Promega Corporation , 2800 Woods Hollow Road, Madison, Wisconsin 53711, United States.
J Proteome Res. 2012 Feb 3;11(2):564-75. doi: 10.1021/pr200459c. Epub 2012 Jan 3.
Efficient determination of protein interactions and cellular localization remains a challenge in higher order eukaryotes and creates a need for robust technologies for functional proteomics studies. To address this, the HaloTag technology was developed for highly efficient and rapid isolation of intracellular complexes and correlative in vivo cellular imaging. Here we demonstrate the strength of this technology by simultaneous capture of human eukaryotic RNA polymerases (RNAP) I, II, and III using a shared subunit, POLR2H, fused to the HaloTag. Affinity purifications showed successful isolation, as determined using quantitative proteomics, of all RNAP core subunits, even at expression levels near endogenous. Transient known RNAP II interacting partners were identified as well as three previously uncharacterized interactors. These interactions were validated and further functionally characterized using cellular imaging. The multiple capabilities of the HaloTag technology demonstrate the ability to efficiently isolate highly challenging multiprotein complexes, discover new interactions, and characterize cellular localization.
高效地确定蛋白质相互作用和细胞定位在高等真核生物中仍然是一个挑战，这就需要强大的技术来进行功能蛋白质组学研究。为了解决这个问题，HaloTag 技术被开发用于高效快速地分离细胞内复合物，并进行相关的体内细胞成像。在这里，我们通过将 POLR2H 融合到 HaloTag 上来同时捕获人类真核 RNA 聚合酶 (RNAP) I、II 和 III 的共享亚基，展示了这项技术的强大之处。使用定量蛋白质组学确定了亲和纯化的成功，即使在接近内源性表达水平时，所有 RNAP 核心亚基都能被成功分离。还鉴定了瞬时已知的 RNAP II 相互作用伙伴以及三个以前未表征的相互作用因子。使用细胞成像验证和进一步功能表征了这些相互作用。HaloTag 技术的多种功能证明了其能够有效地分离极具挑战性的多蛋白复合物、发现新的相互作用，并对细胞定位进行特征描述。
