Scalable purification of recombinant structural proteins, hagfish intermediate filament α and É£, from inclusion bodies for fiber formation.

The purpose of this study was to determine a method to purify recombinant hagfish intermediate filament proteins, alpha and gamma, in a scalable manner. The study succeeded by having an increase in protein recovery of up to 35% when comparing centrifuge purification and the developed tangential flow purification. The proteins were approximately the same purity of 70% pure but further purification increased the purity of the proteins by 16%, based on ImageJ analysis. The developed tangential flow filtration purification and final purification methods could be easily scaled up to meet industry scale purification needs. The scaled-up processes described in this study did not interfere with fiber production or formation, indicating the methods can produce usable proteins for material development.

The next generation of protein super-fibres: robust recombinant production and recovery of hagfish intermediate filament proteins with fibre spinning and mechanical-structural characterizations.

Native hagfish intermediate filament proteins have impressive mechanical properties. However, using these native fibres for any application is impractical, necessitating their recombinant production. In the only literature report on the proteins (denoted α and É£), heterologous expression levels, using E. coli, were low and no attempts were made to optimize expression, explore wet-spinning, or spin the two proteins individually into fibres. Reported here is the high production (~8 g l-1 of dry protein) of the hagfish intermediate filament proteins, with yields orders of magnitude higher (325-1000×) than previous reports. The proteins were spun into fibres individually and in their native-like 1:1 ratio. For all fibres, the hallmark α-helix to ß-sheet conversion occurred after draw-processing. The native-like 1:1 ratio fibres achieved the highest average tensile strength in this study at nearly 200 MPa with an elastic modulus of 5.7 GPa, representing the highest tensile strength reported for these proteins without chemical cross-linking. Interestingly, the recombinant α protein achieved nearly the same mechanical properties when spun as a homopolymeric fibre. These results suggest that varying the two protein ratios beyond the natural 1:1 ratio will allow a high degree of tunability. With robust heterologous expression and purification established, optimizing fibre spinning will be accelerated compared to difficult to produce proteins such as spider silks.

Large scale production of synthetic spider silk proteins in Escherichia coli.

Spider silk, which has remarkable mechanical properties, is a natural protein fiber produced by spiders. Spiders cannot be farmed because of their cannibalistic and territorial nature. Hence, large amounts of spider silk cannot be produced from spiders. Genetic engineering is an alternative approach to produce large quantities of spider silk. Our group has produced synthetic spider silk proteins in E. coli to study structure/function and to produce biomaterials comparable to the silks produced by orb-weaving spiders. Here we give a detailed description of our cloning, expression, and purification methods of synthetic spider silk proteins ranging from ~30 to ~200 kDa. We have cloned the relevant genes of the spider Nephila clavipes and introduced them into bacteria to produce synthetic spider silk proteins using small and large-scale bioreactors. We have optimized the fermentation process, and we have developed protein purification methods as well. The purified proteins are spun into fibers and are used to make alternative materials like films and adhesives with various possible commercial applications.

Silkworms with Spider Silklike Fibers Using Synthetic Silkworm Chow Containing Calcium Lignosulfonate, Carbon Nanotubes, and Graphene.

Silkworm silk has become increasingly relevant for material applications. However, the industry as a whole is retracting because of problems with mass production. One of the key problems is the inconsistent properties of the silk. A means by which to improve the silk material properties is through enhanced sericulture techniques. One possible technique is altering the feed of the silkworms to include single-wall carbon nanotubes (SWNTs) or graphene (GR). Recently published results have demonstrated substantial improvement in fiber mechanical properties. However, the effect of the surfactant used to incorporate those materials into the feed on the fiber mechanical properties in comparison to normal silkworm silk has not been studied or reported. Thus, the total effect of feeding the SWNT and GR in the presence of surfactants on silkworms is not understood. Our study focuses on the surfactant [calcium lignosulfonate (LGS)] and demonstrates that it alone results in appreciable improvement of mechanical properties in comparison to nontreated silkworm silk. Furthermore, our study demonstrates that mixing the LGS, SWNT, and GR directly into the artificial diet of silkworms yields improved mechanical properties without decline below the control silk at high doses of SWNT or GR. Combined, we present evidence that mixing surfactants, in this case LGS, directly with the diet of silkworms creates a high-quality fiber product that can exceed 1 GPa in tensile strength. With the addition of nanocarbons, either SWNT or GR, the improvement is even greater and consistently surpasses control fibers. However, feeding LGS alone is a more economical and practical choice to consistently improve the mechanical properties of silkworm fiber.

Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure.

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order to reproduce the remarkable mechanical properties of spider silks, they must be produced in heterologous hosts as spiders are both territorial and cannibalistic. The synthetic analogs of spider silk also tend to be insoluble in aqueous solutions. Thus, a large percentage of research in recombinant spider silks rely upon organic solvents that are detrimental to large scale production of materials. Our group's method forces the solvation of these recombinant spider silks into water. Remarkably, when these proteins are prepared using this method of heat and pressure, a wide range of material forms can be prepared from the same solution of recombinant spider silk proteins (rSSp) including: films, fibers, sponge, hydrogel, lyogel, and adhesives. This article demonstrates the production of the solvated rSSp and material forms in a manner that is more easily understood than from written materials and methods alone.

Importance of Heat and Pressure for Solubilization of Recombinant Spider Silk Proteins in Aqueous Solution.

The production of recombinant spider silk proteins continues to be a key area of interest for a number of research groups. Several key obstacles exist in their production as well as in their formulation into useable products. The original reported method to solubilize recombinant spider silk proteins (rSSp) in an aqueous solution involved using microwaves to quickly generate heat and pressure inside of a sealed vial containing rSSp and water. Fibers produced from this system are remarkable in their mechanical ability and demonstrate the ability to be stretched and recover 100 times. The microwave method dissolves the rSSPs with dissolution time increasing with higher molecular weight constructs, increasing concentration of rSSPs, protein type, and salt concentration. It has proven successful in solvating a number of different rSSPs including native-like sequences (MaSp1, MaSp2, piriform, and aggregate) as well as chimeric sequences (FlAS) in varied concentrations that have been spun into fibers and formed into films, foams, sponges, gels, coatings, macro and micro spheres and adhesives. The system is effective but inherently unpredictable and difficult to control. Provided that the materials that can be generated from this method of dissolution are impressive, an alternative means of applying heat and pressure that is controllable and predictable has been developed. Results indicate that there are combinations of heat and pressure (135 °C and 140 psi) that result in maximal dissolution without degrading the recombinant MaSp2 protein tested, and that heat and pressure are the key elements to the method of dissolution.

Development of a Process for the Spinning of Synthetic Spider Silk.

Spider silks have unique mechanical properties but current efforts to duplicate those properties with recombinant proteins have been unsuccessful. This study was designed to develop a single process to spin fibers with excellent and consistent mechanical properties. As-spun fibers produced were brittle, but by stretching the fibers the mechanical properties were greatly improved. A water-dip or water-stretch further increased the strength and elongation of the synthetic spider silk fibers. Given the promising results of the water stretch, a mechanical double-stretch system was developed. Both a methanol/water mixture and an isopropanol/water mixture were independently used to stretch the fibers with this system. It was found that the methanol mixture produced fibers with high tensile strength while the isopropanol mixture produced fibers with high elongation.