Novel Highly Soluble Chimeric Recombinant Spidroins with High Yield.

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 ß-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.

Biomimetic spinning of artificial spider silk from a chimeric minispidroin.

Herein we present a chimeric recombinant spider silk protein (spidroin) whose aqueous solubility equals that of native spider silk dope and a spinning device that is based solely on aqueous buffers, shear forces and lowered pH. The process recapitulates the complex molecular mechanisms that dictate native spider silk spinning and is highly efficient; spidroin from one liter of bacterial shake-flask culture is enough to spin a kilometer of the hitherto toughest as-spun artificial spider silk fiber.

Degree of Biomimicry of Artificial Spider Silk Spinning Assessed by NMR Spectroscopy.

Biomimetic spinning of artificial spider silk requires that the terminal domains of designed minispidroins undergo specific structural changes in concert with the ß-sheet conversion of the repetitive region. Herein, we combine solution and solid-state NMR methods to probe domain-specific structural changes in the NT2RepCT minispidroin, which allows us to assess the degree of biomimicry of artificial silk spinning. In addition, we show that the structural effects of post-spinning procedures can be examined. By studying the impact of NT2RepCT fiber drying, we observed a reversible beta-to-alpha conversion. We think that this approach will be useful for guiding the optimization of artificial spider silk fibers.

Characteristics of electrospun membranes in different spidroin/PCL ratios.

Spider silk is a protein fiber with the highest strength and elasticity known in nature, even higher than that of silkworm silk. It was a biological and technical reserve material with great potential. However, the low yield of natural spider silk limits the application of spider silk, and the development of genetic engineering provides opportunities for the mass production of spider silk. We constructed a mini-recombinant spidroin NRC based on spider silk gene fromAraneus ventricosusand successfully expressed it through Prokaryotic expression that provide a high production for application using electrospinning, which is a mature technique to produce micro-nano scale fibers as thin as natural spider silks. By blending the purified and lyophilized NRC with polycaprolactone (PCL) in different mass ratio for electrospinning, different electrospun membranes were obtained, and then characterized in terms of morphology, chemical structure, mechanical and Schwann cell proliferation. Compared the difference between polycaprolactone (PCL) and NRC, the fiber diameter decreased from 1.0779 µm to 0.5785 µm, water contact angel decreased from 104.1 ± 2° to 56.9 ± 5°, and elongation decreased from 240.97 ± 89% to 37.76 ± 13%, while tensile strength increased from 1.74 ± 1.2 MPa to 3.18 ± 0.9 MPa and Young's Module increased from 3.05 ± 1.6 MPa to 16.54 ± 6.7 MPa. In this study, we obtained a thinner fiber, hydrophilicity and high strengthen electrospinning spidroin contained membrane, which can also promote Schwann cell proliferation and adhesion.

Analysis of the Full-Length Pyriform Spidroin Gene Sequence.

Spiders often produce multiple types of silk, each with unique properties suiting them to certain tasks and biological functions. Orb-weaver spiders can generate more than six types of silk fibroins, with pyriform silk used to form attachment discs, adhering silk to other surfaces and substances. The unique higher-order structuring of silk fibroins has been cited as the source of their remarkable biomechanical properties. Even so, only one full-length gene sequence of pyriform silk protein 1 (PySp1) from Argiopeargentata has been reported, and studies on the mechanical properties of natural pyriform silk fibers are also lacking. To better understand the PySp1 family of genes, we used long-distance PCR (LD-PCR) to determine the sequence of PySp1 in the Araneusventricosus species. This full-length PySp1 gene is 11,931 bp in length, encoding for 3976 amino acids residues in non-repetitive N- and C-terminal domains with a central largely repetitive region made up of sixteen remarkably homogeneous units. This was similar to the previously reported A. argentata PySp1 sequence, with PySp1 from A. ventricosus also having a long repetitive N-linker that bridges the N-terminal and repetitive regions. Predictions of secondary structure and hydrophobicity of A. ventricosus PySp1 showed the pyriform silk fiber's functional properties. The amino acid compositions of PySp1 is obviously distinct from other spidroins. Our sequence makes an important contribution to understand pyriform silk protein structure and also provides a new template for recombinant pyriform silk proteins with attractive properties.

Mass spectrometry captures structural intermediates in protein fiber self-assembly.

Self-assembling proteins, the basis for a broad range of biological scaffolds, are challenging to study using most structural biology approaches. Here we show that mass spectrometry (MS) in combination with MD simulations captures structural features of short-lived oligomeric intermediates in spider silk formation, providing direct insights into its complex assembly process.