Tianfu Zhao

Transgenic silkworms (Bombyx mori) produce recombinant spider dragline silk in cocoons

Spider dragline silk is a unique fibrous protein with a combination of tensile strength and elasticity, but the isolation of large amounts of silk from spiders is not feasible. In this study, we generated germline-transgenic silkworms (Bombyx mori) that spun cocoons containing recombinant spider silk. A piggyBac-based transformation vector was constructed that carried spider dragline silk (MaSp1) cDNA driven by the sericin 1 promoter. Silkworm eggs were injected with the vector, producing transgenic silkworms displaying DsRed fluorescence in their eyes. Genotyping analysis confirmed the integration of the MaSp1 gene into the genome of the transgenic silkworms, and silk protein analysis revealed its expression and secretion in the cocoon. Compared with wild-type silk, the recombinant silk displayed a higher tensile strength and elasticity. The results indicate the potential for producing recombinant spider silk in transgenic B. mori.

New and highly efficient expression systems for expressing selectively foreign protein in the silk glands of transgenic silkworm

We constructed three different fibroin H-chain expression systems to estimate the efficacy of producing recombinant proteins in the cocoon of transgenic silkworms. The results showed that the three different EGFP/H-chain fusion genes were all expressed selectively in the posterior silk gland of the transgenic silkworm. The recombinant protein content of transgenic silkworm cocoons is up to 15% (w/w) when using the most highly efficient H-chain expression system. To our knowledge, in comparison with silkworm silk gland expression systems in the literature, the highly efficient expression system developed in this study is the most efficient silkworm silk gland expression system to date. This expression system is the best candidate for foreign gene production and for creation of novel functional silk material. The results suggested the N-terminal domain and the intron of the H-chain gene are important in the secretion of fibroin and its transcription, respectively.

Analysis of a new type of major ampullate spider silk gene, MaSp1s

Spider dragline silk, which is secreted from the major ampullate silk glands, is a unique fibrous protein with a combination of tensile strength and elasticity. Here, we describe a new short type of dragline silk gene, Cyrtophora moluccensis MaSp1s. The full-length gene is only 1320 base pairs (bp), which encodes 439 amino acids that includes the intact non-repetitive N-terminal (149 residues), C-terminal (98 residues) and so-called repetitive regions (192 residues); the deduced molecular weight is approximately 40 kDa. The sequence analysis demonstrated that the two termini are highly homologous to the other characterized dragline silk genes but that the so-called repetitive region is different. Our results suggest that MaSp1s is a possible new characteristic dragline gene; the discovery of this gene should enhance our understanding of the major ampullate spider silk genes.

Silkworm, Bombyx mori larvae expressed the spider silk protein through a novel Bac-to-Bac/BmNPV baculovirus

Abstract:  The silkworm has become an ideal multicellular eukaryotic model system for basic research. The major advantages of expressing foreign genes in silkworm larvae are the low cost of feeding, the extremely high levels of expression achievable compared with expression in cell lines and increased safety because the baculovirus is noninfectious to vertebrates. In this study, we used a recently developed Bombyx mori Nucleopolyhedrovirus (BmNPV) bacmid to express the spider flagelliform silk gene in silkworm larvae. The recombinant bacmid baculoviruses (rBacmid/BmNPV/Flag) were introduced into the first‐day larvae of the fifth instar by subcutaneous injection. The worms presented symptoms typical of NPV infection from 72 h after injection compared with control. The haemolymph was collected from the infected larvae 120 h post‐infection and the recombinant 6× His‐tagged Flag protein was purified by the Ni‐NTA spin kit under denaturing conditions with 8 m urea. A 37.0‐kDa protein was visualized both in rBacmid/BmNPV/Flag‐infected haemolymph and eluting fraction. The results showed that the Bac‐to‐Bac/BmNPV baculovirus expression system is an efficient tool to express the target gene in silkworm larvae, which takes only 7–10 days for generating recombinant baculovirus, compared with the traditional homologous recombination method, which needs at least 40 days for multiple rounds of purification and amplification of viruses.

Characterization of a Cysteine-Rich Protein Specifically Expressed in the Silk Gland of Caddisfly Stenopsyche marmorata (Trichoptera; Stenopsychidae)

A novel protein, Smsp-72k, was found to be selectively expressed in the silk gland of aquatic larvae of the Stenopsychid caddisfly (Stenopsyche marmorata). The protein was characterized by an abundance of cysteine (13.97%) and charged residues (47.21%). Amino acids with hydroxyl side-chains accounted for an additional 10% of the Smsp-72k protein, with serine at 4.4% and threonine at 5.6%. A cysteine-rich repetitive sequence is common to many potential and known underwater adhesive/cement proteins and cell-cell adhesion molecules. We hypothesized that Smsp-72k is an adhesive/cement protein that increases the adhesiveness of the silk fiber of S. marmorata. The hydroxyl groups of Smsp-72k might form a link with the heavy chain fibroin of S. marmorata, removing the weak boundary-water layer and allowing the spreading of the silk protein onto the surface of the substratum during the process of adhesion.

The variability of mechanical properties and molecular conformation among different spider dragline fibers

Spider dragline fiber is a high-performance biomaterial that has received much attention. To screen the outstanding spider dragline fibers, the mechanical properties and microstructures of dragline fibers collected from Nephia clavata, Nephia pilipes, Argiope bruennichi and Argiope amoena were investigated. It was found that the mechanical properties of spider dragline fiber were variable. Among the four different species, the larger spiders did not always extrude thicker dragline fibers and produce fibers with the maximum breaking force. The dragline fibers could sustain one to three times the body weight of the spider at a reeling speed of 20 mm/s. N. clavata dragline fiber showed a stronger breaking stress and initial modulus than that of N. pilipes, A. bruennichi and A. amoena. With an increasing reeling speed, the breaking strain decreased; the initial modulus increased in N. clavata, N. pilipes and A. bruennichi, but the breaking stress exhibited a different tendency. The results also revealed that dragline fiber of N. clavata contained the most β-sheet polypeptides and an excellent orientation of β-sheet molecular chains.