Characterization of Transgenic Silkworm Yielded Biomaterials with Calcium-Binding Activity

RESEARCH ARTICLE Characterization of Transgenic Silkworm Yielded Biomaterials with Calcium-Binding Activity Shaohua Wang1, Yuyu Zhang1, Mingying Yang1, Lupeng Ye1, Lu Gong2, Qiujie Qian1, Yajun Shuai1, Zhengying You1, Yuyin Chen1, Boxiong Zhong1* 1 College of Animal Sciences, Zhejiang University, Hangzhou 310058, P.R. China, 2 College of Life Sciences, Zhejiang University, Hangzhou 310058, P.R. China * a11111 Abstract OPEN ACCESS Citation: Wang S, Zhang Y, Yang M, Ye L, Gong L, Qian Q, et al. (2016) Characterization of Transgenic Silkworm Yielded Biomaterials with Calcium-Binding Activity. PLoS ONE 11(7): e0159111. doi:10.1371/ journal.pone.0159111 Editor: Erjun Ling, Institute of Plant Physiology and Ecology, CHINA Silk fibers have many inherent properties that are suitable for their use in biomaterials. In this study, the silk fibroin was genetically modified by including a Ca-binding sequence, [(AGSGAG)6ASEYDYDDDSDDDDEWD]2 from shell nacreous matrix protein. It can be produced as fibers by transgenic silkworm. The Ca-binding activity and mineralization of the transgenic silk fibroin were examined in vitro. The results showed that this transgenic silk fibroin had relatively higher Ca-binding activity than unmodified silk fibroin. The increased Ca-binding activity could promote the usage of silk fibroin as a biomaterial in the pharmaceutical industry. This study shows the possibility of using silk fibroin as a mineralization accelerating medical material by generating genetically modified transgenic silkworm. Received: April 18, 2016 Accepted: June 27, 2016 Published: July 14, 2016 Copyright: © 2016 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work is supported by the Projects of Zhejiang Provincial Science and Technology Plans (No. 2012C12910), grants from the National Basic Research Program of China (No. 2012CB114601). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Introduction The silkworm Bombyx mori has been used as bioreactor to produce foreign proteins for decades due to its advanced physiological characteristics, such as short life cycle, convenient breeding process, large-scale protein production, and small individuals, which can be maintained in high densities, especially after the germline transformation method for the silkworm was developed using the transposon piggyBac[1–4]. Silk produced by silkworm is a natural protein fiber that contains two main components: fibroin and sericin. Fibroin is synthesized and secreted in the posterior silk gland, coated by sericin when it accumulates in the lumen of the middle silk gland and secreted into the cocoon via the anterior silk gland[5]. Three proteins, a 350 kDa fibroin heavy chain, a 26 kDa fibroin light chain and P25/fibrohexamerin, compose the silk fibroin[6]. The clinical need of materials for bone regeneration is expected to increase, and some essential characteristics of these materials, including biocompatibility, porosity, and appropriate mechanical properties, are important for their application. The silk fibroin from silkworm, with superior mechanical properties such as the ability to be tailored, slow degradation, adequate time permitted for remodeling, and, most importantly, biocompatibility, is an ideal PLOS ONE | DOI:10.1371/journal.pone.0159111 July 14, 2016 1 / 11 Characterization of Transgenic Biomaterials biomaterial for clinical uses. The suture made from the silk fibroin has been used for decades [7]. Sponge made from silk fibroin has been used as a scaffold for chondrocyte distribution and cartilage regeneration; similarly, silk can be used as a Nano carrier for drug delivery or as a ligament to regenerate human bone tissues [8–10]. In addition, cell-adhesive activity increases when the surfaces of plates for mammalian cell cultures are coated by silk fibroin with incorporation of collagen- or fibronectin-derived peptides produced by transgenic silkworm[11]. Bone repair has been explored based on calcium binding silk scaffolds using transgenic silk fibroin produced by transgenic silkworm with Ca-binding sequence [(AGSGAG)4E8AS]4 [12]. The protein [(AGSGAG)3AS(AGSGAG)3ASEYDYDDDSDDDDEWD]2 purified from E. coli was reported to have the ability to bind calcium ions under the certain conditions and can be dip-coated with hydroxyapatite[13]. This peptide sequence was combined with the calcium binding site EYDYDDDSDDDDEWD from the pearl oyster (Pinctada fucata) nacreous layer matrix protein MSI60[14] and the B. mori silk fibroin repetitive domain (AGSGAG)n. In the present study, the transgenic silk fibroin containing the Ca-binding sequence [(AGSGAG)6ASEYDYD DDSDDDDEWD]2 (referred to as CABP) was produced as transgenic silk fibers through the systematic transformation of silkworm. The Ca-binding activity and mineralization of the transgenic silk fibroin was examined in vitro. This silk fibroin-based biomaterial with Ca-binding activity can be produced in large scale by silkworm breeding. Materials and Methods Construction of the Ca-binding protein expression vector Dimerized DNA fragments containing the CABP sequence with certain improvements according to a report described previously[15] were used in this study. The sequence was artificially synthesized by GenScript (Piscataway, NJ, USA) (Fig 1a). The plasmid constructed for Ca-binding protein expression was based on the transposon plasmid pBA3EGFP maintained by our lab. The vector contains two expression frames. One of them is used for CABP expression and contains the fibroin light chain promoter (BGIBMGA009393, from -4021 to -1) (FLP), fibroin light chain signal peptide (FLSP), CABP, and fibroin light chain 3’-flank (BGIBMGA009393, from +12793 to +13299). The other is used for expression of the reporter gene enhanced green fluorescent protein (EGFP) and is composed of the Bombyx mori A3 cytoplasmic actin gene promoter (A3), EGFP, and 3’-untranslated sequences (SV40). Genomic DNA was extracted from silk gland of Qiufeng silkworm strain using a DNA extraction kit (Sangon, Shanghai, China). The fibroin light chain promoter and 3’-flanking region were amplified using high fidelity PrimeSTAR HS DNA Polymerase (TAKARA BIO INC., Otsu, Shiga, Japan). Two clones were sequenced after cloning into the pMD19-T vector (TAKARA BIO INC., Otsu, Shiga, Japan). The signal peptide sequence was synthesized by combining it to the primer used to get fibroin light chain promoter. The structure of the final plasmid used for microinjection is piggyBacFLP-FLSP-CABP-FL-3’polyA+A3-EG (...truncated)