学性能并未发生显著变化,而断裂点应变和断裂能均显著减小,同时也表明无论对于作为救命索还是网丝,拖牵丝的弹性形变性能在与蛛丝相关的微观进化中要优先于塑性形变。这是蜘蛛在能量摄入受到限制时对拖牵丝的投入权衡的结果。     

Structural Characterization of Minor Ampullate Spidroin Domains and Their Distinct Roles in Fibroin Solubility and Fiber Formation

Spider silk is protein fibers with extraordinary mechanical properties. Up to now, it is still poorly understood how silk proteins are kept in a soluble form before spinning into fibers and how the protein molecules are aligned orderly to form fibers. Minor ampullate spidroin is one of the seven types of silk proteins, which consists of four types of domains: N-terminal domain, C-terminal domain (CTD), repetitive domain (RP) and linker domain (LK). Here we report the tertiary structure of CTD and secondary structures of RP and LK in aqueous solution, and their roles in protein stability, solubility and fiber formation. The stability and solubility of individual domains are dramatically different and can be explained by their distinct structures. For the tri-domain miniature fibroin, RP-LK-CTD_Mi, the three domains have no or weak interactions with one another at low protein concentrations (<1 mg/ml). The CTD in RP-LK-CTD_Mi is very stable and soluble, but it cannot stabilize the entire protein against chemical and thermal denaturation while it can keep the entire tri-domain in a highly water-soluble state. In the presence of shear force, protein aggregation is greatly accelerated and the aggregation rate is determined by the stability of folded domains and solubility of the disordered domains. Only the tri-domain RP-LK-CTD_Mi could form silk-like fibers, indicating that all three domains play distinct roles in fiber formation: LK as a nucleation site for assembly of protein molecules, RP for assistance of the assembly and CTD for regulating alignment of the assembled molecules.     

Single-trait and antagonistic index selection for litter size and body weight in mice

Individual selection based on female performance only was conducted in four lines of mice: L⁺ for increased litter size, W⁺ for increased 6-week body weight, L^-W⁺ for a selection index aimed at decreasing litter size and increasing 6-week body weight and L⁺W^- for a selection index aimed at increasing litter size and decreasing 6-week body weight. A fifth line (K) served as an unselected control. All litters were standardized to eight mice at one day of age. Expected heritability was based on twice the regression of offspring on dam (h²_d), which contains additive genetic variance due to direct (σ²_Ao) and maternal (σ²_Am) effects and their covariance (σ_AoAm). Responses and correlated responses were measured either deviated (method 1) or not deviated (method 2) from the control line. Realized heritabilities (h²_R) for litter size were 0.19 ± 0.04 (1) and 0.16 ± 0.03 (2), which were similar to h ²_d of 0.17 ± 0.04. The h² _R for 6-week body weight of 0.55 ± 0.07 (1) and 0.44 ± 0.07 (2) agreed with h²_d of 0.42 ± 0.02. Realized genetic correlations (r*_GR) between litter size and 6-week body weight calculated from the double-selection experiment were 0.52 ± 0.10 (1) and 0.52 ± 0.13 (2), which were not significantly different from the base population estimate of r* _Gd = 0.63 ± 0.14. Divergence (L^-W ⁺ minus L⁺W^-) in the antagonistic index selection lines was 0.21 ± 0.01 index units (I = 0.305 P_W - 0.436 P_L, where P _W and P_L are the phenotypic values for 6-week body weight and litter size, respectively.). The h² _R of index units of 0.14 ± 0.02 calculated from divergence agreed with h²_d of 0.14 ± 0.04. Divergences in litter size (-0.19 ± 0.07) and 6-week body weight (0.46 ± 0.10) were in the expected direction. Antagonistic index selection yielded about one-half the expected divergence in litter size, while divergence in 6-week body weight was only slightly less than expected. Realized genetic correlations indicated that litter size, 6-week body weight and index units each showed positive pleiotropy with 3-week body weight, postweaning gain and weight at vaginal introitus and negative pleiotropy with age at vaginal introitus. Sex ratio and several components of fitness (days from joining to parturition, percent fertile matings and percent perinatal survival) did not change significantly in the selected lines.