Tanida-Miyake Emiko, Koike Masato, Uchiyama Yasuo, Tanida Isei
Department of Cell Biology and Neuroscience, Juntendo University School of Medicine, Tokyo, Japan.
Department of Gastroenterology, Machida Municipal Hospital, Tokyo, Japan.
PLoS One. 2018 Jan 17;13(1):e0191108. doi: 10.1371/journal.pone.0191108. eCollection 2018.
Green fluorescent protein (GFP) is tremendously useful for investigating many cellular and intracellular events. The monomeric GFP mNeonGreen is about 3- to 5-times brighter than GFP and monomeric enhanced GFP and shows high photostability. The maturation half-time of mNeonGreen is about 3-fold faster than that of monomeric enhanced GFP. However, the cDNA sequence encoding mNeonGreen contains some codons that are rarely used in Homo sapiens. For better expression of mNeonGreen in human cells, we synthesized a human-optimized cDNA encoding mNeonGreen and generated an expression plasmid for humanized mNeonGreen under the control of the cytomegalovirus promoter. The resultant plasmid was introduced into HEK293 cells. The fluorescent intensity of humanized mNeonGreen was about 1.4-fold higher than that of the original mNeonGreen. The humanized mNeonGreen with a mitochondria-targeting signal showed mitochondrial distribution of mNeonGreen. We further generated an expression vector of humanized mNeonGreen with 3xFLAG tags at its carboxyl terminus as these tags are useful for immunological analyses. The 3xFLAG-tagged mNeonGreen was recognized well with an anti-FLAG-M2 antibody. These plasmids for the expression of humanized mNeonGreen and mNeonGreen-3xFLAG are useful tools for biological studies in mammalian cells using mNeonGreen.
绿色荧光蛋白（GFP）对于研究许多细胞和细胞内事件非常有用。单体绿色荧光蛋白mNeonGreen比GFP和单体增强型绿色荧光蛋白亮约3至5倍，并且具有高光稳定性。mNeonGreen的成熟半衰期比单体增强型绿色荧光蛋白快约3倍。然而，编码mNeonGreen的cDNA序列包含一些在智人中很少使用的密码子。为了在人类细胞中更好地表达mNeonGreen，我们合成了一种经过人类优化的编码mNeonGreen的cDNA，并在巨细胞病毒启动子的控制下生成了人源化mNeonGreen的表达质粒。将所得质粒导入HEK293细胞。人源化mNeonGreen的荧光强度比原始mNeonGreen高约1.4倍。带有线粒体靶向信号的人源化mNeonGreen显示出mNeonGreen的线粒体分布。我们进一步生成了一种人源化mNeonGreen的表达载体，其羧基末端带有3xFLAG标签，因为这些标签可用于免疫分析。带有3xFLAG标签的mNeonGreen能被抗FLAG-M2抗体很好地识别。这些用于表达人源化mNeonGreen和mNeonGreen-3xFLAG的质粒是在哺乳动物细胞中使用mNeonGreen进行生物学研究的有用工具。
