Biomimetic spider silk and bioactive hydrogels formed by engineered recombinant spider silk proteins
Author: Arndt, Tina
Date: 2022-12-09
Location: Erna Möller Salen, Neo, Karolinska Institutet, Flemingsberg
Time: 10.00
Department: Inst för biovetenskaper och näringslära / Dept of Biosciences and Nutrition
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Thesis (8.505Mb)
Abstract
Spider silk is a unique material and its properties have fascinated material scientists, biologists and physicians for decades. Spider silk display one of the highest toughness found among fibers in nature, is used by spiders for web-spinning, prey-wrapping and cocoon building, while scientists have explored its biomaterials properties for multiple purposes. Spider silk appears to be generally well tolerated when implanted and is biodegrade but it comes with a limited availability and variable quality.
Artificial silk production by recombinant expression of spider silk proteins (spidroins) in heterologous hosts is a promising path to overcome drawbacks associated with natural silk. To recapitulate the elaborate structure of natural silk one must understand the nature of silk formation and mimic this process closely. Previously, an artificial spidroin, NT2RepCT, was developed that can be spun into fibers with impressive mechanical properties in a biomimetic setup. However, NT2RepCT fibers cannot match the properties of natural silk fibers which may be due to incomplete biomimicry of the spidroin, and/or spinning procedure. In paper I, we analyzed to what extent the spidroin solution (dope), from which the silk fiber are spun, recapitulates important features of natural dope and found that it has shear-thinning and viscoelastic behavior and undergoes pH-induced phase-separation and structural changes similar to native dope, but lacks the high viscosity typically seen for natural spinning dope.
In Paper II, we took advantage of insights in the constraints that spidroins have evolved under and used rational protein engineering of the repeat region of NT2RepCT. More specifically, we increased the hydrophobicity of the b-sheet forming poly-Ala regions since hydrophobic amino acid residues side chains are generally more prone to form b-sheets and steric zippers. Such proteins are unlikely to be secreted since the translocon would inserts proteins with hydrophobic sections in the endoplasmic reticulum membrane. Since the NT2RepCT proteins accumulate intracellularly during expression in prokaryotic hosts, we are not confined by these restrictions. When spun into fibers in a biomimetic spinning device, the toughness of fibers spun from several of the engineered proteins improved significantly compared to fibers spun from NT2RepCT. Importantly, one of the fibers had an unprecedented toughness for an as-spun artificial silk fiber. Furthermore, expression of the engineered spidroin in a bioreactor resulted in protein yields that make large-scale production economically feasible.
Paper III explores the surprising finding that the hyper-soluble and stable spidroin N-terminal domain (NT) forms hydrogels when incubated at 37°C, and that gel formation is associated with a conversion of NT into amyloid-like fibrils. The high structural flexibility of NT combined with the presence of amyloidogenic sequences in its a-helices are factors that are important for formation of the gel. Furthermore, by fusing NT to target proteins, we present a novel immobilization platform in which NT is used both as an expression tag for high yield production of soluble fusion proteins and as a fibrillar scaffold in the gel. As hydrogel formation occurred rapidly and under benign conditions also for NT2RepCT, Paper IV focuses on the potential application of NT2RepCT hydrogels as a drug release device and for cell encapsulation. Successful encapsulation and release of active green fluorescent protein suggest that the hydrogels could be suitable candidates for use as drug release devices. An encapsulated cell line released the bioactive molecule progranulin for 31 days to a similar extent as cells cultured under standard conditions. Human mesenchymal stem cells encapsulated in the hydrogels showed high survival but limited proliferation, likely due to restricted space in the dense fibrillar network that is characteristic of the gels. This thesis describes major steps forward in the development of novel spidroin-based materials.
Artificial silk production by recombinant expression of spider silk proteins (spidroins) in heterologous hosts is a promising path to overcome drawbacks associated with natural silk. To recapitulate the elaborate structure of natural silk one must understand the nature of silk formation and mimic this process closely. Previously, an artificial spidroin, NT2RepCT, was developed that can be spun into fibers with impressive mechanical properties in a biomimetic setup. However, NT2RepCT fibers cannot match the properties of natural silk fibers which may be due to incomplete biomimicry of the spidroin, and/or spinning procedure. In paper I, we analyzed to what extent the spidroin solution (dope), from which the silk fiber are spun, recapitulates important features of natural dope and found that it has shear-thinning and viscoelastic behavior and undergoes pH-induced phase-separation and structural changes similar to native dope, but lacks the high viscosity typically seen for natural spinning dope.
In Paper II, we took advantage of insights in the constraints that spidroins have evolved under and used rational protein engineering of the repeat region of NT2RepCT. More specifically, we increased the hydrophobicity of the b-sheet forming poly-Ala regions since hydrophobic amino acid residues side chains are generally more prone to form b-sheets and steric zippers. Such proteins are unlikely to be secreted since the translocon would inserts proteins with hydrophobic sections in the endoplasmic reticulum membrane. Since the NT2RepCT proteins accumulate intracellularly during expression in prokaryotic hosts, we are not confined by these restrictions. When spun into fibers in a biomimetic spinning device, the toughness of fibers spun from several of the engineered proteins improved significantly compared to fibers spun from NT2RepCT. Importantly, one of the fibers had an unprecedented toughness for an as-spun artificial silk fiber. Furthermore, expression of the engineered spidroin in a bioreactor resulted in protein yields that make large-scale production economically feasible.
