Examining the Complexity of Human RNA Polymerase Complexes using HaloTag Technology Coupled to Label Free Quantitative Proteomics
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- 作者：Danette L. Daniels ; Jacqui M茅ndez ; Amber L. Mosley ; Sreenivasa R. Ramisetty ; Nancy Murphy ; H茅l猫ne Benink ; Keith V. Wood ; Marjeta Urh ; Michael P. Washburn
- 刊名：Journal of Proteome Research
- 出版年：2012
- 出版时间：February 3, 2012
- 年：2012
- 卷：11
- 期：2
- 页码：564-575
- 全文大小：526K
- 年卷期：v.11,no.2(February 3, 2012)
- ISSN：1535-3907
文摘
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.