Production and Properties of Triple Chimeric Spidroins

Abstract

All spider silk proteins (spidroins) are composed of N- and C-terminal domains (NT and CT) that act as regulators of silk solubility and assembly and a central repetitive region, which confers mechanical properties to the fiber. Among the seven types of spider silks, aciniform silk has the highest toughness. Herein, we fused NT and CT domains from major and minor ampullate spidroins (MaSps and MiSps), respectively, to 1-4 repeat domains (W) from another type of spidroin, aciniform spidroin 1(AcSp1). Although the three domains originate from distantly related spidroin types, they keep their respective characteristics in the chimeric spidroins. Furthermore, all chimeric spidroins could form silk-like fibers by manual-drawing. In contrast to fibers made in the same manner from W domains only, NTWCT fibers show superior mechanical properties. Our results suggest that chimeric spidroins with NT, CT, and repeat domains can be designed to form fibers with various mechanical properties.

Novel Highly Soluble Chimeric Recombinant Spidroins with High Yield

“…The iN1RC spidroin showed the highest yield, i.e., 254 mg per liter shake flask culture, while that of iN4RC was 89 mg/L. By concentrating in 20 mM Tris buffer at pH 8.0, the solubility of the iNnRC spidroins showed a negative correlation to protein size (Table 1), in other words, with increasing repetitive unit R, the solubility of the spidroins gradually declined from more than 500 mg/mL for The yield of the iNnRC spidroins was negatively correlated to the protein molecular weight (Table 1), which is in line with previous reports on the productivity of recombinant spidroins [19,21]. The iN1RC spidroin showed the highest yield, i.e., 254 mg per liter shake flask culture, while that of iN4RC was 89 mg/L.…”

“…The SDS-PAGE analysis (Figure 1a) showed that the purities of the four spidroins were all above 90%, and the result of western-blot ( Figure 1b) using anti-His-tag confirmed their identities. The yield of the iNnRC spidroins was negatively correlated to the protein molecular weight (Table 1), which is in line with previous reports on the productivity of recombinant spidroins [19,21]. The iN1RC spidroin showed the highest yield, i.e., 254 mg per liter shake flask culture, while that of iN4RC was 89 mg/L.…”

“…By concentrating in 20 mM Tris buffer at pH 8.0, the solubility of the iNnRC spidroins showed a negative correlation to protein size (Table 1), in other words, with increasing repetitive unit R, the solubility of the spidroins gradually declined from more than 500 mg/mL for iN1RC to 134 mg/mL for the largest spidroin, iN4RC. Significantly, the solubility of iN1RC in aqueous buffer (565 mg/mL) was equal to or even higher than the known stored concentration of most natural spidroins and the previously reported recombinant spidroins such as NT2RepCT [22] and NTW 1 CT [21]. Low yield and low aqueous solubility have been two major obstacles to the biomimetic spinning of recombinant spidroins at a large scale [12,29].…”

“…The exceptional performance is thanks to its distinctive protein structure, especially the repetitive domain [17], which usually has long repetitive units, i.e., mostly longer than 200 amino acids with motifs like (A)2, (S)2-5 [9] and high Gly, Ala, and Ser contents [7,14], while lacking short repetitive motifs, such as polyGA, GGX, GPGGX, and polyA [18]. In the previous attempts to synthesize new, high performance and sustainable recombinant spidroin materials, researchers produced many recombinant spidroins, including multiple repetitive motifs [19,20] fused with or without nonrepetitive regions [21][22][23][24][25][26][27][28]. However, the yield of these spidroins were usually too low to meet the needs of large scale application, and only rarely could they reach a level of solubility that was comparable to the storage concentration of natural spidroin in the spider gland, most likely resulting in inferior mechanical properties [3].…”

“…In the previous attempts to synthesize new, high performance and sustainable recombinant spidroin materials, researchers produced many recombinant spidroins, including multiple repetitive motifs [19,20] fused with or without nonrepetitive regions [21][22][23][24][25][26][27][28]. However, the yield of these spidroins were usually too low to meet the needs of large scale application, and only rarely could they reach a level of solubility that was comparable to the storage concentration of natural spidroin in the spider gland, most likely resulting in inferior mechanical properties [3].…”