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Partitioning and characterization of tyrosine-tagged green fluorescent proteins in aqueous two-phase systems

机译：水性两相系统中酪氨酸标记的绿色荧光蛋白的分区和表征

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The green fluorescent protein GFPuv has been genetically engineered to investigate the influence of N-terminal tyrosine extensions in aqueous two-phase systems. Fusions in the N-terminus affected the protein expression, and tags containing three tyrosines and prolines influenced the expression favorably. This effect is probably due to changes in mRNA stability, because the amounts of corresponding mRNAs correlated with the amounts of GFPuv proteins. The partitioning was investigated in two different aqueous two-phase systems, a two-polymer system composed of EO30PO70/dextran and a PEG/salt system with potassium phosphate. Partitioning in the PEG/salt system generally was more favorable than in the EO30PO70/dextran system. Tags with three tyrosines resulted in higher partitioning toward the EO30PO70- and PEG-rich phases, respectively. The effect of adding proline residues to the tag was also investigated, and the partitioning effect of the tag was enhanced when prolines were included in the tags with three tyrosines. The best tyrosine tag, Y3P2, increased the partition coefficient 5 times in the PEG/salt system. Thermoseparation of the EO30PO70 phase allowed recovery of 83% Y3P2-GFPuv protein in a water phase.
机译：已经对绿色荧光蛋白GFPuv进行了基因工程研究，以研究在水相两相系统中N末端酪氨酸延伸的影响。 N末端的融合蛋白影响蛋白质表达，含有三个酪氨酸和脯氨酸的标签对蛋白质表达有良好的影响。这种作用可能是由于mRNA稳定性的变化，因为相应的mRNA的量与GFPuv蛋白的量相关。在两个不同的水性两相系统中研究了分配，这是一个由EO30PO70 /葡聚糖组成的二元聚合物系统，以及一个具有磷酸钾的PEG /盐系统。通常，在PEG /盐系统中进行分区比在EO30PO70 /葡聚糖系统中进行分区更有利。具有三个酪氨酸的标签分别导致分别向富含EO30PO70和PEG的较高相分配。还研究了向标签中添加脯氨酸残基的效果，并且当脯氨酸包含在带有三个酪氨酸的标签中时，标签的分配效果会增强。最好的酪氨酸标签Y3P2在PEG /盐系统中将分配系数提高了5倍。 EO30PO70相的热分离允许在水相中回收83％的Y3P2-GFPuv蛋白。

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