Recombinant Spider Silk Protein and Delignified Wood Form a Strong Adhesive System

Lemetti, Laura; Tersteegen, Jennifer; Sammaljärvi, Juuso; Aranko, A. Sesilja

doi:10.1021/acssuschemeng.1c07043

Cited by 17 publications

(20 citation statements)

References 212 publications

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“…We first studied cross-reactivity with model proteins and no cross-reactivity between the SpyCather/Tag and SilkCather/Tag pairs was observed (Figure S4). Next, we constructed fusion proteins of the ADF3 repeat sequence [43] with the SilkCather and a cellulose binding module (CBM) from the Clostridium thermocellum cellulosome [44] Fusion with CBM improves solubility of the silk protein and allows utilizing it as a composite material with cellulose as well as an adhesive on delignified cellulose [45,46] The fusion protein could be expressed high yields and purified by heat precipitation at 70°C (Figure S5). Attempt to use SnoopCatcher as a reference showed that the combination with ADF3 was not soluble and was therefore not pursued further.…”

Section: Ligation Of Spider Silk Proteinsmentioning

confidence: 99%

Engineering a minimal SilkCatcher/Tag pair compatible with SpyCatcher/Tag pair for the production of native-sized spider silk

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Hakanpää

Elfving³

et al. 2022

Preprint

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Protein/peptide pairs, called Catcher/Tag pairs, are applied for biological isopeptide-bond mediated Click-reactions. Covalent protein ligation using Catcher/Tag pairs has turned out to be a valuable tool in biotechnology and biomedicines. It is essential to increase the current toolbox of Catcher/Tag pairs to expand the range of applications further, e.g., for multiple-fragment ligation that requires several orthogonal ligases. We report here engineering of novel Catcher/Tag pairs for protein ligation, aided by a new crystal structure of a minimal CnaB domain fromLactobacillus plantarum. We engineer several split variants, characterize in detail one of them, named SilkCatcher/Tag pair, and show that the newly engineered SilkCatcher/Tag pair is orthogonal and compatible with the widely used SpyCatcher/Tag pair. Finally, we demonstrate the use of the new SilkCatcher/Tag pair in the production of native-sized highly repetitive spider-silk-like proteins with >90% purity, which is not possible with the traditional recombinant production.

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Section: Ligation Of Spider Silk Proteinsmentioning

confidence: 99%

Engineering a minimal SilkCatcher/Tag pair compatible with SpyCatcher/Tag pair for the production of native-sized spider silk

Fan¹,

Hakanpää

Elfving³

et al. 2022

Preprint

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“…Liquid–liquid phase separation (LLPS) known as coacervation is involved in the biogenesis of many extracellular materials, including various silks 1 – 3 , wings of butterfly 4 , squid beak 5 , and marine adhesives 6 – 8 . Marine mussels make their adhesive byssus by secreting a family of mussel foot proteins (Mfps) with a microfluidic-like fabrication process using an organ called the mussel foot 9 , 10 .…”

Section: Introductionmentioning

confidence: 99%

Hydrogen-bonds mediate liquid-liquid phase separation of mussel derived adhesive peptides

et al. 2022

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Marine mussels achieve strong underwater adhesion by depositing mussel foot proteins (Mfps) that form coacervates during the protein secretion. However, the molecular mechanisms that govern the phase separation behaviors of the Mfps are still not fully understood. Here, we report that GK-16*, a peptide derived from the primary adhesive protein Mfp-5, forms coacervate in seawater conditions. Molecular dynamics simulations combined with point mutation experiments demonstrate that Dopa- and Gly- mediated hydrogen-bonding interactions are essential in the coacervation process. The properties of GK-16* coacervates could be controlled by tuning the strength of the electrostatic and Dopa-mediated hydrogen bond interactions via controlling the pH and salt concentration of the solution. The GK-16* coacervate undergoes a pH induced liquid-to-gel transition, which can be utilized for the underwater delivery and curing of the adhesives. Our study provides useful molecular design principles for the development of mussel-inspired peptidyl coacervate adhesives with tunable properties.

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“… 6 , 17 We found coacervation to be an essential intermediate step for the fiber formation of the silk-like molecule 6 and that the coacervated solution could be used as an adhesive. 11 , 12 The combination of computational and experimental data indicated that both the weak dimerization of the terminal CBM domains 13 and weak interactions mediated by “sticker” regions in the repetitive middle part affect the assembly. 14 The protein studied was 85 kDa in size, which is approximately only a third of the size of the native spider silks that have a size between 250 and 350 kDa.…”

Section: Introductionmentioning

confidence: 99%

“…We have previously reported and characterized the assembly of engineered recombinant silk-like molecules consisting of an engineered spider silk-repeat sequence (eADF3) flanked by two cellulose-binding modules (CBMs), called CBM-eADF3-CBM. , We found coacervation to be an essential intermediate step for the fiber formation of the silk-like molecule and that the coacervated solution could be used as an adhesive. , The combination of computational and experimental data indicated that both the weak dimerization of the terminal CBM domains and weak interactions mediated by “sticker” regions in the repetitive middle part affect the assembly . The protein studied was 85 kDa in size, which is approximately only a third of the size of the native spider silks that have a size between 250 and 350 kDa. , This prompted us to ask how extending the size close to that of the native spider silks would affect the assembly and phase transitions of the silk-mimicking proteins.…”

Section: Introductionmentioning

confidence: 99%

Liquid–Liquid Phase Separation and Assembly of Silk-like Proteins is Dependent on the Polymer Length

et al. 2022

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Phase transitions have an essential role in the assembly of nature’s protein-based materials into hierarchically organized structures, yet many of the underlying mechanisms and interactions remain to be resolved. A central question for designing proteins for materials is how the protein architecture and sequence affects the nature of the phase transitions and resulting assembly. In this work, we produced 82 kDa (1×), 143 kDa (2×), and 204 kDa (3×) silk-mimicking proteins by taking advantage of protein ligation by SpyCatcher/Tag protein-peptide pair. We show that the three silk proteins all undergo a phase transition from homogeneous solution to assembly formation. In the assembly phase, a length- and concentration-dependent transition between two distinct assembly morphologies, one forming aggregates and another coacervates, exists. The coacervates showed properties that were dependent on the protein size. Computational modeling of the proteins by a bead-spring model supports the experimental results and provides us a possible mechanistic origin for the assembly transitions based on architectures and interactions.

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Recombinant Spider Silk Protein and Delignified Wood Form a Strong Adhesive System

Cited by 17 publications

References 212 publications

Engineering a minimal SilkCatcher/Tag pair compatible with SpyCatcher/Tag pair for the production of native-sized spider silk

Engineering a minimal SilkCatcher/Tag pair compatible with SpyCatcher/Tag pair for the production of native-sized spider silk

Hydrogen-bonds mediate liquid-liquid phase separation of mussel derived adhesive peptides

Liquid–Liquid Phase Separation and Assembly of Silk-like Proteins is Dependent on the Polymer Length

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