Florence Teulé

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

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

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

The extreme strength and elasticity of spider silks originate from the modular nature of their repetitive proteins. To exploit such materials and mimic spider silks, comprehensive strategies to produce and spin recombinant fibrous proteins are necessary. This protocol describes silk gene design and cloning, protein expression in bacteria, recombinant protein purification and fiber formation. With an improved gene construction and cloning scheme, this technique is adaptable for the production of any repetitive fibrous proteins, and ensures the exact reproduction of native repeat sequences, analogs or chimeric versions. The proteins are solubilized in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at 25–30% (wt/vol) for extrusion into fibers. This protocol, routinely used to spin single micrometer-size fibers from several recombinant silk-like proteins from different spider species, is a powerful tool to generate protein libraries with corresponding fibers for structure–function relationship investigations in protein-based biomaterials. This protocol may be completed in 40 d.

Combining flagelliform and dragline spider silk motifs to produce tunable synthetic biopolymer fibers.

The two Flag/MaSp 2 silk proteins produced recombinantly were based on the basic consensus repeat of the dragline silk spidroin 2 protein (MaSp 2) from the Nephila clavipes orb weaving spider. However, the proline‐containing pentapeptides juxtaposed to the polyalanine segments resembled those found in the flagelliform silk protein (Flag) composing the web spiral: (GPGGX1 GPGGX2)2 with X1/X2 = A/A or Y/S. Fibers were formed from protein films in aqueous solutions or extruded from resolubilized protein dopes in organic conditions when the Flag motif was (GPGGX1 GPGGX2)2 with X1/X2 = Y/S or A/A, respectively. Post‐fiber processing involved similar drawing ratios (2–2.5×) before or after water‐treatment. Structural (ssNMR and XRD) and morphological (SEM) changes in the fibers were compared to the mechanical properties of the fibers at each step. Nuclear magnetic resonance indicated that the fraction of β‐sheet nanocrystals in the polyalanine regions formed upon extrusion, increased during stretching, and was maximized after water‐treatment. X‐ray diffraction showed that nanocrystallite orientation parallel to the fiber axis increased the ultimate strength and initial stiffness of the fibers. Water furthered nanocrystal orientation and three‐dimensional growth while plasticizing the amorphous regions, thus producing tougher fibers due to increased extensibility. These fibers were highly hygroscopic and had similar internal network organization, thus similar range of mechanical properties that depended on their diameters. The overall structure of the consensus repeat of the silk‐like protein dictated the mechanical properties of the fibers while protein molecular weight limited these same properties. Subtle structural motif re‐design impacted protein self‐assembly mechanisms and requirements for fiber formation.

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

Flagelliform spider silk is the most extensible silk fiber produced by orb weaver spiders, though not as strong as the dragline silk of the spider. The motifs found in the core of the Nephila clavipes flagelliform Flag protein are GGX, spacer, and GPGGX. Flag does not contain the polyalanine motif known to provide the strength of dragline silk. To investigate the source of flagelliform fiber strength, four recombinant proteins were produced containing variations of the three core motifs of the Nephila clavipes flagelliform Flag protein that produces this type of fiber. The as-spun fibers were processed in 80% aqueous isopropanol using a standardized process for all four fiber types, which produced improved mechanical properties. Mechanical testing of the recombinant proteins determined that the GGX motif contributes extensibility and the spacer motif contributes strength to the recombinant fibers. Recombinant protein fibers containing the spacer motif were stronger than the proteins constructed without the spacer that contained only the GGX motif or the combination of the GGX and GPGGX motifs. The mechanical and structural X-ray diffraction analysis of the recombinant fibers provide data that suggests a functional role of the spacer motif that produces tensile strength, though the spacer motif is not clearly defined structurally. These results indicate that the spacer is likely a primary contributor of strength, with the GGX motif supplying mobility to the protein network of native N. clavipes flagelliform silk fibers.

Effects of different post-spin stretching conditions on the mechanical properties of synthetic spider silk fibers

Spider silk is a biomaterial with impressive mechanical properties, resulting in various potential applications. Recent research has focused on producing synthetic spider silk fibers with the same mechanical properties as the native fibers. For this study, three proteins based on the Argiope aurantia Major ampullate Spidroin 2 consensus repeat sequence were expressed, purified and spun into fibers. A number of post-spin draw conditions were tested to determine the effect of each condition on the mechanical properties of the fiber. In all cases, post-spin stretching improved the mechanical properties of the fibers. Aqueous isopropanol was the most effective solution for increasing extensibility, while other solutions worked best for each fiber type for increasing tensile strength. The strain values of the stretched fibers correlated with the length of the proline-rich protein sequence. Structural analysis, including X-ray diffraction and Raman spectroscopy, showed surprisingly little change in the initial as-spun fibers compared with the post-spin stretched fibers.

Modular Spider Silk Fibers: Defining New Modules and Optimizing Fiber Properties

Orb-web weaving spiders use multiple silk fibers to accomplish different tasks, combining repetitive peptide modules to produce different properties in each fiber. Each fiber is the product of a distinct gland, but is subject to a common spinning paradigm to produce an insoluble fiber from an aqueous-soluble protein dope. We start by presenting the cloning of the last of the six silks used by Nephila clavipes, the piriform silk spidroin. This piriform fiber presents a unique set of protein modules, which are used to attach other silk fibers to surfaces and to each other. Fiber spinning studies using major ampullate, minor ampullate, and flagelliform modules responsible for distinct secondary structures and therefore fiber properties will be presented. The properties of various synthetic fibers such as the initial (Young’s) modulus, tensile strength at break, strain at break, and toughness will be presented for a N. clavipes flagelliform/major ampullate hybrid synthetic fiber series, and an Argiope aurantia flagelliform/major ampullate hybrid synthetic fiber. Then, an N. clavipes major ampullate protein 1 synthetic fiber will be compared to itself in terms of how the fiber reacts to a post-spin draw in terms of properties and secondary structure. Finally, two flagelliform/major ampullate hybrid fibers made from slightly different elastic modules will be compared to show how minor changes in a single peptide module can change artificial spinning parameters substantially. Post-spin draw regimens on each fiber will demonstrate the importance of such procedures in optimizing fiber properties to take advantage of the modular protein sequences. Secondary structure studies at different stages of spinning will demonstrate the recruitment of secondary structures that greatly influence fiber properties.