One-step, non-denaturing isolation of an RNA polymerase enzyme complex using an improved multi-use affinity probe resin.
The rapid isolation of protein complexes is critical to the goal of establishing protein interaction networks. High-throughput methods for identifying protein binding partners in a way suitable for mass spectrometric identification and structural analysis are required and small molecule/peptide interactions provide the key. We have now shown that a redesigned resin derivatized with a bisarsenical dye can be used to isolate the Shewanella oneidensis RNA polymerase core enzyme with a tetracysteine-tagged RNA polymerase A as bait protein. A critical advantage of this method is the ability to release the intact complex using a mild, one-step procedure with a competing dithiol. In addition to the identification of the core complex, additional interaction partners, including universal stress protein, were identified. These results provide a path forward to identifying how changes in critical protein complexes over time modulate cell function.
Uljana Mayer
M
M
Cell Biology and Biochemistry Group, Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99452, USA. Uljana.Mayer@pnl.gov
Shi
Liang
L
Squier
Thomas C
TC
D003545:Cysteine / Q000737:chemistry; D013058:Mass Spectrometry; D012318:RNA Polymerase I / Q000302:isolation & purification*; D012117:Resins, Synthetic; D020592:Shewanella / Q000737:chemistry*; D014050:Toluene / Q000031:analogs & derivatives / Q000737:chemistry
D012117:Resins, Synthetic; D014050:Toluene; D012318:RNA Polymerase I; D003545:Cysteine; C004848:dithiol