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

100%

Dai C., Cao Z., Wu Y., Yi H., Jiang D., Li W.

Cellular and Molecular Biology Letters

2007

tom 12

nr 3

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.

Ograniczanie wyników

1 Cellular and Molecular Biology Letters

1 Cao Z.

1 Dai C.

1 Jiang D.

1 Li W.

1 Wu Y.

1 Yi H.

1 2007

Wyniki wyszukiwania

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