Development of an artificial silk protein on the basis of a lacewing egg stalk protein

Silks are widely used in textile industry as clothing and furnishings due to their tensile strength, smoothness, soft texture, lustre, and drape. Most commonly silk of the mulberry silkworm Bombyx mori (B. mori) is used in such applications, however, silks evolved independently in many different arthropods for various purposes.1 During evolution the different silks were optimised for their task-specific uses over millions of years, e.g. adopting different mechanical properties. The mechanical properties mainly derive from the protein secondary structure and its higher order arrangement in silk fibres. Spider silk, for example, is known for its tensile properties surpassing nylon, Kevlar®, silkworm silk, and high-tensile steel.2-5 Beyond their mechanical properties, some silks are also reported to be biocompatible and non-immunogenic.6 One beneficial feature of silk proteins is the possibility to process them into various morphologies.7, 8 Several of these silk features make them interesting for material scientists, intending to produce silks with tuneable properties depending on the desired application, ranging from technical ones such as high performance fibres to medical ones such as drug delivery. This thesis deals with the characterisation and reproduction of a less explored silk, the lacewing egg stalk silk. Mechanical testing revealed a strong dependence on the relative humidity. In the dry state at 30% relative humidity, the stalks are quite rigid and break at an elongation of 2% whereas at 70% and 100% relative humidity they elongate up to 434%. This extension is accompanied by a secondary structure change from cross-ß to parallel-ß. The cross-ß structure in unstretched stalks provides bending stiffness and rigidity to the stalk, and this bending stiffness gets lost when the stalks are stretched. In this thesis a model is proposed which explains these differences at various relative humidity on the molecular level, wherein changes in the strength of hydrogen bonds upon exposure to water (a hydrogen bond donor/acceptor) in combination with multiple disulphide cross-links (which are not affected by water) act together and are responsible for this behaviour. Based on consensus sequences of published sequence data (derived from MalXB2 an egg stalk protein of Mallada signata (M. signata)),9 an engineered egg stalk protein named N[AS]8C was recombinantly produced. To produce an artificial stalk, a droplet of a solution of purified N[AS]8C was placed on a substrate, and tweezers were used to pull out a fibre. After drying, and post treatment, the properties of the artificial stalks were investigated in comparison to the natural ones. Mechanical testing revealed similar behaviour at 30% relative humidity, but at 70% and 100% relative humidity the artificial stalks were not as extensible as the natural ones. This corresponds to the fact, that no cross-ß structure was formed, and, therefore, no rearrangement into parallel-ß structure was possible. Subsequently, N[AS]8C was processed into non-fibrous morphologies. It was possible to produce capsules, hydrogels, foams, and films. The foams show an interesting micro and nano structure which differs from that of recombinant spider silk. The cavities are filled with a mesh of nano fibres building a 3D scaffold. Films are a morphology with potential for application in cell culture. Fibroblast attachment on N[AS]8C films is quite poor. Therefore, we tried to induce guided fibroblast growth on patterned protein films. A first layer of the films was cast from ntagCysC16-c(RGDfK), an engineered spider silk protein coupled with the integrin recognition motif RGD to provide a protein layer to which fibroblasts attached well. The second protein layer was produced using a PDMS (polydimethylsiloxane) template and N[AS]8C. Fibroblasts grown on these films adhere only to the RGD modified spider silk and not to the N[AS]8C areas. A second feature of such films is to orient the fibroblasts on films with alternating lines of the two proteins. Such films might be useful for tissue engineering to control cell adhesion and get a structured cell pattern. This is essential for many tissues such as bones, muscles, and epithelia tissue. The low cell adhesion properties of N[AS]8C films might be interesting for coatings for applications where cell adhesion is not desired such as stents or catheters.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Universität Bayreuth

Year

2013

Author dc:creator

• Bauer, Felix

Contributors dc:contributor

• Scheibel, Thomas

Repository record source_url

https://epub.uni-bayreuth.de/id/eprint/72/

OAI identifier oai:identifier

oai:epub.uni-bayreuth.de:72