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

• Autorzy: Dai C , Cao Z. , Wu Y. , Yi H. , Jiang D. , Li W.
• Języki publikacji: EN

Abstrakty

EN

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.

Słowa kluczowe

EN

green fluorescent protein; enhanced green fluorescent protein; gene expression; reserve transcript polymerase chain reaction; mRNA; mutation; multiple cloning site

• Wydawca: -
• Czasopismo: Cellular and Molecular Biology Letters
• Rocznik: 2007
• Tom: 12
• Numer: 3
• Opis fizyczny: p.362-369,fig.,ref.

Twórcy

autor

Dai C

• Wuhan University, Wuhan, 430072, P.R.China

autor

Cao Z.

autor

Wu Y.

autor

Yi H.

autor

Jiang D.

autor

Li W.

Bibliografia

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