optimizing-evaluating-hand-drawing-wet-spinning-for-recombinant

Optimizing and Evaluating Hand-drawing and Wet-spinning for Recombinant Spider Silk Fiber Production

Spider silks are renowned for their mechanical properties, with hallmark high strength, high extensibility, or a combination of these leading to high ...

Dalhousie University

Proteins are biomolecules with potential applications in agriculture, food sciences, pharmaceutics, biotechnology, and drug delivery. Interactions of hydrophilic and biocompatible polymers with proteins may impart proteolytic stability, improving the therapeutic effects of biomolecules and also acting as excipients for the prolonged storage of proteins under harsh conditions. The interactions of hydrophilic and stealth polymers such as poly(ethylene glycol), poly(trehalose), and zwitterionic polymers with various proteins are well studied. This study evaluates the molecular interactions of hydrophilic and optically active poly(vitamin B5 analogous methacrylamide) (poly(B5AMA)) with model proteins by fluorescence spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and circular dichroism (CD) spectroscopy analysis. The optically active hydrophilic polymers prepared using chiral monomers of -(+)- and -(-)-B5AMA by the photo-iniferter reversible addition fragmentation chain transfer (RAFT) polymerization showed concentration-dependent weak interactions of the polymers with bovine serum albumin and lysozyme proteins. Poly(B5AMA) also exhibited a concentration-dependent protein stabilizing effect at elevated temperatures, and no effect of the stereoisomers of polymers on protein thermal stability was observed. NMR analysis, however, showed poly(B5AMA) stereoisomer-dependent changes in the secondary structure of proteins.

Elucidating the Role of Optical Activity of Polymers in Protein-Polymer Interactions.

Spider silks are natural protein-based biomaterials which are renowned for their mechanical properties and hold great promise for applications ranging from high-performance textiles to regenerative medicine. While some spiders can produce several different types of silks, most spider silk types - including pyriform and aciniform silks - are relatively unstudied. Pyriform and aciniform silks have distinct mechanical behavior and physicochemical properties, with materials produced using combinations of these silks currently unexplored. Here, we introduce an engineered chimeric fusion protein consisting of two repeat units of pyriform (Py) silk followed by two repeat units of aciniform (W) silk named PyW. This recombinant ∼86.5 kDa protein is amenable to expression and purification from  and exhibits high α-helicity in a fluorinated acid- and alcohol-based solution used to form a dope for wet-spinning. Wet-spinning enables continuous fiber production and post-spin stretching of the wet-spun fibers in air or following submersion in water or ethanol leads to increases in optical anisotropy, consistent with increased molecular alignment along the fiber axis. Mechanical properties of the fibers vary as a function of post-spin stretching condition, with the highest extensibility and strength observed in air-stretched and ethanol-treated fibers, respectively, with mechanics being superior to fibers spun from either constituent protein alone. Notably, the maximum extensibility obtained (∼157 ± 38 %) is of the same magnitude reported for natural flagelliform silks, the class of spider silk most associated with being stretchable. Interestingly, PyW is also water-compatible, unlike its constituent Py. Fiber-state secondary structure correlates well with the observed mechanical properties, with depleted α-helicity and increased β-sheet content in cases of increased strength. PyW fibers thus provide enhanced materials behavior in terms of their mechanics, tunability, and fiber properties, providing new directions for design and development of biomaterials suitable and tunable for disparate applications.

A recombinant chimeric spider pyriform-aciniform silk with highly tunable mechanical performance.

Lactam cross-links have been employed to stabilize the helical secondary structure and enhance the activity and physiological stability of antimicrobial peptides; however, stabilization of β-sheets via lactamization has not been observed. In the present study, lactams between the side chains of C- and N-terminal residues have been used to stabilize the β-sheet conformation in a short ten-residue analogue of chicken angiogenin-4. Designed using a combination of molecular dynamics simulations and Markov state models, the lactam cross-linked peptides are shown to adopt stabilized β-sheet conformations consistent with simulated structures. Replacement of the peptide side-chain Cys-Cys disulfide by a lactam cross-link enhanced the broad-spectrum antibacterial activity compared to the parent peptide and exhibited greater propensity to induce proinflammatory activity in macrophages. The combination of molecular simulations and conformational and biological analyses of the synthetic peptides provides a useful paradigm for the rational design of therapeutically active peptides with constrained β-sheet structures.

Secondary Structure Stabilization of Macrocyclic Antimicrobial Peptides via Cross-Link Swapping.

Spider silk biomaterials have generated significant interest due to their high strength, biocompatibility, and biodegradability. However, the complex multiscale structure of silk fibers creates difficulties in investigating the relationship between the structure and mechanical properties in silks. Previous work has shown that silks at the focus of an ultrafast laser produce a significant second harmonic generation (SHG) signal. This presents an exciting opportunity since polarization-resolved SHG microscopy (PSHG) is a technique that has shown high sensitivity local molecular structure and organization in several biological samples. However, applications of PSHG to silks have been impeded by the lack of a theoretical model relating silk molecular structure and organization to its PSHG response. Here, a theoretical model of PSHG from silk materials is presented, which relates β-sheet organization within silk fibers to experimentally measurable parameters. Based on this, we present evidence that postspin stretching in ethanol induces planar alignment of the β-sheets within recombinant spider silk fibers, and the molecular structure and degree of axial alignment of β-sheets are highly dependent on the level of postspin stretching. Overall, this work demonstrates the significant potential for the application of PSHG to map the local structure of silk fibers, providing opportunities for the investigation of silk-based biomaterials.

Article	Year
Elucidating the Role of Optical Activity of Polymers in Protein-Polymer Interactions. Polymers\| PubMed ID: 38201730	2023
A recombinant chimeric spider pyriform-aciniform silk with highly tunable mechanical performance. Materials today. Bio\| PubMed ID: 38711935	2024
Secondary Structure Stabilization of Macrocyclic Antimicrobial Peptides via Cross-Link Swapping. Journal of medicinal chemistry\| PubMed ID: 38771638	2024
Polarization-Resolved Second Harmonic Generation Microscopy of Silk Fibers Is Sensitive to β-Sheet Orientation and Molecular Structure. ACS applied bio materials\| PubMed ID: 40432206	2025

Anupama Ghimire

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