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


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.



China Center for Type Culture Collection


complementary DNA




Dulbecco’s modified Eagle’s medium

E. coli :

Escherichia coli


enhanced green fluorescent protein


fetal calf serum


green fluorescence protein


multiple cloning site


messenger RNA


reverse transcript polymerase chain reaction


sodium dodecyl sulfate polyacrylamide gel electrophoresis


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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).

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Key words

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