Artificial+Synthesis+of+Spider+Silk

flat =**Why Synthesize Spider Silk?** =

Spider dragline silk is a high-performance fibre that displays extraordinary mechanical properties.[4] It is also biocompatible and biodegradable, which makes it suitable for biomaterial production.[9] In order for scientists to be able to artificially replicate the dragline properties, the protein constituents must be characterized and the silk production pathway pinpointed.[2] The large, repetitive sequences of the genes and corresponding proteins have made production of silk analogs difficult in recombinant systems; genetic instability, prematurely terminated synthesis and poor solubility of produced proteins have been frequently observed.[9]

Spiders are able to spin their silk at near-ambient temperatures and pressures using water as a solvent. They are able to produce this by judiciously controlling the folding and crystallization of the main protein constituents, as well as adding auxiliary compounds to create a composite material of defined structure. The liquid 'dope' that the silk is spun from is liquid crystalline which spiders can draw during extrusion into a hardened fibre.[1]

This process has proven to be difficult to reproduce in an artificial setting. The molecular structure of unspun silk is both complex and extremely long. Though this provides the silk fibres with their desirable properties, it is also what makes replication of the fibre somewhat of a challenge. In order to artificially synthesize spider silk into fibres, there are two areas that must be covered; the synthesis of the feedstock (the unspun silk 'dope' in spiders), and mimicry of the spinning apparatus (the funnel, spinning duct, clamp and spigot in spiders).

=**Obtaining the Necessary Proteins** =

The synthesis of the spider silk 'dope' can be produced much easier (or at least in greater quantities) by extracting the spider silk gene and using genetically modified organisms to produce the spider silk such as E //coli//, caterpillars, silkworms and goats.[3,4,8] However, the silk produced via this method is in liquid form due to the water solubility of the spider silk proteins. The problem is mimicry of the spiders extrusion apparatus in order to spin a very strong fibre. One important feature of spider silk proteins is their high protein concentrations (up to 50% w/v) in the dope without apparent aggregation or assembly. Still, spider silk proteins can rapidly assemble into highly stable fibres when needed. The determination of solubility and self-assembly of recombinant spider silk proteins is therefore important to create commercially available silk fibres. For example, pH-shifts are involved in natural silk assembly, but the exact function of acidification during spider silk assembly has not yet been determined.[8]

=**Construction of Biomimicked Apparatus (Spinning Techniques)** =

Besides the chemical parameters discussed above, several mechanical parameters play important roles in generating silk. There are a few spinning methods that have been developed: microfluidics, electrospinning, and the use of a syringe and needle.

Microfluidics is a method that's currently being developed. The goal is to have a very controllable method to test spin very small volumes of unspun fibre, but development costs are expensive. A patent has been granted in this area for spinning fibres in a method mimicking the process found in nature, and fibres are successfully being spun by Spintec Engineering GmbH in Germany.[9,10]

Electrospinning is a fabrication process that uses an electric field to control the deposition of polymer fibres onto a target substrate. Several proteins have been successfully converted into nanofibres using electrospinning, including silkworm silk, collagen, elastin and fibrinogen. The resulting protein nanofibres could be used to fabricate biological scaffolds for membranes, tissue engineering, hemostatic bandages or wound dressings.[7]

One possible way to produce spider silk threads in vitro is to ‘wet-spin’ recombinant spider silk proteins.[2,7] This is a highly complex technique that tries to mimic the natural process of spider silk production. The spinning solution is forced through tiny holes in a spinneret (or syringe) to extrude protein fibres. Spinning occurs in an aqueous environment and the resulting fibre is subjected to a subsequent stretching or drawing process. The properties of the fibre depend on many factors; the concentration and temperature of the spinning solution and spin bath, the composition, and the force applied during spinning.[4,7] These processes are being developed by Nexia Biotech among several companies.

Natural fibre formation during the spiders spinning process is partly dependent on dehydration and protein alignment.[1] This is recognized in the attempts to construct artificial spinning apparatus that, in these respects, mimic the spiders spinning mechanism and produces synthetic silk fibres.[6] This 'wet-spinning' is performed by expelling a concentrated protein solution through a small needle or similar apparatus, to mimic the elongational flow and shear forces created by the spiders progressively narrowing extrusion duct. The emerging fibre is often allowed to enter a bath with a dehydrating coagulant, such as methanol, ethanol or acetone to reproduce the natural water removal.[2]

Industrial spinning technologies currently use temperatures above 200°C, aggressive organic solvents and high pressures.[2,9] The building blocks for fibres are cheap chemical polymers which are processed at spinning speeds of several kilometers per hour. The manufacturing of chemical fibres has reached a high degree of sophistication and allows the production of high quality fibres at multi-ton scale.[10] Under natural spinning conditions, spiders attach their dragline to an object with glue from the piriform glands, before drawing the silk out by moving away or by descending and using their weight to draw the silk. It is therefore important to monitor the rate of spinning in the lab.[4]

