More Than Just Fibers: An Aqueous Method for the Production of Innovative Recombinant Spider Silk Protein Materials

Abstract

Spider silk is a striking and robust natural material that has an unrivaled combination of strength and elasticity. There are two major problems in creating materials from recombinant spider silk proteins (rSSps): expressing sufficient quantities of the large, highly repetitive proteins and solvating the naturally self-assembling proteins once produced. To address the second problem, we have developed a method to rapidly dissolve rSSps in water in lieu of traditional organic solvents and accomplish nearly 100% solvation and recovery of the protein. Our method involves generating pressure and temperature in a sealed vial by using short, repetitive bursts from a conventional microwave. The method is scalable and has been successful with all rSSps used to date. From these easily generated aqueous solutions of rSSps, a wide variety of materials have been produced. Production of fibers, films, hydrogels, lyogels, sponges, and adhesives and studies of their mechanical and structural properties are reported. To our knowledge, ours is the only method that is cost-effective and scalable for mass production. This solvation method allows a choice of the physical form of product to take advantage of spider silks' mechanical properties without using costly and problematic organic solvents.

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

“…Most studies characterizing post-spin stretching of silk-based fibers have reported carrying out this step manually with the use of forceps or tweezers 9,11,12,42 , with some reports of using controlled post-spin stretching apparatuses (e.g. 16,44 ). An apparatus can certainly be envisioned that incorporates the post-spin stretching as part of an automated fiber production process 16,44 .…”

“…16,44 ). An apparatus can certainly be envisioned that incorporates the post-spin stretching as part of an automated fiber production process 16,44 . However, this requires knowledge of the optimal degree of post-spin stretching.…”

“…Additionally, the tensile properties of these fibers fall within the range of previously reported wet-spun fibers from recombinant spidroin solubilized in HFIP. For other types of spidroin, strength and extensibility have ranged between 7-36 MPa and 1-5% for AS fibers 9,11,12,42,44,59 , and, 14-508 MPa and 3-307% for PS (3-5x) fibers 9-12, 42-44, 59 . They are also consistent with reports of post-spin stretching non-silk-based protein fibers in water, specifically native 65 and regenerated 66 hagfish slime threads as well as recombinant vimentin fibers 67 .…”

Importance of Heat and Pressure for Solubilization of Recombinant Spider Silk Proteins in Aqueous Solution

“…While these production systems can produce soluble forms of the rSSPs, high level expression can induce precipitation as well, as during the purification process, as the rSSPs becomes less dilute and more pure, the rSSPs both spontaneously self-assemble and precipitate or, are salted out of solution as part of that process [25,29,30]. The purified rSSPs can be lyophilized for storage, generally further reducing the aqueous solubility [29,31]. Necessary to forming fibers, and other materials, is that the rSSPs must be dissolved.…”

“…The loss of the web and energy required to rebuild it would be an evolutionary disadvantage. As such, when rSSPs are produced from these same sequences and precipitated, β-sheets form much like in the fiber and the rSSPs are largely water insoluble [31]. Due to this solubility issue, the majority of rSSPs have been solubilized using 1,1,1,3,3,3 Hexafluoroisopropanol (HFIP) [25,26,27,28,29,32,33].…”