A novel MiSp from Steatoda triangulosa: Chimeric fibers with repeat-number-modulated mechanical properties

The modular architecture of spider silk proteins offers a blueprint for engineering high-performance artificial silks. While the major ampullate silk (MaSp) of orb-weaving spiders has been extensively studied, the molecular diversity of minor ampullate silk (MiSp) in non-orb-weaving spiders and its functional implications remain largely unexplored. In this study, we report the first cloning and characterization of a full-length MiSp gene from Steatoda triangulosa. The single-exon gene spans 5496 bp and encodes a 1832-amino-acid protein comprising an N-terminal domain (157 aa), a C-terminal domain (112 aa), and 48 repetitive units. Notably, the repeat region lacks spacer motifs and features dominant GAG/A_n crystalline motifs, a configuration distinct from known MiSp sequences. To investigate the mechanical potential of this architecture, we constructed recombinant chimeras (designated NTnRpCT) containing 2, 4, or 6 selected repeat units, flanked by the highly soluble N- and C-terminal domains, and processed them into fibers via wet-spinning. Among the resulting fibers, the NT6RpCT-H fiber achieved a tensile strength of 480 ± 30 MPa and a toughness of 120 ± 10 MJ/m³-performance metrics comparable to those of high-performance chimeric MaSp fibers. These results reveal the potential of the MiSp-derived chimeric constructs in terms of mechanical properties and provide valuable references for future comparative studies. In this specific chimeric system, our data indicate that the number of repeats can modulate mechanical performance, further supporting previous reports on the effects of repeat number in other spider silk protein systems.