Engineered Large Spider Eggcase Silk Protein for Strong Artificial Fibers

Abstract

Spiders employ different combinations of silk proteins to produce up to seven types of silks with distinct mechanical properties for various purposes, ranging from prey capture to offspring protection in egg cases. [ 1 ] Generally, spider silks are semicrystalline protein polymers that contain both crystalline and amorphous regions. [ 2 ] Silk fi bers have attracted the attention of the popular media because of their extraordinary mechanical properties which are unusual in comparison with synthetic non-protein-based fi bers. [ 3 ] Silk biomaterials have been developed for various biomedical and industrial applications, such as sutures for micro surgery, scaffolds for tissue engineering, silk particles for drug delivery, and other materials which require strength, elasticity, biodegradability and biocompatibility. [ 4 ] However, farming of spiders is limited by their highly cannibalistic and territorial behavior. [ 5 ] Consequently, spinning artifi cial silk from spider silk proteins that are produced by recombinant biotechnology is one of the most promising alternatives. [ 6 ] Studies on spider silk genes have demonstrated that silk proteins consist of multiple repetitive domains and non-repetitive terminal domains and have very high molecular weights (MWs), ranging from ∼ 250 to ∼ 366 kDa, [ 7 ] implying that the size of silk proteins is a key determinant of the silk's outstanding mechanical properties. In order to biomimic native spider silks, the recombinant proteins used to spin artifi cial silks should be similar to their native counterparts in size and composition. Due to the difficulties in producing high MW silk proteins in large quantity, attempts to spin artifi cial silks have mainly focused on smallto medium-sized recombinant proteins with simplifi ed amino acid (AA) sequences and MWs of < 120 kDa, which always result in fi bers with much lower tenacity compared to their corresponding natural silks. [ 6a , 8 ] Recently a large silk-like protein ( ∼ 285 kDa), which is similar to the repetitive region of a dragline silk protein in size but different in sequence and composition, was produced in high yield in metabolically engineered Escherichia coli . [ 9 ] The artifi cial fi ber spun from this protein had a tenacity of 508 ± 108 MPa, which is still much lower than its native form's strength of 1215 ± 233 MPa measured from the same spider species. [ 10 ] To overcome the problems in cloning and expression of native-sized spider silk proteins, an inteinbased protein fusion method has been proposed very recently for producing multiple repetitive domains of silk proteins in plant systems. [ 11 ] With this method, a mixture of multimers ranging from two to ten domains was generated, but homogeneous large protein could not be obtained. Similarly, such non-uniform multimers can be synthesized by introducing two cysteine residues into one silk protein fragment and then by chemically linking one cysteine in one domain with one cysteine in another domain. [ 12 ] It was found that the fi bers containing the multim...

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

“…This was also accompanied by a greater tenacity for the recombinant fiber, which may be due to the relatively large size of recombinant TuSp1 protein construct (~378 kDa) that was employed. Perhaps of note, the TuSp1 repetitive domain is more similar in architecture to that of AcSp1 than the other spidroins, with repeats of ~180 amino acids; however, TuSp1 is distinctive in having both a low Gly content and disparate mechanical properties 59,68 . The tertiary structure of the TuSp1 repeat unit in solution is also distinct from that of the W unit, in that it is a tightly packed orthogonal 6-helix bundle 69 .…”

“…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 .…”

“…Interestingly, Lin et al demonstrated that wet-spun recombinant tubuliform spidroin 1 (TuSp1) exhibited a much lower extensibility than the native counterpart 59 . This was also accompanied by a greater tenacity for the recombinant fiber, which may be due to the relatively large size of recombinant TuSp1 protein construct (~378 kDa) that was employed.…”

Silk Spinning in Silkworms and Spiders

“…Expression of highly repetitive sequences such as the MaSp repetitive region is difficult in heterologous hosts, and therefore most constructs have been considerably smaller than the native spidroins, often including only a small part of the repetitive region and lacking one or both of the terminal domains. The water solubility levels of such constructs have still been very low (around 1% w / v [101,102]), and recombinant spidroins have therefore been dissolved in denaturing agents (reaching solubility levels of 5%–25% w / v [103,104,105]), after which spinning into tough fibers has proven difficult. In the same way that regenerated (denatured) silkworm silk cannot form fibers that are similar to native silk, it is highly unlikely that artificial fibers spun from denatured recombinant spidroins can capture the true structure and toughness of native spider silk.…”

Development of New Smart Materials and Spinning Systems Inspired by Natural Silks and Their Applications

“…At least three different proteins have been found in tubuliform silk: tubuliform spidroin 1 (TuSp1), egg case protein 1 and 2 (ECP-1 and ECP-2). Tutuliform silks is the second most studied spider fiber following the dragline silk and the primary sequence and structure of tobuliform silk proteins have been clearly demonstrated (Lin et al, 2009(Lin et al, , 2013Gnesa et al, 2012). Lin et al (2013) produced TuSp1 consisting of both repetitive and conserved terminal domains using recombinant DNA technology, and the artificial fibers spun from the recombinant TuSp1 showed outstanding mechanical properties.…”