Optimizing plant transporter expression in Xenopus oocytes.
Optimizing plant transporter expression in Xenopus oocytes.
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优化非洲爪蟾卵母细胞中的植物转运蛋白表达
DOI：
10.1186/1746-4811-9-48
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发表时间：
2013-12-20
期刊：
影响因子：
5.1
通讯作者：
Miller AJ
中科院分区：
文献类型：
--
作者：
Feng H;Xia X;Fan X;Xu G;Miller AJ
背景 DNA 合成技术的快速改进正在彻底改变基因克隆及其编码蛋白的表征。非洲爪蟾细胞是膜蛋白表达和功能表征常用的异源系统。对于许多植物蛋白，特别是转运蛋白，低水平的表达会限制这些细胞的功能活性，从而难以表征蛋白质。合成 DNA 技术的改进现在使得优化爪蟾植物 cDNA 的密码子使用变得快速、简单且相对便宜。我们测试了这种优化过程是否可以提高卵母细胞中检测的双组分植物硝酸盐转运蛋白的功能活性。结果我们使用通用软件（http://www.kazusa.or.jp/codon/；http://genomes.urv.es/OPTIMIZER/）来预测植物基因的 DNA 序列，更适合非洲爪蟾。 RiceOsNAR2.1和OsNRT2.3aDNA优化序列是商业合成的，用于爪蟾表达。模板DNA用于使用市售试剂盒合成cRNA。向卵母细胞注射优化的和原始OsNAR2.1和OsNRT2.3a的cRNA混合物。在 0.5 mM Na15NO3 中孵育 16 小时后，注射使用优化 DNA 模板获得的 cRNA 的卵母细胞可以积累比原始基因显着更多的 NO3。卵母细胞的两电极电压钳分析证实，与原始水稻cDNA相比，密码子优化的模板产生了明显更大的电流。结论通过基因的DNA密码子优化，提高了卵母细胞中水稻高亲和力硝酸盐转运蛋白的功能活性。该方法为非洲爪蟾细胞系统中植物蛋白的表达改善和后续更好的功能表征提供了前景。
BackgroundRapid improvements in DNA synthesis technology are revolutionizing gene cloning and the characterization of their encoded proteins.Xenopus laevisoocytes are a commonly used heterologous system for the expression and functional characterization of membrane proteins. For many plant proteins, particularly transporters, low levels of expression can limit functional activity in these cells making it difficult to characterize the protein. Improvements in synthetic DNA technology now make it quick, easy and relatively cheap to optimize the codon usage of plant cDNAs forXenopus.We have tested if this optimization process can improve the functional activity of a two-component plant nitrate transporter assayed in oocytes.ResultsWe used the generally available software ( http://www.kazusa.or.jp/codon/ ; http://genomes.urv.es/OPTIMIZER/ ) to predict a DNA sequence for the plant gene that is better suited forXenopus laevis. RiceOsNAR2.1andOsNRT2.3aDNA optimized sequences were commercially synthesized forXenopusexpression. The template DNA was used to synthesize cRNA using a commercially available kit. Oocytes were injected with cRNA mixture of optimized and originalOsNAR2.1andOsNRT2.3a. Oocytes injected with cRNA obtained from using the optimized DNA template could accumulate significantly more NO3-than the original genes after 16 h incubation in 0.5 mM Na15NO3. Two-electrode voltage clamp analysis of the oocytes confirmed that the codon optimized template resulted in significantly larger currents when compared with the original rice cDNA.ConclusionThe functional activity of a rice high affinity nitrate transporter in oocytes was improved by DNA codon optimization of the genes. This methodology offers the prospect for improved expression and better subsequent functional characterization of plant proteins in theXenopusoocyte system.
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影响因子：
3.7
作者：
Fath S;Bauer AP;Liss M;Spriestersbach A;Maertens B;Hahn P;Ludwig C;Schäfer F;Graf M;Wagner R
通讯作者：
Wagner R
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