Extraordinary Mechanical Properties of Composite Silk Through Hereditable Transgenic Silkworm Expressing Recombinant Major Ampullate Spidroin

www.nature.com/scientificreports OPEN Received: 6 December 2017 Accepted: 2 July 2018 Published: xx xx xxxx Extraordinary Mechanical Properties of Composite Silk Through Hereditable Transgenic Silkworm Expressing Recombinant Major Ampullate Spidroin Zhengying You, Xiaogang Ye, Lupeng Ye, Qiujie Qian, Meiyu Wu, Jia Song, Jiaqian Che & Boxiong Zhong Spider dragline silk is a remarkable material that shows excellent mechanical properties, diverse applications, biocompatibility and biodegradability. Transgenic silkworm technology was used to obtain four types of chimeric silkworm/spider (termed composite) silk fibres, including different lengths of recombinant Major ampullate Spidroin1 (re-MaSp1) or recombinant Major ampullate Spidroin2 (re-MaSp2) from the black widow spider, Latrodectus hesperus. The results showed that the overall mechanical properties of composite silk fibres improved as the re-MaSp1 chain length increased, and there were significant linear relationships between the mechanical properties and the re-MaSp1 chain length (p < 0.01). Additionally, a stronger tensile strength was observed for the composite silk fibres that included re-MaSp1, which only contained one type of repetitive motif, (GA)n/An, to provide tensile strength, compared with the silk fibres that includedre-MaSp2, which has the same protein chain length as re-MaSp1 but contains multiple types of repetitive motifs, GPGXX and (GA)n/An. Therefore, the results indicated that the nature of various repetitive motifs in the primary structure played an important role in imparting excellent mechanical properties to the protein-based silk fibres. A silk protein with a single type of repetitive motif and sufficiently long chains was determined to be an additional indispensable factor. Thus, this study forms a foundation for designing and optimizing the structure of re-silk protein using a heterologous expression system. The protein-based silk fibres, produced by spiders and silkworms, have fascinated humans for many years due to their excellent mechanical properties; diverse applications in textiles, optics, and biomedicine; and their biocompatibility and biodegradability1,2. Spiders can produce up to seven types of silks or glues that are used for various purposes. The diverse uses of spider silks derive from their excellent physical properties, which are tailored for specific purposes, resulting in diverse of mechanical properties. Dragline silk is of interest primarily because it displays both high tensile strength and extensibility, rendering it tougher than almost all other natural or man-made synthetic materials3. The major components of dragline silk are two highly conserved spidroins, MaSp1 and MaSp2. The two spidroins possess two notable structural features: both of them contain a number of repetitive motifs in the primary structure, and both have very long protein chains with a molecular weight (MW) up to 320 kDa4–6. According to previous research, the (GA)n/An motifs can impart tensile strength to dragline silk through forming ß-sheets crystalline domain structures, and the GPGXX motif can form ß-turns yielding a spiral structure to impart the elasticity to dragline silk7. MaSp1 is primarily composed of (GA)n/An and GGX motifs, while a large proportion of the GPGXX motif is additionally present in MaSp28. The silk of the best known mulberry silkworm, Bombyx mori, is of specific scientific interest because of its industrial-scale production and excellent mechanical properties. The B. mori silk is wound into a cocoon to protect the pupa during metamorphosis, which requires the silk fibre to be tough rather than strong9. In B. mori, College of Animal Science, Zhejiang University, Hangzhou, 310058, P. R. China. Correspondence and requests for materials should be addressed to B.Z. (email: ) Scientific REPOrTS | (2018) 8:15956 | DOI:10.1038/s41598-018-34150-y 1 www.nature.com/scientificreports/ the silk fibre is composed of two proteins: fibroin and sericin. The silk fibroin consists of a heavy (H) chain of 390 kDa10 and a light (L) chain of 26 kDa with a disulfide bond, and a glycoprotein called P25 (30 kDa). The H chain also exhibits repetitive motif of (GA)nGX, primarily forming ß-sheet structures to form the large crystalline/ semicrystalline domains in the silk fibres11. In conclusion, spider and silkworm silks are composed of fibroin that typically consists of an iterated repetitive central part flanked by smaller non-repetitive domains; meanwhile, the excellent mechanical properties of silk originate from the unique and highly repetitive sequence in the silk protein, along with its molecular organization and self-assembly at the nanoscale12,13, which are occur under physiological and ambient conditions to ensure the structural hierarchy and excellent mechanical properties of the silk fibre14. In the past few years, various heterologous host systems, including bacteria15,16, yeast17, insects18,19, mammalian cell lines20, plants21,22 and animals23–25, were used as platforms to express recombinant spider silk protein for spider-like silk production. Additionally, several attempts have been made to mimic the natural process of producing silk filaments26; however, the resulting silk fibres were considerably weaker than native silk. Thus, understanding the relationship between the fibroin protein structure and its mechanical properties is a key step to mimicking the natural silk and to using silk fibre for specific applications. We speculate that a long protein chain and repetitive motifs in the silk protein could be key factors that are responsible for the extraordinary mechanical properties of silk fibres. Previous studies have indicated that the molecular weight influenced the mechanical properties of polymer silk fibre27,28. To investigate the relationship among protein chain length, repetitive motifs and the mechanical properties of dragline silk, the present study generated four types of composite silk fibres using a silkworm silk-gland bioreactor, including different lengths of re-MaSp1 orre-MaSp2 derived from the corresponding dragline silk proteins of L. hesperus. The results showed that the overall mechanical properties of the composite silk fibres improved with an increasing in the chain length of the recombinant silk protein. This indicated that that the existence of various repetitive motifs in the primary structure and the presence of the same type of repetitive motif and a long protein chain in the silk protein were indispensable important factors for the outstanding mechanical properties of silk fibre. Based on our research results, we speculate that if an artificial spider silk gene expressing proteins with longer length is introduced into the composite silk fibre, then silk fibres with improved mechanical properties would be generated. Results Transgenic vector design and screening of positive transgenic silkworm lineages. The MaSp1 gene of L. hesperus only has a sing (...truncated)