Versatile toolbox for high throughput biochemical and functional studies with fluorescent fusion proteins.
Versatile toolbox for high throughput biochemical and functional studies with fluorescent fusion proteins.
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DOI：
10.1371/journal.pone.0036967
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Hake SB
中科院分区：
文献类型：
--
作者：
Pichler G;Jack A;Wolf P;Hake SB
荧光融合蛋白被广泛用于研究活细胞中蛋白质的定位和相互作用动力学。然而，为了充分表征蛋白质并了解其功能，确定生物化学特性（如酶活性和结合特异性）至关重要。在这里，我们展示了一个简单，可靠和通用的中/高通量的方法来研究荧光融合蛋白的生化和功能特性。使用一个新的系统的基础上96孔微孔板，包括一个固定的GFP结合蛋白（GFP-mulitTrap），我们进行了快速和有效的一步纯化不同的GFP和YFP融合蛋白从粗细胞裂解液。固定后，我们测定了细胞表达的GFP融合蛋白与组蛋白尾肽、DNA或选定的RFP融合蛋白的高度可重复的结合率。特别是，我们发现Cbx 1优先结合到二和三甲基化的H3 K9被废除的磷酸化的相邻的丝氨酸。DNA结合分析表明，MeCP 2的MBD结构域区分完全甲基化的DNA和未甲基化的DNA，蛋白质-蛋白质相互作用研究表明，Dnmt 1的PBD结构域对于结合PCNA是必需的。此外，使用ELISA为基础的方法，我们检测到内源性PCNA和组蛋白H3结合在GFP融合。此外，我们定量了含有不同组蛋白变体的核小体上H3 K4 me 2的水平。总之，我们提出了一种创新的中/高通量方法来分析荧光标记的融合蛋白的结合特异性，并在体外以快速可靠的方式检测内源性相互作用因子。
Fluorescent fusion proteins are widely used to study protein localization and interaction dynamics in living cells. However, to fully characterize proteins and to understand their function it is crucial to determine biochemical characteristics such as enzymatic activity and binding specificity. Here we demonstrate an easy, reliable and versatile medium/high-throughput method to study biochemical and functional characteristics of fluorescent fusion proteins. Using a new system based on 96-well micro plates comprising an immobilized GFP-binding protein (GFP-mulitTrap), we performed fast and efficient one-step purification of different GFP- and YFP-fusion proteins from crude cell lysate. After immobilization we determined highly reproducible binding ratios of cellular expressed GFP-fusion proteins to histone-tail peptides, DNA or selected RFP-fusion proteins. In particular, we found Cbx1 preferentially binding to di-and trimethylated H3K9 that is abolished by phosphorylation of the adjacent serine. DNA binding assays showed, that the MBD domain of MeCP2 discriminates between fully methylated over unmethylated DNA and protein-protein interactions studies demonstrate, that the PBD domain of Dnmt1 is essential for binding to PCNA. Moreover, using an ELISA-based approach, we detected endogenous PCNA and histone H3 bound at GFP-fusions. In addition, we quantified the level of H3K4me2 on nucleosomes containing different histone variants. In summary, we present an innovative medium/high-throughput approach to analyse binding specificities of fluroescently labeled fusion proteins and to detect endogenous interacting factors in a fast and reliable manner in vitro.
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DOI：
10.1083/jcb.149.2.271
发表时间：
2000-04-17
期刊：
The Journal of cell biology
影响因子：
--
作者：
Leonhardt H;Rahn HP;Weinzierl P;Sporbert A;Cremer T;Zink D;Cardoso MC
通讯作者：
Cardoso MC
影响因子：
4.8
作者：
Free, A;Wakefield, RID;Bird, AP
通讯作者：
Bird, AP
影响因子：
5.3
作者：
DUBRIDGE, RB;TANG, P;CALOS, MP
通讯作者：
CALOS, MP
影响因子：
--
作者：
Rottach, Andrea;Kremmer, Elisabeth;Cardoso, M. Cristina
通讯作者：
Cardoso, M. Cristina
影响因子：
56.9
作者：
CHALFIE, M;TU, Y;PRASHER, DC
通讯作者：
PRASHER, DC