Production Of Spider Silk Proteins Research Articles

Review of Spider Silk Applications in Biomedical and Tissue Engineering.

This review will present the latest research related to the production and application of spider silk and silk-based materials in reconstructive and regenerative medicine and tissue engineering, with a focus on musculoskeletal tissues, and including skin regeneration and tissue repair of bone and cartilage, ligaments, muscle tissue, peripheral nerves, and artificial blood vessels. Natural spider silk synthesis is reviewed, and the further recombinant production of spider silk proteins. Research insights into possible spider silk structures, like fibers (1D), coatings (2D), and 3D constructs, including porous structures, hydrogels, and organ-on-chip designs, have been reviewed considering a design of bioactive materials for smart medical implants and drug delivery systems. Silk is one of the toughest natural materials, with high strain at failure and mechanical strength. Novel biomaterials with silk fibroin can mimic the tissue structure and promote regeneration and new tissue growth. Silk proteins are important in designing tissue-on-chip or organ-on-chip technologies and micro devices for the precise engineering of artificial tissues and organs, disease modeling, and the further selection of adequate medical treatments. Recent research indicates that silk (films, hydrogels, capsules, or liposomes coated with silk proteins) has the potential to provide controlled drug release at the target destination. However, even with clear advantages, there are still challenges that need further research, including clinical trials.

Engineering Circularized mRNAs for the Production of Spider Silk Proteins.

Biomaterials offer unique properties that make them irreplaceable for next-generation applications. Fibrous proteins, such as various caterpillar silks and especially spider silk, have strength and toughness not found in human-made materials. In early studies, proteins containing long tandem repeats, such as major ampullate spidroin 1 (MaSp1) and flagelliform silk protein (FSLP), were produced using a large DNA template composed of many tandem repeats. The hierarchical DNA assembly of the DNA template is very time-consuming and labor-intensive, which makes the fibrous proteins difficult to study and engineer. In this study, we designed a circularized mRNA (cmRNA) employing the RNA cyclase ribozyme mechanism. cmRNAs encoding spider silk protein MaSp1 and FSLP were designed based on only one unit of the template sequence but provide ribosomes with a circular and infinite translation template for production of long peptides containing tandem repeats. Using this technique, cmRNAs of MaSp1 and FSLP were successfully generated with circularization efficiencies of 8.5% and 36.7%, respectively, which supported the production of recombinant MaSp1 and FSLP larger than 110 and 88 kDa, containing tens of repeat units. Western blot analysis and mass spectrometry confirmed the authenticity of MaSp1 and FSLP, which were produced at titers of 22.1 and 81.5 mg · liter-1, respectively. IMPORTANCE Spider silk is a biomaterial with superior properties. However, its heterologous expression template is hard to construct. The cmRNA technique simplifies the construction and expression strategy by proving the ribosome a circular translation template for expression of long peptides containing tandem repeats. This revolutionary technique will allow researchers to easily build, study, and experiment with any fiber proteins with sequences either from natural genes or artificial designs. We expect a significantly accelerated development of fibrous protein-based biomaterials with the cmRNA technique.

Secretory production of spider silk proteins in metabolically engineered Corynebacterium glutamicum for spinning into tough fibers

Spider dragline silk is a remarkable fiber made of unique proteins—spidroins—secreted and stored as a concentrated aqueous dope in the major ampullate gland of spiders. This feat has inspired engineering of microbes to secrete spidroins for spinning into tough synthetic fibers, which remains a challenge due to the aggregation-prone feature of the spidroins and low secretory capacity of the expression hosts. Here we report metabolic engineering of Corynebacterium glutamicum to efficiently secrete recombinant spidroins. Using a model spidroin MaSpI16 composed of 16 consensus repeats of the major ampullate spidroin 1 of spider Trichonephila clavipes, we first identified the general Sec protein export pathway for its secretion via N-terminal fusion of a translocation signal peptide. Next we improved the spidroin secretion levels by selection of more suitable signal peptides, multiplexed engineering of the bacterial host, and by high cell density cultivation of the resultant recombinant strains. The high abundance (>65.8%) and titer (554.7 mg L–1) of MaSpI16 in the culture medium facilitated facile, chromatography-free recovery of the spidroin with a purity of 93.0%. The high solubility of the purified spidroin enabled preparation of highly concentrated aqueous dope (up to 66%) amenable for spinning into synthetic fibers with an appreciable toughness of 70.0 MJ m−3. The above metabolic and processing strategies were also found applicable for secretory production of the higher molecular weight spidroin MaSpI64 (64 consensus repeats) to yield similarly tough fibers. These results suggest the good potential of secretory production of protein polymers for sustainable supply of fibrous materials.

Spider Silk for Tissue Engineering Applications.

