pHluorin2: an enhanced, ratiometric, pH-sensitive green florescent protein.
pHluorin2: an enhanced, ratiometric, pH-sensitive green florescent protein.
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DOI：
10.4236/abb.2011.23021
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发表时间：
2011-06
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影响因子：
--
通讯作者：
Mahon MJ
中科院分区：
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作者：
Mahon MJ
对pH值变化敏感的绿色荧光蛋白（GFP）变体是分析与细胞内和胞外泡运输相关的蛋白质动力学的宝贵试剂。比值型氟利昂是一种GFP变体，显示双峰激发光谱，峰位于395和475 nm，最大发射峰位于509 nm。酸化后，395 nm处的phorin激发降低，475 nm处的激发相应增加。GFP2是一种含有哺乳动物化密码子和折叠增强突变F64L的GFP变体，在395 nm激发时显示出比pHluorin高8倍的荧光。以GFP2为模板，构建了一个增强的比例型pHluorin2结构体，该结构体包含完全哺乳动物化的密码子、F64L突变和13个phluorin13个特异性突变中的10个。结果表明，pHluorin2在保持比值ph敏感性的同时显示出比phorin更高的荧光。与天然的pHluorin不同，pHluorin2在甲状旁腺激素1受体的配体结合区域中表达，通过共聚焦显微镜可以很容易地检测到，并且在配体诱导的细胞内囊泡内吞噬过程中，pHluorin2的荧光明显增加。因此，pHluorin2在保持比例ph敏感性的同时增强了荧光，这代表了该方法的重大改进。
Green florescent protein (GFP) variants that are sensitive to changes in pH are invaluable reagents for the analysis of protein dynamics associated with both endo- and exocytotic vesicular trafficking. Ratiometric pHluorin is a GFP variant that displays a bimodal excitation spectrum with peaks at 395 and 475 nm and an emission maximum at 509 nm. Upon acidification, pHluorin excitation at 395 nm decreases with a corresponding increase in the excitation at 475 nm. GFP2, a GFP variant that contains mammalianized codons and the folding enhancing mutation F64L, displays ~8-fold higher florescence compared to pHluorin upon excitation at 395 nm. Using GFP2 as a template, an enhanced ratiometric pHluorin (pHluorin2) construct was developed to contain fully mammalianized codons, the F64L mutation and ten of the thirteen pHluorin-specific mutations. As a result, pHluorin2 displays markedly higher florescence when compared to pHluorin while maintaining the ratiometric pH-sensitivity. Unlike native pHluorin, pHluorin2 expressed in the ligand-binding domain of the parathyroid hormone 1 receptor is readily detectable by confocal microscopy and displays a marked increase in florescence upon ligand-induced endocytosis to intracellular vesicles. Thus, pHluorin2’s enhanced florescence while sustaining ratiometric pH-sensitivity represents a significant improvement for this methodological approach.