Ha D S, Schwarz J K, Turco S J, Beverley S M
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Mol Biochem Parasitol. 1996 Apr;77(1):57-64. doi: 10.1016/0166-6851(96)02580-7.
We have tested the suitability of the green fluorescent protein (GFP) of Aequorea victoria as a marker for studies of gene expression and protein targeting in the trypanosomatid parasite Leishmania. Leishmania promastigotes expressing GFP from episomal pXG vectors showed a bright green fluorescence distributed throughout the cell, readily distinguishable from control parasites. Transfection of a modified GFP gene containing GC-rich synonymous codons and the S65T mutation (GFP+) yielded a much higher fluorescence. FACS analysis revealed a clear quantitative separation between GFP-transfected and control parasites, with pXG-GFP+ transfectants showing fluorescence signals more than 100-fold background. Episomal DNAs could be recovered from small numbers of fixed cells, showing that GFP could be used as a convenient screenable marker for FACS separations. GFP was fused to the C-terminus of the LPG1 protein, which retained its ability to restore LPG expression when expressed in the lpg- R2D2 mutant of L. donovani. The LPG1(GFP) fusion was localized to a region situated between the nucleus and kinetoplast; its pattern was similar to that of LPG2, which is known to be located in the Golgi apparatus. This is notable as LPG1 participates in the biosynthesis of the glycan core of the LPG GPI anchor, whereas protein GPI anchor biosynthesis occurs in the endoplasmic reticulum. These studies suggest that the GFP will be a broadly useful marker in Leishmania.
我们测试了维多利亚多管水母的绿色荧光蛋白（GFP）作为利什曼原虫寄生虫基因表达和蛋白质靶向研究标记的适用性。从游离型pXG载体表达GFP的利什曼原鞭毛体显示出明亮的绿色荧光，遍布整个细胞，很容易与对照寄生虫区分开来。转染含有富含GC的同义密码子和S65T突变的修饰GFP基因（GFP+）产生了更高的荧光。流式细胞仪分析显示，GFP转染的寄生虫和对照寄生虫之间有明显的定量分离，pXG-GFP+转染体显示的荧光信号比背景高100倍以上。可以从少量固定细胞中回收游离型DNA，表明GFP可作为流式细胞仪分离的方便筛选标记。GFP与LPG1蛋白的C末端融合，当在杜氏利什曼原虫的lpg-R2D2突变体中表达时，LPG1蛋白仍保留恢复LPG表达的能力。LPG1（GFP）融合蛋白定位于细胞核和动基体之间的区域；其模式与已知位于高尔基体的LPG2相似。这很值得注意，因为LPG1参与LPG GPI锚定聚糖核心的生物合成，而蛋白质GPI锚定生物合成发生在内质网中。这些研究表明，GFP将成为利什曼原虫中广泛有用的标记。
