| 1. |
- Arndt, Tina
(författare)
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Biomimetic spider silk and bioactive hydrogels formed by engineered recombinant spider silk proteins
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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.
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| 2. |
- Arndt, Tina, et al.
(författare)
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Engineered Spider Silk Proteins for Biomimetic Spinning of Fibers with Toughness Equal to Dragline Silks
- 2022
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Ingår i: Advanced Functional Materials. - : Wiley. - 1616-301X .- 1616-3028. ; 32:23
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Tidskriftsartikel (refereegranskat)abstract
- Spider silk is the toughest fiber found in nature, and bulk production of artificial spider silk that matches its mechanical properties remains elusive. Development of miniature spider silk proteins (mini-spidroins) has made large-scale fiber production economically feasible, but the fibers’ mechanical properties are inferior to native silk. The spider silk fiber's tensile strength is conferred by poly-alanine stretches that are zipped together by tight side chain packing in β-sheet crystals. Spidroins are secreted so they must be void of long stretches of hydrophobic residues, since such segments get inserted into the endoplasmic reticulum membrane. At the same time, hydrophobic residues have high β-strand propensity and can mediate tight inter-β-sheet interactions, features that are attractive for generation of strong artificial silks. Protein production in prokaryotes can circumvent biological laws that spiders, being eukaryotic organisms, must obey, and the authors thus design mini-spidroins that are predicted to more avidly form stronger β-sheets than the wildtype protein. Biomimetic spinning of the engineered mini-spidroins indeed results in fibers with increased tensile strength and two fiber types display toughness equal to native dragline silks. Bioreactor expression and purification result in a protein yield of ≈9 g L−1 which is in line with requirements for economically feasible bulk scale production.
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| 3. |
- Arndt, Tina, et al.
(författare)
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Spidroin N-terminal domain forms amyloid-like fibril based hydrogels and provides a protein immobilization platform
- 2022
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- Recombinant spider silk proteins (spidroins) have multiple potential applications in development of novel biomaterials, but their multimodal and aggregation-prone nature have complicated production and straightforward applications. Here, we report that recombinant miniature spidroins, and importantly also the N-terminal domain (NT) on its own, rapidly form self-supporting and transparent hydrogels at 37 °C. The gelation is caused by NT α-helix to β-sheet conversion and formation of amyloid-like fibrils, and fusion proteins composed of NT and green fluorescent protein or purine nucleoside phosphorylase form hydrogels with intact functions of the fusion moieties. Our findings demonstrate that recombinant NT and fusion proteins give high expression yields and bestow attractive properties to hydrogels, e.g., transparency, cross-linker free gelation and straightforward immobilization of active proteins at high density.
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| 4. |
- Greco, Gabriele, et al.
(författare)
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Properties of Biomimetic Artificial Spider Silk Fibers Tuned by PostSpin Bath Incubation
- 2020
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Ingår i: Molecules. - : MDPI AG. - 1431-5157 .- 1420-3049. ; 25:14
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Tidskriftsartikel (refereegranskat)abstract
- Efficient production of artificial spider silk fibers with properties that match its natural counterpart has still not been achieved. Recently, a biomimetic process for spinning recombinant spider silk proteins (spidroins) was presented, in which important molecular mechanisms involved in native spider silk spinning were recapitulated. However, drawbacks of these fibers included inferior mechanical properties and problems with low resistance to aqueous environments. In this work, we show that >= 5 h incubation of the fibers, in a collection bath of 500 mM NaAc and 200 mM NaCl, at pH 5 results in fibers that do not dissolve in water or phosphate buffered saline, which implies that the fibers can be used for applications that involve wet/humid conditions. Furthermore, incubation in the collection bath improved the strain at break and was associated with increased beta-sheet content, but did not affect the fiber morphology. In summary, we present a simple way to improve artificial spider silk fiber strain at break and resistance to aqueous solvents.
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| 5. |
- Greco, Gabriele, et al.
(författare)
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Tyrosine residues mediate supercontraction in biomimetic spider silk
- 2021
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Ingår i: Communications materials. - : Springer Science and Business Media LLC. - 2662-4443. ; 2:1
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Tidskriftsartikel (refereegranskat)abstract
- Exposing spider silk to wet conditions can cause supercontraction. Here, tyrosine amino acid residues within the amorphous regions are found to contribute to supercontraction, which can be controlled by protein engineering. Water and humidity severely affect the material properties of spider major ampullate silk, causing the fiber to become plasticized, contract, swell and undergo torsion. Several amino acid residue types have been proposed to be involved in this process, but the complex composition of the native fiber complicates detailed investigations. Here, we observe supercontraction in biomimetically produced artificial spider silk fibers composed of defined proteins. We found experimental evidence that proline is not the sole residue responsible for supercontraction and that tyrosine residues in the amorphous regions of the silk fiber play an important role. Furthermore, we show that the response of artificial silk fibers to humidity can be tuned, which is important for the development of materials for applications in wet environments, eg producing water resistant fibers with maximal strain at break and toughness modulus.
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| 6. |
- Hansson, Magnus L., et al.
