An optimized sericin-1 expression system for mass-producing recombinant proteins in the middle silk glands of transgenic silkworms

Abstract

The middle silk gland (MSG) of silkworm is thought to be a potential host for mass-producing valuable recombinant proteins. Transgenic MSG expression systems based on the usage of promoter of sericin1 gene (sericin-1 expression system) have been established to produce various recombinant proteins in MSG. However, further modifying the activity of the sericin-1 expression system to yield higher amounts of recombinant proteins is still necessary. In this study, we provide an alternative modification strategy to construct an efficient sericin-1 expression system by using the hr3 enhancer (hr3 CQ) from a Chongqing strain of the Bombyx mori nuclear polyhedrosis virus (BmNPV) and the 3'UTRs of the fibroin heavy chain (Fib-HPA), the fibroin light chain (Fib-LPA), and Sericin1 (Ser1PA) genes. We first analyzed the effects of these DNA elements on expression of luciferase, and found that the combination of hr3 CQ and Ser1PA was most effective to increase the activity of luciferase. Then, hr3 CQ and Ser1PA were used to modify the sericin1 expression system. Transgenic silkworms bearing these modified sericin1 expression vectors were generated by a piggyBac transposon mediated genetic transformation method. Our results showed that mRNA level of DsRed reporter gene in transgenic silkworms containing hr3 CQ and Ser1PA significantly increased by 9 fold to approximately 83 % of that of endogenous sericin1. As the results of that, the production of recombinant RFP increased by 16 fold to 9.5 % (w/w) of cocoon shell weight. We conclude that this modified sericin-1 expression system is efficient and will contribute to the MSG as host to mass produce valuable recombinant proteins.

Advanced silk material spun by a transgenic silkworm promotes cell proliferation for biomedical application

“…2E-H and 3C and D), indicating that the recombinant protein synthesized in MSG cells was secreted into the sericin layer of the MSG lumen, then spun into silk, which was distributed in the sericin layer of the silk. The transport and localization of recombinant hFGF1 in the transgenic silkworm revealed that the transgenic sericin1 expression system in this study functioned similarly to the previously established ones [27,40].…”

“…The optimized hFGF1 gene with a silkworm codon bias fusing a His 6 tag in the C terminus was synthesized commercially (by GenScript) and inserted into an intermediate vector pSL1180{hSer1spDsRedSv40} [27] by BamHI and NotI to replace the DsRed, then the open reading frame (ORF) of the resulting vector was released by AscI and inserted into the pBac{3xp3EGFPaf} [39] basic transgenic vector cut by the same AscI to generate the final transgenic vector phShFGF1Sv40.…”

“…We first generated a transgenic silkworm line which specifically synthesizes the recombinant hFGF1 in the MSG of the larval silkworm and spins them into the sericin layer of silk using our previously established sericin1 expression system [27]. To achieve this, a piggyBac-based transgenic vector phShFGF1Sv40 was constructed (Fig.…”

“…Various transgenic expression systems based on the usage of fib-H, fib-L, P25 and sericin 1 promoters have been established for the production of exogenous proteins in the silk of transgenic silkworms [27,28]. These transgenic resources make it possible to create genetically modified silk materials with improved mechanical properties and new biofunctionalities.…”

“…It participates in a wide range of biological processes [35], and has potential for clinical applications in wound healing [36] and the treatment of ischemia via its mitogenic activity [37,38]. We generated a transgenic silkworm hSFSV which could specifically synthesize the recombinant hFGF1 proteins in the middle silk gland (MSG) of silkworms and spin them into the sericin layer of silk using our previously established sericin1 expression system [27]. The hSFSV raw silk was directly used to culture NIH/3T3 cells and was found to be biologically active in that it significantly promoted the proliferation of the NIH/3T3 cells.…”