Skip to main content
SpringerLink
Log in

Secretory fluorescent protein, a secretion green fluorescent fusion protein with alkaline phosphatase activity as a sensitive and traceable reporter in baculovirus expression system

  • Original Research Paper
  • Published:
Biotechnology Letters Aims and scope Submit manuscript

Abstract

The advantages of using traceable fluorescent protein (enhanced green fluorescent protein; EGFP) and a secretory alkaline phosphatase (SEAP) have been used to generate a reporter gene: the secretory fluorescent protein (SEFP). Sf21 cells, infected with the recombinant baculovirus containing the SEFP gene, revealed both traceable fluorescence and easily detectable alkaline phosphatase activity in the culture medium. The distribution of SEFP within the cells revealed that it was excluded from the nucleus, implying that the accumulation of SEFP in a secretory pathway, similar to that of the secretion signal-tagged FPs. Furthermore, the time- and dose-dependent release from the blockage of brefeldin A (BFA) confirmed that the secretion of SEFP was mediated by the secretion pathway and excluded leakage from viral infection. This SEFP reporter gene with traceable fluorescence and alkaline phosphatase activity may become a useful tool for studies on secretory protein production.

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

Similar content being viewed by others

References

  • Abbate J, Lacayo JC, Prichard M, Pari G, McVoy MA (2001) Bifunctional protein conferring enhanced green fluorescence and puromycin resistance. BioTechniques 31:336–340

    PubMed  CAS  Google Scholar 

  • Ailor E, Betenbaugh MJ (1999) Modifying secretion and post-translational processing in insect cells. Curr Opin Biotechnol 10:42–145

    Article  Google Scholar 

  • Bennett RP, Cox CA, Hoeffler JP (1998) Fusion of green fluorescent protein with the Zeocin-resistance marker allows visual screening and drug selection of transfected eukaryotic cells. BioTechniques 24:478–482

    PubMed  CAS  Google Scholar 

  • Chen YJ, Chen WS, Wu TY (2005) Development of a bi-cistronic baculovirus expression vector by the Rhopalosiphum padi virus 5′ internal ribosome entry site. Biochem Biophys Res Commun 335:616–623

    Article  PubMed  CAS  Google Scholar 

  • Giepmans BN, Adams SR, Ellisman MH, Tsien RY (2006) The fluorescent toolbox for assessing protein location and function. Science 312:217–224

    Article  PubMed  CAS  Google Scholar 

  • Klausner RD, Donaldson JG, Lippincott-Schwartz J (1992) Brefeldin A: insights into the control of membrane traffic and organelle structure. J Cell Biol 116:1071–1080

    Article  PubMed  CAS  Google Scholar 

  • Kost TA, Condreay JP, Jarvis D (2005) Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol 23:567–575

    Article  PubMed  CAS  Google Scholar 

  • Laukkanen ML, Oker-Blom C, Keinanen K (1996) Secretion of green fluorescent protein from recombinant baculovirus-infected insect cells. Biochem Biophys Res Commun 226:755–761

    Article  PubMed  CAS  Google Scholar 

  • Lee JC, Wu TY, Huang CF, Yang FM, Shih SR, Hsu JT (2005) High-efficiency protein expression mediated by enterovirus 71 internal ribosome entry site. Biotechnology and bioengineering 90:656–662

    Article  PubMed  CAS  Google Scholar 

  • Liu J, Szalay WY, Escher A (2000) Visualizing and quantifying protein secretion using a Renilla luciferase-GFP fusion protein. Luminescence 15:45–49

    Article  PubMed  Google Scholar 

  • Misumi Y, Misumi Y, Miki K, Takatsuki A, Tamura G, Ikehara Y (1986) Novel blockade by brefeldin A of intracellular transport of secretory proteins in cultured rat hepatocytes. J Biol Chem 261:11398–11403

    PubMed  CAS  Google Scholar 

  • Naylor LH (1999) Reporter gene technology: the future looks bright. Biochemical pharmacology 58:749–757

    Article  PubMed  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (2001) Molecular cloning: a laboratory manual, 3nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544

    Article  PubMed  CAS  Google Scholar 

  • Yun EY, Goo TW, Kim SW, Choi KH, Hwang JS, Kang SW, Kwon OY (2005) Changes in cellular secretory processing during baculovirus infection. Biotechnol Lett 27:1041–1045

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by a grant NSC-94-2317-B-033-001 from the National Science Council of Taiwan. And support from The Center-of-Excellence Program on Membrane Technology, the Ministry of Education of Taiwan.

Author information

Authors and Affiliations

  1. Department of Bioscience Technology and Center for Nanotechnology, Chung Yuan Christian University, Chung-Li, Taiwan

    Chao-Yi Teng & Tzong-Yuan Wu

  2. R&D Center for Membrane Technology, CYCU, Chung Yuan Christian University, Chung-Li, Taiwan

    Tzong-Yuan Wu

Authors
  1. Chao-Yi Teng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tzong-Yuan Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tzong-Yuan Wu.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Teng, CY., Wu, TY. Secretory fluorescent protein, a secretion green fluorescent fusion protein with alkaline phosphatase activity as a sensitive and traceable reporter in baculovirus expression system. Biotechnol Lett 29, 1019–1024 (2007). https://doi.org/10.1007/s10529-007-9349-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10529-007-9349-y

Keywords

Search

Navigation