Engineered Spider Silk Protein Research Articles

Recombinant Spider Silk Proteins for Applications in Biomaterials

Due to their extraordinary mechanical and biochemical properties, silks have long been in focus of research. In vivo, fibers are formed from silk proteins, in vitro, however, a variety of materials can be produced in addition to fibers including capsules, particles, films, foams, and gels. The versatility of silk proteins, along with their biocompatibility, biodegradability, and potential for processing in aqueous solution under ambient conditions make silk-based materials good candidates for biomedical applications such as drug delivery systems and scaffolds for tissue engineering. Here, we summarize recent progress in research employing recombinantly produced engineered spider silk proteins with a focus on the fundamentals of silk protein processing. We highlight recombinant spider silk films and particles as morphologies that represent model systems with adjustable material properties controlled by process parameters.

Structural characterization and functionalization of engineered spider silk films

Due to their biocompatibility, their extraordinary mechanical properties and the ability to be processed into various shapes, natural polymers like spider silk proteins are promising candidates for materials' applications. However, for many applications, additional specific functionalization is necessary. Here, we present recombinantly produced engineered spider silk proteins based on one dragline silk component of the European garden spider Araneus diadematus. The proteins have been engineered in order to incorporate cysteine which allows site-specific functionalization. These cysteine containing variant silk proteins are characterized in terms of structure, assembly and chemical reactivity in solution. Further, films composed of these proteins were structurally investigated by CD- and FTIR-spectroscopy. Comparison of the variants with the original cysteine-free silk protein revealed no apparent differences in solution and in the films. Functionalization of the thiol groups of these silk protein-based films with molecules such as nanogold, dyes, biotin and β-galactosidase demonstrates the potential of such films for a broad range of applications which opens up new possibilities in materials research based on silk polymers.

Interfacial rheological properties of recombinant spider-silk proteins

Freestanding protein films are interesting for many applications ranging from microencapsulation to tissue engineering. Here, the authors use interfacial rheology to characterize the adsorption kinetics and the rheology of spider-silk films formed at an oil water interface. The high surface activity of the engineered spider-silk proteins results in a fast formation of highly stable films, which can be modified by the addition of phosphate ions to the solution.

Single molecule force measurements delineate salt, pH and surface effects on biopolymer adhesion

In this paper we probe the influence of surface properties, pH and salt on the adhesion of recombinant spider silk proteins onto solid substrates with single molecule force spectroscopy. A single engineered spider silk protein (monomeric C16 or dimeric (QAQ)8NR3) is covalently bound with one end to an AFM tip, which assures long-time measurements for hours with one and the same protein. The tip with the protein is brought into contact with various substrates at various buffer conditions and then retracted to desorb the protein. We observe a linear dependence of the adhesion force on the concentration of three selected salts (NaCl, NaH2PO4 and NaI) and a Hofmeister series both for anions and cations. As expected, the more hydrophobic C16 shows a higher adhesion force than (QAQ)8NR3, and the adhesion force rises with the hydrophobicity of the substrate. Unexpected is the magnitude of the dependences—we never observe a change of more than 30%, suggesting a surprisingly well-regulated balance between dispersive forces, water-structure-induced forces as well as co-solute-induced forces in biopolymer adhesion.

Biomimetic Spinning of Recombinant Silk Proteins

AbstractIn the past, we have successfully designed and produced a variety of engineered spider silk-like proteins (eADF3 and eADF4) based upon the primary sequence of the natural dragline proteins ADF3 and ADF4 from the spider Araneus diadematus [1]. Genetically engineered spider silk proteins can be modified at the molecular level to optimize the biochemical and mechanical properties of the final product. Although engineered spider silk proteins can be processed into fibers using different spinning methods, our group is interested in the technical realization of a biomimetic approach. Here, we present an overview over our biomimetic fiber production process.

An Engineered Spider Silk Protein Forms Microspheres

An Engineered Spider Silk Protein Forms Microspheres

Processing Conditions for the Formation of Spider Silk Microspheres

Spider silk is a material consisting of very large (>200 kDa) proteins and has a high potential for biomedical applications as a result of its biocompatibility and biodegradability. We report on the influence of physicochemical factors on structure formation of the engineered spider silk protein eADF4(C16), which mimics the known sequence of the dragline protein ADF4 from the spider Araneus diadematus. Under certain experimental conditions, eADF4(C16) forms stable microspheres that have been analyzed with respect to sphere size, size distribution, and surface inertness upon different preparation methods (dialysis, pipette and micromixing). As a result of their material strength, biocompatibility, and the possibility of functionalization, spider silk microspheres have a high potential for the development of targeted drug-delivery systems.

Structural changes of thin films from recombinant spider silk proteins upon post-treatment

Engineering of spider silk proteins offers the possibility to control their molecular sequence and thus their material properties. Spin coating was used to prepare films of engineered spider silk protein derived from the garden spider’s (Araneus diadematus) dragline silk protein ADF-4. A conformational transition from α-helix to β-sheet-rich structures upon methanol treatment of the films was detected by external reflection IR spectroscopy. We present direct evidence for this structural transformation using grazing-incidence X-ray diffraction (GIXRD) and small-angle scattering (GISAXS). The protein film structure after the methanol treatment consists mainly of β-sheet polyalanine crystals dispersed in an amorphous protein matrix. The GIXRD intensity profiles show Bragg peaks from β-sheet polyalanine crystallites having an average size of 7.5 nm. The non-uniform and large crystal size distributions within the film were explained based on the protein composition. The effect of the chemical nature of the interface on the protein film structure was investigated as well.