De Munter Sofie, Görnemann Janina, Derua Rita, Lesage Bart, Qian Junbin, Heroes Ewald, Waelkens Etienne, Van Eynde Aleyde, Beullens Monique, Bollen Mathieu
Laboratory of Biosignaling & Therapeutics, KU Leuven Department of Cellular and Molecular Medicine, University of Leuven, Belgium.
Protein Phosphorylation & Proteomics Lab, KU Leuven Department of Cellular and Molecular Medicine, University of Leuven, Belgium.
FEBS Lett. 2017 Jan;591(2):415-424. doi: 10.1002/1873-3468.12548. Epub 2017 Jan 12.
The biotin identification (BioID) protocol uses a mutant of the biotin ligase BirA (BirA*) fused to a protein-of-interest to biotinylate proximate proteins in intact cells. Here, we show that two inactive halves of BirA* separately fused to a catalytic and regulatory subunit of protein phosphatase PP1 reconstitute a functional BirA* enzyme upon heterodimerization of the phosphatase subunits. We also demonstrate that this BirA* fragment complementation approach, termed split-BioID, can be used to screen for substrates and other protein interactors of PP1 holoenzymes. Split-BioID is a novel and versatile tool for the identification of (transient) interactors of protein dimers.
生物素识别（BioID）实验方案利用与目标蛋白融合的生物素连接酶BirA的突变体（BirA*），在完整细胞中对邻近蛋白进行生物素化标记。在此，我们表明，分别与蛋白磷酸酶PP1的催化亚基和调节亚基融合的BirA的两个无活性片段，在磷酸酶亚基异二聚化时可重新构成功能性BirA酶。我们还证明，这种称为分裂BioID的BirA*片段互补方法，可用于筛选PP1全酶的底物和其他蛋白相互作用分子。分裂BioID是一种用于鉴定蛋白质二聚体（瞬时）相互作用分子的新型通用工具。
