Open Access

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

  • Chao Dai1,
  • Zhijian Cao1,
  • Yingliang Wu1,
  • Hong Yi1,
  • Dahe Jiang1 and
  • Wenxin Li1Email author
Cellular & Molecular Biology LettersAn International Journal200712:8

DOI: 10.2478/s11658-007-0008-z

Received: 17 October 2006

Accepted: 27 November 2006

Published: 23 February 2007

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.

Key words

EGFP Fusion protein expression Subcellular protein localization Scorpion toxin

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