(författare)
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Artificial spider silk supports and guides neurite extension in vitro
- 2021
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Ingår i: The FASEB Journal. - : John Wiley & Sons. - 0892-6638 .- 1530-6860. ; 35:11
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Tidskriftsartikel (refereegranskat)abstract
- Surgical intervention with the use of autografts is considered the gold standard to treat peripheral nerve injuries. However, a biomaterial that supports and guides nerve growth would be an attractive alternative to overcome problems with limited availability, morbidity at the site of harvest, and nerve mismatches related to autografts. Native spider silk is a promising material for construction of nerve guidance conduit (NGC), as it enables regeneration of cm-long nerve injuries in sheep, but regulatory requirements for medical devices demand synthetic materials. Here, we use a recombinant spider silk protein (NT2RepCT) and a functionalized variant carrying a peptide derived from vitronectin (VN-NT2RepCT) as substrates for nerve growth support and neurite extension, using a dorsal root ganglion cell line, ND7/23. Two-dimensional coatings were benchmarked against poly-d-lysine and recombinant laminins. Both spider silk coatings performed as the control substrates with regards to proliferation, survival, and neurite growth. Furthermore, NT2RepCT and VN-NT2RepCT spun into continuous fibers in a biomimetic spinning set-up support cell survival, neurite growth, and guidance to an even larger extent than native spider silk. Thus, artificial spider silk is a promising biomaterial for development of NGCs.
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| 7. |
- Jafari, Mohammad Javad, et al.
(författare)
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Force-Induced Structural Changes in Spider Silk Fibers Introduced by ATR-FTIR Spectroscopy
- 2023
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Ingår i: ACS applied polymer materials. - : American Chemical Society. - 2637-6105. ; 5:11, s. 9433-9444
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Tidskriftsartikel (refereegranskat)abstract
- Silk fibers have unique mechanical properties, and many studies of silk aim at understanding how these properties are related to secondary structure content, which often is determined by infrared spectroscopy. We report significant method-induced irreversible structural changes to both natural and synthetic spider silk fibers, derived from the widely used attenuated total reflection Fourier-transform infrared (ATR-FTIR) technique. By varying the force used to bring fibers into contact with the internal reflection elements of ATR-FTIR accessories, we observed correlated and largely irreversible changes in the secondary structure, with shape relaxation under pressure occurring within minutes. Fitting of spectral components shows that these changes agree with transformations from the alpha-helix to the beta-sheet secondary structure with possible contributions from other secondary structure elements. We further confirm the findings with IR microspectroscopy, where similar differences were seen between the pressed and unaffected regions of spider silk fibers. Our findings show that ATR-FTIR spectroscopy requires care in its use and in the interpretation of the results.
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| 8. |
- Leppert, Axel, et al.
(författare)
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Liquid-Liquid Phase Separation Primes Spider Silk Proteins for Fiber Formation via a Conditional Sticker Domain
- 2023
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Ingår i: Nano Letters. - : American Chemical Society (ACS). - 1530-6984 .- 1530-6992. ; 23:12, s. 5836-5841
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Many protein condensates can convert to fibrillar aggregates, but the underlying mechanisms are unclear. Liquid-liquid phase separation (LLPS) of spider silk proteins, spidroins, suggests a regulatory switch between both states. Here, we combine microscopy and native mass spectrometry to investigate the influence of protein sequence, ions, and regulatory domains on spidroin LLPS. We find that salting out-effects drive LLPS via low-affinity stickers in the repeat domains. Interestingly, conditions that enable LLPS simultaneously cause dissociation of the dimeric C-terminal domain (CTD), priming it for aggregation. Since the CTD enhances LLPS of spidroins but is also required for their conversion into amyloid-like fibers, we expand the stickers and spacers-model of phase separation with the concept of folded domains as conditional stickers that represent regulatory units.
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| 9. |
- Sahin, Cagla, et al.
(författare)
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Mass Spectrometry of RNA-Binding Proteins during Liquid-Liquid Phase Separation Reveals Distinct Assembly Mechanisms and Droplet Architectures
- 2023
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 145:19, s. 10659-10668
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Tidskriftsartikel (refereegranskat)abstract
- Liquid-liquid phase separation (LLPS) of hetero-geneous ribonucleoproteins (hnRNPs) drives the formation of membraneless organelles, but structural information about their assembled states is still lacking. Here, we address this challenge through a combination of protein engineering, native ion mobility mass spectrometry, and molecular dynamics simulations. We used an LLPS-compatible spider silk domain and pH changes to control the self-assembly of the hnRNPs FUS, TDP-43, and hCPEB3, which are implicated in neurodegeneration, cancer, and memory storage. By releasing the proteins inside the mass spectrometer from their native assemblies, we could monitor conformational changes associated with liquid-liquid phase separation. We find that FUS monomers undergo an unfolded-to-globular transition, whereas TDP-43 oligomerizes into partially disordered dimers and trimers. hCPEB3, on the other hand, remains fully disordered with a preference for fibrillar aggregation over LLPS. The divergent assembly mechanisms revealed by ion mobility mass spectrometry of soluble protein species that exist under LLPS conditions suggest structurally distinct complexes inside liquid droplets that may impact RNA processing and translation depending on biological context.
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