Skip to main content

Advertisement

SpringerLink
Log in

Construction of a binary transgenic gene expression system for recombinant protein production in the middle silk gland of the silkworm Bombyx mori

  • Original Paper
  • Published:
Transgenic Research Aims and scope Submit manuscript

Abstract

To construct an efficient system for the production of recombinant proteins in silkworm (Bombyx mori), we investigated the promoter activity of the silkworm sericin 1, 2, and 3 genes (Ser1, Ser2, and Ser3) using a GAL4/UAS binary gene expression system in transgenic silkworm. The promoter activity of the upstream region of Ser1 was strong, yielding high expression of an enhanced green fluorescent protein (EGFP) transgene in the middle and posterior regions of the middle silk gland (MSG) after day 2 of the fifth instar. The Ser3 upstream region exhibited moderate promoter activity in the anterior MSG, but the Ser2 upstream region did not exhibit any promoter activity. Since the strongest promoter activity was observed for Ser1, we devised a system for the production of recombinant proteins using a GAL4–Ser1 promoter construct (Ser1-GAL4). Transgenic silkworms harboring both the Ser1-GAL4 construct and the previously reported upstream activating sequence (UAS)–EGFP construct, which contains the TATA box region of the Drosophila hsp70 gene, yielded approximately 100 μg EGFP per larva. When we then analyzed the TATA box region, signal peptide, and intron sequences for their effects on production from the UAS-EGFP construct, we found that the optimization of these sequences effectively increased production to an average of 500 μg EGFP protein per transgenic larva. We conclude that this binary system is a useful tool for the mass production of recombinant proteins of biomedical and pharmaceutical interest in silkworm.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  • Adachi T, Tomita M, Shimizu K, Ogawa S, Yoshizato K (2006) Generation of hybrid transgenic silkworms that express Bombyx mori prolyl-hydroxylase alpha-subunits and human collagens in posterior silk glands: production of cocoons that contained collagens with hydroxylated proline residues. J Biotechnol 126:205–219. doi:10.1016/j.jbiotec.2006.04.035

    Article  CAS  PubMed  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 124:431–440. doi:10.1016/0012-1606(87)90496-9

    Article  CAS  PubMed  Google Scholar 

  • Dyck MK, Lacroix D, Pothier F, Sirard M-A (2003) Making recombinant proteins in animals—different systems, different applications. Trends Biotechnol 21:394–399. doi:10.1016/S0167-7799(03)00190-2

    Article  CAS  PubMed  Google Scholar 

  • Guo X, Zhang Y, Zhang X, Wang S, Lu C (2008) Recognition of signal peptide by protein translocation machinery in middle silk gland of silkworm Bombyx mori. Acta Biochim Biophys Sin 40:38–46. doi:10.1111/j.1745-7270.2008.00376.x

    CAS  PubMed  Google Scholar 

  • Hidalgo A, Brand H (1997) Targeted neuronal ablation: the role of pioneer neurons in guidance and fasciculation in the CNS of Drosophila. Development 124:3253–3262

    CAS  PubMed  Google Scholar 

  • Hino R, Tomita M, Yoshizato K (2006) The generation of germline transgenic silkworms for the production of biologically active recombinant fusion proteins of fibroin and human basic fibroblast growth factor. Biomaterials 27:5715–5724. doi:10.1016/j.biomaterials.2006.07.028

    Article  CAS  PubMed  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 105:595–603. doi:10.1263/jbb.105.595

    Article  CAS  PubMed  Google Scholar 

  • Imamura M, Nakai J, Inoue S, Quan GX, Kanda T, Tamura T (2003) Targeted gene expression using the GAL4/UAS system in the silkworm Bombyx mori. Genetics 165:1329–1340

    CAS  PubMed  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 275:40517–40528. doi:10.1074/jbc.M006897200

    Article  CAS  PubMed  Google Scholar 

  • Inoue S, Kanda T, Imamura M, Quan GX, Kojima K, Tanaka H, Tomita M, Hino R, Yoshizato K, Mizuno S, Tamura T (2005) A fibroin secretion-deficient silkworm mutant, Nd-sD, provides an efficient system for producing recombinant proteins. Insect Biochem Mol Biol 35:51–59. doi:10.1016/j.ibmb.2004.10.002

    Article  CAS  PubMed  Google Scholar 

  • Kojima K, Kuwana Y, Sezutsu H, Kobayashi I, Uchino K, Tamura T, Tamada Y (2007) A new method for the modification of fibroin heavy chain protein in the transgenic silkworm. Biosci Biotechnol Biochem 71:2943–2951. doi:10.1271/bbb.70353

    Article  CAS  PubMed  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 355:976–980. doi:10.1016/j.bbrc.2007.02.055

    Article  CAS  PubMed  Google Scholar 

  • Lienard D, Sourrouille C, Gomord V, Faye L (2007) Pharming and transgenic plants. Biotechnol Annu Rev 13:115–147. doi:10.1016/S1387-2656(07)13006-4

    Article  CAS  PubMed  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 342:273–279. doi:10.1016/j.bbrc.2006.01.140

    Article  CAS  PubMed  Google Scholar 

  • Matsuno K, Hui CC, Takiya S, Suzuki T, Ueno K, Suzuki Y (1989) Transcription signals and protein binding sites for sericin gene transcription in vitro. J Biol Chem 264:18707–18713

    CAS  PubMed  Google Scholar 

  • Michaille JJ, Garel A, Prudhomme JC (1989) The expression of five middle silk gland specific genes is territorially regulated during the larval development of Bombyx mori. Insect Biochem 19:19–27. doi:10.1016/0020-1790(89)90005-X

