Novel Highly Soluble Chimeric Recombinant Spidroins with High Yield

Abstract

Spider silk has been a hotspot in the study of biomaterials for more than two decades due to its outstanding mechanical properties. Given that spiders cannot be farmed, and their low silk productivity, many attempts have been made to produce recombinant spidroins as an alternative. Herein, we present novel chimeric recombinant spidroins composed of 1 to 4 repetitive units of aciniform spidroin (AcSp) flanked by the nonrepetitive N- and C-terminal domains of the minor ampullate spidroin (MiSp), all from Araneus ventricosus. The spidroins were expressed in the form of inclusion body in E. coli with high yield. Remarkably, the aqueous solubility of the four spidroins ranged from 13.4% to over 50% (m/v). The four spidroins could self-assemble into silk-like fibers by hand-drawing. The secondary structures of these proteins, determined by circular dichroism spectrum (CD) and Fourier transform infrared spectrum (FTIR), indicated a prominent transformation from α-helix to β-sheet after fiber formation. The mechanical properties of the hand-drawn fibers showed a positive correlation with the spidroin molecular weight. In summary, this study describes promising biomaterials for further study and wide application.

Physical Properties of the Second Type of Aciniform Spidroin (AcSp2) from Neoscona theisi Reveal a pH-Dependent Self-Assembly Repetitive Domain

“…48−50 Compared with the fibrous silks with A. trifasciata AcSp1 repeats, the silks spun from recombinant spidroins containing A. ventricosus AcSp1 repeats have higher β-sheet content, strength, overall toughness, indicating that the AcSp repetitive domains from different species could assemble into silk fibers with distinct secondary structure components and mechanical properties. 49,50 For these three N. theisi AcSp repeats, their fibers exhibit different mechanical properties but similar secondary structure contents, implying that their differences in mechanical features could result from other factors. Given the higher contents of lysine, histidine, and aspartic acid in AcSp2 than the other two AcSp1 proteins, the better strength of AcSp2-R fibers is likely based on these charged amino acids (the resulting ionic interactions are generally stronger than the strength of covalent ones) in its repetitive domain.…”

“…trifasciata AcSp1 repeats, the silks spun from recombinant spidroins containing A. ventricosus AcSp1 repeats have higher β-sheet content, strength, and overall toughness, indicating that the AcSp repetitive domains from different species could assemble into silk fibers with distinct secondary structure components and mechanical properties. , For these three N. theisi AcSp repeats, their fibers exhibit different mechanical properties but similar secondary structure contents, implying that their differences in mechanical features could result from other factors.…”

“…The mechanical properties of spider silks are largely dependent on the spidroin repetitive domain. Former studies revealed that at least two AcSp1 repeats or a single AcSp repeat with terminal domains could be induced to form silk-like fibers with a diameter of 1–4 μm by shear forces. − Compared with the fibrous silks with A. trifasciata AcSp1 repeats, the silks spun from recombinant spidroins containing A.…”

Adenylate Kinase Fused to Spidroin as a Catalyst for Decreasing Leakage out of 3D-Bioprinted Hydrogels and for ATP Regeneration

“…Due to their high solubility, C-terminal domains, relatively conserved in amino acid sequences, are thought to play an important role in preventing the disordered aggregation of spidroins in high concentration by remaining outside the oligomer core, formed by Rep domains in storage, and directing the ordered self-assembly of spidroins to form silk fibers . In the absence of the NT domains, which are crucial for spidroin storage in silk glands and fiber formation, truncated miniature spidroins, with only 1–4 repetitive units of Rep domains fused to CT domains, could be self-assembled to form fiber wells . The NT domains could also be substituted by enzymes to form a chimeric spidroin, obtaining both the properties of self-assembly and corresponding enzymatic activity and finally leading to the creation of enzyme-silk materials …”

Nerve Growth Factor‐Binding Engineered Silk Films Promote Neuronal Attachment and Neurite Outgrowth

“…[1][2] The first reported use of silk as a through gene sequencing and protein biochemistry in several species. [16][17][18][19] A variety of recombinant AcSp1 constructs can be expressed in Escherichia coli [20][21][22][23] and these silks can be handdrawn [20][21][22][23] or wet-spun [24][25] into fibers that exhibit high tensile strength combined with high extensibility. Additionally, recombinant AcSp1 is amenable to site-directed mutagenesis [26] and production as a fusion protein, [21][22] making it favorable for biomaterial applications.…”