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Improved fusion protein expression of EGFP via the mutation of both Kozak and the initial ATG codon

Abstract

Since its discovery, green fluorescence protein (GFP) has been used as a reporter in a broad range of applications, including the determination of gene expresion in diverse organisms, and subcellular protein localization. pEGFP-N1 is a eukayotic expression vector encoding EGFP, the MCS of which locates at the N terminus of EGFP. In this study, the cDNA sequence of scorpion toxin BmKK2 was inserted into the XhoI-HindIII cut of pEGFP-N1 to construct a toxin-EGFP fusion gene (named pEGFP-BmKK2). Fluorescence imaging revealed that HEK 293T cells that were transfected by pEGFP-BmKK2 emitted green fluorescence. Transcription of pEGFP-BmKK2 was confirmed by RT-PCR. However, western blotting analysis showed that the transfected HEK 293T cells expressed mostly EGFP, but little toxin-EGFP fusion protein, implying that pEGFP-N1 cannot be used as a fusion expression vector for subcellular protein localization for the BmKK2 gene. Consequently, two modified recombinant vectors (pEGFP-BmKK2-M1 and pEGFP-BmKK2-M2) were constructed based on pEGFP-BmKK2. This greatly improved the expression of toxin-EGFP fusion protein from pEGFP-BmKK2-M2.

Abbreviations

CCTCC:

China Center for Type Culture Collection

cDNA:

complementary DNA

DAB:

diaminobenzidine

DMEM:

Dulbecco’s modified Eagle’s medium

E. coli :

Escherichia coli

EGFP:

enhanced green fluorescent protein

FCS:

fetal calf serum

GFP:

green fluorescence protein

MCS:

multiple cloning site

mRNA:

messenger RNA

RT-PCR:

reverse transcript polymerase chain reaction

SDS-PAGE:

sodium dodecyl sulfate polyacrylamide gel electrophoresis

References

  1. 1.

    Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W. and Prasher, D.C. Green fluorescent protein as a marker for gene expression. Science 263 (1994) 802–805.

    PubMed  Article  CAS  Google Scholar 

  2. 2.

    Gill, S.K., Bhattacharya, M., Ferguson, S.S. and Rylett, R.J. Identification of a novel nuclear localization signal common to 69-and 82-kDa human choline acetyltransferase. J. Biol. Chem. 278 (2003) 20217–20224.

    PubMed  Article  CAS  Google Scholar 

  3. 3.

    Kamei, H. Intracellular localization of histidyl-tRNA synthetase/Jo-1 antigen in T24 cells and some other cells. J. Autoimmun. 22 (2004) 201–210.

    PubMed  Article  CAS  Google Scholar 

  4. 4.

    Chen, T., Li, X., Yang, Y. and Church, R.L. Localization of lens intrinsic membrane protein MP19 and mutant protein MP19(To3) using fluorescent expression vectors. Mol. Vis. 8 (2002) 372–388.

    PubMed  CAS  Google Scholar 

  5. 5.

    Sun, D., Landowski, C.P., Chu, X., Wallsten, R., Komorowski, T.E., Fleisher, D. and Amidon, G.L. Drug inhibition of Gly-Sar uptake and hPepT1 localization using hPepT1-GFP fusion protein. AAPS Pharm. Sci. 3 (2001) E2.

    Article  CAS  Google Scholar 

  6. 6.

    Cormack, B.P., Valdivia, R.H. and Falkow, S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173 (1996) 33–38.

    PubMed  Article  CAS  Google Scholar 

  7. 7.

    Li, M.H., Zhang, N.X., Chen, X.Q., Wu, G., Wu, H., Hu, G.Y. Purification and pharmacological characterization of BmKK2 (alpha-KTx 14.2), a novel potassium channel-blocking peptide, from the venom of Asian scorpion Buthus martensi Karsch. Toxicon 43 (2004) 895–900

    PubMed  Article  CAS  Google Scholar 

  8. 8.

    Rodriguez, D.A., Vega, R.C., Possani, L.D. Current views on scorpion toxins specific for K+-channels. Toxicon 43 (2004) 865–75.

    Article  CAS  Google Scholar 

  9. 9.

    Zeng, X.C., Peng, F., Luo, F., Zhu, S.Y., Liu, H. and Li, W.X. Molecular cloning and characterization of four scorpion K(+)-toxin-like peptides: a new subfamily of venom peptides (alpha-KTx14) and genomic analysis of a member. Biochimie 83 (2001) 883–889.

    PubMed  Article  CAS  Google Scholar 

  10. 10.

    Gray, T.A., Saitoh, S. and Nicholls, R.D. An imprinted, mammalian bicistronic transcript encodes two independent proteins. Proc. Natl. Acad. Sci. USA 96 (1999) 5616–5621.

    PubMed  Article  CAS  Google Scholar 

  11. 11.

    Sloan, J., Kinghorn, J.R. and Unkles, S.E. The two subunits of human molybdopterin synthase: evidence for a bicistronic messenger RNA with overlapping reading frames. Nucleic Acids Res. 27 (1999) 854–858.

    PubMed  Article  CAS  Google Scholar 

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Correspondence to Wenxin Li.

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These authors contributed equally to this article

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Dai, C., Cao, Z., Wu, Y. et al. Improved fusion protein expression of EGFP via the mutation of both Kozak and the initial ATG codon. Cell Mol Biol Lett 12, 362–369 (2007). https://doi.org/10.2478/s11658-007-0008-z

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  • DOI: https://doi.org/10.2478/s11658-007-0008-z

Key words

  • EGFP
  • Fusion protein expression
  • Subcellular protein localization
  • Scorpion toxin