Production and Properties of Triple Chimeric Spidroins

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, NTW_1–4CT 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

Morphology and Composition of the Spider Major Ampullate Gland and Dragline Silk

Spider silk is made of unique proteinsspidroinssecreted and stored as a protein solution (dope) in specialized glands. The major ampullate gland, source of the dragline silk, is composed of a tail, a sac and an elongated duct. For this gland, several different types of epithelial cells and granules have been described, but it is largely unknown how they correlate with spidroin production. It is also not settled what parts of the large spidroins end up in the final silk, and it has been suggested that the N-terminal domain (NT) is lacking. Here we show that NT is present in the dope and throughout dragline silk fibers, including the skin layer, and that the major ampullate tail and sac consist of three different and sharply demarcated zones (A–C), each with a distinct epithelial cell type. Finally, we show that spidroins are produced in the A and B zone epithelia, while the C zone granules lack spidroins

Morphology and Composition of the Spider Major Ampullate Gland and Dragline Silk

Spider silk is made of unique proteinsspidroinssecreted and stored as a protein solution (dope) in specialized glands. The major ampullate gland, source of the dragline silk, is composed of a tail, a sac and an elongated duct. For this gland, several different types of epithelial cells and granules have been described, but it is largely unknown how they correlate with spidroin production. It is also not settled what parts of the large spidroins end up in the final silk, and it has been suggested that the N-terminal domain (NT) is lacking. Here we show that NT is present in the dope and throughout dragline silk fibers, including the skin layer, and that the major ampullate tail and sac consist of three different and sharply demarcated zones (A–C), each with a distinct epithelial cell type. Finally, we show that spidroins are produced in the A and B zone epithelia, while the C zone granules lack spidroins

Morphology and Composition of the Spider Major Ampullate Gland and Dragline Silk

Spider silk is made of unique proteinsspidroinssecreted and stored as a protein solution (dope) in specialized glands. The major ampullate gland, source of the dragline silk, is composed of a tail, a sac and an elongated duct. For this gland, several different types of epithelial cells and granules have been described, but it is largely unknown how they correlate with spidroin production. It is also not settled what parts of the large spidroins end up in the final silk, and it has been suggested that the N-terminal domain (NT) is lacking. Here we show that NT is present in the dope and throughout dragline silk fibers, including the skin layer, and that the major ampullate tail and sac consist of three different and sharply demarcated zones (A–C), each with a distinct epithelial cell type. Finally, we show that spidroins are produced in the A and B zone epithelia, while the C zone granules lack spidroins

Kyte and Doolittle hydropathicity plots for <i>A. ventricosus</i> MiSp.

<p>Positive scores indicate hydrophobicity. NT = N-terminal region, RT =  repetitive region, S =  spacer and CT =  C-terminal region.</p

Primers used for intron identification.

<p>Primers used for intron identification.</p

Complete amino acid sequence for <i>A. ventricosus</i> minor ampullate spidroin

<p>(<b>MiSp</b>)<b>.</b> The sequence is read from left to right and top to bottom. The first methionine (Met) marks the start position and the asterisk denotes the stop position. The protein is dominated by poly-A (red), GGX (blue and fuchsia), GGGX (fuchsia) and GX (red and cyan) motifs. The protein sequence can be divided into nonrepetitive N- and C-terminal regions and a predominantly repetitive region. The repetitive region is interrupted by two nonrepetitive spacer regions which have 100% identity. Gaps (–) have been inserted in order to align repeat units within a type. Non-repetitive N- and C-terminal regions are in black, spacers are in green and linker regions are in gray. The green arrow marks the intron insertion position.</p

Alignment of genomic DNA upstream sequences.

<p>Aligned DNA ranges approximately 210 nucleotides upstream of the predicted transcription start (indicated by a box and a black triangle). The red triangle indicates <i>A. ventricosus</i> MiSp upstream sequence. The translation start ATG codons, and the conserved motifs CACG and TATA are boxed. The sequences shown are the following: NimFlag, <i>N. inaurata madagascariensis</i> flagelliform (Flag) spidroin gene, upstream (GenBank accession no. AF218623S1); NimMaSp2, <i>N. inaurata madagascariensis</i> major ampullate spidroin 2-like gene (GenBank accession no. DQ059135); LhMaSp1, <i>L. hesperus</i> major ampullate spidroin 1 (GenBank accession no. EF595246) and 2 (GenBank accession no. EF595245) genes; LgMaSp1, <i>L. geometricus</i> major ampullate spidroin 1-like gene (GenBank accession no. DQ059133S1); AtMaSp2, <i>A. trifasciata</i> major ampullate spidroin 2 gene (GenBank accession no. DQ059136S1); AvMiSp, <i>A. ventricosus</i> minor ampullate spidroin gene (GenBank accession no. JX513956).</p

Architectures of <i>A. ventricosus</i> MiSp, <i>A. trifasciata</i> MaSp2, <i>Nephila</i> Flag, <i>L. hesperus</i> MaSp1 and MaSp2 genes.

<p>Arrangement of exons (red) interrupted by introns (grey light green) in the spidroin genes. Due to the huge variability in size the lengths of the different segments are not drawn to scale.</p

Characterized full-length spider silk gene sequences.

<p>Characterized full-length spider silk gene sequences.</p