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

Cellular & Molecular Biology Letters volume 12, pages 362–369 (2007)Cite this article

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

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Authors and Affiliations

  1. State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, P R China

    Chao Dai, Zhijian Cao, Yingliang Wu, Hong Yi, Dahe Jiang & Wenxin Li

Authors
  1. Chao Dai
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  2. Zhijian Cao
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  3. Yingliang Wu
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  4. Hong Yi
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  5. Dahe Jiang
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  6. Wenxin Li
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Corresponding author

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

Cellular & Molecular Biology Letters

ISSN: 1689-1392

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