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

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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.

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References

  • Adachi T, Wang XB, Murata T, Obara M, Akutsu H, Machida M, Umezawa A, Tomita M (2010) Production of a non-triple helical collagen alpha chain in transgenic silkworms and its evaluation as a gelatin substitute for cell culture. Biotechnol Bioeng 106(6):860–870. doi:10.1002/Bit.22752

    Article  PubMed  CAS  Google Scholar 

  • Ali S, Taylor WC (2001) The 3′ non-coding region of a C4 photosynthesis gene increases transgene expression when combined with heterologous promoters. Plant Mol Biol 46(3):325–333

    Article  PubMed  CAS  Google Scholar 

  • Cary LC, Goebel M, Corsaro BG, Wang HG, Rosen E, Fraser MJ (1989) Transposon mutagenesis of baculoviruses—analysis of trichoplusia-Ni transposon Ifp2 insertions within the Fp-Locus of nuclear polyhedrosis viruses. Virology 172(1):156–169. doi:10.1016/0042-6822(89)90117-7

    Article  PubMed  CAS  Google Scholar 

  • Couble P, Michaille JJ, Garel A, Couble ML, Prudhomme JC (1987) Developmental switches of sericin mRNA splicing in individual cells of Bombyx mori silkgland. Dev Biol 124(2):431–440

    Article  PubMed  CAS  Google Scholar 

  • Garel A, Deleage G, Prudhomme JC (1997) Structure and organization of the bombyx mori sericin 1 gene and of the sericins 1 deduced from the sequence of the Ser 1B cDNA. Insect Biochem Mol Biol 27(5):469–477

    Article  PubMed  CAS  Google Scholar 

  • Grzelak K (1995) Control of expression of silk protein genes. Comp Biochem Physiol B: Biochem Mol Biol 110(4):671–681

    Article  CAS  Google Scholar 

  • Horn C, Wimmer EA (2000) A versatile vector set for animal transgenesis. Dev Genes Evol 210(12):630–637

    Article  PubMed  CAS  Google Scholar 

  • Iizuka M, Tomita M, Shimizu K, Kikuchi Y, Yoshizato K (2008) Translational enhancement of recombinant protein synthesis in transgenic silkworms by a 5′-untranslated region of polyhedrin gene of bombyx mori nucleopolyhedrovirus. J Biosci Bioeng 105(6):595–603. doi:10.1263/jbb.105.595

    Article  PubMed  CAS  Google Scholar 

  • Iizuka M, Ogawa S, Takeuchi A, Nakakita S, Kubo Y, Miyawaki Y, Hirabayashi J, Tomita M (2009) Production of a recombinant mouse monoclonal antibody in transgenic silkworm cocoons. FEBS J 276(20):5806–5820. doi:10.1111/j.1742-4658.2009.07262.x

    Article  PubMed  CAS  Google Scholar 

  • Inoue S, Tanaka K, Arisaka F, Kimura S, Ohtomo K, Mizuno S (2000) Silk fibroin of bombyx mori is secreted, assembling a high molecular mass elementary unit consisting of H-chain, L-chain, and P25, with a 6:6:1 molar ratio. J Biol Chem 275(51):40517–40528. doi:10.1074/jbc.M006897200

    Article  PubMed  CAS  Google Scholar 

  • Ito T, Tahara SM, Lai MM (1998) The 3′-untranslated region of hepatitis C virus RNA enhances translation from an internal ribosomal entry site. J Virol 72(11):8789–8796

    PubMed  CAS  Google Scholar 

  • Komuro T, Yomota C, Hasegawa R, Isaka H, Kimura T (1988) Studies on the dissolution behaviors of indomethacin extended-release capsules by the flow-through method. Eisei Shikenjo Hokoku 106:87–90

    PubMed  Google Scholar 

  • Kuersten S, Goodwin EB (2003) The power of the 3′ UTR: translational control and development. Nat Rev Genet 4(8):626–637. doi:10.1038/nrg1125

    Article  PubMed  CAS  Google Scholar 

  • Kurihara H, Sezutsu H, Tamura T, Yamada K (2007) Production of an active feline interferon in the cocoon of transgenic silkworms using the fibroin H-chain expression system. Biochem Biophys Res Commun 355(4):976–980. doi:10.1016/j.bbrc.2007.02.055

    Article  PubMed  CAS  Google Scholar 

  • Lee JM, Takahashi M, Mon H, Mitsunobu H, Koga K, Kawaguchi Y, Nakajima Y, Kusakabe T (2008) Construction of gene expression systems in insect cell lines using promoters from the silkworm. Bombyx Mori J Biotechnol 133(1):9–17. doi:10.1016/j.jbiotec.2007.08.033

    CAS  Google Scholar 

  • Liu Y, Yu L, Guo X, Guo T, Wang S, Lu C (2006) Analysis of tissue-specific region in sericin 1 gene promoter of Bombyx mori. Biochem Biophys Res Commun 342(1):273–279. doi:10.1016/j.bbrc.2006.01.140

    Article  PubMed  CAS  Google Scholar 

  • Lu M, Farrell PJ, Johnson R, Iatrou K (1997) A baculovirus (bombyx mori nuclear polyhedrosis virus) repeat element functions as a powerful constitutive enhancer in transfected insect cells. J Biol Chem 272(49):30724–30728

    Article  PubMed  CAS  Google Scholar 

  • Majima K, Kobara R, Maeda S (1993) Divergence and evolution of homologous regions of Bombyx mori nuclear polyhedrosis virus. J Virol 67(12):7513–7521

    PubMed  CAS  Google Scholar 

  • Mange A, Couble P, Prudhomme JC (1996) Two alternative promoters drive the expression of the cytoplasmic actin A4 gene of Bombyx mori. Gene 183(1–2):191–199

    Article  PubMed  CAS  Google Scholar 

  • Mazumder B, Seshadri V, Fox PL (2003) Translational control by the 3′-UTR: the ends specify the means. Trends Biochem Sci 28(2):91–98

    Article  PubMed  CAS  Google Scholar 

  • Mignone F, Gissi C, Liuni S, Pesole G (2002) Untranslated regions of mRNAs. Genome Biol 3:3 REVIEWS0004

    Article  Google Scholar 

  • Ogawa S, Tomita M, Shimizu K, Yoshizato K (2007) Generation of a transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon: production of recombinant human serum albumin. J Biotechnol 128(3):531–544. doi:10.1016/j.jbiotec.2006.10.019

    Article  PubMed  CAS  Google Scholar 

  • Royer C, Jalabert A, Da Rocha M, Grenier AM, Mauchamp B, Couble P, Chavancy G (2005) Biosynthesis and cocoon-export of a recombinant globular protein in transgenic silkworms. Transgenic Res 14(4):463–472

    Article  PubMed  CAS  Google Scholar 

  • Takasu Y, Yamada H, Tsubouchi K (2002) Isolation of three main sericin components from the cocoon of the silkworm. Bombyx mori Biosci Biotechnol Biochem 66(12):2715–2718

    Article  CAS  Google Scholar 

  • Tamura T, Thibert C, Royer C, Kanda T, Abraham E, Kamba M, Komoto N, Thomas JL, Mauchamp B, Chavancy G, Shirk P, Fraser M, Prudhomme JC, Couble P (2000) Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector. Nat Biotechnol 18(1):81–84. doi:10.1038/71978

    Article  PubMed  CAS  Google Scholar 

  • Tangphatsornruang S, Birch-Machin I, Newell CA, Gray JC (2011) The effect of different 3′ untranslated regions on the accumulation and stability of transcripts of a gfp transgene in chloroplasts of transplastomic tobacco. Plant Mol Biol 76(3–5):385–396. doi:10.1007/s11103-010-9689-1

    Article  PubMed  CAS  Google Scholar 

  • Tatematsu K, Kobayashi I, Uchino K, Sezutsu H, Iizuka T, Yonemura N, Tamura T (2010) Construction of a binary transgenic gene expression system for recombinant protein production in the middle silk gland of the silkworm Bombyx mori. Transgenic Res 19(3):473–487. doi:10.1007/s11248-009-9328-2

    Article  PubMed  CAS  Google Scholar 

  • Thomas JL, Da Rocha M, Besse A, Mauchamp B, Chavancy G (2002) 3xP3-EGFP marker facilitates screening for transgenic silkworm Bombyx MoriL. from the embryonic stage onwards. Insect Biochem Mol Biol 32(3):247–253

    Article  PubMed  CAS  Google Scholar 

  • Tomita M (2011) Transgenic silkworms that weave recombinant proteins into silk cocoons. Biotechnol Lett 33(4):645–654. doi:10.1007/s10529-010-0498-z

    Article  PubMed  CAS  Google Scholar 

  • Tomita M, Munetsuna H, Sato T, Adachi T, Hino R, Hayashi M, Shimizu K, Nakamura N, Tamura T, Yoshizato K (2003a) Transgenic silkworms produce recombinant human type III procollagen in cocoons. Nat Biotechnol 21(1):52–56. doi:10.1038/nbt771

