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.
Dai
Chao
C
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, P R China.
Cao
Zhijian
Z
Wu
Yingliang
Y
Yi
Hong
H
Jiang
Dahe
D
Li
Wenxin
W
D001483:Base Sequence; D003062:Codon / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D016254:Mutagenesis, Insertional / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012604:Scorpion Venoms / Q000378:metabolism; D014162:Transfection
D003062:Codon; C445369:KK toxin, Buthus martensi; D011993:Recombinant Fusion Proteins; D012604:Scorpion Venoms; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins