Development of a molecularly evolved, highly sensitive CaMKII FRET sensor with improved expression pattern.
Development of a molecularly evolved, highly sensitive CaMKII FRET sensor with improved expression pattern.
复制标题
DOI：
10.1371/journal.pone.0121109
复制
发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Murakoshi H
中科院分区：
文献类型：
--
作者：
Shibata AC;Maebashi HK;Nakahata Y;Nabekura J;Murakoshi H
基因编码荧光共振能量转移（FRET）生物传感器已经成功地用于可视化活细胞中的蛋白质活性。FRET测量的灵敏度和准确性直接取决于生物传感器折叠效率，表达模式，灵敏度和动态范围。为了提高Ca2+/钙调素依赖性蛋白激酶IIα (CaMKIIα) FRET生物传感器的折叠效率，我们利用易出错的聚合酶链反应（PCR）扩增CaMKIIα基因的关联结构域，并在细菌表达载体中将其融合到mCherry的n端。我们还基于先前报道的荧光蛋白折叠报告方法创建了大肠杆菌表达文库，并发现一个亮红色荧光集落包含四个突变的关联结构域（F394L, I419V， A430T和I434T）。使用纯化突变蛋白的体外实验证实了下游荧光蛋白的折叠动力学改善，但不是关联结构域本身。此外，我们将这些突变引入到先前报道的CaMKIIα FRET传感器中，并使用2光子荧光寿命成像显微镜（2pFLIM）监测其在HeLa细胞中的Ca2+/钙调素依赖性激活，发现突变传感器的表达模式和信号可再现性大大提高，而不影响自磷酸化功能和融入低聚CaMKIIα。我们相信我们改进的CaMKIIα FRET传感器将适用于各种类型的细胞和组织，提供高精度和可重复性的数据。此外，这里描述的方法也可用于改善所有现有的FRET传感器的性能。
Genetically encoded fluorescence resonance energy transfer (FRET) biosensors have been successfully used to visualize protein activity in living cells. The sensitivity and accuracy of FRET measurements directly depend on biosensor folding efficiency, expression pattern, sensitivity, and dynamic range. Here, to improve the folding efficiency of the Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) FRET biosensor, we amplified the association domain of the CaMKIIα gene using error-prone polymerase chain reaction (PCR) and fused it to the N-terminus of mCherry in a bacterial expression vector. We also created an Escherichia coli expression library based on a previously reported fluorescent protein folding reporter method, and found a bright red fluorescent colony that contained the association domain with four mutations (F394L, I419V, A430T, and I434T). In vitro assays using the purified mutant protein confirmed improved folding kinetics of the downstream fluorescent protein, but not of the association domain itself. Furthermore, we introduced these mutations into the previously reported CaMKIIα FRET sensor and monitored its Ca2+/calmodulin-dependent activation in HeLa cells using 2-photon fluorescence lifetime imaging microscopy (2pFLIM), and found that the expression pattern and signal reproducibility of the mutant sensor were greatly improved without affecting the autophosphorylation function and incorporation into oligomeric CaMKIIα. We believe that our improved CaMKIIα FRET sensor would be useful in various types of cells and tissues, providing data with high accuracy and reproducibility. In addition, the method described here may also be applicable for improving the performance of all currently available FRET sensors.
登录
查看更多内容
测量树突状刺中的CaMKII浓度。
影响因子：
3
作者：
Otmakhov, Nikolai;Lisman, John
通讯作者：
Lisman, John
影响因子：
34.7
作者：
Lisman, John;Yasuda, Ryohei;Raghavachari, Sridhar
通讯作者：
Raghavachari, Sridhar
影响因子：
3.9
作者：
Pologruto, Thomas A.;Sabatini, Bernardo L.;Svoboda, Karel
通讯作者：
Svoboda, Karel
影响因子：
64.8
作者：
Lee, Seok-Jin R.;Escobedo-Lozoya, Yasmin;Szatmari, Erzsebet M.;Yasuda, Ryohei
通讯作者：
Yasuda, Ryohei
DOI：
10.1016/j.bbrc.2008.02.070
发表时间：
2008-05-02
影响因子：
3.1
作者：
Kwok, Showining;Lee, Claudia;Hayashi, Yasunori
通讯作者：
Hayashi, Yasunori