Due to its properties, such as biodegradability, low density, excellent biocompatibility and unique mechanics, spider silk has been used as a natural biomaterial for a myriad of applications. First clinical applications of spider silk as suture material go back to the 18th century. Nowadays, since natural production using spiders is limited due to problems with farming spiders, recombinant production of spider silk proteins seems to be the best way to produce material in sufficient quantities. The availability of recombinantly produced spider silk proteins, as well as their good processability has opened the path towards modern biomedical applications. Here, we highlight the research on spider silk-based materials in the field of tissue engineering and summarize various two-dimensional (2D) and three-dimensional (3D) scaffolds made of spider silk. Finally, different applications of spider silk-based materials are reviewed in the field of tissue engineering in vitro and in vivo.

Microbial production of amino acid-modified spider dragline silk protein with intensively improved mechanical properties

ABSTRACTSpider dragline silk is a remarkably strong fiber with impressive mechanical properties, which were thought to result from the specific structures of the underlying proteins and their molecular size. In this study, silk protein 11R26 from the dragline silk protein of Nephila clavipes was used to analyze the potential effects of the special amino acids on the function of 11R26. Three protein derivatives, ZF4, ZF5, and ZF6, were obtained by site-directed mutagenesis, based on the sequence of 11R26, and among these derivatives, serine was replaced with cysteine, isoleucine, and arginine, respectively. After these were expressed and purified, the mechanical performance of the fibers derived from the four proteins was tested. Both hardness and average elastic modulus of ZF4 fiber increased 2.2 times compared with those of 11R26. The number of disulfide bonds in ZF4 protein was 4.67 times that of 11R26, which implied that disulfide bonds outside the poly-Ala region affect the mechanical properties of spider silk more efficiently. The results indicated that the mechanical performances of spider silk proteins with small molecular size can be enhanced by modification of the amino acids residues. Our research not only has shown the feasibility of large-scale production of spider silk proteins but also provides valuable information for protein rational design.

Recombinant DNA production of spider silk proteins.

Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks.

Spider Silk Capsules as Protective Reaction Containers for Enzymes

Spider silk fibres are well known for their high tensile strength in combination with high elasticity. Based on the possibility of recombinant production of spider silk proteins, technical applications of spider silk materials are nowadays feasible. The engineered recombinant spider silk protein eADF4(C16) is based on the sequence of ADF4 (Araneus diadematus fibroin), one out of at least three proteins of the dragline silk of the European garden spider A. diadematus. The protein eADF4(C16) can be processed into different morphologies. Here, capsules of eADF4(C16) are assembled at an oil/water interface. These microcapsules are mechanically stable and can be used as a transport system for higher molecular weight compounds such as enzymes or chemical catalysts. Further, they can be regarded as a small enclosed reaction chamber with a semi‐permeable membrane. Reactions can be initiated by diffusion of the reactants through the silk membrane. The eADF4(C16) capsules protect the enzyme β‐galactosidase, used as model, against proteolysis. Functional α‐complementation of β‐galactosidase visualizes the controllable activation of an enzyme within such spider silk capsule, highlighting the broad applicability thereof as reaction containers, e.g., for enzymes.

Impact of initial solvent on thermal stability and mechanical properties of recombinant spider silk films

Spider silk fibers are one of the most remarkable biopolymers displaying a unique combination of mechanical properties, biocompatibility and biodegradability. The recombinant production of spider silk proteins now allows the processing of silk proteins into novel materials with the aim of future applications. Here, we analyzed films made of a recombinantly produced, engineered spider silk protein with a sequence derived from the dragline silk protein ADF4 of the European garden spider Araneus diadematus. An influence of different initial solvents (aqueous buffer, hexafluoro-2-propanol and formic acid) on certain film properties was identified in as cast as well as methanol post-treated films: while no significant effects on the films' thermal stability were observed, a significant influence on their mechanical properties could be shown. Interestingly, solvent-induced effects were sustained after methanol post-treatment and could be correlated with the presence and arrangement of secondary structure elements. Insights into molecular orientation of individual structural elements within the films upon applied load were revealed by combined polarized IR spectroscopy and mechanical measurements.

Production of spider silk proteins in tobacco seeds

Production of spider silk proteins in tobacco seeds

Production of spider silk proteins in tobacco and potato.

Spider dragline silk is a proteinaceous fiber with remarkable mechanical properties that make it attractive for technical applications. Unfortunately, the material cannot be obtained in large quantities from spiders. We have therefore generated transgenic tobacco and potato plants that express remarkable amounts of recombinant Nephila clavipes dragline proteins. Using a gene synthesis approach, the recombinant proteins exhibit homologies of >90% compared to their native models. Here, we demonstrate the accumulation of recombinant silk proteins, which are encoded by synthetic genes of 420-3,600 base pairs, up to a level of at least 2% of total soluble protein in the endoplasmic reticulum (ER) of tobacco and potato leaves and potato tubers, respectively. Using the present expression system, spider silk proteins up to 100 kDa could be detected in plant tissues. When produced in plants, the recombinant spidroins exhibit extreme heat stability-a property that is used to purify the spidroins by a simple and efficient procedure.