Expression of EGFP-spider dragline silk fusion protein in BmN cells and larvae of silkworm showed the solubility is primary limit for dragline proteins yield

Abstract

Spider dragline silk is a unique fibrous protein with combination of tensile strength and elasticity, but the isolation of large amount of silk from spiders is not feasible. In this paper, we used a newly established Bac-to-Bac/BmNPV Baculovirus expression system to express the recombinant spider (Nephila clavata) dragline silk protein (MaSp1) fused EGFP in BmN cells and larvae of silkworm. A 70 kDa fusion protein was visualized after rBacmid/BmNPV/drag infection by SDS-PAGE and immunoblotting analysis. Fusion protein expressed in the BmN cells probably occupied five percent of the cell total protein; In a silkworm larva, approximately 6 mg fusion proteins were expressed. Solubility analysis of the expressed spider dragline silk protein indicated that 60% fusion protein is insoluble. EGFP fluorescence showed that fusion protein is tend to form aggregate by self assemblage. The results indicated the solubility is the primary limit for spider dragline proteins yield. It also suggested that directly produce fibrous spider silk in the secreting-silk organs of the transgenic silkworm larvae might be a better method.

Silk for Drug Delivery Applications: Opportunities and Challenges

“…[1,22] Silk fibers are spun by arthropods; [23] the fibers derived from silkworms are most commonly used for biomedical sutures, while solutions are prepared and studied for drug delivery. Spiders are more challenging to raise for silk production; thus, typically Nephila clavipesÀ or Araneus diadematusÀinspired protein sequences are adopted and expressed in various hosts such as yeast, [24] metabolically engineered E. coli (to elevate the glycyl-tRNA pool), [25] plants, [26] mammalian cells, [27] insects [28] or transgenic ani- mals, including mice [29] and goats, [30] where the protein was expressed in the mammary gland and secreted into the milk (reviewed in reference [31]). The large size of naturally occurring spider silks and the highly repetitive nature of these proteins poses a challenge during expression, in part due to the limits of the glycyl-tRNA pools in the expression host.…”

Recombinant Spider Silks—Biopolymers with Potential for Future Applications

“…This has been carried further through the use of silkworms as production hosts; as these are natural silk producers they should have the capacity to deal with the large spider silk proteins. Silkworms have been infected with a baculovirus containing a MaSp1 fragment (70 kDa), producing reasonable yields up to 3% of the total soluble protein in the BmN cells of silkworms [36]. A more recent approach is the first example of transgenic B. mori silkworms, which have been transformed with an 83 kDa fragment of the MaSp1 gene from N. clavata.…”

Research Article Improving the PCR protocol to amplify a repetitive DNA sequence.

“…Its protein is characterized by elasticity and high tensile strength, more than any synthetic material (Lewis, 2006). Spiders, because of their aggressive and territorial nature, are difficult to be farm, and they also produce low amounts of silk (Rising et al, 2011); hence, the dragline silk is already being expressed in some host systems, like mice, silkworms, yeasts, plants, and bacteria (Fahnestock and Bedzyk, 1997;Scheller et al, 2001;Xu et al, 2007;Zhang et al, 2008;Xia et al, 2010;Peng et al, 2016). However, this heterologous expression is quite difficult because of the high GC content, sequence length, specific codon usage in spiders, and the repetitive nature of the sequence (Scheibel, 2004).…”