How to Increase Brightness of Near-Infrared Fluorescent Proteins in Mammalian Cells.
How to Increase Brightness of Near-Infrared Fluorescent Proteins in Mammalian Cells.
复制标题
DOI：
10.1016/j.chembiol.2017.05.018
复制
发表时间：
2017-06-22
影响因子：
8.6
通讯作者：
Verkhusha VV
中科院分区：
文献类型：
--
作者：
Shemetov AA;Oliinyk OS;Verkhusha VV
许多近红外（NIR）荧光蛋白（FP）最近被工程从细菌的光感受器，但缺乏系统的比较，使研究人员的选择相当困难。在这里，我们并排评估了几种现代近红外FP，如蓝移的smURFP和miRFP670，以及红移的mIFP和miRFP703。我们发现，在所有NIR FP中，miRFP670在各种哺乳动物细胞中具有最高的荧光强度。例如，在常见的HeLa细胞中，miRFP703、mIFP和smURFP比miRFP670二聚2倍、9倍和53倍。无论是血红素加氧酶的共表达或与血红素前体细胞孵育弱影响近红外荧光，然而，在后一种情况下，升高的细胞自发荧光。外源添加的发色团显著增加了smURFP亮度，但仅略微增强了其他NIR FP的亮度。mIFP表现出中等水平，而单体miRFP670和miRFP703表现出内源性胆绿素发色团的高结合效率。这一特征使它们易于用作GFP样蛋白，用于与可见光范围的FP进行光谱复用。对活哺乳动物细胞中的现代近红外荧光蛋白进行了系统的侧边比较。Shemetov等人发现，蛋白质初始分子工程的质量是决定其在细胞中荧光的关键因素。
Numerous near-infrared (NIR) fluorescent proteins (FPs) were recently engineered from bacterial photoreceptors but lack of their systematic comparison makes researcher’s choice rather difficult. Here we evaluated side-by-side several modern NIR FPs, such as blue-shifted smURFP and miRFP670, and red-shifted mIFP and miRFP703. We found that among all NIR FPs, miRFP670 had the highest fluorescence intensity in various mammalian cells. For instance in common HeLa cells miRFP703, mIFP and smURFP were 2-, 9- and 53-fold dimmer than miRFP670. Either co-expression of heme oxygenase or incubation of cells with heme precursor weakly affected NIR fluorescence, however, in the latter case elevated cellular autofluorescence. Exogenously added chromophore substantially increased smURFP brightness but only slightly enhanced brightness of other NIR FPs. mIFP showed intermediate while monomeric miRFP670 and miRFP703 exhibited high binding efficiency of endogenous biliverdin chromophore. This feature makes them easy to use as GFP-like proteins for spectral multiplexing with FPs of visible range. A systematic side-to-side comparison of the modern near-infrared fluorescent proteins in live mammalian cells was performed. Shemetov et al. found that the quality of the initial molecular engineering of the proteins is the key factor that determines their fluorescence in cells.
登录
查看更多内容
影响因子：
2.4
作者：
Tran MT;Tanaka J;Hamada M;Sugiyama Y;Sakaguchi S;Nakamura M;Takahashi S;Miwa Y
通讯作者：
Miwa Y
影响因子：
3.7
作者：
Moelzer, Christine;Pfleger, Barbara;Putz, Elisabeth;Rossmann, Antonia;Schwarz, Ursula;Wallner, Marlies;Bulmer, Andrew C.;Wagner, Karl-Heinz
通讯作者：
Wagner, Karl-Heinz
影响因子：
4.6
作者：
Rumyantsev KA;Turoverov KK;Verkhusha VV
通讯作者：
Verkhusha VV
影响因子：
16
作者：
Nakatogawa, H;Ito, K
通讯作者：
Ito, K
影响因子：
15
作者：
Fujii, H;Zhang, XH;Yoshida, T
通讯作者：
Yoshida, T