Paper III explores the surprising finding that the hyper-soluble and stable spidroin N-terminal domain (NT) forms hydrogels when incubated at 37°C, and that gel formation is associated with a conversion of NT into amyloid-like fibrils. The high structural flexibility of NT combined with the presence of amyloidogenic sequences in its a-helices are factors that are important for formation of the gel. Furthermore, by fusing NT to target proteins, we present a novel immobilization platform in which NT is used both as an expression tag for high yield production of soluble fusion proteins and as a fibrillar scaffold in the gel. As hydrogel formation occurred rapidly and under benign conditions also for NT2RepCT, Paper IV focuses on the potential application of NT2RepCT hydrogels as a drug release device and for cell encapsulation. Successful encapsulation and release of active green fluorescent protein suggest that the hydrogels could be suitable candidates for use as drug release devices. An encapsulated cell line released the bioactive molecule progranulin for 31 days to a similar extent as cells cultured under standard conditions. Human mesenchymal stem cells encapsulated in the hydrogels showed high survival but limited proliferation, likely due to restricted space in the dense fibrillar network that is characteristic of the gels. This thesis describes major steps forward in the development of novel spidroin-based materials.
List of papers:
I. Native-like flow properties of an artificial spider silk dope. Arndt, T., Laity, P. R., Johansson, J., Holland, C., Rising, A. ACS Biomaterials Science & Engineering. (2021);7(2), 462-471.
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II. Engineered Spider Silk Proteins for Biomimetic Spinning of Fibers with Toughness Equal to Dragline Silks. Arndt, T., Greco, G., Schmuck, B., Bunz, J., Shilkova, O., Francis, J., Pugno, N. M., Jaudzems, K., Barth, A., Johansson, J., Rising, A. Advanced Functional Materials. (2022);2200986.
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III. Spidroin N-terminal domain forms amyloid-like fibril based hydrogels and provides a protein immobilization platform. Arndt, T., Jaudzems, K., Shilkova, O., Francis, J., Johansson, M., Laity, P. R., Sahin, C., Chatterjee, U., Kronqvist, N., Barajas-Ledesma, E., Kumar, R., Chen, G., Strömberg, R., Abelein, A., Langton, M., Landreh, M., Barth, A., Holland, C., Johansson, J., Rising, A. Nature communications. (2022);13(1), 1-14.
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IV. Tuneable recombinant spider silk protein hydrogels for drug release and 3D cell culture. Arndt, T.*, Chatterjee, U.*, Shilkova, O., Francis, J., Lundkvist, J., Johansson, D., Schmuck, B. , Ekblad Nordberg, Å., Altskär, A., Lorén, N., Li, Y., Wahlberg L. U., Langton M., Johansson, J., Götherström, C., Rising, A. *These authors contributed equally to this work. [Manuscript]
I. Native-like flow properties of an artificial spider silk dope. Arndt, T., Laity, P. R., Johansson, J., Holland, C., Rising, A. ACS Biomaterials Science & Engineering. (2021);7(2), 462-471.
Fulltext (DOI)
Pubmed
View record in Web of Science®
II. Engineered Spider Silk Proteins for Biomimetic Spinning of Fibers with Toughness Equal to Dragline Silks. Arndt, T., Greco, G., Schmuck, B., Bunz, J., Shilkova, O., Francis, J., Pugno, N. M., Jaudzems, K., Barth, A., Johansson, J., Rising, A. Advanced Functional Materials. (2022);2200986.
Fulltext (DOI)
Pubmed
View record in Web of Science®
III. Spidroin N-terminal domain forms amyloid-like fibril based hydrogels and provides a protein immobilization platform. Arndt, T., Jaudzems, K., Shilkova, O., Francis, J., Johansson, M., Laity, P. R., Sahin, C., Chatterjee, U., Kronqvist, N., Barajas-Ledesma, E., Kumar, R., Chen, G., Strömberg, R., Abelein, A., Langton, M., Landreh, M., Barth, A., Holland, C., Johansson, J., Rising, A. Nature communications. (2022);13(1), 1-14.
Fulltext (DOI)
Pubmed
View record in Web of Science®
IV. Tuneable recombinant spider silk protein hydrogels for drug release and 3D cell culture. Arndt, T.*, Chatterjee, U.*, Shilkova, O., Francis, J., Lundkvist, J., Johansson, D., Schmuck, B. , Ekblad Nordberg, Å., Altskär, A., Lorén, N., Li, Y., Wahlberg L. U., Langton M., Johansson, J., Götherström, C., Rising, A. *These authors contributed equally to this work. [Manuscript]
Institution: Karolinska Institutet
Supervisor: Rising, Anna
Co-supervisor: Johansson, Jan
Issue date: 2022-11-18
Rights:
Publication year: 2022
ISBN: 978-91-8016-831-1
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