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Akitsu Masuda,Jian Xu,Kosuke Minamihata,Genki Kagawa,Yusei Hamada,Yoshiki Morifuji,Takumi Yano,Masato Hino,Daisuke Morokuma,Noriko Karasaki,Hiroaki Mon,Noriho Kamiya,Takahiro Kusakabe,이재만 한국응용곤충학회 2018 Journal of Asia-Pacific Entomology Vol.21 No.2
As a therapeutic treatment, recombinant human basic fibroblast growth factor (rhbFGF) is usually employed in tissue regeneration, and as an essential component in culture medium for maintaining the induced pluripotent stem (iPS) cell and embryonic stem (ES) cell in an undifferentiated state. Therefore, a large amount of biologically active rhbFGF is required. In this study, silkworm-baculovirus expression vector system (silkworm-BEVS) is employed to achieve a high productivity of recombinant rhbFGF with two small affinity tags (His-tag and STREP-tag) at the N or C-terminus. It is observed that rhbFGF with 30 K signal peptide of silkworm were successfully expressed but are not sufficiently secreted into the culture medium of cultured insect cells. Then we purified the N- or C-tagged intracellular rhbFGF protein and obtained a yield of about 0.7 mg/larva and 1.2 mg/ larva, respectively. Although the final yield of the C-tagged rhbFGF is higher than that of the N-tagged, rhbFGF with N-tag demonstrated promising and comparable biological activity, which is evaluated through a mammalian cell proliferation assay. Taken together, these results indicate that silkworm-BEVS could contribute to the mass-production of the biologically active rhbFGF for medical uses.
Nagai Ryo,Ebihara Takeru,Kakino Kohei,Masuda Akitsu,Xu Jian,Minamihata Kosuke,Kamiya Noriho,Kongkrongtong Tatphon,Kawahara Masahiro,Mon Hiroaki,Fujii Tsuguru,Kusakabe Takahiro,Lee Jae Man 한국응용곤충학회 2021 Journal of Asia-Pacific Entomology Vol.24 No.3
Due to the biological significance and therapeutic potential of Interleukin-3 (IL-3) secreted mainly by activated T cells, various protein expression systems have been challenged to produce recombinant IL3 to meet the increasing demands worldwide. Recently, we established an updated silkworm-based baculovirus expression vector system (silkworm-BEVS), which in most cases, produces eukaryotic proteins in biological or enzymatical active forms with considerable amounts. We attempted to reconstruct and express a recombinant mouse IL-3 (rMmIL-3) with C-terminal His8-Strep tags in silkworm-BEVS in the current study. From our results, we gained an active glycosylated rMmIL-3 protein in a substantial amount and quality. As compared with the E. coli expression system, silkworm-BEVS is a better choice regarding the glycosylations attached in rMmIL-3 and upscalable system in case that a commercial amount is required in the future. Collectively, our method shares an excellent model to produce interleukin molecular for approaching pharmaceutical applications.
Masahiko Kobayashi,Jian Xu,Kohei Kakino,Akitsu Masuda,Masato Hino,Naoki Fujimoto,Kosuke Minamihata,Noriho Kamiya,Hiroaki Mon,Hiroshi Iida,Masateru Takahashi,Takahiro Kusakabe,이재만 한국응용곤충학회 2020 Journal of Asia-Pacific Entomology Vol.23 No.1
Interleukine-4 (IL-4) is a cytokine that plays an important role in the immune system and recognized as a biological medicine. Therefore, there is a demand for the production of IL-4 with high performance. The expression of a recombinant IL-4 protein in the prokaryotic system usually results in the formation of an inclusion body. To date, the solution to obtain those active products without the refolding process remains to be established. In this study, we tried to acquire a biologically active recombinant Mus musculus IL-4 (rMmIL-4) using a silkworm-baculovirus expression vector system (silkworm-BEVS). We constructed two recombinant baculoviruses coding rMmIL-4 with the distinct location of affinity purification tags and succeeded in the expression and purification of rMmIL-4 proteins directly without the refolding process. Both purified proteins displayed comparable biological activity to the commercial proteins produced by the E. coli expression system. Besides, we performed screening of silkworm strains to seek optimal hosts for the mass-production of rMmIL-4. Intriguingly, we found that some silkworm strains showed significantly higher secretion levels of rMmIL-4 in silkworm sera. Our study provides meaningful insights into the industrial-scale production of rMmIL-4 with high productivity for pharmaceutical applications in the future.