DWORF序列增强GFP荧光强度

The DWORF Sequence Enhances Fluorescence Intensity of Green Fluorescent Proteins

荧光蛋白质是重要且常用的标签蛋白质,但是在没有强启动子的情况下表达量低,会影响检测的灵敏度。DWORF(dwarf open reading frame)是由NONMMUG026737编码的长度为34个氨基酸的短肽,其mRNA 5′端能够启动下游基因表达。Kozak序列是现今唯一一个位于成熟mRNA 5′端的增强子,能够提高基因在蛋白质水平的表达,但对某些基因在蛋白质水平的表达增强效果不理想。为了检测DWORF mRNA 5′端(命名为DW)是否能够通过提高荧光蛋白质基因在蛋白质水平的表达,从而增强荧光强度,我们设计了如下实验:将DWORF mRNA 5′端与绿色荧光蛋白(green fluorescent protein, GFP)构建到表达载体中(命名为DW-GFP),转染细胞后检测荧光强度,发现与对照组相比,转染DW-GFP质粒的细胞荧光强度显著提高(56.82±1.77 vs. 45.97±0.44,P<0.05)。为了便于后续应用,将DW截短到18个碱基(命名为DW18),仍能显著提高GFP的荧光强度(33.57±1.11 vs.25.34±0.98,P<0.05),且比Kozak序列提高6.80%。同时,我们发现DW18与Kozak序列同时存在能够进一步提高GFP的荧光强度,比Kozak序列单独存在时提高13.58%。本研究证明DWORF序列及其截短形式(DW18)能够有效提高表达效率,为研究低丰度蛋白质的生物学功能提供了可能的方法和工具。

Green fluorescent proteins ( GFPs) are important and commonly used tag proteins, but low expression levels without strong promoters can affect the sensitivity of experimental assays. So far, the Kozak sequence is the only enhancer at the 5' end of mature mRNA, which can increase the expression of genes at the protein level, but the expression enhancement of some genes at the protein level is insufficient. DWORF ( dwarf open reading frame) is a short peptide of 34 amino acids encoded by NONMMUG026737, and of which the 5' end of mRNA can initiate downstream gene expression. To investigate whether the 5' end of DWORF mRNA ( named DW ) can enhance fluorescence intensity by increasing the expression of fluorescent protein genes at the protein level, we designed the following experiment: compared with the control cell line ( only transfected with GFP plasmids), the fluorescence intensity of the cell line transfected with the DW-GFP plasmid was significantly increased ( 56. 82 ± 1. 77 vs. 45. 97 ± 0. 44, P < 0. 05 ). For subsequent application, DW was truncated to 18 bases ( named DW18), which significantly increased the fluorescence intensity of GFP compared to the control group (only transfected with GFP plasmid)( 33. 57 ± 1.11 vs. 25. 34 ± 0.98, P < 0. 05 ). Moreover, the DW 18 sequence increased the fluorescence intensity of GFP by 6. 80% compared to the Kozak sequence, and the fluorescence intensity of GFP enhanced 13. 58% at the presence of DW 18 and Kozak sequence compared with at presence of Kozak sequence alone. This study demonstrates that the DWORF sequence and its truncated form ( DW 18 ) can effectively increase the expression efficiency of GFP at protein levels and provide a possible method and tool for studying the biological function of low?abundance proteins.