    Article  PubMed  CAS  Google Scholar 

  • Tomita M, Munetsuna H, Sato T, Adachi T, Hino R, Hayashi M, Shimizu K, Nakamura N, Tamura T, Yoshizato K (2003b) Transgenic silkworms produce recombinant human type III procollagen in cocoons. Nat Biotechnol 21(1):52–56. doi:10.1038/Nbt771

    Article  PubMed  CAS  Google Scholar 

  • Tomita M, Hino R, Ogawa S, Iizuka M, Adachi T, Shimizu K, Sotoshiro H, Yoshizato K (2007) A germline transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon. Transgenic Res 16(4):449–465. doi:10.1007/s11248-007-9087-x

    Article  PubMed  CAS  Google Scholar 

  • Xia Q, Zhou Z, Lu C, Cheng D, Dai F, Li B, Zhao P, Zha X, Cheng T, Chai C, Pan G, Xu J, Liu C, Lin Y, Qian J, Hou Y, Wu Z, Li G, Pan M, Li C, Shen Y, Lan X, Yuan L, Li T, Xu H, Yang G, Wan Y, Zhu Y, Yu M, Shen W, Wu D, Xiang Z, Yu J, Wang J, Li R, Shi J, Li H, Su J, Wang X, Zhang Z, Wu Q, Li J, Zhang Q, Wei N, Sun H, Dong L, Liu D, Zhao S, Zhao X, Meng Q, Lan F, Huang X, Li Y, Fang L, Li D, Sun Y, Yang Z, Huang Y, Xi Y, Qi Q, He D, Huang H, Zhang X, Wang Z, Li W, Cao Y, Yu Y, Yu H, Ye J, Chen H, Zhou Y, Liu B, Ji H, Li S, Ni P, Zhang J, Zhang Y, Zheng H, Mao B, Wang W, Ye C, Wong GK, Yang H (2004) A draft sequence for the genome of the domesticated silkworm (Bombyx mori). Science 306(5703):1937–1940. doi:10.1126/science.1102210

    Article  PubMed  Google Scholar 

  • Yang L, Wakasa Y, Kawakatsu T, Takaiwa F (2009) The 3′-untranslated region of rice glutelin GluB-1 affects accumulation of heterologous protein in transgenic rice. Biotechnol Lett 31(10):1625–1631. doi:10.1007/s10529-009-0056-8

    Article  PubMed  CAS  Google Scholar 

  • Zhao A, Zhao T, Zhang Y, Xia Q, Lu C, Zhou Z, Xiang Z, Nakagaki M (2010) New and highly efficient expression systems for expressing selectively foreign protein in the silk glands of transgenic silkworm. Transgenic Res 19(1):29–44. doi:10.1007/s11248-009-9295-7

    Article  PubMed  Google Scholar 

  • Zhou CZ, Confalonieri F, Medina N, Zivanovic Y, Esnault C, Yang T, Jacquet M, Janin J, Duguet M, Perasso R, Li ZG (2000) Fine organization of Bombyx Morifibroin heavy chain gene. Nucleic Acids Res 28(12):2413–2419

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We are grateful for Prof. Hongjuan Cui, Dr. Fei Wang for the critical comments on the manuscript. We also thank Prof. Takahiro Kusakabe for the kind gift of vector and Dr. Jun Zhang for the supply of cell lines. This work was supported by the Grant (2012CB114600) from the National Basic Research Program of China, Grant (31000981) from National Natural Science Foundation of China, Grant (2010BB1144) from Chongqing Natural Science Foundation and Grants (kb2011008, kb2010002 and ky2009002) from the doctoral Innovation Fund of Southwest University.

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Authors and Affiliations

  1. State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, 400715, People’s Republic of China

    Feng Wang, Hanfu Xu, Lin Yuan, Sanyuan Ma, Yuancheng Wang, Xiaoli Duan, Jianping Duan, Zhonghuai Xiang & Qingyou Xia

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  1. Feng Wang
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  9. Qingyou Xia
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Correspondence to Qingyou Xia.

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Feng Wang and Hanfu Xu are contributed equally to this work.

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Wang, F., Xu, H., Yuan, L. et al. An optimized sericin-1 expression system for mass-producing recombinant proteins in the middle silk glands of transgenic silkworms. Transgenic Res 22, 925–938 (2013). https://doi.org/10.1007/s11248-013-9695-6

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