Identification of Wet-Spinning and Post-Spin Stretching Methods Amenable to Recombinant Spider Aciniform Silk

Abstract

Spider silks are outstanding biomaterials with mechanical properties that outperform synthetic materials. Of the six fibrillar spider silks, aciniform (or wrapping) silk is the toughest through a unique combination of strength and extensibility. In this study, a wet-spinning method for recombinant Argiope trifasciata aciniform spidroin (AcSp1) is introduced. Recombinant AcSp1 comprising three 200 amino acid repeat units was solubilized in a 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP)/water mixture, forming a viscous α-helix-enriched spinning dope, and wet-spun into an ethanol/water coagulation bath allowing continuous fiber production. Post-spin stretching of the resulting wet-spun fibers in water significantly improved fiber strength, enriched β-sheet conformation without complete α-helix depletion, and enhanced birefringence. These methods allow reproducible aciniform silk fiber formation, albeit with lower extensibility than native silk, requiring conditions and methods distinct from those previously reported for other silk proteins. This provides an essential starting point for tailoring wet-spinning of aciniform silk to achieve desired properties. Graphical Abstract*

Recombinant Silk Fiber Properties Correlate to Prefibrillar Self‐Assembly

“…W 3 protein secondary structuring in each dope solution was first evaluated by far‐UV circular dichroism (CD) spectroscopy ( Figure ). The W 3 protein in the TFA/TFE spinning dope exhibited a positive band at ≈193 nm and negative bands at ≈208 and 222 nm consistent with significant α‐helical content, as previously observed in 50 × 10 −3 m potassium phosphate at pH 7.5, 20 × 10 −3 m sodium acetate at pH5.0, HFIP spinning dope, and native aciniform gland . This is also consistent with the typical α‐helix stabilization behavior of fluorinated alcohol solvents, although the significant TFA content of the spinning dope is certainly a distinction relative to the fluorinated solvent/water mixtures that have been characterized in depth.…”

“…This conclusion is also supported by the characteristics of the observed Raman amide III signal (Figure S3, Supporting Information). Such post‐spin stretching‐induced structural conversion and alignment has also been observed in our previous studies employing W 3 in HFIP dope and with other protein fibers and is suggested to promote greater mechanical strength, coupling with the improved mechanical properties observed upon post‐spin stretching.…”

“…HFIP AS fiber surfaces certainly exhibit globular structures, but without alignment to form a clear fibrillar surface morphology perpendicular to the long‐axis at the sub‐µm scale. Instead, multi‐µm‐scale fibrous units with grooves separating them (Figure , larger in scale in our previous study) are observed running parallel to the long‐axis.…”

“…A TFA/TFE/H 2 O solvent mixture (in a ratio of 3/1/1 v/v/v; denoted as TFA/TFE dope, herein) solubilizes W 3 instantly at a high concentration (10%, w/v). The resulting spinning dope is amenable to continuous wet‐spinning of silk fibers (denoted TFA/TFE fibers herein) using the method we previously introduced with an HFIP/H 2 O (7/3 v/v)‐based spinning dope (denoted as HFIP dope and fibers, as appropriate, herein) . Strikingly, the strength, extensibility, toughness, and Young's modulus for the “as‐spun” (AS) fibers collected by spooling of the W 3 silk directly following passage through a 95% ethanol coagulation bath were all dramatically improved relative to those of HFIP AS fibers ( Table 1 and Figure ).…”

“…We previously reported wet‐spinning of fibers from a dope of W 3 dissolved in a 1,1,1,3,3,3‐hexafluoro‐2‐propanol/H 2 O (HFIP/H 2 O) solvent mixture. Unfortunately, the resulting silk fibers exhibited a low extensibility and, correspondingly, low toughness . Here, we introduce a solvent mixture of trifluoroacetic acid/trifluoroethanol/H 2 O (TFA/TFE/H 2 O) also capable of solubilizing W 3 into a spinning dope and compare the resulting silk fiber ultrastructure, secondary structuring, and mechanical behavior, all of which are improved relative to fibers formed from the HFIP/H 2 O dope.…”