Nature ‘operates’ under constant energy constraints, but had endless amounts of time to evolve complex biological polymers through trial-and-error (evolution) which are able to self-assemble into ordered, macromolecular structures. Compared to industrial processes, biological self-assembly requires low energy input and works at room temperature, normal pressure and purely in water without organic solvents. Spintec scientists mimicked the biological spinning process into a biomimetic endless fibre spinning machine which operates with conditions as similar as possible to those evolved by nature.[10] (Refer to figure 1)

 **￼Figure 1. Biomimetic extrusion apparatus (Sections numbered 1-4 from left to right)[1,10]**

<span style="font-family: 'Times New Roman',Times,serif; font-size: 16px;">1. Funnel: The funnel allows acceleration and prealignment of the spider's spinning dope (silk proteins) in the direction of flow.

<span style="font-family: 'Times New Roman',Times,serif; font-size: 16px;">2. Spinning Duct: The duct has a thin cuticle which acts as a dialysis membrane and allows water and sodium ions out of the lumen and potassium ions, surfactants and lubricants into the lumen to facilitate thread formation.

<span style="font-family: 'Times New Roman',Times,serif; font-size: 16px;">3. Clamp: The muscle controlled clamp allows the spider to grip the thread, for example for controlled downward roping. The clamp appears also to play a role as a ‘ratchet’ or ‘pump‘, to restart spinning after internal rupture of a filament.

<span style="font-family: 'Times New Roman',Times,serif; font-size: 16px;">4. Spigot: Finally, the thread is gripped by the flexible and elastic lips of the spigot through which it passes to the outside world. The spigot strips off the last of the aqueous phase surrounding the tread, thus helping to retain water in the spider, and also places the tread under tension for the final air-drawing step.

=<span style="font-family: 'Times New Roman',Times,serif; font-size: 16px;">**Current Results** =

<span style="font-family: 'Times New Roman',Times,serif; font-size: 16px;">So far, synthetic materials with mechanical properties matching the dragline silk have not been produced. Processes are currently getting closer to producing a fibre that is as strong, tough and fine as spider silk, but recombinant synthesis is not there yet. Extrusion has produced silk fibres of diameters ranging from 10 to 60 μm, compared to diameters of 2.5 to 4 μm for natural spider silk.[2,8] These silks exhibited toughness comparable to native dragline silk, but with lower tenacity.[2,8] A further consideration is the long-term durability of these silk fibres. Under natural conditions these silks are only required for a short period of time, which spiders are able to produce as needed. As newer and better methods are developed to produce dragline silk these considerations will become more relevant. Still, the results found are good indication that these processes are headed in the right direction, and that there is much more to this cutting-edge field of study.

<span style="font-family: 'Times New Roman',Times,serif; font-size: 16px;">[1] Vollrath F., Knight, D.P. (2001). Liquid crystalline spinning of spider silk. Nature. 410 (6828): 541–548. <span style="font-family: 'Times New Roman',Times,serif; font-size: 16px;">[2] Lazaris A., et al. (2002). Spider silk fibers spun from soluble recombinant silk produced in mammalian cells. Science. 295: 472–476. <span style="font-family: 'Times New Roman',Times,serif; font-size: 16px;"> [3]Xia X.X., et al. (2010). Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber. Proceedings of the National Academy of Sciences of the United States of America. 107: 14059–14063. <span style="font-family: 'Times New Roman',Times,serif; font-size: 16px;"> [4]Gould P. (2002). Exploiting Spiders Silk. Materials today. 5(12): 42-47. <span style="font-family: 'Times New Roman',Times,serif; font-size: 16px;"> [5]Seidel A., et al. (2000). "Regenerated spider silk: Processing, properties, and structure". Macromolecules. 33: 775–780. <span style="font-family: 'Times New Roman',Times,serif; font-size: 16px;"> [6]Seidel A., Liivak O., Jelinski, L.W. (1998). Artificial spinning of spider silk. Macromolecules. 31: 6733-6736. <span style="font-family: 'Times New Roman',Times,serif; font-size: 16px;"> [7]Scheibel T. (2005). Protein fibers as performance proteins: new technologies and applications. Current Opinion in Biotechnology. 16: 427–433. <span style="font-family: 'Times New Roman',Times,serif; font-size: 16px;"> [8]Scheibel T. (2004). Spider silks: recombinant synthesis, assembly, spinning, and engineering of synthetic proteins. Microbial Cell Factories. 3: 14. <span style="font-family: 'Times New Roman',Times,serif; font-size: 16px;"> [9]Stark M., Grip S., Rising A., Hedhammar M., Engström W., Hjälm G., Johansson J. (2007). Macroscopic fibers self-assembled from recombinant miniature spider silk proteins. Biomacromolecules. 8: 1695-1701. <span style="font-family: 'Times New Roman',Times,serif; font-size: 16px;"> [10]http://www.spintec-engineering.de/spintec-engineering.de/Home_E.html