DWORF序列增强GFP荧光强度
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摘要
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 DW18 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 DW18 and Kozak sequence compared with at presence of Kozak sequence alone. This study demonstrates that the DWORF sequence and its truncated form(DW18) 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.
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 DW18 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 DW18 and Kozak sequence compared with at presence of Kozak sequence alone. This study demonstrates that the DWORF sequence and its truncated form(DW18) 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.
引文
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[19] Jacobson EM,Concepcion E,Oashi T,et al.A Graves’ disease-associated Kozak sequence single-nucleotide polymorphism enhances the efficiency of CD40 gene translation:a case for translational pathophysiology[J].Endocrinology,2005,146(6):2684-2691
[20] Kozak M.Point mutations close to the AUG initiator codon affect the efficiency of translation of rat preproinsulin in vivo[J].Nature,1984,308(5956):241-246
[21] 李巧彦,刘宗智,张婷,等.Kozak序列对nNOS基因表达及神经细胞分化的影响[J].石河子大学学报:自然科学版(Li QY,Liu ZZ,Zhang T,et al.The effect of kozak sequence on the expression of nNOS and neuronal cell differentiation[J].J Shihezi Univ:Nat Sci),2017,35(2):157-162
[2] Shaner NC,Campbell RE,Steinbach PA,et al.Improved monomeric red,orange and yellow fluorescent proteins derived from Discosoma sp.red fluorescent protein[J].Nat Biotechnol,2004,22(12):1567-1572
[3] Merian J,Gravier J,Navarro F,et al.Fluorescent nanoprobes dedicated to in vivo imaging:from preclinical validations to clinical translation[J].Molecules,2012,17(5):5564-5591
[4] Abe T,Sakaue-Sawano A,Kiyonari H,et al.Visualization of cell cycle in mouse embryos with Fucci2 reporter directed by Rosa26 promoter[J].Development,2013,140(1):237-246
[5] Chu J,Haynes RD,Corbel SY,et al.Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein[J].Nat Methods,2014,11(5):572-578
[6] Cai D,Cohen KB,Luo T,et al.Improved tools for the Brainbow toolbox[J].Nat Methods,2013,10(6):540-547
[7] Enterina JR,Wu L,Campbell RE.Emerging fluorescent protein technologies[J].Curr Opin Chem Biol,2015,27:10-17
[8] Shaner NC,Steinbach PA,Tsien RY.A guide to choosing fluorescent proteins[J].Nat Methods,2005,2(12):905-909
[9] Tsien RY.Constructing and exploiting the fluorescent protein paintbox(Nobel Lecture)[J].Angew Chem Int Ed Engl,2009,48(31):5612-5626
[10] Campbell RE,Tour O,Palmer AE,et al.A monomeric red fluorescent protein[J].Proc Natl Acad Sci U S A,2002,99(12):7877-7882
[11] Jianwei D,Qianqian Z,Songcai L,et al.The combination of a synthetic promoter and a CMV promoter improves foreign gene expression efficiency in myocytes[J].J Biotechnol,2012,158(3):91-96
[12] 章倩倩,刘松财,戴建威,等.CMV与SP双启动子增强外源基因在小鼠骨骼肌中的表达效率[J].中国生物化学与分子生物学报(Zhang QQ,Liu SC,Dai JW,et al.Two promoters CMV and SP enhance the expression efficacy of an exogenous gene in skeletal muscle of mice[J].Chin J Biochem Mol Biol),2007,23(7):554-559
[13] Klein R,Ruttkowski B,Knapp E,et al.WPRE-mediated enhancement of gene expression is promoter and cell line specific[J].Gene,2006,372(1):153-161
[14] Kozak M.An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs[J].Nucleic Acids Res,1987,15(20):8125-8148
[15] Nelson BR,Makarewich CA,Anderson DM,et al.A peptide encoded by a transcript annotated as long noncoding RNA enhances SERCA activity in muscle[J].Science,2016,351(6270):271-275
[16] Ramezani A,Hawley TS,Hawley RG.Lentiviral vectors for enhanced gene expression in human hematopoietic cells[J].Mol Ther,2000,2(5):458-469
[17] Schambach A,Wodrich H,Hildinger M,et al.Context dependence of different modules for posttranscriptional enhancement of gene expression from retroviral vectors[J].Mol Ther,2000,2(5):435-445
[18] Nakai J,Ohkura M,Imoto K.A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein[J].Nat Biotechnol,2001,19(2):137-141
[19] Jacobson EM,Concepcion E,Oashi T,et al.A Graves’ disease-associated Kozak sequence single-nucleotide polymorphism enhances the efficiency of CD40 gene translation:a case for translational pathophysiology[J].Endocrinology,2005,146(6):2684-2691
[20] Kozak M.Point mutations close to the AUG initiator codon affect the efficiency of translation of rat preproinsulin in vivo[J].Nature,1984,308(5956):241-246
[21] 李巧彦,刘宗智,张婷,等.Kozak序列对nNOS基因表达及神经细胞分化的影响[J].石河子大学学报:自然科学版(Li QY,Liu ZZ,Zhang T,et al.The effect of kozak sequence on the expression of nNOS and neuronal cell differentiation[J].J Shihezi Univ:Nat Sci),2017,35(2):157-162