    Article  Google Scholar 

  • Michaille JJ, Garel A, Prudhomme JC (1990) Cloning and characterization of the highly polymorphic Ser2 gene of Bombyx mori. Gene 86:177–184. doi:10.1016/0378-1119(90)90277-X

    Article  CAS  PubMed  Google Scholar 

  • Mori K, Tanaka K, Kikuchi Y, Waga M, Waga S, Mizuno S (1995) Production of a chimeric fibroin light-chain polypeptide in a fibroin secretion-deficient naked pupa mutant of the silkworm Bombyx mori. J Mol Biol 251:217–228. doi:10.1006/jmbi.1995.0429

    Article  CAS  PubMed  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 128:531–544. doi:10.1016/j.jbiotec.2006.10.019

    Article  CAS  PubMed  Google Scholar 

  • Okamoto H, Ishikawa E, Suzuki Y (1982) Structural analysis of sericin genes. Homologies with fibroin gene in the 5′ flanking nucleotide sequences. J Biol Chem 257:15192–15199

    CAS  PubMed  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 14:463–472. doi:10.1007/s11248-005-4351-4

    Article  CAS  PubMed  Google Scholar 

  • Sakudoh T, Sezutsu H, Nakashima T, Kobayashi I, Fujimoto H, Uchino K, Banno Y, Iwano H, Maekawa H, Tamura T, Kataoka H, Tsuchida K (2007) Carotenoid silk coloration is controlled by a carotenoid-binding protein, a product of the Yellow blood gene. Proc Natl Acad Sci USA 104:8941–8946. doi:10.1073/pnas.0702860104

    Article  CAS  PubMed  Google Scholar 

  • Takasu Y, Yamada H, Tamura T, Sezutsu H, Mita K, Tsubouchi K (2007) Identification and characterization of a novel sericin gene expressed in the anterior middle silk gland of the silkworm Bombyx mori. Insect Biochem Mol Biol 37:1234–1240. doi:10.1016/j.ibmb.2007.07.009

    Article  CAS  PubMed  Google Scholar 

  • Takei F, Oyama F, Kimura K, Hyodo A, Mizuno S, Shimura K (1984) Reduced level of secretion and absence of subunit combination for the fibroin synthesized by a mutant silkworm, Nd(2). J Cell Biol 99:2005–2010

    Article  CAS  PubMed  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 18:81–84. doi:10.1038/71978

    Article  CAS  PubMed  Google Scholar 

  • Tamura T, Kuwabara N, Uchino K, Kobayashi I, Kanda T (2007) An improved DNA injection method for silkworm eggs drastically increases the efficiency of producing transgenic silkworms. J Insect Biotechnol Sericol 76:155–159

    CAS  Google Scholar 

  • Thomas JL, Da Rocha M, Besse A, Mauchamp B, Chavancy G (2002) 3×P3-EGFP marker facilitates screening for transgenic silkworm Bombyx mori L. from the embryonic stage onwards. Insect Biochem Mol Biol 32:247–253. doi:10.1016/S0965-1748(01)00150-3

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  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 16:449–465. doi:10.1007/s11248-007-9087-x

    Article  CAS  PubMed  Google Scholar 

  • Uchino K, Imamura M, Sezutsu H, Kobayashi I, Kojima K, Kanda T, Tamura T (2006) Evaluating promoter sequences for trapping an enhancer activity in the silkworm Bombyx mori. J Insect Biotechnol Sericol 75:89–97

    CAS  Google Scholar 

  • Wurm FM (2003) Human therapeutic proteins from silkworms. Nat Biotechnol 21:34–35. doi:10.1038/nbt0103-34

    Article  CAS  PubMed  Google Scholar 

  • Yanagisawa S, Zhu Z, Kobayashi I, Uchino K, Tamada Y, Tamura T, Asakura T (2007) Improving cell-adhesive properties of recombinant Bombyx mori silk by incorporation of collagen or fibronectin derived peptides produced by transgenic silkworms. Biomacromolecules 8:3487–3492. doi:10.1021/bm700646f

    Article  CAS  PubMed  Google Scholar 

  • Zhao A, Zhao T, Zhang Y, Xia Q, Lu C, Zhou Z, Xiang Z, Nakagaki M (2009) New and highly efficient expression systems for expressing selectively foreign protein in the silk glands of transgenic silkworm. Transgenic Res. doi:10.1007/s11248-009-9295-7

Download references

Acknowledgments

We thank Mr. Kaoru Nakamura and Mr. Koji Hashimoto for maintaining the silkworms. We thank also to Miss Sayaka Kobayashi for very useful supports and advices for experiments. This work was partly supported by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Integrated research project for plant, insect and animal using genome technology INSECT-2202)

Author information

Authors and Affiliations

  1. Transgenic Silkworm Research Center, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, 305-8634, Japan

    Ken-ichiro Tatematsu, Isao Kobayashi, Keiro Uchino, Hideki Sezutsu, Tetsuya Iizuka, Naoyuki Yonemura & Toshiki Tamura

Authors
  1. Ken-ichiro Tatematsu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Isao Kobayashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Keiro Uchino
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hideki Sezutsu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tetsuya Iizuka
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Naoyuki Yonemura
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Toshiki Tamura
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Toshiki Tamura.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tatematsu, Ki., Kobayashi, I., Uchino, K. et al. 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, 473–487 (2010). https://doi.org/10.1007/s11248-009-9328-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11248-009-9328-2

Keywords

Search

Navigation